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...

Brome mosaic virus (BMV) packages its genomic and subgenomic RNAs into three separate viral particles. BMV purified from barley, wheat and tobacco have distinct relative abundances of the encapsidated RNAs. We seek to identify the basis for the host-dependent differences in viral RNA encapsidation. Sequencing of the viral RNAs revealed recombination events in the 3′ untranslated region of RNA1 of BMV purified from barley and wheat, but not from tobacco. However, the relative amounts of the BMV RNAs that accumulated in barley and wheat are similar and RNA accumulation is not sufficient to account for the difference in RNA encapsidation. Virions purified from barley and wheat were found to differ in their isoelectric points, resistance to proteolysis, and contacts between the capsid residues and the RNA. Mass spectrometric analyses revealed that virions from the three hosts had different post-translational modifications that should impact the physiochemical properties of the virions. Another major source of variation in RNA encapsidation was due to the purification of BMV particles to homogeneity. Highly enriched BMV present in lysates had a surprising range of sizes, buoyant densities, and distinct relative amounts of encapsidated RNAs. These results show that the encapsidated BMV RNAs reflect a combination of host effects on the physiochemical properties of the viral capsids and the enrichment of a subset of virions. The previously unexpected heterogeneity in BMV should influence the timing of the infection and also the host innate immune responses.

Toll-like Receptor 3 (TLR3) detects double-stranded (ds) RNAs to activate innate immune responses. While poly(I:C) is an excellent agonist for TLR3 in several cell lines and in human peripheral blood mononuclear cells, viral dsRNAs tend to be poor agonists, leading to the hypothesis that additional factor(s) are likely required to allow TLR3 to respond to viral dsRNAs. TLR3 signaling was examined in a lung epithelial cell line by quantifying cytokine production and in human embryonic kidney cells by quantifying luciferase reporter levels. Recombinant 1b hepatitis C virus polymerase was found to enhance TLR3 signaling in the lung epithelial BEAS-2B cells when added to the media along with either poly(I:C) or viral dsRNAs. The polymerase from the genotype 2a JFH-1 HCV was a poor enhancer of TLR3 signaling until it was mutated to favor a conformation that could bind better to a partially duplexed RNA. The 1b polymerase also co-localizes with TLR3 in endosomes. RNA-binding capsid proteins (CPs) from two positive-strand RNA viruses and the hepadenavirus hepatitis B virus (HBV) were also potent enhancers of TLR3 signaling by poly(I:C) or viral dsRNAs. A truncated version of the HBV CP that lacked an arginine-rich RNA-binding domain was unable to enhance TLR3 signaling. These results demonstrate that several viral RNA-binding proteins can enhance the dsRNA-dependent innate immune response initiated by TLR3.

bBrome mosaic virus (BMV) is a model positive-strand RNA virus whose replication has been studied in a number of surrogate hosts. In transiently transfected human cells, the BMV polymerase 2a activated signaling by the innate immune receptor RIG-I, which recognizes de novo-initiated non-self-RNAs. Active-site mutations in 2a abolished RIG-I activation, and coexpression of the BMV 1a protein stimulated 2a activity. Mutations previously shown to abolish 1a and 2a interaction prevented the 1a-dependent enhancement of 2a activity. New insights into 1a-2a interaction include the findings that helicase active site of 1a is required to enhance 2a polymerase activity and that negatively charged amino acid residues between positions 110 and 120 of 2a contribute to interaction with the 1a helicase-like domain but not to the intrinsic polymerase activity. Confocal fluorescence microscopy revealed that the BMV 1a and 2a colocalized to perinuclear region in human cells. However, no perinuclear spherule-like structures were detected in human cells by immunoelectron microscopy. Sequencing of the RNAs coimmunoprecipitated with RIG-I revealed that the 2a-synthesized short RNAs are derived from the message used to translate 2a. That is, 2a exhibits a strong cis preference for BMV RNA2. Strikingly, the 2a RNA products had initiation sequences (5=-GUAAA-3=) identical to those from the 5= sequence of the BMV genomic RNA2 and RNA3. These results show that the BMV 2a polymerase does not require other BMV proteins to initiate RNA synthesis but that the 1a helicase domain, and likely helicase activity, can affect RNA synthesis by 2a. Brome mosaic virus (BMV), a member of the alphavirus-like superfamily of RNA viruses, is a model system for studies of viral RNA replication. In addition to being able to infect monocot and dicot plant hosts, BMV can replicate and transcribe its genome in yeast, Saccharomyces cerevisiae (9,11,12,32). Studies in S. cerevisiae have led to the identification of a number of cellular pathways that impact BMV replication (10) and contributed to our understanding of the membrane-encased BMV RNA replication factories, so-called spherules (38).The BMV genome is composed of three capped, messengersense RNAs. RNA3 encodes two proteins required for cell-to-cell movement and for RNA encapsidation. RNA1 and RNA2 encode nonstructural proteins 1a and 2a, respectively (24, 38). 1a and 2a expressed from mRNAs that lack the cis-acting sequences required for RNA replication can replicate RNA3 (1, 39).The multifunctional 1a protein containing an N-terminal domain with m 7 G methyltransferase activity required for viral RNA capping (4, 16) and a C-terminal half that has sequence motifs of DEAD box RNA helicases exhibits ATPase and GTPase activities (17, 47). 1a is necessary and sufficient to form the BMV replication factories in the perinuclear endoplasmic reticulum of barley cells and in the surrogate host, S. cerevisiae (33,34,37,38). It also recruits RNA2 and RNA3 to the replication factory (7,8). Mapping studies using 1a mut...