Research Article
Structural Mimicry Drives HIV-1 Rev-Mediated HERV-K Expression

https://doi.org/10.1016/j.jmb.2020.11.010Get rights and content

Highlights

  • HIV-1 Rev binds to and mediates the nuclear export of the HERV-K RcRE.

  • The topological structure of the HERV-K RcRE resembles that of the HIV-1 RRE.

  • The unique long stem oscillates between folded and extended conformations.

  • RcRE variants defective of Rev-binding and nuclear export activity have distorted structures.

  • The first topological structure for a HERV molecule is reported.

Abstract

Expression of the Human Endogenous Retrovirus Type K (HERV-K), the youngest and most active HERV, has been associated with various cancers and neurodegenerative diseases. As in all retroviruses, a fraction of HERV-K transcripts is exported from the nucleus in unspliced or incompletely spliced forms to serve as templates for translation of viral proteins. In a fraction of HERV-K loci (Type 2 proviruses), nuclear export of the unspliced HERV-K mRNA appears to be mediated by a cis-acting signal on the mRNA, the RcRE, and the protein Rec—these are analogous to the RRE-Rev system in HIV-1. Interestingly, the HIV-1 Rev protein is able to mediate the nuclear export of the HERV-K RcRE, contributing to elevated HERV-K expression in HIV-infected patients. We aimed to understand the structural basis for HIV Rev-HERV-K RcRE recognition. We examined the conformation of the RcRE RNA in solution using small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM). We found that the 433-nt long RcRE can assume folded or extended conformations as observed by AFM. SAXS analysis of a truncated RcRE variant revealed an “A”-shaped topological structure similar to the one previously reported for the HIV-1 RRE. The effect of the overall topology was examined using several deletion variants. SAXS and biochemical analyses demonstrated that the “A” shape is necessary for efficient Rev-RcRE complex formation in vitro and nuclear export activity in cell culture. The findings provide insight into the mechanism of HERV-K expression and a structural explanation for HIV-1 Rev-mediated expression of HERV-K in HIV-infected patients.

Importance

Expression of the human endogenous retrovirus type K (HERV-K) has been associated with various cancers and autoimmune diseases. Nuclear export of both HIV-1 and HERV-K mRNAs is dependent on the interaction between a small viral protein (Rev in HIV-1 and Rec in HERV-K) and a region on the mRNA (RRE in HIV-1 and RcRE in HERV-K). HIV-1 Rev is able to mediate the nuclear export of RcRE-containing HERV-K mRNAs, which contributes to elevated production of HERV-K proteins in HIV-infected patients. We report the solution conformation of the RcRE RNA—the first three-dimensional topological structure for a HERV molecule—and find that the RcRE resembles the HIV-1 nuclear export signal, RRE. The finding reveals the structural basis for the increased HERV-K expression observed in HIV-infected patients. Elevated HERV expression, mediated by HIV infection or other stressors, can have various HERV-related biological consequences. The findings provide structural insight for regulation of HERV-K expression.

Introduction

Retroviruses constitute an ancient and ubiquitous family of RNA viruses that replicate via a DNA intermediate, which integrates into the host’s genome. When retroviruses infect germ cells, the resulting integrated sequence is inherited by the offspring in a Mendelian manner and becomes endogenized—HERVs are a result of multiple ancient germ-line infections1 and constitute ∼8% of the human genome.2 A growing number of studies attribute HERVs with important physiological roles in placental development,3 embryonic immunity,4 and muscle formation.[5], [6] However, HERVs have also been associated with a number of diseases, most notably germline cancers and autoimmune diseases. We direct the reader to several reviews on the role of HERVs in human health and disease.[7], [8], [9], [10], [11], [12]

HERV-K entered the human lineage relatively recently (less than one million years ago).13 The full-length HERV-K genome encodes four genes (gag, pro, pol, and env) flanked by long terminal repeat (LTR) sequences that contain regulatory elements (Figure 1). Many HERV-K loci have deletions or inactivating mutations. Deletion events, most of which occur during homologous recombination, have resulted in about 1000 solo LTRs distributed throughout the genome.14 Nevertheless, a number of the HERV-K loci encode functional proteins and LTRs can exert regulatory activity on neighboring genes.[9], [15] The function of the HERV-K protein products is the subject of ongoing research. The HERV-K Gag protein can package HERV-K sequences into viral particles.[16], [17] HERV-K Gag is homologous to the retrotransposon capsid-forming protein Arc, which encapsidates and transmits cellular mRNAs between neurons.18 The Gag-Pro-Pol polyprotein is cleaved into the catalytic enzymes—protease, reverse transcriptase, and integrase. Env is a glycoprotein that has been proposed to facilitate oncogenesis via its ability to promote cell fusion and suppress immune responses (reviewed in8).

In addition to the core elements, complex retroviruses also code for smaller, accessory proteins, which are products of alternative splicing (Figure 1). Synthesis of Gag, Gag-Pro-Pol, and Env polyproteins requires that unspliced or incompletely spliced HERV-K mRNAs are exported from the nucleus to the cytoplasm. Improperly processed cellular mRNAs are typically (though not always19 targeted for degradation, and viruses tend to evolve in order to evade these cellular quality control pathways.[20], [21] Complex retroviruses code for a small protein—the product of a fully spliced message—which enters the nucleus upon translation and binds to a cis-acting region on the viral genome.[21], [22] In HIV, the small protein is called Rev, and it binds to the Rev-response element (RRE) present on unspliced or incompletely spliced mRNAs. Rev-RRE binding initiates Rev oligomerization onto the RRE and this complex recruits the Crm-1/Ran-GTP cellular factors for nuclear transport (reviewed in21). Cargo molecules are transported through nuclear core complexes that can mediate the egress and ingress of molecules that range widely in size, including large ribosomal units.24 Other complex retroviruses, including HIV-2,23 HERV-K (see below), Human T-cell Lymphotropic Virus Type I (HTLV-I),24 Jaagsiekte Sheep Retrovirus (JSRV),25 Equine Infectious Anemia Virus (EIAV),26 and Mouse Mammary Tumor Virus (MMTV),[27], [28] use analogous protein-RNA pairs to mediate nuclear export of intron-containing mRNAs.

There are approximately 100 HERV-K loci in the human genome, some of which (known as type 2 HERV-K) code for a Rev/RRE-like system referred to as Rec and Rec-response element (RcRE) (the terms K-Rev, K-RRE, and cORF have also been used).[29], [30], [31], [32], [33] The Rec protein interacts with the RcRE RNA in vitro,31 and in cell culture this interaction is necessary for Crm1-dependent nuclear export of RcRE-containing RNAs.[31], [32], [33] Successful nuclear export of unspliced or incompletely spliced mRNAs enables translation of the viral Gag, Gag-Pro-Pol, and Env polyproteins.32 Functional cross-talk studies between HIV-1 and HERV-K have demonstrated a non-reciprocal relationship between the two retroviral systems; namely, HIV-1 Rev can mediate nuclear export of RcRE-containing mRNAs,[29], [31] but HERV-K Rec cannot induce transport HIV-1 RRE-containing mRNAs.31 HIV-1 Rev-mediated trafficking of RcRE-containing mRNAs appears to vary with different RcRE loci.34 Regardless, expression of the Rev protein in HIV-infected individuals (or in lentivirus-based therapeutic approaches) could alter the molecular composition of the cell by mediating the expression of both type 1 and type 2 HERV-K loci and neighboring genes.

Although the HIV-1 RRE and HERVK RcRE RNAs share little sequence similarity (Supplemental Figure 1), they have similar secondary structures characterized by a long stem and a cluster of several small stems centered around a central junction (Figure 1).[29], [35], [36], [37], [38] We previously solved the three-dimensional topological structure of the HIV-1 RRE and found that it has a unique, flat and extended shape that resembles the letter “A”.39 Based on the similarity of the secondary structures, we hypothesized that the two molecules, the RRE and the RcRE RNAs, have conserved three-dimensional structures allowing Rev to recognize both elements. To understand the structural basis of HIV Rev/HERV-K RcRE recognition, we examined the three-dimensional topological structure of the RcRE RNA in solution using small angle X-ray scattering (SAXS) and atomic force microscopy (AFM). We found that the RcRE exhibits striking similarities to the HIV-1 RRE attesting to the significance of the three-dimensional structure. The preservation of the three-dimensional topology was critical for Rev-binding in vitro and RcRE nuclear export activity in cell culture. The findings reveal the structural basis for the interaction between HIV-1 Rev and the HERV-K RcRE RNA and provide insight into the mechanism of Rev-mediated HERV-K expression.

Section snippets

Results

HIV-1 Rev binds to and mediates the nuclear export of RcRE-containing mRNAs. Previous studies have demonstrated that the HIV-1 Rev protein is able to mediate the nuclear export of RcRE-containing mRNAs.[29], [34], [40] To compare the binding affinity of HIV-1 Rev to the RcRE versus the RRE, we performed electrophoretic mobility shift assays (EMSAs). The HERV-K 108 sequence (7p22.1) was chosen due to its use in previous RcRE (K-RRE) studies,[29], [31] it is relatively well-studied, and its Env

Discussion

The expanding field of study on human endogenous retroviruses is increasingly shedding light on the little understood roles of HERVs in human health and disease. In this work, we report the solution conformation of the HERV-K RcRE RNA (also known as K-RRE), the first three-dimensional topological structure of a HERV molecule. We found that: (1) the overall shape, assessed by SAXS and AFM, is flat and extended and resembles that of the HIV-1 RRE, (2) the long Stem I confers flexibility to the

Materials and Methods

Plasmid construction and RNA preparation. A plasmid containing the 433-nucleotide long HERV-K 108 RcRE (K-RRE) sequence (GenBank accession number AF17922529 flanked by the T7 promoter and a XhoI site was synthesized by Integrated DNA Technologies, Inc. Deletions were introduced by PCR mutagenesis using primers flanking the regions to be deleted. The RcRE was truncated by first deleting nucleotides 1–43 using the primers 5′-/Phos/TCCCTAATCTCAAGTACCCAGGG-3′ and 5′-CCTATAGTGAGTCGTATTATGGACTAGC-3′,

Small-Angle X-Ray Scattering (SAXS) Data Collection and Analysis

SAXS measurements were performed at the 12ID-B beamline of the Advanced Photon Source, Argonne National Laboratory. Photon energy was 13.3-keV and sample-to-detector distance was 1.9 m to achieve a useable q range of 0.005 < q < 0.88 Å−1, where q = (4π/λ)sinθ, and 2θ is the scattering angle. Concentration series measurements for the same sample were carried out to remove the scattering contribution due to interparticle interactions and to extrapolate the data to infinite dilution. The sample

CRediT authorship contribution statement

Ina P. O’Carroll: Conceptualization, Methodology, Investigation, Formal analysis, Writing - original draft, Writing - review & editing, Visualization, Project administration, Funding acquisition. Lixin Fan: Methodology, Software, Validation, Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review & editing. Tomáš Kroupa: Conceptualization, Methodology, Investigation, Visualization, Writing - original draft, Writing - review & editing. Erin K. McShane:

Acknowledgements

We are grateful to Demetria Harvin, Dr. Xiaobing Zuo, Dr. Marzena Dyba, Leah Duke, LCDR Douglas Marks, MIDN 1/C Benjamin Phelps, and Janine Avante for technical assistance; Dr. Leighanne Basta for providing the pET28a vector for cloning and E.coli BL21(DE3) cells; Dr. Virginia Smith for thoughtful review of the manuscript and Dr. Danny Morse for insightful discussions. The work was supported by a Defense Threat Research Agency CB Technologies Service Academy Research Initiative grant to I.P.O

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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    These authors contributed equally.

    Current address: Stanford School of Medicine, Stanford, CA 94305, USA.

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