Nodavirus-Induced Membrane Rearrangement in Replication Complex Assembly Requires Replicase Protein A, RNA Templates, and Polymerase Activity

Self Cite

, and Dhh1 control the ratio of subgenomic RNA3 to genomic RNA1 production, a key feature in the FHV life cycle mediated by a long-distance base pairing within RNA1. Depletion of LSM1, PAT1, or DHH1 dramatically increased RNA3 accumulation during replication. This was not caused by differences between RNA1 and RNA3 steady-state levels in the absence of replication. Importantly, coimmunoprecipitation assays indicated that LSm1-7, Pat1, and Dhh1 interact with the FHV RNA genome and the viral polymerase. By using a strategy that allows dissecting different stages of the replication process, we found that LSm1-7, Pat1, and Dhh1 did not affect the early replication steps of RNA1 recruitment to the replication complex or RNA1 synthesis. Furthermore, their function on RNA3/RNA1 ratios was independent of the membrane compartment, where replication occurs and requires ATPase activity of the Dhh1 helicase. Together, these results support that LSm1-7, Pat1, and Dhh1 control RNA3 synthesis. Their described function in mediating cellular mRNP rearrangements suggests a parallel role in mediating key viral RNP transitions, such as the one required to maintain the balance between the alternative FHV RNA1 conformations that control RNA3 synthesis.

Section: Lsm1supporting

confidence: 57%

The Cellular Decapping Activators LSm1, Pat1, and Dhh1 Control the Ratio of Subgenomic to Genomic Flock House Virus RNAs

Giménez‐Barcons

Alves-Rodrigues

Jungfleisch

et al. 2013

Self Cite

“…Similarly, BMV 1a protein induces replication complex-like spherules in which replication template RNA is recruited and sequestered, even in the absence of the 2a polymerase (47). In contrast, RNA polymerase activity is required for the formation of replication complex spherules by Flock House virus and Semliki Forest virus (50,51). In a certain host mutant, negative-strand RNA of tomato bushy stunt virus accumulates to levels similar to that in wild-type cells, but the negative-strand RNA is sensitive to nucleases, unlike in wild-type cells (52).…”

Section: Discussionmentioning

confidence: 99%

The Resistance Protein Tm-1 Inhibits Formation of a Tomato Mosaic Virus Replication Protein-Host Membrane Protein Complex

Ishibashi

Ishikawa

2013

The Tm-1 gene of tomato confers resistance to Tomato mosaic virus (ToMV). Tm-1 encodes a protein that binds ToMV replication proteins and inhibits the RNA-dependent RNA replication of ToMV. The replication proteins of resistance-breaking mutants of ToMV do not bind Tm-1, indicating that the binding is important for inhibition. In this study, we analyzed how Tm-1 inhibits ToMV RNA replication in a cell-free system using evacuolated tobacco protoplast extracts. In this system, ToMV RNA replication is catalyzed by replication proteins bound to membranes, and the RNA polymerase activity is unaffected by treatment with 0.5 M NaCl-containing buffer and remains associated with membranes. We show that in the presence of Tm-1, negative-strand RNA synthesis is inhibited; the replication proteins associate with membranes with binding that is sensitive to 0.5 M NaCl; the viral genomic RNA used as a translation template is not protected from nuclease digestion; and host membrane proteins TOM1, TOM2A, and ARL8 are not copurified with the membrane-bound 130K replication protein. Deletion of the polymerase read-through domain or of the 3= untranslated region (UTR) of the genome did not prevent the formation of complexes between the 130K protein and the host membrane proteins, the 0.5 M NaCl-resistant binding of the replication proteins to membranes, and the protection of the genomic RNA from nucleases. These results indicate that Tm-1 binds ToMV replication proteins to inhibit key events in replication complex formation on membranes that precede negative-strand RNA synthesis. Tomato mosaic virus (ToMV) is a positive-strand RNA virus in the genus Tobamovirus, family Virgaviridae (1). The genome of a tobamovirus encodes at least four proteins (2). The 130K protein and its read-through product, the 180K protein, are involved in RNA replication and thus are referred to as replication proteins (3). The 130K protein contains the methyltransferase and helicase domains, and the read-through region of the 180K protein contains the polymerase domain. The other two tobamoviral proteins, movement protein and coat protein, are not required for RNA replication (4, 5).When the genomic RNA of a positive-strand RNA virus enters host cells, the replication proteins are translated from the genomic RNA, recognize the genomic RNA as a template for replication, and form replication complexes on intracellular membranes. Replication of all known eukaryotic positive-strand RNA viruses occurs in replication complexes formed on host membranes (6-10), which contain viral genomic RNAs, replication proteins, and host factors (11). To avoid elicitation of host defense systems triggered by viral double-stranded RNA (12), the activity of eukaryotic positive-strand RNA virus RNA-dependent RNA polymerases (RdRps) must be strictly regulated so that they synthesize negative-strand RNA only in the replication complexes that are sequestered from the cytoplasm. Many host factors, including chaperones and enzymes, and cellular membranes are hijacked by viruses for rep...

“…There is precedence for the involvement of the viral RNA in the RC maturation process. For example, it has been shown that the viral RNA along with the NSPs is necessary for formation of RCs in Semliki Forest virus-infected cells (38), and this is also the case for nodaviruses (16). In testing our hypothesis that CP chaperones P200, we examined the steps in P200 maturation that we had been studying in this and previous studies (29,30), finding no difference in processing efficiency, P150-P90 interaction, or membrane association by P200 or P150 in the presence or absence of CP.…”

Section: Discussionmentioning

confidence: 99%

Determinants in the Maturation of Rubella Virus P200 Replicase Polyprotein Precursor

Matthews

Tzeng

Frey

2012

Rubella virus (RUBV), a positive-strand RNA virus, replicates its RNA within membrane-associated replication complexes (RCs) in the cytoplasm of infected cells. RNA synthesis is mediated by the nonstructural proteins (NSPs) P200 and its cleavage products, P150 and P90 (N and C terminal within P200, respectively), which are processed by a protease residing at the C terminus of P150. In this study of NSP maturation, we found that early NSP localization into foci appeared to target the membranes of the endoplasmic reticulum. During maturation, P150 and P90 likely interact within the context of P200 and remain in a complex after cleavage. We found that P150-P90 interactions were blocked by mutational disruption of an alpha helix at the N terminus (amino acids [aa] 36 to 49) of P200 and that these mutations also had an effect on NSP targeting, processing, and membrane association. While the P150-P90 interaction also required residues 1700 to 1900 within P90, focus formation required the entire RNA-dependent RNA polymerase (aa 1700 to 2116). Surprisingly, the RUBV capsid protein (CP) rescued RNA synthesis by several alanine-scanning mutations in the N-terminal alpha helix, and packaged replicon assays showed that rescue could be mediated by CP in the virus particle. We hypothesize that CP rescues these mutations as well as internal deletions of the Q domain within P150 and mutations in the 5′ and 3′ cis -acting elements in the genomic RNA by chaperoning the maturation of P200. CP's ability to properly target the otherwise aggregated plasmid-expressed P200 provides support for this hypothesis.