RNA Synthesis by the Brome Mosaic Virus RNA-Dependent RNA Polymerase in Human Cells Reveals Requirements for
            <i>De Novo</i>
            Initiation and Protein-Protein Interaction

Subba-Reddy, Chennareddy V.; Tragesser, Brady; Xu, Zhili; Stein, Barry D.; Ranjith-Kumar, C. T.; Kao, C. Cheng

doi:10.1128/jvi.00069-12

Cited by 9 publications

(6 citation statements)

References 48 publications

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“…These results suggest that 2a alone is sufficient to initiate synthesis of (ϩ) strands on (Ϫ)-strand templates. Similar observations were recently reported while examining the activity of BMV 1a and 2a in transiently transfected human cells (65). Keeping these results and the fact that viral RdRp recruits (ϩ)-strand templates to spherules for replication in perspective, it is possible that synthesis of B3(ϩ)-strand templates from ectopically expressed B3(Ϫ)-strand templates is initiated by the 2a protein associated with the cytoplasm prior to spherule recruitment.…”

Section: Discussionsupporting

confidence: 84%

Emergence of Distinct Brome Mosaic Virus Recombinants Is Determined by the Polarity of the Inoculum RNA

Kwon¹,

Rao²

2012

J Virol

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Despite overwhelming interest in the impact exerted by recombination during evolution of RNA viruses, the relative contribution of the polarity of inoculum templates remains poorly understood. Here, by agroinfiltrating Nicotiana benthamiana leaves, we show that brome mosaic virus (BMV) replicase is competent to initiate positive-strand [(؉)-strand] synthesis on an ectopically expressed RNA3 negative strand [(؊) strand] and faithfully complete the replication cycle. Consequently, we sought to examine the role of RNA polarity in BMV recombination by expressing a series of replicationdefective mutants of BMV RNA3 in (؉) or (؊) polarity. Temporal analysis of progeny sequences revealed that the genetic makeup of the primary recombinant pool is determined by the polarity of the inoculum template. When the polarity of the inoculum template was (؉), the recombinant pool that accumulated during early phases of replication was a mixture of nonhomologous recombinants. These are longer than the inoculum template length, and a nascent 3= untranslated region (UTR) of wild-type (WT) RNA1 or RNA2 was added to the input mutant RNA3 3= UTR due to end-to-end template switching by BMV replicase during (؊)-strand synthesis. In contrast, when the polarity of the inoculum was (؊), the progeny contained a pool of native-length homologous recombinants generated by template switching of BMV replicase with a nascent UTR from WT RNA1 or RNA2 during (؉)-strand synthesis. Repair of a point mutation caused by polymerase error occurred only when the polarity of the inoculum template was (؉). These results contribute to the explanation of the functional role of RNA polarity in recombination mediated by copy choice mechanisms.A mong a wide range of events, genome replication is considered to be the fundamental aspect in the biology of positivestrand [(ϩ)-strand] RNA viruses pathogenic to humans, animals, or plants. Production of copious amounts of infectious (ϩ)-strand progeny occurs in two distinct phases that are preferentially modulated by virus-encoded RNA-dependent RNA polymerase (RdRp): initiation and synthesis of complementary negativestrand [(Ϫ)-strand] RNA using genomic (ϩ)-strand RNA as a template, followed by the synthesis of progeny (ϩ)-strand RNA using a (Ϫ)-strand RNA as the template (53). The accuracy with which the viral genomes are copied by viral RdRp plays an important role in establishing a successful infection in susceptible hosts (20). However, due to the lack of inherent proofreading activity during RNA synthesis by viral RdRp, polymerase errors result in the emergence of highly heterogeneous populations (quasispecies) held together in a dynamic equilibrium (20,22,56). In addition to polymerase error, RNA recombination has been shown to be one of the major driving forces toward genetic variation in RNA viruses (15,20,36,37,50,56). Whether it is polymerase error or RNA recombination, natural selection is likely to maintain the most favorable consensus sequences for optimal replication of viral genomes (20). Ever since the f...

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Section: Discussionsupporting

confidence: 84%

Emergence of Distinct Brome Mosaic Virus Recombinants Is Determined by the Polarity of the Inoculum RNA

Kwon¹,

Rao²

2012

J Virol

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“…Bands in the blots were quantified using ImageQuant software. Reverse transcription and quantitative PCR (RT-qPCR) to quantify minus-strand BMV RNA1 and RNA2 used 500 ng total RNA to synthesize the cDNA by strand-specific primers that recognized either nucleotides (nt) 100 to 120 on RNA1 or nt 371 to 392 on RNA2 as described by Subba-Reddy et al (17). The cDNA was detected using pairs of primers that recognized nt 100 to 232 of RNA1 and nt 371 to 499 of RNA2.…”

Section: Methodsmentioning

confidence: 99%

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

Vaughan

Tragesser

et al. 2014

J Virol

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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...

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“…Monocistronic RNA1 and RNA2 each encode one protein required for BMV replication. 7,8 Dicistronic RNA3 encodes the movement protein required for cell to cell spread and the coat protein (CP) that encapsidates BMV RNAs. The CP is translated from subgenomic RNA4 that is transcribed from the minus-strand RNA3.…”

Section: Introductionmentioning

confidence: 99%

The Plant Host Can Affect the Encapsidation of Brome Mosaic Virus (BMV) RNA: BMV Virions Are Surprisingly Heterogeneous

Vaughan

Tragesser

et al. 2014

Journal of Molecular Biology

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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.

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RNA Synthesis by the Brome Mosaic Virus RNA-Dependent RNA Polymerase in Human Cells Reveals Requirements for De Novo Initiation and Protein-Protein Interaction

Cited by 9 publications

References 48 publications

Emergence of Distinct Brome Mosaic Virus Recombinants Is Determined by the Polarity of the Inoculum RNA

Emergence of Distinct Brome Mosaic Virus Recombinants Is Determined by the Polarity of the Inoculum RNA

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

The Plant Host Can Affect the Encapsidation of Brome Mosaic Virus (BMV) RNA: BMV Virions Are Surprisingly Heterogeneous

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