Rotavirus VP2 Core Shell Regions Critical for Viral Polymerase Activation

Abstract

The innermost VP2 core shell of the triple-layered, icosahedral rotavirus particle surrounds the viral genome and RNA processing enzymes, including the RNA-dependent RNA polymerase (VP1). In addition to anchoring VP1 within the core, VP2 is also an essential cofactor that triggers the polymerase to initiate double-stranded RNA (dsRNA) synthesis using packaged plus-strand RNA templates. The VP2 requirement effectively couples packaging with genome replication and ensures that VP1 makes dsRNA only within an assembling previrion particle. However, the mechanism by which the rotavirus core shell protein activates the viral polymerase remains very poorly understood. In the current study, we sought to elucidate VP2 regions critical for VP1-mediated in vitro dsRNA synthesis. By comparing the functions of proteins from several different rotaviruses, we found that polymerase activation by the core shell protein is specific. Through truncation and chimera mutagenesis, we demonstrate that the VP2 amino terminus, which forms a decameric, internal hub underneath each 5-fold axis, plays an important but nonspecific role in VP1 activation. Our results indicate that the VP2 residues correlating with polymerase activation specificity are located on the inner face of the core shell, distinct from the amino terminus. Based on these findings, we predict that several regions of VP2 engage the polymerase during the concerted processes of rotavirus core assembly and genome replication.

Norovirus RNA Synthesis Is Modulated by an Interaction between the Viral RNA-Dependent RNA Polymerase and the Major Capsid Protein, VP1

“…The AMV CP can regulate RNA synthesis by binding to the 3= ends of Alfamovirus and Ilarvirus RNAs to activate genome replication (6,23). In rotavirus, VP2 can serve as a scaffold for the viral polymerase as well as act as a cofactor for VP1 to initiate genome replication (32,38). The CPs of the plant-infecting brome mosaic virus and the bacteriophage MS2 play regulatory roles in binding to RNA elements that regulate RNA synthesis (46,50,51).…”

A Temperature-Sensitive Lesion in the N-Terminal Domain of the Rotavirus Polymerase Affects Its Intracellular Localization and Enzymatic Activity

“…The BaculoDirect expression system (Life Technologies) was used in accordance with the manufacturer's protocol to create His-tagged recombinant VP1 containing the L138P lesion (rVP1 L138P ). Briefly, outward PCR and sitedirected mutagenesis were used to engineer the L138P mutation into the SA11-4F VP1 ORF of the pENTR-SA11 VP1 vector (18). The entire VP1 L138P ORF was then transferred into the BaculoDirect C-Term linear DNA by recombination with LR Clonase II.…”

“…The ORF sequences of WT or L138P mutant VP1 were cloned into the pEGFP-N1 vector (Clontech) to express the polymerases with enhanced GFP fused to their C termini (VP1 WT -GFP or VP1 L138P -GFP, respectively). The WT SA11-4F VP1 ORF was amplified from the pENTR-SA11 VP1 vector by PCR (18). Primer-generated restriction sites (5= SacI and 3= KpnI) were used to subclone the WT SA11-4F VP1 ORF into the pEGFP-N1 vector, thereby creating pEGFP-SA11-VP1 WT , which expresses VP1 WT -GFP.…”

“…Binding of VP1 by NSP2 and/or NSP5 localizes the polymerase to viroplasms, sites of genome replication and early particle assembly in the cell cytosol (20,21). Within the viroplasm, binding of VP1 by core shell protein VP2 triggers the polymerase to initiate genome replication (i.e., dsRNA synthesis) in the context of subviral assembly intermediates (18,19,22). Because the VP1 protein of SA11-tsC has an L138P mutation in the N-terminal domain, we wondered whether it affects the abilities of the polymerase to localize to viroplasms, interact with other viral proteins, and synthesize viral dsRNA in a temperature-dependent manner.…”

“…This region of VP1 helps form a template RNA entry tunnel, and it may also aid in polymerase regulation by serving as a binding platform for other viral proteins. VP1 is known to engage at least three other viral proteins (VP2, NSP2, and NSP5) during the RV life cycle, though the interaction interfaces are poorly defined (16)(17)(18)(19). Binding of VP1 by NSP2 and/or NSP5 localizes the polymerase to viroplasms, sites of genome replication and early particle assembly in the cell cytosol (20,21).…”