Deletion Mutants of VPg Reveal New Cytopathology Determinants in a Picornavirus

Arias, Armando; Perales, Celia; Escarmı́s, Cristina; Domingo, Esteban

doi:10.1371/journal.pone.0010735

Cited by 20 publications

(21 citation statements)

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“…An investigation has shown non-structural protein 2C, 3A, and VPg as key determinants for modulating cytopathology in cell culture [29]. In fact, the replacements either in 2C or 3A are associated with altered virulence, cell tropism or host range in several picornaviruses [23,[30][31][32][33][34].…”

Section: Discussionmentioning

confidence: 99%

A mutant of infectious Asia 1 serotype foot-and-mouth disease virus with the deletion of 10-amino-acid in the 3A protein

Gao

Zhang

et al. 2010

Virus Genes

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Foot-and-mouth disease virus (FMDV) serotype Asia 1 is one of the most predominant endemic serotypes in China. Our previous study has generated a full-length cDNA clone (pBSAs) of an Asia 1 serotype FMDV (As1/CHA/05) isolated from bovine. To further study the properties of this virus, a mutant in the 3A region of the cDNA clone (pBSAs-3A10D), containing the deletion at position 93-102 of the 3A protein of As1/CHA/05, was generated by PCR and cloning. After synthesis of RNA in vitro and transfection, the recombinant rvAs-3A10D virus was recovered from BHK-21 cells. Characterization of the rvAs-3A10D revealed that the infectivity, immunoreactivity, and replication kinetics in BHK-21 and PK-15 cells and virulence in mice of the rvAs-3A10D were similar to that of its parent virus. Notably, while wild-type and recombinant viruses containing full-length sequence of the 3A replicated well in primary calf kidney cells, the mutant rvAs-3A10D failed to replicate in primary calf kidney cells in vitro. Apparently, the full-length sequence of 3A in As1/CHA/05 is a necessary component for its replication in calf kidney cells. The availability of this 3A deletion infectious cDNA clone may help in further investigating the virulent determinants of FMDV and potentially developing FMDV vaccines.

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Section: Discussionmentioning

confidence: 99%

A mutant of infectious Asia 1 serotype foot-and-mouth disease virus with the deletion of 10-amino-acid in the 3A protein

Gao

Zhang

et al. 2010

Virus Genes

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“…We typically use BSR-T7 cells since they grow faster than the parental BHK clone line. Cells are infected with fowlpox (FPV) encoding for T7 RNA polymerase (FPV-T7) 18 which functions as a helper virus to drive expression of the viral RNA and subsequent recovery of infectious virus (Figure 4). Although BSR-T7 cells constitutively express T7 RNA polymerase, this expression is not sufficient to rescue infectious MNV after transfection of pT7:MNV 3'Rz in the absence of helper FPV-T7.…”

Section: Direct Recovery Of Infectious Mnv From Cdna In Cells Expressmentioning

confidence: 99%

“…However it is worth noting that new preparations of helper virus grown in primary fibroblasts are functionally titrated to determine the dose required for efficient virus recovery. Protocols for the propagation and titration of FPV-T7 have been previously described 18 . 3.…”

Section: Direct Recovery Of Infectious Mnv From Cdna In Cells Expressmentioning

confidence: 99%

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Arias

Ureña

Thorne

et al. 2012

JoVE

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Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided 1,2 . During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture 3 . The recent isolation of a murine norovirus (MNV), closely related to human norovirus 4 but which can be propagated in cells 5 has opened new avenues for the investigation of these pathogens 6,7 .MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) ( Figure 1). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) 8 .Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery [9][10][11] . Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs [12][13][14] . This sophisticated mechanism of translation is likely to be a major factor in the limited efficiency of viral recovery by conventional reverse genetics approaches.Here we report two different strategies based on the generation of murine norovirus-1 (referred to as MNV herewith) transcripts capped at the 5' end. One of the methods involves both in vitro synthesis and capping of viral RNA, whereas the second approach entails the transcription of MNV cDNA in cells expressing T7 RNA polymerase. The availability of these reverse genetics systems for the study of MNV and a small animal model has provided an unprecedented ability to dissect the role of viral sequences in replication and pathogenesis [15][16][17] . Video LinkThe video component of this article can be found at https://www.jove.com/video/4145/ Protocol RNA Transcription and Capping for The Recovery of Infectious MNVThis protocol is designed to allow the efficient recovery of infectious MNV from cDNA via in vitro transcription and its subsequent in vitro capping (section 1.1). The resulting capped transcripts are then transfected into cells to recover infectious MNV (sections 1.2 and ...

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“…However, they also found that viruses lacking 3B3 were not viable and proposed that this was due to aberrant processing at the novel 3B2/3C junction generated by deleting 3B3 [49]. Subsequently, Pacheco et al [55] and Arias et al [56] reported that viruses lacking both 3B1 and 3B2 were viable, with the former study reporting normal growth phenotypes in BHK-21 cells. Arias et al went on to suggest that although a virus with a single 3B had near normal RNA replication in BHK-21 cells, virus particle release was severely compromised [56].…”

Section: Introductionmentioning

confidence: 99%

Genetic economy in picornaviruses: Foot-and-mouth disease virus replication exploits alternative precursor cleavage pathways

et al. 2017

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The RNA genomes of picornaviruses are translated into single polyproteins which are subsequently cleaved into structural and non-structural protein products. For genetic economy, proteins and processing intermediates have evolved to perform distinct functions. The picornavirus precursor protein, P3, is cleaved to produce membrane-associated 3A, primer peptide 3B, protease 3Cpro and polymerase 3Dpol. Uniquely, foot-and-mouth disease virus (FMDV) encodes three similar copies of 3B (3B1-3), thus providing a convenient natural system to explore the role(s) of 3B in the processing cascade. Using a replicon system, we confirmed by genetic deletion or functional inactivation that each copy of 3B appears to function independently to prime FMDV RNA replication. However, we also show that deletion of 3B3 prevents replication and that this could be reversed by introducing mutations at the C-terminus of 3B2 that restored the natural sequence at the 3B3-3C cleavage site. In vitro translation studies showed that precursors with 3B3 deleted were rapidly cleaved to produce 3CD but that no polymerase, 3Dpol, was detected. Complementation assays, using distinguishable replicons bearing different inactivating mutations, showed that replicons with mutations within 3Dpol could be recovered by 3Dpol derived from “helper” replicons (incorporating inactivation mutations in all three copies of 3B). However, complementation was not observed when the natural 3B-3C cleavage site was altered in the “helper” replicon, again suggesting that a processing abnormality at this position prevented the production of 3Dpol. When mutations affecting polyprotein processing were introduced into an infectious clone, viable viruses were recovered but these had acquired compensatory mutations in the 3B-3C cleavage site. These mutations were shown to restore the wild-type processing characteristics when analysed in an in vitro processing assay. Overall, this study demonstrates a dual functional role of the small primer peptide 3B3, further highlighting how picornaviruses increase genetic economy.

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Deletion Mutants of VPg Reveal New Cytopathology Determinants in a Picornavirus

Cited by 20 publications

References 61 publications

A mutant of infectious Asia 1 serotype foot-and-mouth disease virus with the deletion of 10-amino-acid in the 3A protein

A mutant of infectious Asia 1 serotype foot-and-mouth disease virus with the deletion of 10-amino-acid in the 3A protein

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Genetic economy in picornaviruses: Foot-and-mouth disease virus replication exploits alternative precursor cleavage pathways

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