Independent replication of cowpea mosaic virus bottom component RNA: In vivo instability of the viral RNAs

Varennes, Amarílis de; Maule, Andrew J.

doi:10.1016/0042-6822(85)90289-2

Cited by 11 publications

(5 citation statements)

References 21 publications

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“…In addition, with probes spanning different regions of RNA1 or RNA2, no specific signal corresponding to minus-strand RNA was observed (data not shown). This suggests that in CPMV-infected protoplasts, minus-strand RNA accumulates much less than does plus-strand RNA, a suggestion in line with earlier findings obtained in Northern hybridization studies (11).…”

Section: Localization Of Cpmv Rna In Infected Protoplastssupporting

confidence: 80%

“…Strikingly, almost no fluorescence was present in the cytoplasm at the periphery of the cells, which was the site where the viral particles were shown to accumulate, indicating that only unencapsidated plus-strand RNA hybridizes with the fluorescent probe. It has been shown previously that under conditions that leave viral RNA unprotected by coat protein, the genomic RNA is unstable (11), suggesting that in CPMV-infected cells, the large majority of plus-strand viral RNA is present in viral particles whereas only a small proportion is present as unencapsidated RNA. However, we observed no clear differences in the plus-strand RNA1 signal in protoplasts infected with RNA1 alone and in protoplasts coinfected with RNA2, which encodes the capsid proteins.…”

Section: Discussionmentioning

confidence: 99%

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Coalescence of the Sites of Cowpea Mosaic Virus RNA Replication into a Cytopathic Structure

Carette

Gühl

Wellink

et al. 2002

J Virol

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Cowpea mosaic virus (CPMV) replication induces an extensive proliferation of endoplasmic reticulum (ER) membranes, leading to the formation of small membranous vesicles where viral RNA replication takes place. Using fluorescent in situ hybridization, we found that early in the infection of cowpea protoplasts, CPMV plus-strand RNA accumulates at numerous distinct subcellular sites distributed randomly throughout the cytoplasm which rapidly coalesce into a large body located in the center of the cell, often near the nucleus. The combined use of immunostaining and a green fluorescent protein ER marker revealed that during the course of an infection, CPMV RNA colocalizes with the 110-kDa viral polymerase and other replication proteins and is always found in close association with proliferated ER membranes, indicating that these sites correspond to the membranous site of viral replication. Experiments with the cytoskeleton inhibitors oryzalin and latrunculin B point to a role of actin and not tubulin in establishing the large central structure. The induction of ER membrane proliferations in CPMV-infected protoplasts did not coincide with increased levels of BiP mRNA, indicating that the unfolded-protein response is not involved in this process.Infection with positive-stranded RNA viruses often causes extensive membrane rearrangements in the host cell, establishing a distinct compartment where viral RNA synthesis occurs. The viral replication complexes are associated with these membranes, which can originate from different intracellular membranes including the late and early endomembrane system (21,22,27,29,30,33). Despite the central role of such a virusinduced membranous compartment in the replicative cycle, the cellular components involved in the formation of this compartment are largely unknown.Cowpea mosaic virus (CPMV), a bipartite positive-stranded RNA virus, is the type member of the comoviruses, which bear strong resemblance to animal picornaviruses in both the gene organization and the amino acid sequence of replication proteins (1, 14). Both RNA1 and RNA2 are translated into large polyproteins, which are proteolytically cleaved into the different cleavage products by the 24-kDa proteinase (24K) (Fig. 1). The proteins encoded by RNA1 are necessary and sufficient for replication, whereas RNA2 codes for the capsid proteins and the movement protein. The RNA1-encoded 87-kDa protein (87K) contains a domain specific to RNA-dependent RNA polymerases; however, a 110-kDa protein (110K; 87K plus 24K) is the only viral protein present in highly purified, RNAdependent RNA polymerase preparations capable of elongating nascent RNA chains, suggesting that fusion to 24K is required for replicase activity (13).Upon infection of cowpea plants with CPMV, a typical cytopathic structure is formed, often adjacent to the nucleus, consisting of an amorphous matrix of electron-dense material that is traversed by rays of small membranous vesicles (12). Autoradiography in conjunction with electron microscopy on sections of CPMV-infect...

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Section: Localization Of Cpmv Rna In Infected Protoplastssupporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

Coalescence of the Sites of Cowpea Mosaic Virus RNA Replication into a Cytopathic Structure

Carette

Gühl

Wellink

et al. 2002

J Virol

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“…This property of the interaction between the proteins and the RNA could be related to a protection of the RNA against the degradation by cell ribonucleases. In fact, in the absence of RNA 2 (which codes for the coat proteins), RNA 1 accumulates to a much lower level than it does in a full infection (de Varennes & Maulé, 1985), probably because of its increased turnover when unprotected by the proteins. On the other hand, the irreversibility of the dissociation of the top component leads to the question of how empty capsids can be formed in vivo.…”

Section: Discussionmentioning

confidence: 99%

Differences in Pressure Stability of the Three Components of Cowpea Mosaic Virus: Implications for Virus Assembly and Disassembly

Poian¹,

Johnson²,

Silva³

1994

Biochemistry

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A comparison of pressure stability of empty capsids and ribonucleoprotein particles of cowpea mosaic virus (CPMV) is presented. A combination of high pressure and subdenaturing concentrations of urea was utilized to promote dissociation and denaturation. We found that RNA plays an important role in stabilizing the particles as well as in conferring reversibility to the pressure-induced denaturation. Dissociation and denaturation of the top component (empty capsid) was observed at 2.5 kbar and in the presence of 2.5 M urea. The pressure-dissociated state of the capsid protein had the characteristics of a denatured conformation as suggested by fluorescence spectra, lifetime of tryptophans, and binding of bis-ANS. The properties of the dissociated capsid protein were more similar to those of a molten-globule conformation, different from the more drastically unfolded state obtained using high concentrations of urea. Whereas the fluorescence of bis-ANS increased for the pressure-dissociated protein (1.5 M urea and 2.5 kbar), it decreased for the virus denatured by 6.0 M urea. Middle and bottom components underwent less than 50% change in center of spectral mass at 2.5 kbar and 2.5 M urea. The particles containing RNA could be fully affected by pressures of 2.5 kbar--as measured by the spectral shift--only in the presence of 5.0 M urea. RNA-containing capsids denatured by pressure did not bind bis-ANS, suggesting that the capsid protein continues to be bound to the RNA after the protein-protein contacts are broken by pressure. Reassembly of the nucleoprotein particles was obtained after decompression, reinforcing the idea that proteins had not dissociated from RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…Overall, the results presented here provide compelling evidence that the suppressor activity of the CPMV S protein requires the C-terminal region, and aa 198-213 in particular. The data explain the debilitation of mutants harbouring deletions in this region (Taylor et al, 1999) and the low level of replication often found when RNA-1 replicates on its own in protoplasts (de Varennes & Maule, 1985). The fact that the native CPMV sequence cannot be functionally substituted by the equivalent region of BPMV also explains the phenotype of the infection caused by pCP-Hybrid and why CPMV chimaeras with the natural C-terminal amino acids replaced by foreign epitopes grow extremely poorly (Taylor, 1997).…”

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confidence: 99%

Surface-exposed C-terminal amino acids of the small coat protein of Cowpea mosaic virus are required for suppression of silencing

Cañizares

Taylor

Lomonossoff

2004

Journal of General Virology

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The small (S) coat protein of Cowpea mosaic virus (CPMV) has been identified previously as a virus-encoded suppressor of post-transcriptional gene silencing (PTGS). Deletions within the C-terminal 24 aa of this protein affect the yield and systemic spread of the virus, suggesting that the C-terminal amino acids of the S protein, which are exposed on the surface of assembled virus particles, may be responsible for the suppressor activity. To investigate this, versions of CPMV RNA-2 with deletions at the C terminus of the S protein were tested for their ability to counteract PTGS in leaf-patch tests. The results showed that the C-terminal 16 aa of the S protein are particularly important for suppressing PTGS and that these amino acids are virus-specific and cannot be substituted by the equivalent sequence from the related virus Bean pod mottle virus.The genome of Cowpea mosaic virus (CPMV) consists of two molecules of separately encapsidated single-stranded, positive-sense RNA, termed RNA-1 and RNA-2. Both of these RNAs are translated to give precursor polyproteins, which are processed to give the mature viral proteins. RNA-1 encodes proteins involved in RNA replication and RNA-2, which is dependent on RNA-1 for its replication, encodes the viral movement protein and the two viral coat proteins, large (L) and small (S). Like many plant viruses, CPMV encodes a suppressor of post-transcriptional gene silencing (PTGS), though its activity appears to be relatively weak (Voinnet et al., 1999). This suppressor function was subsequently mapped to the S coat protein (Liu et al., 2004).CPMV particles consist of 60 copies each of the L and S coat proteins arranged with pseudo T=3 (P=3) symmetry (Lin & Johnson, 2003). The only portion of the S protein not visible in the X-ray structure consists of the C-terminal 24 aa (aa 190-213). This sequence is exposed on the virus surface, is mobile and is frequently lost by proteolysis without affecting particle stability (Lin & Johnson, 2003). Analysis of mutants with deletions in the C-terminal 24 aa region showed that it played an important role in efficient growth and spread of the virus (Taylor et al., 1999). Deletion of the entire 24 aa sequence (mutant DM4) reduced virus yield to less than 5 % of the wild-type level in inoculated leaves and substantially delayed systemic spread. Furthermore, lesions on the inoculated leaves were surrounded by rings of necrosis and virion preparations contained a dramatically increased proportion (70 %) of protein-only shells (empty capsids). Smaller deletions of 16 or 7 aa from the C terminus (mutants DM5 and DM6, respectively) again reduced virus yield and delayed systemic spread, but to a lesser extent than in DM4, the degree of debilitation being approximately proportional to the size of the deletion (Taylor et al., 1999). Moreover, no necrosis was observed with DM5 and DM6 and virus preparations contained wildtype levels (5-10 %) of empty particles. Taken together, these results indicated that aa 190-197 of the S protein are involved in the ...

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Independent replication of cowpea mosaic virus bottom component RNA: In vivo instability of the viral RNAs

Cited by 11 publications

References 21 publications

Coalescence of the Sites of Cowpea Mosaic Virus RNA Replication into a Cytopathic Structure

Coalescence of the Sites of Cowpea Mosaic Virus RNA Replication into a Cytopathic Structure

Differences in Pressure Stability of the Three Components of Cowpea Mosaic Virus: Implications for Virus Assembly and Disassembly

Surface-exposed C-terminal amino acids of the small coat protein of Cowpea mosaic virus are required for suppression of silencing

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