Alfalfa mosaic virus (AMV) RNA replication requires the viral coat protein (CP). AMV CP is an integral component of the viral replicase; moreover, it binds to the viral RNA 3'-termini and induces the formation of multiple new base pairs that organize the RNA conformation. The results described here suggest that AMV coat protein binding defines template selection by organizing the 3'-terminal RNA conformation and by positioning the RNA-dependent RNA polymerase (RdRp) at the initiation site for minus strand synthesis. RNA-protein interactions were analyzed by using a modified Northwestern blotting protocol that included both viral coat protein and labeled RNA in the probe solution ("far-Northwestern blotting"). We observed that labeled RNA alone bound the replicase proteins poorly; however, complex formation was enhanced significantly in the presence of AMV CP. The RNA-replicase bridging function of the AMV CP may represent a mechanism for accurate de novo initiation in the absence of canonical 3' transfer RNA signals.

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 ...

The alfalfa mosaic virus (AMV) RNAs are infectious only in the presence of the viral coat protein; however, the mechanisms describing coat protein's role during replication are disputed. We reasoned that mechanistic details might be revealed by identifying RNA mutations in the 3-terminal coat protein binding domain that increased or decreased RNA replication without affecting coat protein binding. Degenerate (doped) in vitro genetic selection, based on a pool of randomized 39-mers, was used to select 30 variant RNAs that bound coat protein with high affinity. AUGC sequences that are conserved among AMV and ilarvirus RNAs were among the invariant nucleotides in the selected RNAs. Five representative clones were analyzed in functional assays, revealing diminished viral RNA expression resulting from apparent defects in replication and/or translation. These data identify a set of mutations, including G-U wobble pairs and nucleotide mismatches in the 5 hairpin, which affect viral RNA functions without significant impact on coat protein binding. Because the mutations associated with diminished function were scattered over the 3-terminal nucleotides, we considered the possibility that RNA conformational changes rather than disruption of a precise motif might limit activity. Native polyacrylamide gel electrophoresis experiments showed that the 3 RNA conformation was indeed altered by nucleotide substitutions. One interpretation of the data is that coat protein binding to the AUGC sequences determines the orientation of the 3 hairpins relative to one another, while local structural features within these hairpins are also critical determinants of functional activity.Alfalfa mosaic virus (AMV) has a number of exceptional features that make it a useful system for studying RNA-protein interactions (reviewed in references 6 and 17). The three positive-sense genomic AMV RNAs (RNAs 1 to 3) and the subgenomic coat protein mRNA (RNA 4) are packaged individually into four bacilliform, aphid-transmitted particles. The viral genomic RNAs are infectious only in the presence of the viral coat protein (7), which binds specifically to the RNA 3Ј termini. Unlike the arginine-rich RNA binding domains of the human immunodeficiency virus Tat and Rev proteins, the AMV coat protein RNA binding domain is lysine rich (3); however, it includes an arginine residue whose position and side chain identity are crucial for specific, high-affinity RNA binding (2). The AMV RNAs lack a poly(A) tail and are further distinguished from closely related plant viruses (e.g., brome mosaic virus) by the absence of a canonical CCA 3Ј terminus and the inability to be charged by aminoacyl synthetases, both of which are defining features of tRNA-like ends (12).The relationship between the formation of the AMV coat protein-RNA complex and functional activity in viral RNA translation, replication, and assembly is poorly understood. Previous biochemical characterization has defined specific binding determinants in both the RNA and coat protein (1,3,14,15,18,(26)(27)(28...

bFlock House virus (FHV) is a positive-sense RNA insect virus with a bipartite genome. RNA1 encodes the RNA-dependent RNA polymerase, and RNA2 encodes the capsid protein. A third protein, B2, is translated from a subgenomic RNA3 derived from the 3= end of RNA1. B2 is a double-stranded RNA (dsRNA) binding protein that inhibits RNA silencing, a major antiviral defense pathway in insects. FHV is conveniently propagated in Drosophila melanogaster cells but can also be grown in mammalian cells. It was previously reported that B2 is dispensable for FHV RNA replication in BHK21 cells; therefore, we chose this cell line to generate a viral mutant that lacked the ability to produce B2. Consistent with published results, we found that RNA replication was indeed vigorous but the yield of progeny virus was negligible. Closer inspection revealed that infected cells contained very small amounts of coat protein despite an abundance of RNA2. B2 mutants that had reduced affinity for dsRNA produced analogous results, suggesting that the dsRNA binding capacity of B2 somehow played a role in coat protein synthesis. Using fluorescence in situ hybridization of FHV RNAs, we discovered that RNA2 is recruited into large cytoplasmic granules in the absence of B2, whereas the distribution of RNA1 remains largely unaffected. We conclude that B2, by binding to double-stranded regions in progeny RNA2, prevents recruitment of RNA2 into cellular structures, where it is translationally silenced. This represents a novel function of B2 that further contributes to successful completion of the nodaviral life cycle.T he nodaviruses are a family of positive-strand RNA viruses that naturally infect insects and fish. They have a bipartite genome that contains approximately 4,500 bases and encodes three proteins. The most thoroughly studied nodavirus is the insect virus Flock House virus (FHV), which has served as an important model system to investigate the structural and molecular basis of RNA replication, virus assembly, and inhibition of antiviral host responses (1). The larger of the two genomic RNAs, RNA1 (3.1 kb), encodes the RNAdependent RNA polymerase (RdRp; 112 kDa), which is located on the outer membrane of mitochondria in infected cells (2, 3). Viral RNA synthesis induces so-called spherules (4), i.e., membrane invaginations, which are thought to sequester the replication complexes and double-stranded RNA (dsRNA) intermediates to protect them from RNA silencing, a major antiviral pathway activated in insects upon infection with RNA viruses (5). Further protection from RNA silencing is afforded by FHV protein B2 (11.6 kDa), a dsRNA binding protein that is translated from RNA3 (387 nucleotides), a subgenomic RNA derived from the 3= end of RNA1 (6-8). The capsid protein, protein alpha (43 kDa), is translated from RNA2 (1.4 kb), the second genomic RNA segment.Although a seemingly simple virus, FHV uses a sophisticated regulatory system to control its gene expression. This regulation occurs at several levels and is currently incompletely understo...

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Managing the risks associated with laboratory use of biological agents is necessary to ensure the health and safety of laboratory personnel, the public, and the environment. Inadvertent or deliberate release of infectious materials risks the health of people, animals, or plants and can potentially undermine national and international security and stability. Effective management of the risks associated with laboratory use of biological agents is a process in which the components of the research environment associated with biological agents are examined to determine if they can cause harm, whether the appropriate precautions are in place to mitigate the harm, and, if not, to determine what additional steps are required. Application of risk mitigation measures must be based on a thorough assessment of risk, consistent with the specific risks at the facility and cost‐effective. Mitigation measures must not unduly impede life sciences research. This process of managing laboratory risk is the shared responsibility of all those engaged in the life sciences, including the researcher, institution, local community, national government, and international community.