Coat Protein Activation of Alfalfa Mosaic Virus Replication Is Concentration Dependent

Bamboo mosaic virus (BaMV) is a positive-sense RNA virus belonging to the genus Potexvirus. Open reading frame 1 (ORF1) encodes the viral replication protein that consists of a capping enzyme domain, a helicase-like domain (HLD), and an RNA-dependent RNA polymerase domain from the N to C terminus. ORF5 encodes the viral coat protein (CP) required for genome encapsidation and the virus movement in plants. In this study, application of a yeast-two hybrid assay detected an interaction between the viral HLD and CP. However, the interaction did not affect the NTPase activity of the HLD. To identify the critical amino acids of CP interacting with the HLD, a random mutational library of CP was created using error-prone PCR, and the mutations adversely affecting the interaction were screened by a bacterial two-hybrid system. As a result, the mutations A209G and N210S in CP were found to weaken the interaction. To determine the significance of the interaction, the mutations were introduced into a BaMV infectious clone, and the mutational effects on viral replication, movement, and genome encapsidation were investigated. There was no effect on accumulations of BaMV CP and genomic RNAs within protoplasts; however, the virus cell-to-cell movement in plants was restricted. Sequence alignment revealed that A209 of BaMV CP is conserved in many potexviruses. Mutation of the corresponding residue in Foxtail mosaic virus CP also reduced the viral HLD-CP interaction and restricted the virus movement, suggesting that interaction between CP and a widely conserved HLD in the potexviral replication protein is crucial for viral trafficking through plasmodesmata.To spread throughout hosts, plant viruses have evolved a number of pathways to allow their progeny to pass across plasmodesmata into neighboring cells and travel along the vascular system (8, 26). The virus-encoded movement proteins play a pivotal role through diverse mechanisms in these cellto-cell and vascular transports. Ancillary proteins, for example, the viral coat proteins (CPs) in some cases, and host factors may also participate in these processes. Numerous studies have been conducted to elucidate the movement mechanisms. Many of the results have been summarized in a number of recent reviews (23,28,30). They provided in-depth discussions on issues such as the identification and characterization of the involved viral and host proteins and the transport models for some exemplified viruses, such as Tobacco mosaic virus (TMV) and Potato virus X (PVX). Despite these efforts, many details of the processes remain elusive.Members of the genus Potexvirus have a positive-strand RNA genome that contains five open reading frames (ORFs), a 5Ј methyl cap, and a 3Ј poly(A) tail. ORF1 encodes the viral replication protein, consisting of a capping enzyme domain, a helicase-like domain (HLD), and an RNA-dependent RNA polymerase domain (RdRp) from the N terminus to the C terminus (16,17). The HLD has RNA 5Ј-triphosphatase and nucleoside triphosphatase (NTPase) activities (18). With the co...

Section: Discussionmentioning

confidence: 77%

The Interaction between Bamboo Mosaic Virus Replication Protein and Coat Protein Is Critical for Virus Movement in Plant Hosts

Lee

et al. 2011

“…We propose that the differences in functional data from the two laboratories may be traced to the use of wild-type cells (described here) versus in vitro analysis and the use of transgenic cells that overexpress replicase proteins (22). The accumulated evidence presented here, along with functional data presented in the accompanying paper (12) and the crystal structure of the AMV RNA-peptide complex (11), suggest that CP binding organizes the 3Ј termini of the viral RNAs for activation of early viral RNA replication.…”

mentioning

confidence: 99%

Evaluation of the Conformational Switch Model for Alfalfa Mosaic Virus RNA Replication

Petrillo

Rocheleau

Kelley-Clarke

et al. 2005

Self Cite

Key elements of the conformational switch model describing regulation of alfalfa mosaic virus (AMV) replication (R. C. Olsthoorn, S. Mertens, F. T. Brederode, and J. F. Bol, EMBO J. 18:4856-4864, 1999) have been tested using biochemical assays and functional studies in nontransgenic protoplasts. Although comparative sequence analysis suggests that the 3 untranslated regions of AMV and ilarvirus RNAs have the potential to fold into pseudoknots, we were unable to confirm that a proposed pseudoknot forms or has a functional role in regulating coat protein-RNA binding or viral RNA replication. Published work has suggested that the pseudoknot is part of a tRNA-like structure (TLS); however, we argue that the canonical sequence and functional features that define the TLS are absent. We suggest here that the absence of the TLS correlates directly with the distinctive requirement for coat protein to activate replication in these viruses. Experimental data are evidence that elevated magnesium concentrations proposed to stabilize the pseudoknot structure do not block coat protein binding. Additionally, covarying nucleotide changes proposed to reestablish pseudoknot pairings do not rescue replication. Furthermore, as described in the accompanying paper (L. M. Guogas, S. M. Laforest, and L. Gehrke, J. Virol. 79:5752-5761, 2005), coat protein is not, by definition, inhibitory to minus-strand RNA synthesis. Rather, the activation of viral RNA replication by coat protein is shown to be concentration dependent. We describe the 3 organization model as an alternate model of AMV replication that offers an improved fit to the available data.Regulation of the switch between translation and replication is a fundamental problem in understanding the biology of positive-stranded RNA viruses. Olsthoorn et al. (22) published the conformational switch model to propose a mechanism for regulating the switch in alfalfa mosaic virus (AMV). The conformational switch model asserts that the 3Ј termini of the viral RNAs fold into two mutually exclusive conformations that have distinct functions, that is, pseudoknotted (coat protein [CP]-free) and extended (CP-bound) forms. In the absence of viral CP, the RNA 3Ј terminus is said to adopt a pseudoknotted tRNA-like structure (TLS) that would make it structurally homologous to many other bromovirus RNAs. Regions 1 and 2 of the viral RNA (see Fig. 1B) have the potential to form a pseudoknot, and nucleotide variations across the ilarviruses suggest covarying substitutions that would maintain the longrange pseudoknot pairing (22). Equally interesting, however, is the fact that the nucleotides in region 3 of the viral RNAs also covary with changes in region 2, thereby also maintaining the potential to form the downstream hairpin by short-range folding (hairpin from nucleotides 869 through 877) (Fig. 1B). Recent data confirm that hairpin from nucleotides 869 through 877 is present in the crystal structure of the 39-nucleotide 3Ј-terminal RNA fragment in complex with the RNA binding domain of the viral CP ...

“…Neeleman et al (36) reported that the coat protein (CP) of alfalfa mosaic virus (AMV), a member of the genus Alfamovirus in the family Bromoviridae, can bind to the 3= end of viral RNA and enhance subgenomic RNA4 translation. The AMV CP can regulate RNA synthesis by binding to the 3= ends of Alfamovirus and Ilarvirus RNAs to activate genome replication (6,23). In rotavirus, VP2 can serve as a scaffold for the viral polymerase as well as act as a cofactor for VP1 to initiate genome replication (32,38).…”

Section: R E T R a C T E Dmentioning

confidence: 99%

Norovirus RNA Synthesis Is Modulated by an Interaction between the Viral RNA-Dependent RNA Polymerase and the Major Capsid Protein, VP1

Subba-Reddy

Yunus

Goodfellow

et al. 2012

Using a cell-based assay for RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of noroviruses, we previously observed that VP1, the major structural protein of the human GII.4 norovirus, enhanced the GII.4 RdRp activity but not that of the related murine norovirus (MNV) or other unrelated RNA viruses (C. V. Subba-Reddy, I. Goodfellow, and C. C. Kao, J. Virol. 85:13027–13037, 2011). Here, we examine the mechanism of VP1 enhancement of RdRp activity and the mechanism of mouse norovirus replication. We determined that the GII.4 and MNV VP1 proteins can enhance cognate RdRp activities in a concentration-dependent manner. The VP1 proteins coimmunoprecipitated with their cognate RdRps. Coexpression of individual domains of VP1 with the viral RdRps showed that the VP1 shell domain (SD) was sufficient to enhance polymerase activity. Using SD chimeras from GII.4 and MNV, three loops connecting the central β-barrel structure were found to be responsible for the species-specific enhancement of RdRp activity. A differential scanning fluorimetry assay showed that recombinant SDs can bind to the purified RdRpsin vitro. An MNV replicon with a frameshift mutation in open reading frame 2 (ORF2) that disrupts VP1 expression was defective for RNA replication, as quantified by luciferase reporter assay and real-time quantitative reverse transcription-PCR (qRT-PCR).Trans-complementation of VP1 or its SD significantly recovered the VP1 knockout MNV replicon replication, and the presence or absence of VP1 affected the kinetics of viral RNA synthesis. The results document a regulatory role for VP1 in the norovirus replication cycle, further highlighting the paradigm of viral structural proteins playing additional functional roles in the virus life cycle.