The Tripartite Virions of the Brome Mosaic Virus Have Distinct Physical Properties That Affect the Timing of the Infection Process

Abstract

The three subsets of virions that comprise the Brome mosaic virus (BMV) were previously thought to be indistinguishable. This work tested the hypothesis that distinct capsid-RNA interactions in the BMV virions allow different rates of viral RNA release. Several results support distinct interactions between the capsid and the BMV genomic RNAs. First, the deletion of the first eight residues of the BMV coat protein (CP) resulted in the RNA1-containing particles having altered morphologies, while those containing RNA2 were unaffected. Second, subsets of the BMV particles separated by density gradients into a pool enriched for RNA1 (B1) and for RNA2 and RNA3/4 (B2.3/4) were found to have different physiochemical properties. Compared to the B2.3/4 particles, the B1 particles were more sensitive to protease digestion and had greater resistivity to nanoindentation by atomic force microscopy and increased susceptibility to nuclease digestion. Mapping studies showed that portions of the arginine-rich N-terminal tail of the CP could interact with RNA1. Mutational analysis in the putative RNA1-contacting residues severely reduced encapsidation of BMV RNA1 without affecting the encapsidation of RNA2. Finally, during infection of plants, the more easily released RNA1 accumulated to higher levels early in the infection. IMPORTANCEViruses with genomes packaged in distinct virions could theoretically release the genomes at different times to regulate the timing of gene expression. Using an RNA virus composed of three particles, we demonstrated that the RNA in one of the virions is released more easily than the other two in vitro. The differential RNA release is due to distinct interactions between the viral capsid protein and the RNAs. The ease of RNA release is also correlated with the more rapid accumulation of that RNA in infected plants. Our study identified a novel role for capsid-RNA interactions in the regulation of a viral infection. Viruses use multiple strategies to maximize their fitness (1, 2). For viruses with genomes consisting of multiple RNA segments, their genomes can reassort with each infection to increase the ability to gain new combinations of traits (3, 4). A multipartite genome can also allow differential regulation for the expression of each segment of the genome. This could impact the timing of viral gene expression and limit exposure of the viral genome to attacks by host defenses (5, 6).Brome mosaic virus (BMV) is an excellent model system for a virus with a multipartite genome. BMV has three positivestranded genomic RNAs (RNA1, RNA2, and RNA3) and a subgenomic RNA (RNA4) (7,8). The genomic RNAs are required for successful infection and are encapsidated into three separate virions. Subgenomic RNA4 is coencapsidated with RNA3. All three subsets of BMV virions contain 180 copies of the BMV coat protein (CP) and are indistinguishable in electron micrographs. Indeed, a mixture of three virions is used to grow crystals for structural studies (9, 10).Contrary to the idea that subpopulations of BMV p...

Phosphorylation of the Brome Mosaic Virus Capsid Regulates the Timing of Viral Infection

“…The minus-strand BMV RNA1 and RNA2 synthesized during BMV replication were quantified using quantitative RT-PCR. RNA1 has been demonstrated to preferentially accumulate to higher levels earlier in infection than RNA2 (9). We observed this in the mock-treated virions (Fig.…”

“…Both have an intrinsically disordered N-terminal arm rich in positively charged residues followed by sequences that fold into a globular domain (7). The N-terminal arm contributes to the differential encapsidation of BMV RNA, has the ability to translocate from the internal cavity to the outside of the virion, and is preferentially cleaved during proteolysis (8,9). Virions formed by CPs that lack the first 8 residues were found to encapsidate RNA2 well but were labile for the encapsidation of RNA1 (8).…”

“…The B1 virions release RNA1 more rapidly than the B2.3/4 virions, likely to enable the more rapid expression of the protein needed to form the viral replication factory (9,10). The B2.3/4 virions are much more stable than the B1 virions, and the capsid appears to interact more stably with the encapsidated RNAs in B2.3/4 virions (8,9), raising the question of whether the release of RNA2, RNA3, and RNA4 requires an additional layer of control.…”

“…The BMV virions can be separated by their density into two populations, one enriched for virions containing RNA1, called B1 virions, and the other enriched for virions that encapsidate RNA2 and RNA3/4, called B2.3/4 virions (9). The B1 virions release RNA1 more rapidly than the B2.3/4 virions, likely to enable the more rapid expression of the protein needed to form the viral replication factory (9,10).…”

<div>Phosphorylation of the viral coat protein regulates RNA virus infection</div>

“…This likely regulates the timing of BMV RNA release in the cell and the timing of BMV protein synthesis. 40 Notably, RNA sequences found to bind the CP include regulatory motifs in the RNAs that affect BMV translation and replication. Replacement of a threonine in the flexible N-terminal arm of the BMV CP with a phosphomimetic glutamate altered the ratios of encapsidated RNA, affected the kinetics of replication, and decreased the time needed for translation of the BMV CP.…”