An Alternate Pathway for Recruiting Template RNA to the Brome Mosaic Virus RNA Replication Complex

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Poliovirus infection remodels intracellular membranes, creating a large number of membranous vesicles on which viral RNA replication occurs. Poliovirus-induced vesicles display hallmarks of cellular autophagosomes, including delimiting double membranes surrounding the cytosolic lumen, acquisition of the endosomal marker LAMP-1, and recruitment of the 18-kDa host protein LC3. Autophagy results in the covalent lipidation of LC3, conferring the property of membrane association to this previously microtubule-associated protein and providing a biochemical marker for the induction of autophagy. Here, we report that a similar modification of LC3 occurs both during poliovirus infection and following expression of a single viral protein, a stable precursor termed 2BC. Therefore, one of the early steps in cellular autophagy, LC3 modification, can be genetically separated from the induction of double-membraned vesicles that contain the modified LC3, which requires both viral proteins 2BC and 3A. The existence of viral inducers that promote a distinct aspect of the formation of autophagosome-like membranes both facilitates the dissection of this cellular process and supports the hypothesis that this branch of the innate immune response is directly subverted by poliovirus.All positive-strand RNA viruses of eukaryotes replicate their genomes on cytoplasmic membranes, and infections are often accompanied by dramatic reorganization of those membranes. We and others have argued that one function of membrane localization of viral RNA replication proteins is to create twodimensional surfaces to promote the oligomerization of viral proteins (6, 31). Yet the detailed cell biology of the targeted membranes and the mechanisms by which they are altered differ greatly from virus to virus (40,53).Poliovirus infection rearranges intracellular membranes, creating membranous vesicles, 200 to 400 nm in diameter, that accumulate in the cytoplasm (9, 42, 47). Immunoelectron microscopy revealed that the cytoplasmic surfaces of these membranous vesicles, not the interior lumen, are the sites of viral RNA replication (1, 7). The specific morphology of these membranes has proven somewhat controversial, because they are rich in lipids (17,33) and their apparent morphology is therefore sensitive to the fixation method used in electron microscopy (43, 46). Using high-pressure cryopreservation methods, we have consistently observed double-membraned vesicles (42, 47), as did Dales et al. using more conventional fixation techniques (9). Double-membraned vesicular structures have also been associated with viral RNA replication complexes in cells infected with several other positive-strand RNA viruses, including equine arterivirus (37), murine hepatitis virus (16), and sudden acute respiratory syndrome-associated virus (15). Therefore, understanding the mechanism(s) by which viral proteins induce the formation of double-membrane vesicles is of interest for understanding the formation of RNA replication complexes of several kinds of RNA viruses.Several of ...

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confidence: 99%

Modification of Cellular Autophagy Protein LC3 by Poliovirus

Taylor

Kirkegaard

2007

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149

“…In barley cells, the deletion of the B box resulted in defects in BMV replication and transcription (35,50). In yeasts the B box in RNA2 binds the 1a protein in a process postulated to be required for translocating RNA2 into membranes, presumably the location of the BMV replicase and translation (11,12).…”

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confidence: 99%

Replicase-Binding Sites on Plus- and Minus-Strand Brome Mosaic Virus RNAs and Their Roles in RNA Replication in Plant Cells

Choi

Hema

Gopinath

et al. 2004

The cis-acting elements for Brome mosaic virus (BMV) RNA synthesis have been characterized primarily for RNA3. To identify additional replicase-binding elements, nested fragments of all three of the BMV RNAs, both plus-and minus-sense fragments, were constructed and tested for binding enriched BMV replicase in a template competition assay. Ten RNA fragments containing replicase-binding sites were identified; eight were characterized further because they were more effective competitors. All eight mapped to noncoding regions of BMV RNAs, and the positions of seven localized to sequences containing previously characterized core promoter elements ( The replication of positive-strand RNA viruses is essential for pathogenesis. The responsible enzyme, the viral replicase, is a complex that includes the virus-encoded RNA-dependent RNA polymerase, additional viral replicase proteins, and host factors (6, 32). In addition, specific interactions between the viral genomic RNAs and viral replicase are required for replication and/or transcription of the viral RNAs (1, 6). The interaction process is likely to be quite complex because an RNA virus expresses different classes of RNAs at programmed levels and times.Brome mosaic virus (BMV) belongs to the alphavirus-like superfamily of plant and animal positive-strand RNA viruses. The BMV genome is divided into three capped RNAs, designated RNA1, RNA2, and RNA3 (3, 26). RNA1 and RNA2 encode replication-associated proteins, while RNA3 encodes two proteins required for systemic movement of the virus in plants and encapsidation of viral RNAs. Due to the dicistronic nature of RNA3, the second cistron encoding the capsid is translated from subgenomic RNA that is transcribed from the subgenomic promoter. In all, three classes of RNAs must be produced during successful BMV replication: genomic minusstrand, genomic plus-strand, and subgenomic RNAs.cis-acting elements for efficient genomic plus-strand, minusstrand, and subgenomic BMV RNA synthesis have been characterized by a combination of approaches, including RNA synthesis by the BMV replicase in vitro (37), transfection of BMV RNAs into protoplasts (34), analysis in plants (7), and replication in Saccharomyces cerevisiae, which is permissive for BMV replication and transcription (23). Each study generally focused on one cis-acting element and on RNA3, which does not contain functions directly required for replication. Nonetheless, a number of required elements have been identified (Fig. 1).The 3Ј noncoding regions (NCR) of BMV genomic plusstrand RNAs form a tRNA-like structure that directs the initiation of minus-strand RNA synthesis in vitro (10, 15) and in vivo (38, 46). Stem-loop C (SLC) within the tRNA-like structure of RNA3 binds the BMV replicase through an RNA structure called a clamped adenine motif (29). Given the highly similar structures predicted for RNA1 and RNA2, it is likely that the same structures are required to direct their minusstrand replication (Fig. 1). A mutation in SLC in RNA2 was shown to affect RNA replicat...

“…In yeast, 1a could recruit viral RNA2 and RNA3 to the replication complexes in a process that involves an RNA sequence, named the B box, in the intercistronic region of RNA3 and the 5Ј UTR of RNA2 (7, 21). 1a could also interact with the N-terminal portion of the 2a RNA-dependent RNA polymerase to recruit it to the replication factory where RNA synthesis takes place (6,8,10,22,42,43).Recently, an Agrobacterium-mediated T-DNA gene delivery system was established to study BMV replication, gene expression, and RNA packaging in Nicotiana benthamiana (4, 14). The agroinfiltration system provides robust transient expression of viral proteins and allows careful dissection of the cisand trans-acting requirements for BMV RNA replication in plants.…”

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confidence: 99%

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confidence: 99%

Selective Repression of Translation by the Brome Mosaic Virus 1a RNA Replication Protein

Gopinath

Kao

2007

Differential expression of viral replication proteins is essential for successful infection. We report here that overexpression of the brome mosaic virus (BMV) 1a protein can repress viral RNA replication in a dosagedependent manner. Using RNA replication-incompetent reporter constructs, repression of translation from BMV RNA1 and RNA2 was observed, suggesting that the effect on translation of the BMV RNA replication proteins is responsible for the decrease in RNA levels. Furthermore, repression of translation by 1a required the B box in the 5 -untranslated region (5 UTR); BMV RNA3 that lacks a B box in its 5 UTR is not subject to 1a-mediated translational inhibition. Mutations in either the methyltransferase or the helicase-like domains of 1a reduced the repression of replication and translation. These results suggest that in addition to its known functions in BMV RNA synthesis, 1a also regulates viral gene expression.Viruses must produce their gene products in the proper amounts and with the appropriate timing. Failure to do so can lead to innate defense responses from the host that may act on RNA replication intermediates (47). A number of mechanisms can contribute to the differential expression of viral products, such as ribosome shunting, leaky scanning, frameshifting, functional recoding, and reinitiation (11, 12). For Sindbis virus and Tobacco mosaic virus, with single genomic RNAs, the expression of the RNA-dependent RNA polymerase is decreased by readthrough of leaky termination codons (27,37,44,45). Translational control also plays an important role in regulating viral gene expression in negative-sense RNA viruses and retroviruses (19,28,31,41,49). Regulation of protein production from segmented plus-strand RNA viruses is less well understood.Brome mosaic virus (BMV), a member of the alphavirus-like superfamily of RNA viruses, is a model segmented positivestrand RNA virus. The BMV genome consists of three capped, messenger-sense genomic RNAs which have a tRNA-like structure within the 3Ј-untranslated region (3Ј UTR). Genomic RNA1 and RNA2 encode nonstructural proteins 1a and 2a, respectively, which direct RNA replication (33). Genomic RNA3 is a bicistronic RNA encoding the cell-to-cell movement protein (MP) and the coat protein (CP). The MP is translated from RNA3, whereas the 3Ј-proximal coat protein is translated from a subgenomic RNA4 that is made using minus-strand RNA3 as the template (33). In addition to replication in various plant species, BMV can replicate and transcribe its genome in Saccharomyces cerevisiae (20).The multifunctional 1a protein contains two domains separated by a proline-rich sequence. The N-terminal domain contains activities for 7-methyl-guanosine methyltransferase and covalent GTP binding required for viral RNA capping (2, 23). The C-terminal domain contains all of the motifs of the DEAD box RNA helicase domain, with ATPase and GTPase activities (24, 48). 1a is the primary viral protein determinant for the subcellular localization of the BMV RNA replication complex (9, 39, ...