Host Factors in Positive-Strand RNA Virus Genome Replication

Ahlquist, Paul; Noueiry, Amine; Lee, Wai-Ming; Kushner, David; Dye, Billy T.

doi:10.1128/jvi.77.15.8181-8186.2003

Cited by 449 publications

(368 citation statements)

References 45 publications

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“…Several yeast proteins have been identified to regulate BMV replication and translation, such as Lsm1p-7p/Pat1p deadenylation-dependent mRNA-decapping factors and DED1. Mutations in these host proteins affected viral replication in yeast at the levels of both viral replication and gene expression (1,17,25,26,30,32,34,46). If 1a represses translation through affecting cellular proteins, then these proteins are prime candidates for the B box binding proteins.…”

Section: Discussionmentioning

confidence: 99%

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

Gopinath

Kao

2007

J Virol

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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, ...

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Section: Discussionmentioning

confidence: 99%

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

Gopinath

Kao

2007

J Virol

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“…It is known that VRCs are composed of virus-encoded replicationrelated proteins, viral RNAs and host factors. Furthermore, VRCs can anchor to a cellular membrane to form a specialized compartment, which protects viral RNA from degradation by host defence mechanisms, such as RNA silencing (David et al, 1992;Ahlquist et al, 2003;Ding et al, 2004;Mackenzie, 2005;Nagy & Pogany, 2008;Novoa et al, 2005;Waigmann et al, 2007). For example, the VRCs of TMV are macromolecular structures capable of replicating genomic and subgenomic RNAs, and contain viral replicase proteins, viral RNAs, and host proteins and membranes (Kawakami et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

Subcellular localization and membrane association of the replicase protein of grapevine rupestris stem pitting-associated virus, family Betaflexiviridae

Prosser

Xiao

et al. 2015

Journal of General Virology

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As a member of the newly established Betaflexiviridae family, grapevine rupestris stem pittingassociated virus (GRSPaV) has an RNA genome containing five ORFs. ORF1 encodes a putative replicase polyprotein typical of the alphavirus superfamily of positive-strand ssRNA viruses. Several viruses of this superfamily have been demonstrated to replicate in structures designated viral replication complexes associated with intracellular membranes. However, structure and cellular localization of the replicase complex have not been studied for members of Betaflexiviridae, a family of mostly woody plant viruses. As a first step towards the elucidation of the replication complex of GRSPaV, we investigated the subcellular localization of full-length and truncated versions of its replicase polyprotein via fluorescent tagging, followed by fluorescence microscopy. We found that the replicase polyprotein formed distinctive punctate bodies in both Nicotiana benthamiana leaf cells and tobacco protoplasts. We further mapped a region of 76 amino acids in the methyl-transferase domain responsible for the formation of these punctate structures. The punctate structures are distributed in close proximity to the endoplasmic reticulum network. Membrane flotation and biochemical analyses demonstrate that the N-terminal region responsible for punctate structure formation associated with cellular membrane is likely through an amphipathic a helix serving as an in-plane anchor. The identity of this membrane is yet to be determined. This is, to our knowledge, the first report on the localization and membrane association of the replicase proteins of a member of the family Betaflexiviridae.

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“…In addition to the viral replication proteins, they rely on the host-cell environment for replication. All known positive-sense ssRNA viruses replicate in association with host endomembranes (Mackenzie, 2005), and different host factors participate in the process (Ahlquist et al, 2003;Whitham & Wang, 2004). Numerous studies have described the importance of post-translational modifications in regulating viral protein function during host-virus interplay, including phosphorylation, glycosylation and lipid modifications.…”

Section: Introductionmentioning

confidence: 99%

Purification of viral genome-linked protein VPg from potato virus A-infected plants reveals several post-translationally modified forms of the protein

Hafrén

Mäkinen

2008

Journal of General Virology

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In order to be able to analyse post-translational modifications and protein interactions of viral genome-linked protein VPg taking place during potato virus A (PVA) infection, an affinity tag-based purification system was developed by inserting a sequence encoding a six-histidine and haemagglutinin (HisHA) tag to the 3′ end of the VPg coding sequence within the infectious cDNA clone of PVA. The engineered virus was fully functional and the HisHA tag-encoding sequence remained stable in the PVA genome throughout the infection process. Purification under denaturing conditions resulted in a protein sample that contained multiple VPg and NIa forms carrying post-translational modifications that altered their isoelectric points. Non-modified tagged VPg (pI 8) was a minor product in the protein sample derived from total leaf proteins, but when the replication-associated membranes were used as starting material, its relative amount increased. Further characterization demonstrated that some of the PVA VPg isoforms were modified by multiple phosphorylation events. Purity of the proteins derived from the native purifications with either of the tags was evaluated. A clearly purer VPg sample was obtained by performing tandem affinity purification utilizing both tags sequentially. NIb, CI and HC-Pro co-purified in an affinity-tagged VPg-dependent manner, indicating that the system was able to isolate protein complexes operating during PVA infection.

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Host Factors in Positive-Strand RNA Virus Genome Replication

Cited by 449 publications

References 45 publications

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

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

Subcellular localization and membrane association of the replicase protein of grapevine rupestris stem pitting-associated virus, family Betaflexiviridae

Purification of viral genome-linked protein VPg from potato virus A-infected plants reveals several post-translationally modified forms of the protein

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