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  • Longitudinal within-host evolution of HIV Nef-mediated CD4, HLA and SERINC5 downregulation activity: a case study
    Retrovirology (IF 3.744) Pub Date : 2020-01-09
    Hanwei Sudderuddin; Natalie N. Kinloch; Steven W. Jin; Rachel L. Miller; Bradley R. Jones; Chanson J. Brumme; Jeffrey B. Joy; Mark A. Brockman; Zabrina L. Brumme

    The HIV accessory protein Nef downregulates the viral entry receptor CD4, the Human Leukocyte Antigen (HLA)-A and -B molecules, the Serine incorporator 5 (SERINC5) protein and other molecules from the infected cell surface, thereby promoting viral infectivity, replication and immune evasion. The nef locus also represents one of the most genetically variable regions in the HIV genome, and nef sequences undergo substantial evolution within a single individual over the course of infection. Few studies however have simultaneously characterized the impact of within-host nef sequence evolution on Nef protein function over prolonged timescales. Here, we isolated 50 unique Nef clones by single-genome amplification over an 11-year period from the plasma of an individual who was largely naïve to antiretroviral treatment during this time. Together, these clones harbored nonsynonymous substitutions at 13% of nef’s codons. We assessed their ability to downregulate cell-surface CD4, HLA and SERINC5 and observed that all three Nef functions declined modestly over time, where the reductions in CD4 and HLA downregulation (an average of 0.6% and 2.0% per year, respectively) achieved statistical significance. The results from this case study support all three Nef activities as being important to maintain throughout untreated HIV infection, but nevertheless suggest that, despite nef’s mutational plasticity, within-host viral evolution can compromise Nef function, albeit modestly, over prolonged periods.

    更新日期:2020-01-11
  • HTLV-1 bZIP factor: the key viral gene for pathogenesis
    Retrovirology (IF 3.744) Pub Date : 2020-01-08
    Masao Matsuoka; Jean-Michel Mesnard

    Human T cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATL) and inflammatory diseases. The HTLV-1 bZIP factor (HBZ) gene is constantly expressed in HTLV-1 infected cells and ATL cells. HBZ protein suppresses transcription of the tax gene through blocking the LTR recruitment of not only ATF/CREB factors but also CBP/p300. HBZ promotes transcription of Foxp3, CCR4, and T-cell immunoreceptor with Ig and ITIM domains (TIGIT). Thus, HBZ is critical for the immunophenotype of infected cells and ATL cells. HBZ also functions in its RNA form. HBZ RNA suppresses apoptosis and promotes proliferation of T cells. Since HBZ RNA is not recognized by cytotoxic T cells, HTLV-1 has a clever strategy for avoiding immune detection. HBZ plays central roles in maintaining infected T cells in vivo and determining their immunophenotype.

    更新日期:2020-01-09
  • An insight to HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) pathogenesis; evidence from high-throughput data integration and meta-analysis
    Retrovirology (IF 3.744) Pub Date : 2019-12-30
    Sayed-Hamidreza Mozhgani; Mehran Piran; Mohadeseh Zarei-Ghobadi; Mohieddin Jafari; Seyed-Mohammad Jazayeri; Talat Mokhtari-Azad; Majid Teymoori-Rad; Narges Valizadeh; Hamid Farajifard; Mehdi Mirzaie; Azam Khamseh; Houshang Rafatpanah; Seyed-Abdolrahim Rezaee; Mehdi Norouzi

    Human T-lymphotropic virus 1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a progressive disease of the central nervous system that significantly affected spinal cord, nevertheless, the pathogenesis pathway and reliable biomarkers have not been well determined. This study aimed to employ high throughput meta-analysis to find major genes that are possibly involved in the pathogenesis of HAM/TSP. High-throughput statistical analyses identified 832, 49, and 22 differentially expressed genes for normal vs. ACs, normal vs. HAM/TSP, and ACs vs. HAM/TSP groups, respectively. The protein–protein interactions between DEGs were identified in STRING and further network analyses highlighted 24 and 6 hub genes for normal vs. HAM/TSP and ACs vs. HAM/TSP groups, respectively. Moreover, four biologically meaningful modules including 251 genes were identified for normal vs. ACs. Biological network analyses indicated the involvement of hub genes in many vital pathways like JAK-STAT signaling pathway, interferon, Interleukins, and immune pathways in the normal vs. HAM/TSP group and Metabolism of RNA, Viral mRNA Translation, Human T cell leukemia virus 1 infection, and Cell cycle in the normal vs. ACs group. Moreover, three major genes including STAT1, TAP1, and PSMB8 were identified by network analysis. Real-time PCR revealed the meaningful down-regulation of STAT1 in HAM/TSP samples than AC and normal samples (P = 0.01 and P = 0.02, respectively), up-regulation of PSMB8 in HAM/TSP samples than AC and normal samples (P = 0.04 and P = 0.01, respectively), and down-regulation of TAP1 in HAM/TSP samples than those in AC and normal samples (P = 0.008 and P = 0.02, respectively). No significant difference was found among three groups in terms of the percentage of T helper and cytotoxic T lymphocytes (P = 0.55 and P = 0.12). High-throughput data integration disclosed novel hub genes involved in important pathways in virus infection and immune systems. The comprehensive studies are needed to improve our knowledge about the pathogenesis pathways and also biomarkers of complex diseases.

    更新日期:2019-12-31
  • HTLV-1 infection of myeloid cells: from transmission to immune alterations
    Retrovirology (IF 3.744) Pub Date : 2019-12-23
    Brenda Rocamonde; Auriane Carcone; Renaud Mahieux; Hélène Dutartre

    Human T cell leukemia virus type 1 (HTLV-1), the etiological agent of adult T-cell leukemia/lymphoma (ATLL) and the demyelinating neuroinflammatory disease known as HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP), was the first human retrovirus to be discovered. T-cells, which represent the main reservoir for HTLV-1, have been the main focus of studies aimed at understanding viral transmission and disease progression. However, other cell types such as myeloid cells are also target of HTLV-1 infection and display functional alterations as a consequence. In this work, we review the current investigations that shed light on infection, transmission and functional alterations subsequent to HTLV-1 infection of the different myeloid cells types, and we highlight the lack of knowledge in this regard.

    更新日期:2019-12-23
  • HTLV-1 CTCF-binding site is dispensable for in vitro immortalization and persistent infection in vivo
    Retrovirology (IF 3.744) Pub Date : 2019-12-21
    Michael P. Martinez; Xiaogang Cheng; Ancy Joseph; Jacob Al-Saleem; Amanda R. Panfil; Marilly Palettas; Wessel P. Dirksen; Lee Ratner; Patrick L. Green

    Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATL) and the neurological disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The exact mechanism(s) through which latency and disease progression are regulated are not fully understood. CCCTC-binding factor (CTCF) is an 11-zinc finger, sequence-specific, DNA-binding protein with thousands of binding sites throughout mammalian genomes. CTCF has been shown to play a role in organization of higher-order chromatin structure, gene expression, genomic imprinting, and serve as a barrier to epigenetic modification. A viral CTCF-binding site (vCTCF-BS) was previously identified within the overlapping p12 (sense) and Hbz (antisense) genes of the HTLV-1 genome. Thus, upon integration, HTLV-1 randomly inserts a vCTCF-BS into the host genome. vCTCF-BS studies to date have focused primarily on HTLV-1 chronically infected or tumor-derived cell lines. In these studies, HTLV-1 was shown to alter the structure and transcription of the surrounding host chromatin through the newly inserted vCTCF-BS. However, the effects of CTCF binding in the early stages of HTLV-1 infection remains unexplored. This study examines the effects of the vCTCF-BS on HTLV-1-induced in vitro immortalization and in vivo viral persistence in infected rabbits. HTLV-1 and HTLV-1∆CTCF LTR-transactivation, viral particle production, and immortalization capacity were comparable in vitro. The total lymphocyte count, proviral load, and Hbz gene expression were not significantly different between HTLV-1 and HTLV-1∆CTCF-infected rabbits throughout a 12 week study. However, HTLV-1∆CTCF-infected rabbits displayed a significantly decreased HTLV-1-specific antibody response compared to HTLV-1-infected rabbits. Mutation of the HTLV-1 vCTCF-BS does not significantly alter T-lymphocyte transformation capacity or early in vivo virus persistence, but results in a decreased HTLV-1-specific antibody response during early infection in rabbits. Ultimately, understanding epigenetic regulation of HTLV-1 gene expression and pathogenesis could provide meaningful insights into mechanisms of immune evasion and novel therapeutic targets.

    更新日期:2019-12-21
  • p30 protein: a critical regulator of HTLV-1 viral latency and host immunity
    Retrovirology (IF 3.744) Pub Date : 2019-12-18
    Ramona Moles; Sarkis Sarkis; Veronica Galli; Maria Omsland; Damian F. J. Purcell; David Yurick; Georges Khoury; Cynthia A. Pise-Masison; Genoveffa Franchini

    The extraordinarily high prevalence of HTLV-1 subtype C (HTLV-1C) in some isolated indigenous communities in Oceania and the severity of the health conditions associated with the virus impress the great need for basic and translational research to prevent and treat HTLV-1 infection. The genome of the virus’s most common subtype, HTLV-1A, encodes structural, enzymatic, and regulatory proteins that contribute to viral persistence and pathogenesis. Among these is the p30 protein encoded by the doubly spliced Tax-orf II mRNA, a nuclear/nucleolar protein with both transcriptional and post-transcriptional activity. The p30 protein inhibits the productive replication cycle via nuclear retention of the mRNA that encodes for both the viral transcriptional trans-activator Tax, and the Rex proteins that regulate the transport of incompletely spliced viral mRNA to the cytoplasm. In myeloid cells, p30 inhibits the PU-1 transcription factor that regulates interferon expression and is a critical mediator of innate and adaptive immunity. Furthermore, p30 alters gene expression, cell cycle progression, and DNA damage responses in T-cells, raising the hypothesis that p30 may directly contribute to T cell transformation. By fine-tuning viral expression while also inhibiting host innate responses, p30 is likely essential for viral infection and persistence. This concept is supported by the finding that macaques, a natural host for the closely genetically related simian T-cell leukemia virus 1 (STLV-1), exposed to an HTLV-1 knockout for p30 expression by a single point mutation do not became infected unless reversion and selection of the wild type HTLV-1 genotype occurs. All together, these data suggest that inhibition of p30 may help to curb and eventually eradicate viral infection by exposing infected cells to an effective host immune response.

    更新日期:2019-12-19
  • Role of HTLV-1 orf-I encoded proteins in viral transmission and persistence
    Retrovirology (IF 3.744) Pub Date : 2019-12-18
    Sarkis Sarkis; Veronica Galli; Ramona Moles; David Yurick; Georges Khoury; Damian F. J. Purcell; Genoveffa Franchini; Cynthia A. Pise-Masison

    The human T cell leukemia virus type 1 (HTVL-1), first reported in 1980 by Robert Gallo’s group, is the etiologic agent of both cancer and inflammatory diseases. Despite approximately 40 years of investigation, the prognosis for afflicted patients remains poor with no effective treatments. The virus persists in the infected host by evading the host immune response and inducing proliferation of infected CD4+ T-cells. Here, we will review the role that viral orf-I protein products play in altering intracellular signaling, protein expression and cell–cell communication in order to escape immune recognition and promote T-cell proliferation. We will also review studies of orf-I mutations found in infected patients and their potential impact on viral load, transmission and persistence. Finally, we will compare the orf-I gene in HTLV-1 subtypes as well as related STLV-1.

    更新日期:2019-12-19
  • HTLV-1 contains a high CG dinucleotide content and is susceptible to the host antiviral protein ZAP
    Retrovirology (IF 3.744) Pub Date : 2019-12-16
    Paola Miyazato; Misaki Matsuo; Benjy J. Y. Tan; Michiyo Tokunaga; Hiroo Katsuya; Saiful Islam; Jumpei Ito; Yasuhiro Murakawa; Yorifumi Satou

    Human T cell leukaemia virus type 1 (HTLV-1) is a retrovirus associated with human diseases such as adult T-cell leukaemia/lymphoma and HTLV-1 associated myelopathy/tropical spastic paraparesis. In contrast to another human retrovirus, human immunodeficiency virus type 1 (HIV-1), HTLV-1 persists in the host not via vigorous virus production but mainly via proliferation and/or long-term survival in the form of silent proviruses in infected host cells. As a result, HTLV-1-infected cells rarely produce virus particles in vivo even without anti-retroviral treatment. That should be an advantage for the virus to escape from the host immune surveillance by minimizing the expression of viral antigens in host cells. However, why HIV-1 and HTLV-1 behave so differently during natural infection is not fully understood. We performed cap analysis of gene expression (CAGE) using total RNAs and nascent, chromatin-associated, RNAs in the nucleus and found that HTLV-1 RNAs were processed post-transcriptionally in infected cells. RNA processing was evident for the sense viral transcripts but not the anti-sense ones. We also found a higher proportion of CG di-nucleotides in proviral sequences of HTLV-1-infected cells, when compared to the HIV-1 genomic sequence. It has been reported recently that CG dinucleotide content of viral sequence is associated with susceptibility to the antiviral ZC3HAV1 (ZAP), suggesting the involvement of this protein in the regulation of HTLV-1 transcripts. To analyse the effect of ZAP on HTLV-1 transcripts, we over-expressed it in HTLV-1-infected cells. We found there was a dose-dependent reduction in virus production with ZAP expression. We further knocked down endogenous ZAP with two independent targeting siRNAs and observed a significant increase in virus production in the culture supernatant. Other delta-type retroviruses such as simian T-cell leukaemia virus and bovine leukaemia virus, also contain high CG-dinucleotide contents in their viral genomes, suggesting that ZAP-mediated suppression of viral transcripts might be a common feature of delta-type retroviruses, which cause minimal viremia in their natural hosts. The post-transcriptional regulatory mechanism involving ZAP might allow HTLV-1 to maintain a delicate balance required for prolonged survival in infected individuals.

    更新日期:2019-12-17
  • Molecular epidemiology, genetic variability and evolution of HTLV-1 with special emphasis on African genotypes
    Retrovirology (IF 3.744) Pub Date : 2019-12-16
    Philippe V. Afonso; Olivier Cassar; Antoine Gessain

    Human T cell leukemia virus (HTLV-1) is an oncoretrovirus that infects at least 10 million people worldwide. HTLV-1 exhibits a remarkable genetic stability, however, viral strains have been classified in several genotypes and subgroups, which often mirror the geographic origin of the viral strain. The Cosmopolitan genotype HTLV-1a, can be subdivided into geographically related subgroups, e.g. Transcontinental (a-TC), Japanese (a-Jpn), West-African (a-WA), North-African (a-NA), and Senegalese (a-Sen). Within each subgroup, the genetic diversity is low. Genotype HTLV-1b is found in Central Africa; it is the major genotype in Gabon, Cameroon and Democratic Republic of Congo. While strains from the HTLV-1d genotype represent only a few percent of the strains present in Central African countries, genotypes -e, -f, and -g have been only reported sporadically in particular in Cameroon Gabon, and Central African Republic. HTLV-1c genotype, which is found exclusively in Australo-Melanesia, is the most divergent genotype. This reflects an ancient speciation, with a long period of isolation of the infected populations in the different islands of this region (Australia, Papua New Guinea, Solomon Islands and Vanuatu archipelago). Until now, no viral genotype or subgroup is associated with a specific HTLV-1-associated disease. HTLV-1 originates from a simian reservoir (STLV-1); it derives from interspecies zoonotic transmission from non-human primates to humans (ancient or recent). In this review, we describe the genetic diversity of HTLV-1, and analyze the molecular mechanisms that are at play in HTLV-1 evolution. Similar to other retroviruses, HTLV-1 evolves either through accumulation of point mutations or recombination. Molecular studies point to a fairly low evolution rate of HTLV-1 (between 5.6E−7 and 1.5E−6 substitutions/site/year), supposedly because the virus persists within the host via clonal expansion (instead of new infectious cycles that use reverse transcriptase).

    更新日期:2019-12-17
  • HIV-1 Rev interacts with HERV-K RcREs present in the human genome and promotes export of unspliced HERV-K proviral RNA
    Retrovirology (IF 3.744) Pub Date : 2019-12-16
    Laurie R. Gray; Rachel E. Jackson; Patrick E. H. Jackson; Stefan Bekiranov; David Rekosh; Marie-Louise Hammarskjöld

    The HERV-K (HML-2) viruses are the youngest of the human endogenous retroviruses. They are present as several almost complete proviral copies and numerous fragments in the human genome. Many HERV-K proviruses express a regulatory protein Rec, which binds to an element present in HERV-K mRNAs called the RcRE. This interaction is necessary for the nucleo-cytoplasmic export and expression of HERV-K mRNAs that retain introns and plays a role analogous to that of Rev and the RRE in HIV replication. There are over 900 HERV-K RcREs distributed throughout the human genome. Thus, it was of interest to determine if Rev could functionally interact with selected RcRE elements that map either to HERV-K proviruses or human gene regions. This interaction would have the potential to alter the expression of both HERV-K mRNAs and cellular mRNAs during HIV-1 infection. In this study we employed a combination of RNAseq, bioinformatics and cell-based functional assays. Potential RcREs were identified through a number of bioinformatic approaches. They were then tested for their ability to promote export and translation of a reporter mRNA with a retained intron in conjunction with Rev or Rec. Some of the selected elements functioned well with either Rev, Rec or both, whereas some showed little or no function. Rev function on individual RcREs varied and was also dependent on the Rev sequence. We also performed RNAseq on total and cytoplasmic RNA isolated from SupT1 cells expressing HIV Rev, with or without Tat, or HERV-K Rec. Proviral mRNA from three HERV-K loci (4p16.1b, 22q11.23 and most significantly 3q12.3) accumulated in the cytoplasm in the presence of Rev or Tat and Rev, but not Rec. Consistent with this, the 3′ RcRE from 3q12.3 functioned well with HIV-Rev in our reporter assay. In contrast, this RcRE showed little or no function with Rec. The HIV Rev protein can functionally interact with many RcREs present in the human genome, depending on the RcRE sequence, as well as the Rev sequence. This leads to export of some of the HERV-K proviral mRNAs and also has the potential to change the expression of non-viral genes.

    更新日期:2019-12-17
  • STLV-1 as a model for studying HTLV-1 infection
    Retrovirology (IF 3.744) Pub Date : 2019-12-16
    Brice Jégado; Fatah Kashanchi; Hélène Dutartre; Renaud Mahieux

    Few years after HTLV-1 identification and isolation in humans, STLV-1, its simian counterpart, was discovered. It then became clear that STLV-1 is present almost in all simian species. Subsequent molecular epidemiology studies demonstrated that, apart from HTLV-1 subtype A, all human subtypes have a simian homolog. As HTLV-1, STLV-1 is the etiological agent of ATL, while no case of TSP/HAM has been described. Given its similarities with HTLV-1, STLV-1 represents a unique tool used for performing clinical studies, vaccine studies as well as basic science.

    更新日期:2019-12-17
  • Restriction factors in human retrovirus infections and the unprecedented case of CIITA as link of intrinsic and adaptive immunity against HTLV-1
    Retrovirology (IF 3.744) Pub Date : 2019-11-29
    Greta Forlani; Mariam Shallak; Elise Ramia; Alessandra Tedeschi; Roberto S. Accolla

    Immunity against pathogens evolved through complex mechanisms that only for sake of simplicity are defined as innate immunity and adaptive immunity. Indeed innate and adaptive immunity are strongly intertwined each other during evolution. The complexity is further increased by intrinsic mechanisms of immunity that rely on the action of intracellular molecules defined as restriction factors (RFs) that, particularly in virus infections, counteract the action of pathogen gene products acting at different steps of virus life cycle. Here we provide an overview on the nature and the mode of action of restriction factors involved in retrovirus infection, particularly Human T Leukemia/Lymphoma Virus 1 (HTLV-1) infection. As it has been extensively studied by our group, special emphasis is given to the involvement of the MHC class II transactivator CIITA discovered in our laboratory as regulator of adaptive immunity and subsequently as restriction factor against HIV-1 and HTLV-1, a unique example of dual function linking adaptive and intrinsic immunity during evolution. We describe the multiple molecular mechanisms through which CIITA exerts its restriction on retroviruses. Of relevance, we review the unprecedented findings pointing to a concerted action of several restriction factors such as CIITA, TRIM22 and TRIM19/PML in synergizing against retroviral replication. Finally, as CIITA profoundly affects HTLV-1 replication by interacting and inhibiting the function of HTLV-1 Tax-1 molecule, the major viral product associated to the virus oncogenicity, we also put forward the hypothesis of CIITA as counteractor of HTLV-1-mediated cancer initiation.

    更新日期:2019-11-30
  • Immunovirological markers in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP)
    Retrovirology (IF 3.744) Pub Date : 2019-11-29
    Yoshimi Enose-Akahata; Steven Jacobson

    Human T cell lymphotropic virus 1 (HTLV-1) is a human retrovirus and infects approximately 10–20 million people worldwide. While the majority of infected people are asymptomatic carriers of HTLV-1, only 4% of infected people develop HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HAM/TSP is a chronic, progressive, neurological disease which usually progresses slowly without remission, and is characterized by perivascular inflammatory infiltrates in chronic inflammatory lesions of the central nervous system (CNS), primarily affecting the spinal cord. A high HTLV-1 proviral load, high levels of antibodies against HTLV-1 antigens, and elevated concentration of proteins are detected in cerebrospinal fluid (CSF) of HAM/TSP patients. These chronically activated immune responses against HTLV-1 and infiltration of inflammatory cells including HTLV-1 infected cells into the CNS contribute to clinical disability and underlie the pathogenesis of HAM/TSP. Since the disease development of HAM/TSP mainly occurs in adults, with a mean age at onset of 40–50 years, it is important for HTLV-1-infected carriers and HAM/TSP patients to be monitored throughout the disease process. Recent advances in technologies and findings provide new insights to virological and immunological aspects in both the CNS as well as in peripheral blood. In this review, we focus on understanding the inflammatory milieu in the CNS and discuss the immunopathogenic process in HTLV-1-associated neurologic diseases.

    更新日期:2019-11-30
  • Potent suppression of HIV-1 cell attachment by Kudzu root extract
    Retrovirology (IF 3.744) Pub Date : 2018-09-20
    S. Mediouni; J. A. Jablonski; S. Tsuda; A. Richard; C. Kessing; M. V. Andrade; A. Biswas; Y. Even; T. Tellinghuisen; H. Choe; M. Cameron; M. Stevenson; S. T. Valente

    There is a constant need to improve antiretrovirals against HIV since therapy is limited by cost, side effects and the emergence of drug resistance. Kudzu is a climbing vine from which the root extract (Pueraria lobata), rich in isoflavones and saponins, has long been used in traditional Chinese medicine for a variety of purposes, from weight loss to alcoholism prevention. Here we show that Kudzu root extract significantly inhibits HIV-1 entry into cell lines, primary human CD4+T lymphocytes and macrophages, without cell-associated toxicity. Specifically, Kudzu inhibits the initial attachment of the viral particle to the cell surface, a mechanism that depends on the envelope glycoprotein gp120 but is independent from the HIV-1 cell receptor CD4 and co-receptors CXCR4/CCR5. This activity seems selective to lentiviruses since Kudzu inhibits HIV-2 and simian immunodeficiency virus, but does not interfere with Hepatitis C, Influenza, Zika Brazil and adenovirus infection. Importantly, depending on the dose, Kudzu can act synergistically or additively with the current antiretroviral cocktails against HIV-1 and can block viruses resistant to the fusion inhibitor Enfuvirtide. Together our results highlight Kudzu’s root extract value as a supplement to current antiretroviral therapy against HIV.

    更新日期:2019-11-28
  • Cellular RelB interacts with the transactivator Tat and enhance HIV-1 expression
    Retrovirology (IF 3.744) Pub Date : 2018-09-21
    Meng Wang; Wei Yang; Yu Chen; Jian Wang; Juan Tan; Wentao Qiao

    Human immunodeficiency virus type 1 (HIV-1) Tat protein plays an essential role in HIV-1 gene transcription. Tat transactivates HIV-1 long terminal repeat (LTR)-directed gene expression through direct interactions with the transactivation-responsive region (TAR) element and other cis elements in the LTR. The TAR-independent Tat-mediated LTR transactivation is modulated by several host factors, but the mechanism is not fully understood. Here, we report that Tat interacts with the Rel homology domain of RelB through its core region. Furthermore, RelB significantly increases Tat-mediated transcription of the HIV-1 LTR and viral gene expression, which is independent of the TAR. Both Tat and RelB are recruited to the HIV-1 promoter, of which RelB facilitates the recruitment of Tat to the viral LTR. The NF-κB elements are key to the accumulation of Tat and RelB on the LTR. Knockout of RelB reduces the accumulation of RNA polymerase II on the LTR, and decreases HIV-1 gene transcription. Together, our data suggest that RelB contributes to HIV-1 transactivation. Our results demonstrate that RelB interacts with Tat and enhances TAR-independent activation of HIV-1 LTR promoter, which adds new insights into the multi-layered mechanisms of Tat in regulating the gene expression of HIV-1.

    更新日期:2019-11-28
  • Adeno-associated virus gene delivery of broadly neutralizing antibodies as prevention and therapy against HIV-1
    Retrovirology (IF 3.744) Pub Date : 2018-10-01
    Allen Lin; Alejandro B. Balazs

    Vectored gene delivery of HIV-1 broadly neutralizing antibodies (bNAbs) using recombinant adeno-associated virus (rAAV) is a promising alternative to conventional vaccines for preventing new HIV-1 infections and for therapeutically suppressing established HIV-1 infections. Passive infusion of single bNAbs has already shown promise in initial clinical trials to temporarily decrease HIV-1 load in viremic patients, and to delay viral rebound from latent reservoirs in suppressed patients during analytical treatment interruptions of antiretroviral therapy. Long-term, continuous, systemic expression of such bNAbs could be achieved with a single injection of rAAV encoding antibody genes into muscle tissue, which would bypass the challenges of eliciting such bNAbs through traditional vaccination in naïve patients, and of life-long repeated passive transfers of such biologics for therapy. rAAV delivery of single bNAbs has already demonstrated protection from repeated HIV-1 vaginal challenge in humanized mouse models, and phase I clinical trials of this approach are underway. Selection of which individual, or combination of, bNAbs to deliver to counter pre-existing resistance and the rise of escape mutations in the virus remains a challenge, and such choices may differ depending on use of this technology for prevention versus therapy.

    更新日期:2019-11-28
  • RNA-induced epigenetic silencing inhibits HIV-1 reactivation from latency
    Retrovirology (IF 3.744) Pub Date : 2018-10-04
    Catalina Méndez; Scott Ledger; Kathy Petoumenos; Chantelle Ahlenstiel; Anthony D. Kelleher

    Current antiretroviral therapy is effective in controlling HIV-1 infection. However, cessation of therapy is associated with rapid return of viremia from the viral reservoir. Eradicating the HIV-1 reservoir has proven difficult with the limited success of latency reactivation strategies and reflects the complexity of HIV-1 latency. Consequently, there is a growing need for alternate strategies. Here we explore a “block and lock” approach for enforcing latency to render the provirus unable to restart transcription despite exposure to reactivation stimuli. Reactivation of transcription from latent HIV-1 proviruses can be epigenetically blocked using promoter-targeted shRNAs to prevent productive infection. We aimed to determine if independent and combined expression of shRNAs, PromA and 143, induce a repressive epigenetic profile that is sufficiently stable to protect latently infected cells from HIV-1 reactivation when treated with a range of latency reversing agents (LRAs). J-Lat 9.2 cells, a model of HIV-1 latency, expressing shRNAs PromA, 143, PromA/143 or controls were treated with LRAs to evaluate protection from HIV-1 reactivation as determined by levels of GFP expression. Cells expressing shRNA PromA, 143, or both, showed robust resistance to viral reactivation by: TNF, SAHA, SAHA/TNF, Bryostatin/TNF, DZNep, and Chaetocin. Given the physiological importance of TNF, HIV-1 reactivation was induced by TNF (5 ng/mL) and ChIP assays were performed to detect changes in expression of epigenetic markers within chromatin in both sorted GFP− and GFP+ cell populations, harboring latent or reactivated proviruses, respectively. Ordinary two-way ANOVA analysis used to identify interactions between shRNAs and chromatin marks associated with repressive or active chromatin in the integrated provirus revealed significant changes in the levels of H3K27me3, AGO1 and HDAC1 in the LTR, which correlated with the extent of reduced proviral reactivation. The cell line co-expressing shPromA and sh143 consistently showed the least reactivation and greatest enrichment of chromatin compaction indicators. The active maintenance of epigenetic silencing by shRNAs acting on the HIV-1 LTR impedes HIV-1 reactivation from latency. Our “block and lock” approach constitutes a novel way of enforcing HIV-1 “super latency” through a closed chromatin architecture that renders the virus resistant to a range of latency reversing agents.

    更新日期:2019-11-28
  • Friend retrovirus infection induces the development of memory-like natural killer cells
    Retrovirology (IF 3.744) Pub Date : 2018-10-06
    Elisabeth Littwitz-Salomon; Thanh Nguyen; Simone Schimmer; Ulf Dittmer

    Traditionally, NK cells belong to the innate immune system and eliminate virus-infected cells through their germline-encoded receptors. However, NK cells were recently reported to possess memory-like functions that were predominantly provided by hepatic NK cells. Memory properties were mainly documented in contact hypersensitivity models or during cytomegalovirus infections. However, the precise role and the physiologic importance of memory-like NK cells during retroviral infections are still under investigation. Here, we show that Friend retrovirus (FV) infection of mice induced a population of phenotypically memory-like NK cells at 28 days post infection. Upon secondary antigen encounter, these NK cells showed an increased production of the pro-inflammatory cytokines IFNγ and TNFα as well as the death ligand FasL in comparison to naïve NK cells. Furthermore, we found an augmented elimination of antigen-matched but not antigen-mismatched target cells by these memory-like NK cells. In adoptive cell transfer experiments, equal antiviral activities of splenic and hepatic memory-like NK cells during the late phase of acute FV infection were found. Our results strongly imply the existence and antiviral activity of spleen and liver memory-like NK cells in FV infection, which efficiently respond upon secondary exposure to retroviral antigens.

    更新日期:2019-11-28
  • Host SAMHD1 protein restricts endogenous reverse transcription of HIV-1 in nondividing macrophages
    Retrovirology (IF 3.744) Pub Date : 2018-10-13
    Bijan Mahboubi; Christina Gavegnano; Dong-Hyun Kim; Raymond F. Schinazi; Baek Kim

    SAM domain and HD domain containing protein 1 (SAMHD1) is a host anti-HIV-1 restriction factor known to suppress viral reverse transcription in nondividing myeloid cells by its dNTP triphosphorylase activity that depletes cellular dNTPs. However, HIV-2 and some SIV strains rapidly replicate in macrophages due to their accessory protein, viral protein X (Vpx), which proteosomally degrades SAMHD1 and elevates dNTP levels. Endogenous reverse transcription (ERT) of retroviruses is the extra-cellular reverse transcription step that partially synthesizes proviral DNAs within cell-free viral particles before the viruses infect new cells. ERT activity utilizes dNTPs co-packaged during budding from the virus-producing cells, and high ERT activity is known to enhance HIV-1 infectivity in nondividing cells. Here, since Vpx elevates cellular dNTP levels in macrophages, we hypothesize that HIV-2 should contain higher ERT activity than HIV-1 in macrophages, and that the Vpx-mediated dNTP elevation should enhance both ERT activity and infectivity of HIV-1 particles produced in macrophages. Here, we demonstrate that HIV-2 produced from human primary monocyte derived macrophages displays higher ERT activity than HIV-1 produced from macrophages. Also, HIV-1 particles produced from macrophages treated with virus like particles (VLPs) containing Vpx, Vpx (+), displayed large increases of ERT activity with the enhanced copy numbers of early, middle and late reverse transcription products within the viral particles, compared to the viruses produced from macrophages treated with Vpx (−) VLPs. Furthermore, upon the infection with an equal p24 amount to fresh macrophages, the viruses produced from the Vpx (+) VLP treated macrophages demonstrated higher infectivity than the viruses from the Vpx (−) VLP treated macrophages. This finding identifies the viral ERT step as an additional step of HIV-1 replication cycle that SAMHD1 restricts in nondividing myeloid target cells.

    更新日期:2019-11-28
  • The expanding array of HIV broadly neutralizing antibodies
    Retrovirology (IF 3.744) Pub Date : 2018-10-16
    Laura E. McCoy

    A large array of broadly neutralizing antibodies (bnAbs) against HIV have been isolated and described, particularly in the last decade. This continually expanding array of bnAbs has crucially led to the identification of novel epitopes on the HIV envelope protein via which antibodies can block a broad range of HIV strains. Moreover, these studies have produced high-resolution understanding of these sites of vulnerability on the envelope protein. They have also clarified the mechanisms of action of bnAbs and provided detailed descriptions of B cell ontogenies from which they arise. However, it is still not possible to predict which HIV-infected individuals will go onto develop breath nor is it possible to induce neutralization breadth by immunization in humans. This review aims to discuss the major insights gained so far and also to evaluate the requirement to continue isolating and characterizing new bnAbs. While new epitopes may remain to be uncovered, a clearer probable benefit of further bnAb characterization is a greater understanding of key decision points in bnAb development within the anti-HIV immune response. This in turn may lead to new insights into how to trigger bnAbs by immunization and more clearly define the challenges to using bnAbs as therapeutic agents.

    更新日期:2019-11-28
  • The role of integration and clonal expansion in HIV infection: live long and prosper
    Retrovirology (IF 3.744) Pub Date : 2018-10-23
    Elizabeth M. Anderson; Frank Maldarelli

    Integration of viral DNA into the host genome is a central event in the replication cycle and the pathogenesis of retroviruses, including HIV. Although most cells infected with HIV are rapidly eliminated in vivo, HIV also infects long-lived cells that persist during combination antiretroviral therapy (cART). Cells with replication competent HIV proviruses form a reservoir that persists despite cART and such reservoirs are at the center of efforts to eradicate or control infection without cART. The mechanisms of persistence of these chronically infected long-lived cells is uncertain, but recent research has demonstrated that the presence of the HIV provirus has enduring effects on infected cells. Cells with integrated proviruses may persist for many years, undergo clonal expansion, and produce replication competent HIV. Even proviruses with defective genomes can produce HIV RNA and may contribute to ongoing HIV pathogenesis. New analyses of HIV infected cells suggest that over time on cART, there is a shift in the composition of the population of HIV infected cells, with the infected cells that persist over prolonged periods having proviruses integrated in genes associated with regulation of cell growth. In several cases, strong evidence indicates the presence of the provirus in specific genes may determine persistence, proliferation, or both. These data have raised the intriguing possibility that after cART is introduced, a selection process enriches for cells with proviruses integrated in genes associated with cell growth regulation. The dynamic nature of populations of cells infected with HIV during cART is not well understood, but is likely to have a profound influence on the composition of the HIV reservoir with critical consequences for HIV eradication and control strategies. As such, integration studies will shed light on understanding viral persistence and inform eradication and control strategies. Here we review the process of HIV integration, the role that integration plays in persistence, clonal expansion of the HIV reservoir, and highlight current challenges and outstanding questions for future research.

    更新日期:2019-11-28
  • Distinct gene expression signatures induced by viral transactivators of different HTLV-1 subgroups that confer a different risk of HAM/TSP
    Retrovirology (IF 3.744) Pub Date : 2018-11-06
    Tadasuke Naito; Jun-ichirou Yasunaga; Yuichi Mitobe; Kazumasa Shirai; Hiroe Sejima; Hiroshi Ushirogawa; Yuetsu Tanaka; Tatsufumi Nakamura; Kousuke Hanada; Masahiro Fujii; Masao Matsuoka; Mineki Saito

    Among human T cell leukemia virus type 1 (HTLV-1)-infected individuals, there is an association between HTLV-1 tax subgroups (subgroup-A or subgroup-B) and the risk of HAM/TSP in the Japanese population. To investigate the role of HTLV-1 subgroups in viral pathogenesis, we studied the functional difference in the subgroup-specific viral transcriptional regulators Tax and HBZ using microarray analysis, reporter gene assays, and evaluation of viral-host protein–protein interaction. (1) Transcriptional changes in Jurkat Tet-On human T-cells that express each subgroup of Tax or HBZ protein under the control of an inducible promoter revealed different target gene profiles; (2) the number of differentially regulated genes induced by HBZ was 2–3 times higher than that induced by Tax; (3) Tax and HBZ induced the expression of different classes of non-coding RNAs (ncRNAs); (4) the chemokine CXCL10, which has been proposed as a prognostic biomarker for HAM/TSP, was more efficiently induced by subgroup-A Tax (Tax-A) than subgroup-B Tax (Tax-B), in vitro as well as in unmanipulated (ex vivo) PBMCs obtained from HAM/TSP patients; (5) reporter gene assays indicated that although transient Tax expression in an HTLV-1-negative human T-cell line activated the CXCL10 gene promoter through the NF-κB pathway, there was no difference in the ability of each subgroup of Tax to activate the CXCL10 promoter; however, (6) chromatin immunoprecipitation assays showed that the ternary complex containing Tax-A is more efficiently recruited onto the promoter region of CXCL10, which contains two NF-κB binding sites, than that containing Tax-B. Our results indicate that different HTLV-1 subgroups are characterized by different patterns of host gene expression. Differential expression of pathogenesis-related genes by subgroup-specific Tax or HBZ may be associated with the onset of HAM/TSP.

    更新日期:2019-11-28
  • Antibody-mediated prevention and treatment of HIV-1 infection
    Retrovirology (IF 3.744) Pub Date : 2018-11-16
    Henning Gruell; Florian Klein

    Novel broadly neutralizing antibodies targeting HIV-1 hold promise for their use in the prevention and treatment of HIV-1 infection. Pre-clinical results have encouraged the evaluation of these antibodies in healthy and HIV-1-infected humans. In first clinical trials, highly potent broadly neutralizing antibodies have demonstrated their safety and significant antiviral activity by reducing viremia and delaying the time to viral rebound in individuals interrupting antiretroviral therapy. While emerging antibody-resistant viral variants have indicated limitations of antibody monotherapy, strategies to enhance the efficacy of broadly neutralizing antibodies in humans are under investigation. These include the use of antibody combinations to prevent viral escape, antibody modifications to increase the half-life and the co-administration of latency-reversing agents to target the cellular reservoir of HIV-1. We provide an overview of the results of pre-clinical and clinical studies of broadly HIV-1 neutralizing antibodies, discuss their implications and highlight approaches for the ongoing advancement into humans.

    更新日期:2019-11-28
  • HIV-1 immunogens and strategies to drive antibody responses towards neutralization breadth
    Retrovirology (IF 3.744) Pub Date : 2018-11-26
    Jelle van Schooten; Marit J. van Gils

    Despite enormous efforts no HIV-1 vaccine has been developed that elicits broadly neutralizing antibodies (bNAbs) to protect against infection to date. The high antigenic diversity and dense N-linked glycan armor, which covers nearly the entire HIV-1 envelope protein (Env), are major roadblocks for the development of bNAbs by vaccination. In addition, the naive human antibody repertoire features a low frequency of exceptionally long heavy chain complementary determining regions (CDRH3s), which is a typical characteristic that many HIV-1 bNAbs use to penetrate the glycan armor. Native-like Env trimer immunogens can induce potent but strain-specific neutralizing antibody responses in animal models but how to overcome the many obstacles towards the development of bNAbs remains a challenge. Here, we review recent HIV-1 Env immunization studies and discuss strategies to guide strain-specific antibody responses towards neutralization breadth.

    更新日期:2019-11-28
  • MBL2 gene polymorphisms in HHV-8 infection in people living with HIV/AIDS
    Retrovirology (IF 3.744) Pub Date : 2018-11-27
    Viviane Martha Santos de Morais; Elker Lene Santos de Lima; Georgea Gertrudes de Oliveira Mendes Cahú; Thaisa Regina Rocha Lopes; Juliana Prado Gonçales; Maria Tereza Cartaxo Muniz; Maria Rosângela Cunha Duarte Coêlho

    Host genetic factors such as MBL2 gene polymorphisms cause defects in the polymerization of MBL protein and result in a functional deficiency and/or in low serum levels that can influence susceptibility to various viral infections. The aim of this study was to estimate the frequency of alleles, genotypes and haplotypes related to -550, -221 and exon 1 polymorphisms of the MBL2 gene and investigate their association with HHV-8 in people living with HIV/AIDS (PLWHA), as well as the impacts on CD4 cell count and HIV viral load in HIV/HHV-8 coinfected and HIV monoinfected patients. A cross sectional study in PLWHA, with and without HHV-8 infection, exploring associations between different factors, was performed in the outpatient infectious and parasitic diseases clinic at a referral hospital. Genomic DNA extractions from leukocytes were performed using a commercial Wizard® Genomic DNA Purification kit (Promega, Madison, WI). The promoter region (-550 and -221) was genotyped with the TaqMan system (Applied TaqMan Biosystems® genotyping Assays), and the structural region (exon1) was genotyped with Express Sybr Greener Supermix kit (Invitrogen, USA). In total, 124 HIV/HHV-8 coinfected and 213 HIV monoinfected patients were analysed. Median TCD4 counts were significantly lower in HIV/HHV-8 coinfected patients, whereas the mean of the first and last viral load of HIV did not present significant difference. There was no difference in frequency between the LL, YY and AA genotypes between the HIV/HHV-8 coinfected or HIV monoinfected patients. However, in a multivariate analysis, coinfected patients with the intermediate expression haplotype of the MBL2 gene had an odds ratio of 3.1-fold (CI = 1.2–7.6) of their last CD4 cell count being below 350 cells/mm3. Among the coinfected individuals, four developed KS and presented the intermediate expression MBL haplotype, with three being HYA/LXA and one being LYA/LYO. Host genetic factors, such as -550, -221 and exon 1 polymorphisms, can be related to the may modify coinfections and/or to the development clinical manifestations caused by HHV-8, especially in HIV/HHV-8 coinfected patients who present the intermediate expression haplotypes of MBL.

    更新日期:2019-11-28
  • Reduction of inflammation and T cell activation after 6 months of cART initiation during acute, but not in early chronic HIV-1 infection
    Retrovirology (IF 3.744) Pub Date : 2018-12-12
    Hury Hellen Souza de Paula; Ana Cristina Garcia Ferreira; Diogo Gama Caetano; Edson Delatorre; Sylvia Lopes Maia Teixeira; Lara Esteves Coelho; Eduarda Grinsztejn João; Michelle Morata de Andrade; Sandra Wagner Cardoso; Beatriz Grinsztejn; Valdilea Gonçalves Veloso; Mariza Gonçalves Morgado; Monick Lindenmeyer Guimarães; Fernanda Heloise Côrtes

    To investigate the impact of early combined antiretroviral therapy (cART) on inflammation biomarkers and immune activation during acute and early chronic HIV-1 infection. We included 12 acute (AHI), 11 early chronic (EcHI), and 18 late chronic HIV-1-infected (LcHI) individuals who were treated with cART and 18 HIV-1-uninfected (HIV-neg) individuals. Plasmatic levels of inflammation biomarkers, CD8+CD38+HLA-DR+ T cell frequencies, CD4 T cell counts, CD4/CD8 ratio, total HIV-1 DNA and plasmatic viral load were evaluated. Mann–Whitney test, Pearson and Spearman correlation, and linear regression models were used for statistical analyses. IP-10, IL-18, and sCD163 were significantly elevated at pre-ART in the AHI and EcHI groups, showing a significant reduction after 6 months of cART in the AHI group, achieving similar levels to the HIV-neg group. For the EcHI group, the IP-10 and sCD163 levels were also significantly reduced on M6-ART; however, IP-10 levels remained higher than in the HIV-neg group, and no significant reduction of IL-18 levels was observed. The CD8+ T cell activation levels were elevated in the AHI and EcHI groups at pre-ART and showed a significant reduction on M6-ART, but they were similar to levels seen for HIV-neg only after 12 months of cART. At pre-ART, IP-10 levels but not IL-18 levels were positively correlated with HIV-1 viral load in the AHI group. Early initiation of cART in HIV infection can reduce systemic inflammation, but the earlier normalization of the inflammation markers was only observed when cART was initiated in the acute phase of infection. A slower dynamic of reduction was observed for CD8+ T cell activation.

    更新日期:2019-11-28
  • Lipid levels, insulin resistance and cardiovascular risk over 96 weeks of antiretroviral therapy: a randomised controlled trial comparing low-dose stavudine and tenofovir
    Retrovirology (IF 3.744) Pub Date : 2018-12-14
    Alinda G. Vos; Matthew F. Chersich; Kerstin Klipstein-Grobusch; Peter Zuithoff; Michelle A. Moorhouse; Samanta T. Lalla-Edward; Andrew Kambugu; N. Kumarasamy; Diederick E. Grobbee; Roos E. Barth; Willem D. Venter

    HIV infection and antiretroviral treatment are associated with changes in lipid levels, insulin resistance and risk of cardiovascular disease (CVD). We investigated these changes in the first 96 weeks of treatment with low-dose stavudine or tenofovir regimens. This is a secondary analysis of a double blind, randomised controlled trial performed in South-Africa, Uganda and India comparing low-dose stavudine (20 mg twice daily) with tenofovir in combination with efavirenz and lamivudine in antiretroviral-naïve adults (n = 1067) (Clinicaltrials.gov, NCT02670772). Over 96 weeks, data were collected on fasting lipids, glucose and insulin. Insulin resistance was assessed with the HOMA-IR index and 10-year CVD risk with the Framingham risk score (FRS). A generalized linear mixed model was used to estimate trends over time. Participants were on average 35.3 years old, 57.6% female and 91.8% Black African. All lipid levels increased following treatment initiation, with the sharpest increase in the first 24 weeks of treatment. The increase in all lipid subcomponents over 96 weeks was higher among those in the stavudine than the tenofovir group. Insulin resistance increased steadily with no difference detected between study groups. FRS rose from 1.90% (1.84–1.98%) at baseline to 2.06 (1.98–2.15%) at week 96 for the total group, with no difference between treatment arms (p = 0.144). Lipid changes were more marked in Indian than African participants. Lipid levels increased in both groups, with low-dose stavudine resulting in a worse lipid profile compared to tenofovir. Insulin resistance increased, with no difference between regimens. CVD risk increased over time and tended to increase more in the group on stavudine. The low CVD risk across both arms argues against routine lipid and glucose monitoring in the absence of other CVD risk factors. In high risk patients, monitoring may only be appropriate at least a year after treatment initiation.

    更新日期:2019-11-28
  • Natural APOBEC3C variants can elicit differential HIV-1 restriction activity
    Retrovirology (IF 3.744) Pub Date : 2018-12-17
    Brett D. Anderson; Terumasa Ikeda; Seyed Arad Moghadasi; Amber St. Martin; William L. Brown; Reuben S. Harris

    The APOBEC3 (A3) family of DNA cytosine deaminases provides an innate barrier to infection by retroviruses including HIV-1. A total of five enzymes, A3C, A3D, A3F, A3G and A3H, are degraded by the viral accessory protein Vif and expressed at high levels in CD4+ T cells, the primary reservoir for HIV-1 replication in vivo. Apart from A3C, all of these enzymes mediate restriction of Vif-deficient HIV-1. However, a rare variant of human A3C (Ile188) was shown recently to restrict Vif-deficient HIV-1 in a 293T-based single cycle infection system. The potential activity of this naturally occurring A3C variant has yet to be characterized in a T cell-based spreading infection system. Here we employ a combination of Cas9/gRNA disruption and transient and stable protein expression to assess the roles of major Ser188 and minor Ile188 A3C variants in HIV-1 restriction in T cell lines. Cas9-mediated mutation of endogenous A3C in the non-permissive CEM2n T cell line did not alter HIV-1 replication kinetics, and complementation with A3C-Ser188 or A3C-Ile188 was similarly aphenotypic. Stable expression of A3C-Ser188 in the permissive T cell line SupT11 also had little effect. However, stable expression of A3C-Ile188 in SupT11 cells inhibited Vif-deficient virus replication and inflicted G-to-A mutations. A3C-Ile188 is capable of inhibiting Vif-deficient HIV-1 replication in T cells. Although A3C is eclipsed by the dominant anti-viral activities of other A3s in non-permissive T cell lines and primary T lymphocytes, this enzyme may still be able to contribute to HIV-1 diversification in vivo. Our results highlight the functional redundancy in the human A3 family with regards to HIV-1 restriction and the need to consider naturally occurring variants.

    更新日期:2019-11-28
  • The role of exosomal transport of viral agents in persistent HIV pathogenesis
    Retrovirology (IF 3.744) Pub Date : 2018-12-22
    Benjamin J. Patters; Santosh Kumar

    Human immunodeficiency virus (HIV) infection, despite great advances in antiretroviral therapy, remains a lifelong affliction. Though current treatment regimens can effectively suppress viral load to undetectable levels and preserve healthy immune function, they cannot fully alleviate all symptoms caused by the presence of the virus, such as HIV-associated neurocognitive disorders. Exosomes are small vesicles that transport cellular proteins, RNA, and small molecules between cells as a mechanism of intercellular communication. Recent research has shown that HIV proteins and RNA can be packaged into exosomes and transported between cells, to pathogenic effect. This review summarizes the current knowledge on the diverse mechanisms involved in the sorting of viral elements into exosomes and the damage those exosomal agents can inflict. In addition, potential therapeutic options to counteract exosome-mediated HIV pathogenesis are reviewed and considered.

    更新日期:2019-11-28
  • Characterization update of HIV-1 M subtypes diversity and proposal for subtypes A and D sub-subtypes reclassification
    Retrovirology (IF 3.744) Pub Date : 2018-12-22
    Nathalie Désiré; Lorenzo Cerutti; Quentin Le Hingrat; Marine Perrier; Stefan Emler; Vincent Calvez; Diane Descamps; Anne-Geneviève Marcelin; Stéphane Hué; Benoit Visseaux

    The large and constantly evolving HIV-1 pandemic has led to an increasingly complex diversity. Because of some taxonomic difficulties among the most diverse HIV-1 subtypes, and taking advantage of the large amount of sequence data generated in the recent years, we investigated novel lineage patterns among the main HIV-1 subtypes. All HIV full-length genomes available in public databases were analysed (n = 2017). Maximum likelihood phylogenies and pairwise genetic distance were obtained. Clustering patterns and mean distributions of genetic distances were compared within and across the current groups, subtypes and sub-subtypes of HIV-1 to detect and analyse any divergent lineages within previously defined HIV lineages. The level of genetic similarity observed between most HIV clades was deeply consistent with the current classification. However, both subtypes A and D showed evidence of further intra-subtype diversification not fully described by the nomenclature system at the time and could be divided into several distinct sub-subtypes. With this work, we propose an updated nomenclature of sub-types A and D better reflecting their current genetic diversity and evolutionary patterns. Allowing a more accurate nomenclature and classification system is a necessary step for easier subtyping of HIV strains and a better detection or follow-up of viral epidemiology shifts.

    更新日期:2019-11-28
  • Clonal anergy of CD117+chB6+ B cell progenitors induced by avian leukosis virus subgroup J is associated with immunological tolerance
    Retrovirology (IF 3.744) Pub Date : 2019-01-03
    Shuhai He; Gaoying Zheng; Defang Zhou; Gen Li; Mingjun Zhu; Xusheng Du; Jing Zhou; Ziqiang Cheng

    The pathogenesis of immunological tolerance caused by avian leukosis virus subgroup J (ALV-J), an oncogenic retrovirus, is largely unknown. In this study, the development, differentiation, and immunological capability of B cells and their progenitors infected with ALV-J were studied both morphologically and functionally by using a model of ALV-J congenital infection. Compared with posthatch infection, congenital infection of ALV-J resulted in severe immunological tolerance, which was identified as the absence of detectable specific antivirus antibodies. In congenitally infected chickens, immune organs, particularly the bursa of Fabricius, were poorly developed. Moreover, IgM-and IgG-positive cells and total immunoglobulin levels were significantly decreased in these chickens. Large numbers of bursa follicles with no differentiation into cortex and medulla indicated that B cell development was arrested at the early stage. Flow cytometry analysis further confirmed that ALV-J blocked the differentiation of CD117+chB6+ B cell progenitors in the bursa of Fabricius. Furthermore, both the humoral immunity and the immunological capability of B cells and their progenitors were significantly suppressed, as assessed by (a) the antibody titres against sheep red blood cells and the Marek’s disease virus attenuated serotype 1 vaccine; (b) the proliferative response of B cells against thymus-independent antigen lipopolysaccharide (LPS) in the spleen germinal centres; and (c) the capacities for proliferation, differentiation and immunoglobulin gene class-switch recombination of B cell progenitors in response to LPS and interleukin-4(IL-4) in vitro. These findings suggested that the anergy of B cells in congenitally infected chickens is caused by the developmental arrest and dysfunction of B cell progenitors, which is an important factor for the immunological tolerance induced by ALV-J.

    更新日期:2019-11-28
  • Sequential trafficking of Env and Gag to HIV-1 T cell virological synapses revealed by live imaging
    Retrovirology (IF 3.744) Pub Date : 2019-01-15
    Lili Wang; Sudeh Izadmehr; Edwin Kamau; Xiang-Peng Kong; Benjamin K. Chen

    HIV infection is enhanced by cell adhesions that form between infected and uninfected T cells called virological synapses (VS). VS are initiated by an interaction between Env and CD4 on cell surfaces and result in the recruitment of virus assembly to the site of cell–cell contact. However, the recruitment of Env to the VS and its relationship to Gag recruitment is not well defined. To study the trafficking of HIV-1 Env through the VS, we constructed a molecular clone of HIV carrying a green fluorescent protein-Env fusion protein called, HIV Env-isfGFP-∆V1V2. The Env-isfGFP-∆V1V2 fusion protein does not produce virus particles on its own, but can be rescued by cotransfection with full-length HIV constructs and produce virus particles that package the fluorescent Env. These rescued fluorescent Env can participate in VS formation and can be used to directly image CD4-dependent Env transfer across VS from donor to target cells. The movements of fluorescently tagged Gag and Env to the VS and transfer into target cells can be also tracked through live imaging. Time lapse live imaging reveals evidence of limited Env accumulation at the site of cell–cell contact shortly after cell adhesion, followed by Gag re-distribution to contact area. Both Gag and Env can be recruited to form button-like spots characteristic of VS. Env and Gag are recruited to the VS in a coordinated temporal sequence and subsequently transfer together across the synapse into the target cell. Env accumulations, when observed, are earlier than Gag re-distribution to the contact area during formation of VS.

    更新日期:2019-11-28
  • Host mRNA decay proteins influence HIV-1 replication and viral gene expression in primary monocyte-derived macrophages
    Retrovirology (IF 3.744) Pub Date : 2019-02-07
    Shringar Rao; Raquel Amorim; Meijuan Niu; Yann Breton; Michel J. Tremblay; Andrew J. Mouland

    Mammalian cells harbour RNA quality control and degradative machineries such as nonsense-mediated mRNA decay that target cellular mRNAs for clearance from the cell to avoid aberrant gene expression. The role of the host mRNA decay pathways in macrophages in the context of human immunodeficiency virus type 1 (HIV-1) infection is yet to be elucidated. Macrophages are directly infected by HIV-1, mediate the dissemination of the virus and contribute to the chronic activation of the inflammatory response observed in infected individuals. Therefore, we characterized the effects of four host mRNA decay proteins, i.e., UPF1, UPF2, SMG6 and Staufen1, on viral replication in HIV-1-infected primary monocyte-derived macrophages (MDMs). Steady-state expression levels of these host mRNA decay proteins were significantly downregulated in HIV-1-infected MDMs. Moreover, UPF2 and SMG6 inhibited HIV-1 gene expression in macrophages to a similar level achieved by SAMHD1, by directly influencing viral genomic RNA levels. Staufen1, a host protein also involved in UPF1-dependent mRNA decay and that acts at several HIV-1 replication steps, enhanced HIV-1 gene expression in MDMs. These results provide new evidence for roles of host mRNA decay proteins in regulating HIV-1 replication in infected macrophages and can serve as potential targets for broad-spectrum antiviral therapeutics.

    更新日期:2019-11-28
  • Replication competence of virions induced from CD4+ lymphocytes latently infected with HIV
    Retrovirology (IF 3.744) Pub Date : 2019-02-15
    Douglas D. Richman; Karissa Huang; Steven M. Lada; Xiaoying Sun; Sonia Jain; Marta Massanella; Bryson Menke

    Latently infected CD4 lymphocytes preclude cure of HIV infection, even with the most effective antiretroviral therapy. The replication competent latent HIV reservoir has been quantified with the terminal dilution quantitative viral outgrowth assay, which induces virus propagation in CD4+ T cell culture supernatants following cellular activation. Efforts to improve the sensitivity of this inefficient assay have introduced more sensitive p24 ELISA and RNA PCR based endpoints, but these more sensitive endpoints have raised the question whether they are measuring induced replication competent or defective virions. Here we performed parallel terminal dilution assays with CD4 lymphocytes from subjects effectively treated with antiretroviral therapy. An HIV integrase inhibitor was incorporated into one set of parallel cultures to compare the frequency of cells that can be induced to produce virions to those that produce virus that can propagate and amplify with co-culture in permissive cells. The majority of cells that can be induced to generate virus particles are producing replication competent virus, thus justifying more sensitive and faster assays of this reservoir.

    更新日期:2019-11-28
  • Hypericin-photodynamic therapy inhibits the growth of adult T-cell leukemia cells through induction of apoptosis and suppression of viral transcription
    Retrovirology (IF 3.744) Pub Date : 2019-02-19
    Lingling Xu; Xueqing Zhang; Wenzhao Cheng; Yong Wang; Kaining Yi; Zhilong Wang; Yiling Zhang; Linxiang Shao; Tiejun Zhao

    Adult T-cell leukemia (ATL) is an aggressive neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). ATL carries a poor prognosis due to chemotherapy resistance. Thus, it is urgent to develop new treatment strategies. Hypericin (HY) is a new-type of photosensitizer in the context of photodynamic therapy (PDT) due to its excellent photosensitizing properties and anti-tumor activities. In the present study, we investigated the efficacy of hypericin in ATL cells. Clinically achievable concentrations of hypericin in association with PDT induced the inhibition of cell proliferation in ATL cell lines with minimal effect on peripheral blood CD4+ T lymphocytes. Moreover, hypericin-PDT treatment caused apoptosis and G2/M phase cell cycle arrest in leukemic cells. Western blot analyses revealed that hypericin-PDT treatment resulted in downregulation of Bcl-2 and enhanced the expression of Bad, cytochrome C, and AIF. Cleavage of caspases-3/-7/-9/-8, Bid, and PARP was increased in hypericin-PDT-treated ATL cells. In a luciferase assay, hypericin-PDT treatment was able to activate the promoter activity of Bax and p53, resulting in enhanced expression of Bax and p53 proteins. Finally, hypericin-PDT treatment suppressed the expression of viral protein HBZ and Tax by blocking the promoter activity via HTLV-1 5′LTR and 3′LTR. Our results revealed that hypericin-PDT is highly effective against ATL cells by induction of apoptosis and suppression of viral transcription. These studies highlight the promising use of hypericin-PDT as a targeted therapy for ATL.

    更新日期:2019-11-28
  • Origin and recent expansion of an endogenous gammaretroviral lineage in domestic and wild canids
    Retrovirology (IF 3.744) Pub Date : 2019-03-07
    Julia V. Halo; Amanda L. Pendleton; Abigail S. Jarosz; Robert J. Gifford; Malika L. Day; Jeffrey M. Kidd

    Vertebrate genomes contain a record of retroviruses that invaded the germlines of ancestral hosts and are passed to offspring as endogenous retroviruses (ERVs). ERVs can impact host function since they contain the necessary sequences for expression within the host. Dogs are an important system for the study of disease and evolution, yet no substantiated reports of infectious retroviruses in dogs exist. Here, we utilized Illumina whole genome sequence data to assess the origin and evolution of a recently active gammaretroviral lineage in domestic and wild canids. We identified numerous recently integrated loci of a canid-specific ERV-Fc sublineage within Canis, including 58 insertions that were absent from the reference assembly. Insertions were found throughout the dog genome including within and near gene models. By comparison of orthologous occupied sites, we characterized element prevalence across 332 genomes including all nine extant canid species, revealing evolutionary patterns of ERV-Fc segregation among species as well as subpopulations. Sequence analysis revealed common disruptive mutations, suggesting a predominant form of ERV-Fc spread by trans complementation of defective proviruses. ERV-Fc activity included multiple circulating variants that infected canid ancestors from the last 20 million to within 1.6 million years, with recent bursts of germline invasion in the sublineage leading to wolves and dogs.

    更新日期:2019-11-28
  • Obituary: Jonas Blomberg (1944–2019)
    Retrovirology (IF 3.744) Pub Date : 2019-03-14
    Patrik Medstrand; Patric Jern

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    更新日期:2019-11-28
  • Impact of LEDGIN treatment during virus production on residual HIV-1 transcription
    Retrovirology (IF 3.744) Pub Date : 2019-04-02
    Gerlinde Vansant; Lenard S. Vranckx; Irena Zurnic; Dominique Van Looveren; Paulien Van de Velde; Christopher Nobles; Rik Gijsbers; Frauke Christ; Zeger Debyser

    Persistence of latent, replication-competent provirus is the main impediment towards the cure of HIV infection. One of the critical questions concerning HIV latency is the role of integration site selection in HIV expression. Inhibition of the interaction between HIV integrase and its chromatin tethering cofactor LEDGF/p75 is known to reduce integration and to retarget residual provirus to regions resistant to reactivation. LEDGINs, small molecule inhibitors of the interaction between HIV integrase and LEDGF/p75, provide an interesting tool to study the underlying mechanisms. During early infection, LEDGINs block the interaction with LEDGF/p75 and allosterically inhibit the catalytic activity of IN (i.e. the early effect). When present during virus production, LEDGINs interfere with proper maturation due to enhanced IN oligomerization in the progeny virions (i.e. the late effect). We studied the effect of LEDGINs present during virus production on the transcriptional state of the residual virus. Infection of cells with viruses produced in the presence of LEDGINs resulted in a residual reservoir that was refractory to activation. Integration of residual provirus was less favored near epigenetic markers associated with active transcription. However, integration near H3K36me3 and active genes, both targeted by LEDGF/p75, was not affected. Also in primary cells, LEDGIN treatment induced a reservoir resistant to activation due to a combined early and late effect. LEDGINs present a research tool to study the link between integration and transcription, an essential question in retrovirology. LEDGIN treatment during virus production altered integration of residual provirus in a LEDGF/p75-independent manner, resulting in a reservoir that is refractory to activation.

    更新日期:2019-11-28
  • Analysis of herpes simplex type 1 gB, gD, and gH/gL on production of infectious HIV-1: HSV-1 gD restricts HIV-1 by exclusion of HIV-1 Env from maturing viral particles
    Retrovirology (IF 3.744) Pub Date : 2019-04-02
    Sachith Polpitiya Arachchige; Wyatt Henke; Maria Kalamvoki; Edward B. Stephens

    We previously showed that the gM of HSV-1 could restrict the release of infectious HIV-1 from cells. In this study, we analyzed if the four HSV-1 glycoproteins (gD, gB, and gH/gL), which are the minimum glycoproteins required for HSV-1 entry, restricted the release of infectious HIV-1. Of these four glycoproteins, gD and gH/gL restricted the production of infectious HIV-1 from cells transfected with an infectious molecular clone of HIV-1 (strain NL4-3) while gB had no significant effect. Pulse-chase analyses indicated that gD did not affect the biosynthesis and processing of gp160 into gp120/gp41, the transport of the gp120/gp41 to the cell surface, or the release of HIV-1 particles from the cell surface. Our analyses revealed that gD was incorporated into HIV-1 virus particles while gp120/gp41 was excluded from released virus particles. Truncated mutants of gD revealed that the cytoplasmic domain was dispensable but that a membrane bound gD was required for the restriction of release of infectious HIV-1. Finally, cell lines expressing gD also potently restricted the release of infectious virus. Due to its ability to exclude HIV-1 gp120/gp41 from maturing virus, gD may provide a useful tool in deciphering mechanisms of Env incorporation into maturing virus particles.

    更新日期:2019-11-28
  • Functional analysis of the secondary HIV-1 capsid binding site in the host protein cyclophilin A
    Retrovirology (IF 3.744) Pub Date : 2019-04-04
    Wang Peng; Jiong Shi; Chantal L. Márquez; Derrick Lau; James Walsh; K. M. Rifat Faysal; Chang H. Byeon; In-Ja L. Byeon; Christopher Aiken; Till Böcking

    Efficient HIV-1 replication depends on interaction of the viral capsid with the host protein cyclophilin A (CypA). CypA, a peptidylprolyl isomerase, binds to an exposed loop in the viral CA protein via the enzyme’s active site. Recent structural analysis of CypA in complex with CA tubes in conjunction with molecular dynamics simulations identified a secondary CA binding site on CypA that allows a bridging interaction with two hexameric subunits of the assembled CA lattice, leading to capsid stabilization (Liu et al. in Nat Commun 7:10714, 2016). We performed mutational analysis of residues that have been proposed to mediate CA binding at the secondary binding site on CypA (A25, K27, P29 and K30) and tested the effects of the amino acid substitutions using interaction assays and HIV-1 infection assays in cells. The binding of recombinant CypA to self-assembled CA tubes or native HIV-1 capsids was measured in vitro using a quantitative fluorescence microscopy binding assay revealing that affinity and stoichiometry of CypA to the CA lattice was not affected by the substitutions. To test for functionality of the CypA secondary CA-binding site in HIV-1 infection, mutant CypA proteins were expressed in cells in which endogenous CypA was deleted, and the effects on HIV-1 infection were assayed. In normal HeLa-P4 cells, infection with HIV-1 bearing the A92E substitution in CA is inhibited by endogenous CypA and was inhibited to the same extent by expression of CypA mutants in CypA-null HeLa-P4 cells. Expression of the mutant CypA proteins in CypA-null Jurkat cells restored their permissiveness to infection by wild type HIV-1. The amino acid changes at A25, K27, P29 and K30 did not affect the affinity of CypA for the CA lattice and did not impair CypA function in infection assays suggesting that these residues are not part of a secondary CA binding site on CypA.

    更新日期:2019-11-28
  • Low-level alternative tRNA priming of reverse transcription of HIV-1 and SIV in vivo
    Retrovirology (IF 3.744) Pub Date : 2019-04-04
    Christine M. Fennessey; Celine Camus; Taina T. Immonen; Carolyn Reid; Frank Maldarelli; Jeffrey D. Lifson; Brandon F. Keele

    Reverse transcription (RT) of HIV and SIV is initiated by the binding of the acceptor stem of tRNALys3 to the primer binding site (PBS) of the viral RNA genome. Previous studies have suggested that this tRNALys3 is not the only molecule capable of priming reverse transcription, and that at least one other lysyl tRNA, tRNALys5, which has an acceptor stem sequence varying from tRNALys3 by only a single transition mutation resulting in the integration of a thymine (T) at position 8 of the PBS in the viral genome, can prime reverse transcription. We undertook an unbiased approach, evaluating the primer binding site by deep-sequencing of HIV and SIV directly from the plasma of 15 humans and 11 macaques. We found that in humans there are low but measurable levels of viral RNA genomes harboring a PBS containing the noncanonical T at position 8 (PBS-Lys5) corresponding to the tRNAlys5 sequence and representing an average of 0.52% (range 0.07–1.6%) of the total viral population. This value is remarkably consistent with the proportion of PBS-Lys5 we identified in a cross-sectional assessment of the LANL HIV database (0.51%). In macaques chronically infected with SIVmac239, the PBS-Lys5 was also detected but at a frequency 1-log less than seen for HIV, with an average of 0.056% (range 0.01–0.09%). At this proportion, PBS-Lys5 was comparable to other transition mutations, making it impossible to determine whether the mutation observed is a result of use of tRNALys5 as an RT primer at very low levels or merely the product of in vitro cDNA synthesis/PCR error. We also identified two novel PBS sequences in HIV and SIV at low levels in vivo corresponding to tRNALys6 and tRNALys1,2, suggesting that these tRNAs may rarely also be used to prime RT. In vivo reversion of the PBS-Lys5 found in SIVmac239 was rapid and reached background levels by 30 days post-infection. We conclude that while alternative tRNAs can initiate reverse transcription of HIV and SIV in vivo, their overall contributions to the replicating viral population are small.

    更新日期:2019-11-28
  • Interaction of HIV-1 integrase with polypyrimidine tract binding protein and associated splicing factor (PSF) and its impact on HIV-1 replication
    Retrovirology (IF 3.744) Pub Date : 2019-04-29
    Pooja Yadav; Souvik Sur; Dipen Desai; Smita Kulkarni; Vartika Sharma; Vibha Tandon

    The different interactions between viral proteins and cellular host proteins are required for efficient replication of HIV-1. Various reports implicated host cellular proteins as a key factor that either interact directly with HIV-1 integrase (IN) or get involved in the integration process of virus resulting in the modulation of integration step. Polypyrimidine tract binding protein and associated splicing factor (PSF) has diverse functions inside the cell such as transcriptional regulation, DNA repair, acts as nucleic acids binding protein and regulate replication and infectivity of different viruses. The protein binding study identified the association of host protein PSF with HIV-1 integrase. The siRNA knockdown (KD) of PSF resulted in increased viral replication in TZM-bl cells, suggesting PSF has negative influence on viral replication. The quantitative PCR of virus infected PSF knockdown TZM-bl cells showed more integrated DNA and viral cDNA as compared to control cells. We did not observe any significant difference between the amount of early reverse transcription products as well as infectivity of virus in the PSF KD and control TZM-bl cells. Molecular docking study supported the argument that PSF hinders the binding of viral DNA with IN. In an attempt to study the host interacting protein of IN, we have identified a new interacting host protein PSF which is a splicing factor and elucidated its role in integration and viral replication. Experimental as well as in silico analysis inferred that the host protein causes not only change in the integration events but also targets the incoming viral DNA or the integrase-viral DNA complex. The role of PSF was also investigated at early reverse transcript production as well as late stages. The PSF is causing changes in integration events, but it does not over all make any changes in the virus infectivity. MD trajectory analyses provided a strong clue of destabilization of Integrase-viral DNA complex occurred due to PSF interaction with the conserved bases of viral DNA ends that are extremely crucial contact points with integrase and indispensable for integration. Thus our study emphasizes the negative influence of PSF on HIV-1 replication.

    更新日期:2019-11-28
  • Effect of transcription inhibition and generation of suppressive viral non-coding RNAs
    Retrovirology (IF 3.744) Pub Date : 2019-04-29
    Daniel O. Pinto; Tristan A. Scott; Catherine DeMarino; Michelle L. Pleet; Thy T. Vo; Mohammed Saifuddin; Dmytro Kovalskyy; James Erickson; Maria Cowen; Robert A. Barclay; Chen Zeng; Marc S. Weinberg; Fatah Kashanchi

    HIV-1 patients receiving combination antiretroviral therapy (cART) survive infection but require life-long adherence at high expense. In chronic cART-treated patients with undetectable viral titers, cell-associated viral RNA is still detectable, pointing to low-level viral transcriptional leakiness. To date, there are no FDA-approved drugs against HIV-1 transcription. We have previously shown that F07#13, a third generation Tat peptide mimetic with competitive activity against Cdk9/T1-Tat binding sites, inhibits HIV-1 transcription in vitro and in vivo. Here, we demonstrate that increasing concentrations of F07#13 (0.01, 0.1, 1 µM) cause a decrease in Tat levels in a dose-dependent manner by inhibiting the Cdk9/T1-Tat complex formation and subsequent ubiquitin-mediated Tat sequestration and degradation. Our data indicate that complexes I and IV contain distinct patterns of ubiquitinated Tat and that transcriptional inhibition induced by F07#13 causes an overall reduction in Tat levels. This reduction may be triggered by F07#13 but ultimately is mediated by TAR-gag viral RNAs that bind suppressive transcription factors (similar to 7SK, NRON, HOTAIR, and Xist lncRNAs) to enhance transcriptional gene silencing and latency. These RNAs complex with PRC2, Sin3A, and Cul4B, resulting in epigenetic modifications. Finally, we observed an F07#13-mediated decrease of viral burden by targeting the R region of the long terminal repeat (HIV-1 promoter region, LTR), promoting both paused polymerases and increased efficiency of CRISPR/Cas9 editing in infected cells. This implies that gene editing may be best performed under a repressed transcriptional state. Collectively, our results indicate that F07#13, which can terminate RNA Polymerase II at distinct sites, can generate scaffold RNAs, which may assemble into specific sets of “RNA Machines” that contribute to gene regulation. It remains to be seen whether these effects can also be seen in various clades that have varying promoter strength, mutant LTRs, and in patient samples.

    更新日期:2019-11-28
  • Bovine leukemia virus proviral load is more strongly associated with bovine major histocompatibility complex class II DRB3 polymorphism than with DQA1 polymorphism in Holstein cow in Japan
    Retrovirology (IF 3.744) Pub Date : 2019-05-16
    Shin-nosuke Takeshima; Ayumu Ohno; Yoko Aida

    Bovine leukemia virus (BLV) causes enzootic bovine leukosis and is closely related to the human T-lymphotropic virus. Bovine major histocompatibility complex (BoLAs) are used extensively as markers of disease and immunological traits in cattle. For BLV diagnosis, proviral load is a major diagnosis index for the determination of disease progression and transmission risk. Therefore, we investigated the frequency of BoLA-DRB3 alleles, BoLA-DQA1 alleles, and haplotypes of BoLA class II isolated from the heads of 910 BLV-infected cows out of 1290 cows assessed from BLV-positive farms, in a nationwide survey from 2011 to 2014 in Japan. Our aim was to identify BoLA class II polymorphisms associated with the BLV proviral load in the Holstein cow. The study examined 569 cows with a high proviral load and 341 cows with a low proviral load. Using the highest odds ratio (OR) as a comparison index, we confirmed that BoLA-DRB3 was the best marker for determining which cow spread the BLV (OR 13.9 for BoLA-DRB3, OR 11.5 for BoLA-DQA1, and OR 6.2 for BoLA class II haplotype). In addition, DRB3*002:01, *009:02, *012:01, *014:01, and *015:01 were determined as BLV provirus associated alleles. BoLA-DRB3*002:01, *009:02, and *014:01 were determined as resistant alleles (OR > 1), and BoLA-DRB3*012:01 and *015:01 were determined as susceptible alleles (OR < 1). In this study, we showed that BoLA-DRB3 was a good marker for determining which cow spread BLV, and we found not only one resistant allele (BoLA-DRB3*009:02), but also two other disease-resistant alleles and two disease-susceptible alleles. This designation of major alleles as markers of susceptibility or resistance can allow the determination of the susceptibility or resistance of most cows to disease. Overall, the results of this study may be useful in eliminating BLV from farms without having to separate cows into several cowsheds.

    更新日期:2019-11-28
  • CCR5 editing by Staphylococcus aureus Cas9 in human primary CD4+ T cells and hematopoietic stem/progenitor cells promotes HIV-1 resistance and CD4+ T cell enrichment in humanized mice
    Retrovirology (IF 3.744) Pub Date : 2019-06-11
    Qiaoqiao Xiao; Shuliang Chen; Qiankun Wang; Zhepeng Liu; Shuai Liu; Huan Deng; Wei Hou; Dongcheng Wu; Yong Xiong; Jiafu Li; Deyin Guo

    The chemokine receptor CCR5, which belongs to the superfamily of G protein-coupled receptors, is the major co-receptor for HIV-1 entry. Individuals with a homozygous CCR5Δ32 mutation have a long lasting and increased resistance to HIV-1 infection. Therefore, CCR5 represents an optimal target for HIV-1/AIDS gene therapy. The CRISPR/Cas9 system has been developed as one of the most efficacious gene editing tools in mammalian cells and the small-sized version from Staphylococcus aureus (SaCas9) has an advantage of easier delivery compared to the most commonly used version from Streptococcus pyogenes Cas9 (SpCas9). Here, we demonstrated that CCR5 could be specifically and efficiently edited by CRISPR/SaCas9 together with two sgRNAs, which were identified through a screening of 13 sgRNAs. Disruption of CCR5 expression by lentiviral vector-mediated CRISPR/SaCas9 led to increased resistance against HIV-1 infection in human primary CD4+ T cells. Moreover, humanized mice engrafted with CCR5-disrupted CD4+ T cells showed selective survival and enrichment when challenged with CCR5 (R5)-tropic HIV-1 in comparison to mock-treated CD4+ T cells. We also observed CCR5 could be targeted by CRISPR/SaCas9 in human CD34+ hematopoietic stem/progenitor cells without obvious differentiation deficiencies. This work provides an alternative approach to disrupt human CCR5 by CRISPR/SaCas9 for a potential gene therapy strategy against HIV-1/AIDS.

    更新日期:2019-11-28
  • Fused in sarcoma silences HIV gene transcription and maintains viral latency through suppressing AFF4 gene activation
    Retrovirology (IF 3.744) Pub Date : 2019-06-25
    Simona Krasnopolsky; Lital Marom; Rachel A. Victor; Alona Kuzmina; Jacob C. Schwartz; Koh Fujinaga; Ran Taube

    The human immunodeficiency virus (HIV) cell reservoir is currently a main obstacle towards complete eradication of the virus. This infected pool is refractory to anti-viral therapy and harbors integrated proviruses that are transcriptionally repressed but replication competent. As transcription silencing is key for establishing the HIV reservoir, significant efforts have been made to understand the mechanism that regulate HIV gene transcription, and the role of the elongation machinery in promoting this step. However, while the role of the super elongation complex (SEC) in enhancing transcription activation of HIV is well established, the function of SEC in modulating viral latency is less defined and its cell partners are yet to be identified. In this study we identify fused in sarcoma (FUS) as a partner of AFF4 in cells. FUS inhibits the activation of HIV transcription by AFF4 and ELL2, and silences overall HIV gene transcription. Concordantly, depletion of FUS elevates the occupancy of AFF4 and Cdk9 on the viral promoter and activates HIV gene transcription. Live cell imaging demonstrates that FUS co-localizes with AFF4 within nuclear punctuated condensates, which are disrupted upon treating cells with aliphatic alcohol. In HIV infected cells, knockout of FUS delays the gradual entry of HIV into latency, and similarly promotes viral activation in a T cell latency model that is treated with JQ1. Finally, effects of FUS on HIV gene transcription are also exhibited genome wide, where FUS mainly occupies gene promoters at transcription starting sites, while its knockdown leads to an increase in AFF4 and Cdk9 occupancy on gene promoters of FUS affected genes. Towards eliminating the HIV infected reservoir, understanding the mechanisms by which the virus persists in the face of therapy is important. Our observations show that FUS regulates both HIV and global gene transcription and modulates viral latency, thus can potentially serve as a target for future therapy that sets to reactivate HIV from its latent state.

    更新日期:2019-11-28
  • NNRTI-induced HIV-1 protease-mediated cytotoxicity induces rapid death of CD4 T cells during productive infection and latency reversal
    Retrovirology (IF 3.744) Pub Date : 2019-06-26
    Benjamin Trinité; Hongtao Zhang; David N. Levy

    Current efforts towards HIV-1 eradication focus on the reactivation and elimination of the latent viral reservoir, so-called shock and kill therapy. However, work from several groups indicates that infected cell death following virus reactivation is not guaranteed. Thus, it is imperative to develop strategies to foster specific elimination of cells carrying integrated proviruses. It has been shown that some non-nucleoside reverse transcriptase inhibitors (NNRTIs) including efavirenz can induce premature HIV-1 GagPol dimerization in productively infected cells, resulting in intracellular HIV-1 Protease (PR) activation and a reduction in HIV-1 expressing cells. Here, we document that NNRTI-induced PR activation triggers apoptotic death of productively infected resting or activated T cells in as little as 2 h via caspase-dependent and independent pathways. Rilpivirine, efavirenz and etravirine were the most potent NNRTIs, whereas nevirapine had almost no effect. NNRTI-induced cell killing was prevented by inhibitors of HIV-1 Protease (PR) activity including indinavir and nelfinavir. HIV-1 transmitter founder viruses induced cell killing similarly to lab-adapted HIV-1 except when NNRTI resistance conferring mutations were present in reverse transcriptase. Mutations in PR that confer PR inhibitor (PI) resistance restore NNRTI-induced killing in the presence of PI. Finally, we show that NNRTIs can rapidly eliminate cells in which latent viruses are stimulated to active expression. This work supports the notion that select NNRTIs might help promote the elimination of HIV-1 producing cells as an adjuvant during shock and kill therapy.

    更新日期:2019-11-28
  • The autophagy protein ATG9A promotes HIV-1 infectivity
    Retrovirology (IF 3.744) Pub Date : 2019-07-03
    Elodie Mailler; Abdul A. Waheed; Sang-Yoon Park; David C. Gershlick; Eric O. Freed; Juan S. Bonifacino

    Nef is a multifunctional accessory protein encoded by HIV-1, HIV-2 and SIV that plays critical roles in viral pathogenesis, contributing to viral replication, assembly, budding, infectivity and immune evasion, through engagement of various host cell pathways. To gain a better understanding of the role of host proteins in the functions of Nef, we carried out tandem affinity purification-mass spectrometry analysis, and identified over 70 HIV-1 Nef-interacting proteins, including the autophagy-related 9A (ATG9A) protein. ATG9A is a transmembrane component of the machinery for autophagy, a catabolic process in which cytoplasmic components are degraded in lysosomal compartments. Pulldown experiments demonstrated that ATG9A interacts with Nef from not only HIV-1 and but also SIV (cpz, smm and mac). However, expression of HIV-1 Nef had no effect on the levels and localization of ATG9A, and on autophagy, in the host cells. To investigate a possible role for ATG9A in virus replication, we knocked out ATG9A in HeLa cervical carcinoma and Jurkat T cells, and analyzed virus release and infectivity. We observed that ATG9A knockout (KO) had no effect on the release of wild-type (WT) or Nef-defective HIV-1 in these cells. However, the infectivity of WT virus produced from ATG9A-KO HeLa and Jurkat cells was reduced by ~ fourfold and eightfold, respectively, relative to virus produced from WT cells. This reduction in infectivity was independent of the interaction of Nef with ATG9A, and was not due to reduced incorporation of the viral envelope (Env) glycoprotein into the virus. The loss of HIV-1 infectivity was rescued by pseudotyping HIV-1 virions with the vesicular stomatitis virus G glycoprotein. These studies indicate that ATG9A promotes HIV-1 infectivity in an Env-dependent manner. The interaction of Nef with ATG9A, however, is not required for Nef to enhance HIV-1 infectivity. We speculate that ATG9A could promote infectivity by participating in either the removal of a factor that inhibits infectivity or the incorporation of a factor that enhances infectivity of the viral particles. These studies thus identify a novel host cell factor implicated in HIV-1 infectivity, which may be amenable to pharmacologic manipulation for treatment of HIV-1 infection.

    更新日期:2019-11-28
  • On the generation of the MSD-Ѱ class of defective HIV proviruses
    Retrovirology (IF 3.744) Pub Date : 2019-07-11
    Atze T. Das; Alexander O. Pasternak; Ben Berkhout

    Antiretroviral therapy (ART) can effectively suppress ongoing HIV replication and block disease progression, but the infection is never cured due to the persistence of a small pool of latently infected cells hosting integrated replication-competent HIV proviruses. However, the vast majority of HIV proviruses in ART-treated patients are replication-incompetent due to a variety of genetic defects. Most defective proviruses (around 90%) contain large internal deletions or are G-to-A hypermutated, resulting in destruction of most if not all viral open reading frames, which is consistent with the idea that cytotoxic T cells (CTLs) effectively remove cells that produce viral antigens. An intriguing subclass of defective proviruses (around 10%) that are consistently detected in such patients carry a small deletion or a point mutation in a relatively precise and well conserved region near the 5ʹ end of the HIV genome, in the area that encodes the major splice donor (MSD) site and the packaging signal Ѱ in the viral RNA genome. Why this subclass of proviruses is defective has never been properly understood. We now propose a mechanistic scenario for how these MSD-Ѱ mutations can prevent viral protein expression. Based on ample results in literature, we argue that MSD inactivation triggers the activity of the 5ʹ-polyadenylation site, resulting in the production of ultra-short non-protein-coding HIV transcripts.

    更新日期:2019-11-28
  • Correction to: CCR5 editing by Staphylococcus aureus Cas9 in human primary CD4+ T cells and hematopoietic stem/progenitor cells promotes HIV-1 resistance and CD4+ T cell enrichment in humanized mice
    Retrovirology (IF 3.744) Pub Date : 2019-07-23
    Qiaoqiao Xiao; Shuliang Chen; Qiankun Wang; Zhepeng Liu; Shuai Liu; Huan Deng; Wei Hou; Dongcheng Wu; Yong Xiong; Jiafu Li; Deyin Guo

    Following publication of their article [1], the authors realized that they inadvertently omitted the contribution of Dr. Li Wu (Ohio State University) who commented on the manuscript at the early stage of the manuscript preparation and provided one plasmid related to this work.

    更新日期:2019-11-28
  • Comparative virology of HTLV-1 and HTLV-2
    Retrovirology (IF 3.744) Pub Date : 2019-08-07
    Michael P. Martinez; Jacob Al-Saleem; Patrick L. Green

    Human T cell leukemia virus type 1 (HTLV-1) was the first discovered human retrovirus and the etiologic agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. Shortly after the discovery of HTLV-1, human T-cell leukemia virus type 2 (HTLV-2) was isolated from a patient with hairy cell leukemia. Despite possession of similar structural features to HTLV-1, HTLV-2 has not been definitively associated with lymphoproliferative disease. Since their discovery, studies have been performed with the goal of highlighting the differences between HTLV-1 and HTLV-2. A better understanding of these differences will shed light on the specific pathogenic mechanisms of HTLV-1 and lead to novel therapeutic targets. This review will compare and contrast the two oldest human retroviruses with regards to epidemiology, genomic structure, gene products, and pathobiology.

    更新日期:2019-11-28
  • A trip down memory lane with Retrovirology
    Retrovirology (IF 3.744) Pub Date : 2019-08-21
    Monsef Benkirane; Ben Berkhout; Persephone Borrow; Ariberto Fassati; Masahiro Fujii; J. Victor Garcia-Martinez; D. Margolis; Monique Nijhuis; Leslie Parent; Klaus Strebel; François Venter; Frank Kirchhoff; Andrew Lever; Susan Ross; Johnson Mak

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    更新日期:2019-11-28
  • Impact of host immunity on HTLV-1 pathogenesis: potential of Tax-targeted immunotherapy against ATL
    Retrovirology (IF 3.744) Pub Date : 2019-08-22
    Mari Kannagi; Atsuhiko Hasegawa; Yoshiko Nagano; Shuichi Kimpara; Youko Suehiro

    Human T-cell leukemia virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATL), HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and other inflammatory diseases. There is no disease-specific difference in viral strains, and it is unclear how HTLV-1 causes such different diseases manifesting as lymphoproliferation or inflammation. Although some progress has been made in therapies for these diseases, the prognosis for ATL is still dismal and HAM/TSP remains an intractable disease. So far, two regulatory proteins of HTLV-1, Tax and HBZ, have been well studied and shown to have pleiotropic functions implicated in viral pathogenesis. Tax in particular can strongly activate NFκB, which is constitutively activated in HTLV-1-infected cells and considered to contribute to both oncogenesis and inflammation. However, the expression level of Tax is very low in vivo, leading to confusion in understanding its role in viral pathogenesis. A series of studies using IL-2-dependent HTLV-1-infected cells indicated that IL-10, an anti-inflammatory/immune suppressive cytokine, could induce a proliferative phenotype in HTLV-1-infected cells. In addition, type I interferon (IFN) suppresses HTLV-1 expression in a reversible manner. These findings suggest involvement of host innate immunity in the switch between lymphoproliferative and inflammatory diseases as well as the regulation of HTLV-1 expression. Innate immune responses also affect another important host determinant, Tax-specific cytotoxic T lymphocytes (CTLs), which are impaired in ATL patients, while activated in HAM/TSP patients. Activation of Tax-specific CTLs in ATL patients after hematopoietic stem cell transplantation indicates Tax expression and its fluctuation in vivo. A recently developed anti-ATL therapeutic vaccine, consisting of Tax peptide-pulsed dendritic cells, induced Tax-specific CTL responses in ATL patients and exhibited favorable clinical outcomes, unless Tax-defective ATL clones emerged. These findings support the significance of Tax in HTLV-1 pathogenesis, at least in part, and encourage Tax-targeted immunotherapy in ATL. Host innate and acquired immune responses induce host microenvironments that modify HTLV-1-encoded pathogenesis and establish a complicated network for development of diseases in HTLV-1 infection. Both host and viral factors should be taken into consideration in development of therapeutic and prophylactic strategies in HTLV-1 infection.

    更新日期:2019-11-28
  • The KT Jeang Prize 2019: Reuben S. Harris
    Retrovirology (IF 3.744) Pub Date : 2019-08-29

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    更新日期:2019-11-28
  • Silencers of HTLV-1 and HTLV-2: the pX-encoded latency-maintenance factors
    Retrovirology (IF 3.744) Pub Date : 2019-09-06
    Robert Harrod

    Of the members of the primate T cell lymphotropic virus (PTLV) family, only the human T-cell leukemia virus type-1 (HTLV-1) causes disease in humans—as the etiological agent of adult T-cell leukemia/lymphoma (ATLL), HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and other auto-inflammatory disorders. Despite having significant genomic organizational and structural similarities, the closely related human T-cell lymphotropic virus type-2 (HTLV-2) is considered apathogenic and has been linked with benign lymphoproliferation and mild neurological symptoms in certain infected patients. The silencing of proviral gene expression and maintenance of latency are central for the establishment of persistent infections in vivo. The conserved pX sequences of HTLV-1 and HTLV-2 encode several ancillary factors which have been shown to negatively regulate proviral gene expression, while simultaneously activating host cellular proliferative and pro-survival pathways. In particular, the ORF-II proteins, HTLV-1 p30II and HTLV-2 p28II, suppress Tax-dependent transactivation from the viral promoter—whereas p30II also inhibits PU.1-mediated inflammatory-signaling, differentially augments the expression of p53-regulated metabolic/pro-survival genes, and induces lymphoproliferation which could promote mitotic proviral replication. The ubiquitinated form of the HTLV-1 p13II protein localizes to nuclear speckles and interferes with recruitment of the p300 coactivator by the viral transactivator Tax. Further, the antisense-encoded HTLV-1 HBZ and HTLV-2 APH-2 proteins and mRNAs negatively regulate Tax-dependent proviral gene expression and activate inflammatory signaling associated with enhanced T-cell lymphoproliferation. This review will summarize our current understanding of the pX latency-maintenance factors of HTLV-1 and HTLV-2 and discuss how these products may contribute to the differences in pathogenicity between the human PTLVs.

    更新日期:2019-11-28
  • BLV: lessons on vaccine development
    Retrovirology (IF 3.744) Pub Date : 2019-10-07
    Alejandro Abdala; Irene Alvarez; Hélène Brossel; Luis Calvinho; Hugo Carignano; Lautaro Franco; Hélène Gazon; Christelle Gillissen; Malik Hamaidia; Clotilde Hoyos; Jean-Rock Jacques; Thomas Joris; Florent Laval; Marcos Petersen; Florent Porquet; Natalia Porta; Vanesa Ruiz; Roghaiyeh Safari; Guillermo Suárez Archilla; Karina Trono; Luc Willems

    Vaccination against retroviruses is a challenge because of their ability to stably integrate into the host genome, undergo long-term latency in a proportion of infected cells and thereby escape immune response. Since clearance of the virus is almost impossible once infection is established, the primary goal is to achieve sterilizing immunity. Besides efficacy, safety is the major issue since vaccination has been associated with increased infection or reversion to pathogenicity. In this review, we discuss the different issues that we faced during the development of an efficient vaccine against bovine leukemia virus (BLV). We summarize the historical failures of inactivated vaccines, the efficacy and safety of a live-attenuated vaccine and the economical constraints of further industrial development.

    更新日期:2019-11-28
  • Isolation of the Arawete and Asurini Indians keeps the tribes free from HTLV infection during 36 years of follow-up
    Retrovirology (IF 3.744) Pub Date : 2019-10-22
    Antonio C. R. Vallinoto; Mateus I. Otake; Paulo V. N. R. Sousa; Felipe T. Lopes; Eliene R. P. Sacuena; Maria A. F. Queiroz; Greice L. C. Costa; Marluísa O. G. Ishak; Izaura M. V. Cayres-Vallinoto; João F. Guerreiro; Ricardo Ishak

    Arawete and Asurini Indian tribes were revisited after a 36-year follow-up in search of HTLV infections. 46 persons (23 from each tribe) were tested for HTLV-1/2 antibodies and viral DNA. None were positive; this was probably because of their social/cultural isolation from neighboring tribes where HTLV-2c is hyperendemic.

    更新日期:2019-11-28
  • Characterization of resistance to a potent d-peptide HIV entry inhibitor
    Retrovirology (IF 3.744) Pub Date : 2019-10-22
    Amanda R. Smith; Matthew T. Weinstock; Amanda E. Siglin; Frank G. Whitby; J. Nicholas Francis; Christopher P. Hill; Debra M. Eckert; Michael J. Root; Michael S. Kay

    PIE12-trimer is a highly potent d-peptide HIV-1 entry inhibitor that broadly targets group M isolates. It specifically binds the three identical conserved hydrophobic pockets at the base of the gp41 N-trimer with sub-femtomolar affinity. This extremely high affinity for the transiently exposed gp41 trimer provides a reserve of binding energy (resistance capacitor) to prevent the viral resistance pathway of stepwise accumulation of modest affinity-disrupting mutations. Such modest mutations would not affect PIE12-trimer potency and therefore not confer a selective advantage. Viral passaging in the presence of escalating PIE12-trimer concentrations ultimately selected for PIE12-trimer resistant populations, but required an extremely extended timeframe (> 1 year) in comparison to other entry inhibitors. Eventually, HIV developed resistance to PIE12-trimer by mutating Q577 in the gp41 pocket. Using deep sequence analysis, we identified three mutations at Q577 (R, N and K) in our two PIE12-trimer resistant pools. Each point mutant is capable of conferring the majority of PIE12-trimer resistance seen in the polyclonal pools. Surface plasmon resonance studies demonstrated substantial affinity loss between PIE12-trimer and the Q577R-mutated gp41 pocket. A high-resolution X-ray crystal structure of PIE12 bound to the Q577R pocket revealed the loss of two hydrogen bonds, the repositioning of neighboring residues, and a small decrease in buried surface area. The Q577 mutations in an NL4-3 backbone decreased viral growth rates. Fitness was ultimately rescued in resistant viral pools by a suite of compensatory mutations in gp120 and gp41, of which we identified seven candidates from our sequencing data. These data show that PIE12-trimer exhibits a high barrier to resistance, as extended passaging was required to develop resistant virus with normal growth rates. The primary resistance mutation, Q577R/N/K, found in the conserved gp41 pocket, substantially decreases inhibitor affinity but also damages viral fitness, and candidate compensatory mutations in gp160 have been identified.

    更新日期:2019-11-28
  • Effect of induced dNTP pool imbalance on HIV-1 reverse transcription in macrophages
    Retrovirology (IF 3.744) Pub Date : 2019-10-26
    Caitlin Shepard; Joella Xu; Jessica Holler; Dong-Hyun Kim; Louis M. Mansky; Raymond F. Schinazi; Baek Kim

    Terminally differentiated/nondividing macrophages, a key target cell type of HIV-1, harbor extremely low dNTP concentrations established by a host dNTP triphosphohydrolase, SAM domain and HD domain containing protein 1 (SAMHD1). We tested whether the induction of dNTP pool imbalance can affect HIV-1 replication in macrophages. For this test, we induced a large dNTP pool imbalance by treating human primary monocyte derived macrophages with either one or three of the four deoxynucleosides (dNs), which are phosphorylated to dNTPs in cells, to establish two different dNTP imbalance conditions in macrophages. The transduction efficiency and 2-LTR circle copy number of HIV-1 GFP vector were greatly diminished in human primary macrophages treated with the biased dN treatments, compared to the untreated macrophages. We also observed the induced dNTP bias blocked the production of infectious dual tropic HIV-1 89.6 in macrophages. Moreover, biochemical DNA synthesis by HIV-1 reverse transcriptase was significantly inhibited by the induced dNTP pool imbalance. Third, the induced dNTP bias increased the viral mutant rate by approximately 20–30% per a single cycle infection. Finally, unlike HIV-1, the single dN treatment did not significantly affect the transduction of SIVmac239-based GFP vector encoding Vpx in macrophages. This is likely due to Vpx, which can elevate all four dNTP levels even with the single dN treatment. Collectively, these data suggest that the elevated dNTP pool imbalance can induce kinetic block and mutation synthesis of HIV-1 in macrophages.

    更新日期:2019-11-28
  • NHEJ pathway is involved in post-integrational DNA repair due to Ku70 binding to HIV-1 integrase
    Retrovirology (IF 3.744) Pub Date : 2019-11-06
    Ekaterina Knyazhanskaya; Andrey Anisenko; Olga Shadrina; Anastasia Kalinina; Timofei Zatsepin; Arthur Zalevsky; Dmitriy Mazurov; Marina Gottikh

    HIV-1 integration results in genomic DNA gaps that are repaired by cellular DNA repair pathways. This step of the lentiviral life cycle remains poorly understood despite its crucial importance for successful replication. We and others reported that Ku70 protein of the non-homologous end joining pathway (NHEJ) directly binds HIV-1 integrase (IN). Here, we studied the importance of this interaction for post-integrational gap repair and the recruitment of NHEJ factors in this process. We engineered HIV-based pseudovirus with mutant IN defective in Ku70 binding and generated heterozygous Ku70, Ku80 and DNA-PKcs human knockout (KO) cells using CRISPR/Cas9. KO of either of these proteins or inhibition of DNA-PKcs catalytic activity substantially decreased the infectivity of HIV-1 with native IN but not with the mutant one. We used a recently developed qPCR assay for the measurement of gap repair efficiency to show that HIV-1 with mutant IN was defective in DNA post-integrational repair, whereas the wild type virus displayed such a defect only when NHEJ system was disrupted in any way. This effect was present in CRISPR/Cas9 modified 293T cells, in Jurkat and CEM lymphoid lines and in primary human PBMCs. Our data provide evidence that IN recruits DNA-PK to the site of HIV-1 post-integrational repair due to Ku70 binding—a novel finding that explains the involvement of DNA-PK despite the absence of free double stranded DNA breaks. In addition, our data clearly indicate the importance of interactions between HIV-1 IN and Ku70 in HIV-1 replication at the post-integrational repair step.

    更新日期:2019-11-28
Contents have been reproduced by permission of the publishers.
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