Capsid Protein Synthesis from Replicating RNA Directs Specific Packaging of the Genome of a Multipartite, Positive-Strand RNA Virus

Viral inclusion bodies, or viroplasms, that form in rotavirus-infected cells direct replication and packagingof the segmented double-stranded RNA (dsRNA) genome. NSP2, one of two rotavirus proteins needed for viroplasm assembly, possesses NTPase, RNA-binding, and helix-unwinding activities. NSP2 of the rotavirus group causing endemic infantile diarrhea (group A) was shown to self-assemble into large doughnut-shaped octamers with circumferential grooves and deep clefts containing nucleotide-binding histidine triad (HIT)-like motifs. Here, we demonstrate that NSP2 of group C rotavirus, a group that fails to reassort with group A viruses, retains the unique architecture of the group A octamer but differs in surface charge distribution. By using an NSP2-dependent complementation system, we show that the HIT-dependent NTPase activity of NSP2 is necessary for dsRNA synthesis, but not for viroplasm formation. The complementation system also showed that despite the retention of the octamer structure and the HIT-like fold, group C NSP2 failed to rescue replication and viroplasm formation in NSP2-deficient cells infected with group A rotavirus. The distinct differences in the surface charges on the Bristol and SA11 NSP2 octamers suggest that charge complementarity of the viroplasm-forming proteins guides the specificity of viroplasm formation and, possibly, reassortment restriction between rotavirus groups.

Section: Discussionmentioning

confidence: 99%

Structure-Function Analysis of Rotavirus NSP2 Octamer by Using a Novel Complementation System

Taraporewala

Carpió

Jayaram

et al. 2006

“…To estimate the relative viral yield from transfected BHK21 cells, FHV particles were partially purified using a previously described protocol (29) and subjected to quantitative immunoblot analysis using rabbit anti-FHV serum. In brief, the cells were lysed in 0.5% Nonidet P-40, and cell debris was pelleted at maximum speed for 10 min at 4°C in a microcentrifuge.…”

Section: Methodsmentioning

confidence: 99%

Cytoplasmic Granule Formation and Translational Inhibition of Nodaviral RNAs in the Absence of the Double-Stranded RNA Binding Protein B2

Petrillo

Venter

Short

et al. 2013

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bFlock House virus (FHV) is a positive-sense RNA insect virus with a bipartite genome. RNA1 encodes the RNA-dependent RNA polymerase, and RNA2 encodes the capsid protein. A third protein, B2, is translated from a subgenomic RNA3 derived from the 3= end of RNA1. B2 is a double-stranded RNA (dsRNA) binding protein that inhibits RNA silencing, a major antiviral defense pathway in insects. FHV is conveniently propagated in Drosophila melanogaster cells but can also be grown in mammalian cells. It was previously reported that B2 is dispensable for FHV RNA replication in BHK21 cells; therefore, we chose this cell line to generate a viral mutant that lacked the ability to produce B2. Consistent with published results, we found that RNA replication was indeed vigorous but the yield of progeny virus was negligible. Closer inspection revealed that infected cells contained very small amounts of coat protein despite an abundance of RNA2. B2 mutants that had reduced affinity for dsRNA produced analogous results, suggesting that the dsRNA binding capacity of B2 somehow played a role in coat protein synthesis. Using fluorescence in situ hybridization of FHV RNAs, we discovered that RNA2 is recruited into large cytoplasmic granules in the absence of B2, whereas the distribution of RNA1 remains largely unaffected. We conclude that B2, by binding to double-stranded regions in progeny RNA2, prevents recruitment of RNA2 into cellular structures, where it is translationally silenced. This represents a novel function of B2 that further contributes to successful completion of the nodaviral life cycle.T he nodaviruses are a family of positive-strand RNA viruses that naturally infect insects and fish. They have a bipartite genome that contains approximately 4,500 bases and encodes three proteins. The most thoroughly studied nodavirus is the insect virus Flock House virus (FHV), which has served as an important model system to investigate the structural and molecular basis of RNA replication, virus assembly, and inhibition of antiviral host responses (1). The larger of the two genomic RNAs, RNA1 (3.1 kb), encodes the RNAdependent RNA polymerase (RdRp; 112 kDa), which is located on the outer membrane of mitochondria in infected cells (2, 3). Viral RNA synthesis induces so-called spherules (4), i.e., membrane invaginations, which are thought to sequester the replication complexes and double-stranded RNA (dsRNA) intermediates to protect them from RNA silencing, a major antiviral pathway activated in insects upon infection with RNA viruses (5). Further protection from RNA silencing is afforded by FHV protein B2 (11.6 kDa), a dsRNA binding protein that is translated from RNA3 (387 nucleotides), a subgenomic RNA derived from the 3= end of RNA1 (6-8). The capsid protein, protein alpha (43 kDa), is translated from RNA2 (1.4 kb), the second genomic RNA segment.Although a seemingly simple virus, FHV uses a sophisticated regulatory system to control its gene expression. This regulation occurs at several levels and is currently incompletely understo...

“…Monolayers consisting of 8 ϫ 10 6 Sf21 cells in 100-mm dishes were infected at a multiplicity of infection of either 5 or 10 PFU per cell by adding recombinant baculovirus AcR2. AcR2 contains the cDNA of FHV RNA2 under control of the polyhedrin promoter and was described previously (40). Following addition of the virus, cells were incubated at room temperature with rocking for 1 h. Unattached virus was then removed and replaced with 7 ml of complete TC100 growth medium.…”

Section: Preparation Of [ 35 S]methionine-and [mentioning

confidence: 99%

Morphological Changes in the T=3 Capsid of Flock House Virus during Cell Entry

Walukiewicz

Johnson

Schneemann

2006

Self Cite

We report the identification and characterization of a viral intermediate formed during infection of Drosophila cells with the nodavirus Flock House virus (FHV). We observed that even at a very low multiplicity of infection, only 70% of the input virus stayed attached to or entered the cells, while the remaining 30% of the virus eluted from cells after initial binding. The eluted FHV particles did not rebind to Drosophila cells and, thus, could no longer initiate infection by the receptor-mediated entry pathway. FHV virus-like particles with the same capsid composition as native FHV but containing cellular RNA also exhibited formation of eluted particles when incubated with the cells. A maturation cleavage-defective mutant of FHV, however, did not. Compared to naïve FHV particles, i.e., particles that had never been incubated with cells, eluted particles showed an acid-sensitive phenotype and morphological alterations. Furthermore, eluted particles had lost a fraction of the internally located capsid protein gamma. Based on these results, we hypothesize that FHV eluted particles represent an infection intermediate analogous to eluted particles observed for members of the family Picornaviridae.Nonenveloped viruses are stable protein complexes designed to protect and transport the viral genome from cell to cell. During assembly and disassembly, these complexes undergo transitions through meta-stable intermediates. Metastable assembly intermediates known as provirions have previously been identified and characterized for several viruses (9, 15, 19), but less is known about the intermediates formed during viral cell entry. Members of the family Picornaviridae, including poliovirus, human rhinovirus, and coxsackievirus B3, are known to form "eluted particle" intermediates during cell entry (3,8,11,14,22,(27)(28)(29). Eluted particles are virions that, after initial binding to their cognate receptor, have dissociated from the receptor in an altered form. They have lost the internal capsid protein VP4 and sediment at a decreased rate on sucrose gradients. They also display altered antigenic properties, show increased protease susceptibility, and, most importantly, are no longer able to reattach to their receptor (11,13,14,17,27).Here we report that Flock House virus (FHV), a member of the family Nodaviridae, also forms eluted particles during the initial stages of viral infection. FHV is a nonenveloped icosahedral insect virus with a bipartite positive-strand RNA genome (for a review, see references 2 and 37). Its life cycle is confined to the cytoplasm of infected cells. FHV has a wellcharacterized Tϭ3 capsid that is initially assembled from multiple subunits of the single structural precursor protein alpha (16,19). Following assembly, alpha protein undergoes a maturation cleavage, which gives rise to the major coat protein beta and a small peptide, gamma, which remains associated with mature virions and is located inside the virus particle near the packaged RNA (16,19). Previous results have shown that the C-terminal...