Mutual Interference between Genomic RNA Replication and Subgenomic mRNA Transcription in Brome Mosaic Virus

Abstract

Replication by many positive-strand RNA viruses includes genomic RNA amplification and subgenomic mRNA (sgRNA) transcription. For brome mosaic virus (BMV), both processes occur in virus-induced, membrane-associated compartments, require BMV replication factors 1a and 2a, and use negative-strand RNA3 as a template for genomic RNA3 and sgRNA syntheses. To begin elucidating their relations, we examined the interaction of RNA3 replication and sgRNA transcription in Saccharomyces cerevisiae expressing 1a and 2a, which support the full RNA3 replication cycle. Blocking sgRNA transcription stimulated RNA3 replication by up to 350%, implying that sgRNA transcription inhibits RNA3 replication. Such inhibition was independent of the sgRNA-encoded coat protein and operated in cis. We further found that sgRNA transcription inhibited RNA3 replication at a step or steps after negative-strand RNA3 synthesis, implying competition with positivestrand RNA3 synthesis for negative-strand RNA3 templates, viral replication factors, or common host components. Consistent with this, sgRNA transcription was stimulated by up to 400% when mutations inhibiting positive-strand RNA3 synthesis were introduced into the RNA3 5-untranslated region. Thus, BMV subgenomic and genomic RNA syntheses mutually interfered with each other, apparently by competition for one or more common factors. In plant protoplasts replicating all three BMV genomic RNAs, mutations blocking sgRNA transcription often had lesser effects on RNA3 accumulation, possibly because RNA3 also competed with RNA1 and RNA2 replication templates and because any increase in RNA3 replication at the expense of RNA1 and RNA2 would be self-limited by decreased 1a and 2a expression from RNA1 and RNA2.Subgenomic mRNA (sgRNA) transcription is a mechanism used by many eukaryotic RNA viruses to multiply the number of separate genes expressed and to regulate the timing and magnitude of their expression. Numerous positive-strand RNA viruses produce sgRNAs, using diverse transcription mechanisms distinguished by internal initiation or termination, continuous or discontinuous RNA synthesis, etc., to link internal open reading frames (ORFs) to a translatable 5Ј end, producing sgRNAs that are 3Ј-coterminal with the viral genomic RNA (40). Many studies have revealed cis-signals and mechanistic features of sgRNA transcription and genomic RNA replication in different positive-strand RNA viruses, but relatively little is known about how these two processes, which depend on common viral replication factors and RNA templates, are coordinated.Relevant infectious processes, such as RNA replication, sgRNA transcription, and host factor involvement, have been studied for a number of positive-strand RNA viruses, including brome mosaic virus (BMV), a member of the alphavirus superfamily. The BMV genome consists of three genomic RNAs. RNA1 and RNA2 encode viral RNA synthesis factors 1a and 2a, which are required for genome replication and sgRNA synthesis. 2a is the viral RNA polymerase, while 1a is a multifunction...

Interaction between Brome Mosaic Virus Proteins and RNAs: Effects on RNA Replication, Protein Expression, and RNA Stability

“…These diverse systems have yielded useful insights into the mechanism of viral infection. Furthermore, the results from different systems are generally in agreement, but some notable differences have been observed (31,65,86).…”

“…4B). Grdzelishvili et al (31) have proposed that in S. cerevisiae the subgenomic and genomic RNA3 promoters compete for replicase. However, if agroinfiltration produces higher levels of BMV replicase, then a subgenomic promoter mutation should have a more modest effect on RNA3 levels in tobacco, rather than in barley protoplasts.…”

Nuclear Protein Sam68 Interacts with the Enterovirus 71 Internal Ribosome Entry Site and Positively Regulates Viral Protein Translation

“…Knowledge of the interactions between a virus and its host factors could offer insights into viral and cellular functions and provide new antiviral targets for disease control. The identification of such interactions and the associated host factors is thus a major frontier in virology (48,49). In this study, we provided new evidence indicating that the host factor, nuclear protein Sam68, was relocalized to the cytoplasm during EV71 infection and interacted with the IRES of EV71 5=UTR.…”

Distinct Sites on the Sindbis Virus RNA-Dependent RNA Polymerase for Binding to the Promoters for the Synthesis of Genomic and Subgenomic RNA

“…It has been suggested that viral RDRPs may have different sites for the recognition of different promoters (3). If so, it would follow that the RDRPs of RNA viruses that make an SG RNA via internal initiation, such as SV, should have different sites for the recognition of the G and SG promoters.…”