Regulation of rotavirus polymerase activity by inner capsid proteins
Introduction
In the life cycle of RNA viruses, the activity of the viral RNA-dependent RNA polymerase (RdRP) must be carefully regulated to ensure that RNA products are made at levels and in appropriate sites to support genome replication and protein translation. Such regulation is particularly intriguing in the case of rotavirus, as its RdRP (VP1) has a dual role, sometimes acting as a replicase and, at other times, as a transcriptase. The observation that rotavirus dsRNA genome segments are made at equimolar levels, while the viral transcripts are not [1, 2], makes the question of how RdRP activity is regulated even more interesting. A critical discovery made by analysis of intracellular subviral particles [3, 4] and by in vitro assays with recombinant proteins [5••] is that rotavirus dsRNAs and +RNAs are only made by particle-associated VP1 [6••, 7]. These findings have established that rotavirus inner capsid proteins are essential for VP1 polymerase activity and determine whether VP1 acts as a replicase or transcriptase. Regulation of VP1 activity by capsid proteins allows the coordination of genome replication with the packaging of newly made dsRNAs into progeny particles and likely prevents the induction of host antiviral responses to exposed dsRNAs. Here we review insights gained from structural and functional studies into the mechanism by which VP1 polymerase activity is regulated by the virion capsid protein.
Section snippets
Virion architecture
Rotavirus, a member of the Reoviridae family, is a non-enveloped icosahedral triple-layered particle (TLP) with a genome consisting of 11 segments of dsRNA (Figure 1). The outer and intermediate protein layers are formed by trimers of VP7 and VP6, respectively, each organized with T = 13 symmetry [8, 9]. Anchored into the VP6 layer and projecting out of the VP7 layer is the viral attachment protein VP4. The inner layer defines the core shell and is composed of 60 VP2 dimers, organized with T = 1
Viral life cycle
During rotavirus entry, the virion VP4–VP7 outer capsid is disrupted, yielding a double-layered particle (DLP) [20, 21]. Through the activity of its VP1 and VP3 components, the DLP synthesizes 11 capped, nonpolyadenylated +RNAs. These are translated, giving rise to six structural proteins (VP1–VP4, VP6–VP7) and six nonstructural proteins (NSP1–NSP6). Transcriptionally active DLPs become embedded within viral inclusions (viroplasms) formed by NSP2 and NSP5, and containing the core proteins VP1,
Polymerase activities
Several earlier studies provided evidence of a link between VP1 activity and the presence of the viral inner capsid proteins. Among these were results showing that rotavirus replicase and transcriptase activities were only detectable in isolated subviral particles that contained VP1 and VP2, or VP1, VP2, and VP6, respectively [3, 21, 24, 25]. Moreover, analysis of the rotavirus temperature-sensitive mutants, tsF and tsG, indicated that the formation of subviral particles with replicase activity
VP1 structure and function
Studies reporting atomic structures for VP1, both in its apo form and in complex with short viral RNAs, have provided important insights into the mechanism by which VP2 affects rotavirus polymerase activity. These analyses revealed that VP1 has a closed cage-like structure consisting of a central polymerase domain that is sandwiched in between large N-terminal and C-terminal (bracelet) domains (Figure 2) [28••]. The polymerase domain has the classical ‘right-handed’ polymerase architecture that
Regulation of replication
Even when incubated with NTPs and viral +RNAs, rotavirus VP1 lacks polymerase activity unless VP2 is present [5••, 6••]. However, VP1 alone can specifically bind +RNAs [6••, 31•], an interaction mediated by an extensive network of hydrogen bonds formed between residues lining the template entry tunnel of VP1 and the conserved 3′-terminal UGUGACC element of the RNA [28••]. The polymerase forms hydrogen bonds with the bases of the UGUG residues and the ribose-phosphate backbone of the ACC
Regulation of transcription
Structural analysis has revealed the presence of a flexible element (plug) at the C-terminus of VP1 that extends from the surface of the bracelet domain into the dsRNA/−RNA exit tunnel (Figure 3) [28••]. Although the plug was observed within this tunnel in crystal structures of VP1, successful purification of VP1 using a C-terminal polyhistidine affinity tag indicates that the plug can also extend outside the tunnel [28••]. Deletion mutagenesis has indicated that while the plug is not required
Conclusion
Rotavirus polymerase activity requires the inner capsid proteins, a connection that links genome replication with core assembly and transcription with DLP assembly. For either replicase or transcriptase activity, VP1 likely requires the movement of the polymerase's priming loop from a retracted form to an extended form, thereby allowing RNA initiation. Because such movement is dependent on assembled VP2, VP1 alone lacks polymerase activity, even though it can bind viral template RNA. Whether
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We thank Marco Morelli and Kristen Ogden for their help in preparing the manuscript. The authors are supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
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