Mutational Analysis of the Genome-Linked Protein of Cowpea Mosaic Virus

Carette, Jan E.; Kujawa, A.; Gühl, Kerstin; Verver, J.; Wellink, J.; Kammen, A. van

doi:10.1006/viro.2001.1137

Cited by 9 publications

(6 citation statements)

References 44 publications

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“…3). Usually the residue is conserved within the family and cannot be substituted by another residue (Carette et al, 2001;Murphy et al, 1996). It appears that within the sobemovirus genera the RNA linking is species-specific.…”

Section: A Olspert and Others 448mentioning

confidence: 99%

Protein-RNA linkage and post-translational modifications of two sobemovirus VPgs

Olspert

Peil

Hébrard

et al. 2010

Journal of General Virology

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Sobemoviruses possess a viral genome-linked protein (VPg) attached to the 59 end of viral RNA. VPg is processed from the viral polyprotein. In the current study, Cocksfoot mottle virus (CfMV) and Rice yellow mottle virus (RYMV) VPgs were purified from virions and analysed by mass spectrometry. The cleavage sites in the polyprotein and thereof the termini of VPg were experimentally proven. The lengths of the mature VPgs were determined to be 78 and 79 aa residues, respectively. The amino acid residues covalently linked to RNA in the two VPgs were, surprisingly, not conserved; it is a tyrosine at position 5 of CfMV VPg and serine at position 1 of RYMV VPg. Phosphorylations were identified in CfMV and RYMV VPgs with two positionally similar locations T20/S14 and S71/S72, respectively. RYMV VPg contains an additional phosphorylation site at S41. INTRODUCTIONCocksfoot mottle virus (CfMV) and Rice yellow mottle virus (RYMV) are members of the genus Sobemovirus, a group of viruses with small icosahedral virions and a positive-sense ssRNA genome of approximately 4.0-4.5 kb. Like many other genera with an RNA genome, sobemoviruses have a viral genome-linked protein (VPg) attached to the 59 end of the genomic and subgenomic RNAs (Ghosh et al., 1981;Mang et al., 1982).The VPgs of sobemoviruses are translated as part of the polyprotein and cleaved by the viral protease (Nair & Savithri, 2010;van der Wilk et al., 1998). In contrast to potyviruses, the polyprotein processing and VPg maturation of sobemoviruses is poorly described. The specificity of the sobemoviral protease has been proposed as Q, E/T, S, N (Gorbalenya et al., 1988; Mäkinen et al., 2000;Nair & Savithri, 2010;van der Wilk et al., 1998), based on the fact that many different cleavage sites can be predicted for the N and C termini of sobemovirus VPgs. For several sobemoviruses -CfMV, RYMV, Southern bean mosaic virus (SBMV) and Sesbania mosaic virus (SeMV) -the N terminus of VPg has been mapped (Hébrard et al., 2008; Mäkinen et al., 2000;Nair & Savithri, 2010;van der Wilk et al., 1998), while the C terminus of VPg has so far been experimentally proven for only SeMV (Nair & Savithri, 2010). The determined SeMV VPg processing sites corroborate the predicted consensus cleavage sequence. However, sobemoviruses deploy 21 programmed ribosomal frameshifting (21 PRF) for the expression of polyprotein and VPg occupies a position in the polyprotein close to the 21 PRF signal. Therefore, it has been proposed that at least CfMV might express its VPg through the 21 PRF mechanism and as a result even encode VPgs with different C termini (Mäkinen et al., 2000).The VPgs are covalently linked to the 59 end of viral RNA (Ambros & Baltimore, 1978;Rothberg et al., 1978). The VPg is attached to the RNA over a phosphodiester bond formed between the hydroxyl group of the amino acid residue and 59 phosphate group of RNA (Ambros & Baltimore, 1978;Rothberg et al., 1978). The amino acid residue involved in the linkage has been reported to be a tyrosine or a serine (Ambros & Baltimore, 19...

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Section: A Olspert and Others 448mentioning

confidence: 99%

Protein-RNA linkage and post-translational modifications of two sobemovirus VPgs

Olspert

Peil

Hébrard

et al. 2010

Journal of General Virology

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“…VPg probably acts as a primer for RNA transcription. The serine residue linking VPg to the RNA is part of the Gln/Ser cleavage site between 58K and VPg, implying that proteolytic cleavage and RNA attachment may be interrelated (Carette et al ., 2001). Although the 87 kDa protein (87K) has a domain specific for RNA‐dependent RNA polymerases (RdRp), the 110K (87K + 24K) is the only viral protein present in highly purified replicase preparations capable of elongating nascent viral RNA chains (Dorssers et al ., 1984), suggesting that fusion to the 24K proteinase is required for RNA polymerase activity.…”

Section: Introductionmentioning

confidence: 99%

“…This actin‐dependent process may represent a higher order of compartmentalization in the infected cell (Carette et al ., 2002a). It is interesting to note that the distribution of replication proteins in cowpea protoplasts infected with CPMV carrying a particular mutation in VPg (D26E/A27G) was dispersed over the cytoplasm (Carette et al ., 2001). Replication of the mutant was decreased to 50% of wild‐type, which may suggest that the formation of electron‐dense material is beneficial but not necessary for virus replication.…”

Section: Introductionmentioning

confidence: 99%

Cowpea mosaic virus: effects on host cell processes

Pouwels

Carette

Lent³

et al. 2002

Molecular Plant Pathology

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SUMMARYTaxonomy: Cowpea mosaic virus (CPMV) is the type member of the Comoviridae and bears a strong resemblance to animal picornaviruses, both in gene organization and in the amino acid sequence of replication proteins. Little systematic work has been done to compare isolates of the virus from different parts of the world.Physical properties: Purified preparations of virus contain three centrifugal components; empty protein shells without RNA (T) and two nucleoprotein components (M and B), containing 24% and 34% RNA, respectively. The icosahedral particles have with a diameter of 28 nm, consist of 60 copies of two coat proteins, and are heat stable.Hosts: CPMV causes one of the most commonly reported virus diseases of cowpea ( Vigna unguiculata ), in which it produces chlorotic spots with diffuse borders in inoculated primary leaves. Trifoliate leaves develop a bright yellow or light green mosaic of increasing severity in younger leaves. The host range is rather limited, and few hosts are known outside the Leguminosae. The virus is transmitted by various beetles with biting mouthparts. Reported in Africa, the Philippines and Iran. Is apparently absent from North and South America.

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“…The picornavirus and comovirus proteins are relatively smaller (approximately 2 to 6 kDa) than those of the potyviruses and caliciviruses (approximately 13 to 21 kDa). However, a common feature of these VPg proteins is that mutation of the tyrosine or serine involved in the linkage of RNA to the VPg protein is lethal for virus growth and replication (3,21,25). The picornavirus VPg protein is uridylylated by the 3D polymerase to form VPg-pU and VPg-pUpU and functions as a primer for RNA synthesis during replication (24,34).…”

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confidence: 99%

“…Certain mutations in the VPg proteins of poliovirus and cowpea mosaic virus (a comovirus) were shown to affect proteolytic processing (3,16). We examined the effects of the engineered FCV VPg mutations on proteolytic processing of the ORF1 polyprotein.…”

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confidence: 99%

Mutagenesis of Tyrosine 24 in the VPg Protein Is Lethal for Feline Calicivirus

Mitra

Sosnovtsev

Green

2004

J Virol

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The genome of feline calicivirus (FCV) is an ϳ7.7-kb single-stranded positive-sense RNA molecule that is polyadenylated at its 3 end and covalently linked to a VPg protein (calculated mass, 12.6 kDa) at its 5 end. We performed a mutational analysis of the VPg protein in order to identify amino acids potentially involved in linkage to the genome and replication. The tyrosine residues at positions 12, 24, 76, and 104 were changed to alanines by mutagenesis of an infectious FCV cDNA clone. Viruses were recovered when Tyr-12, Tyr-76, or Tyr-104 of the VPg protein was changed to alanine, but virus was not recovered when Tyr-24 was changed to alanine. Growth properties of the recovered viruses were similar to those of the parental virus. We examined whether the amino acids serine, threonine, and phenylalanine could substitute for the tyrosine at position 24, but these mutations were lethal as well. A tyrosine at this relative position is conserved among all calicivirus VPg proteins examined thus far, suggesting that the VPg protein of caliciviruses, like those of picornaviruses and potyviruses, utilizes tyrosine in the formation of a covalent bond with RNA.

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Mutational Analysis of the Genome-Linked Protein of Cowpea Mosaic Virus

Cited by 9 publications

References 44 publications

Protein-RNA linkage and post-translational modifications of two sobemovirus VPgs

Protein-RNA linkage and post-translational modifications of two sobemovirus VPgs

Cowpea mosaic virus: effects on host cell processes

Mutagenesis of Tyrosine 24 in the VPg Protein Is Lethal for Feline Calicivirus

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