Expression of Cowpea Mosaic Virus M RNA in Cowpea Protoplasts

The putative 38 kDa movement protein (P38) gene, located on the RNA2 of grapevine fanleaf nepovirus (GFLV), was cloned in Escherichia coli and expressed as a fusion protein fused to six histidines (6HisP38). This protein was purified and used to produce a specific polyclonal antiserum from which immunoglobulins were isolated by immunoaffinity against the recombinant 6HisP38. Western immunoblot analyses on GFLV RNA2 in vitro maturation products showed that the antibodies were specific for P38 protein. This protein was detected as early as 18 h post-inoculation in GFLVinfected Chenopodium quinoa protoplasts and accumulated to very high levels. Tissue-prints and time course experiments on infected C. quinoa plants confirmed that P38 is present at a high level late in infection and is a final maturation product of the GFLV RNA2 polyprotein in vivo. P94 and P66 intermediates of maturation and polyprotein P2 were also detected in vivo but in very low concentrations. No significant difference was observed in the relative amounts of P66 and P94 detected in vivo, contrary to what occurs in vitro. Subcellular fractionation studies showed that P38, although mainly cytosolic, is also found in association with cell wall and membranes. Thought to be the GFLV movement protein, P38 would thus behave in an 'atypical' manner. IntroductionGrapevine fanleaf virus (GFLV) belongs to the genus Nepovirus in the family Comoviridae (Ward, 1993). Its genetic information is divided over two plus-stranded RNA molecules: RNA1 (7342 nt) (Ritzenthaler et al., 1991) encodes a 253 kDa polyprotein (P1) and RNA2 (3774nt) (Serghini et al., 1990) encodes a 122kDa polyprotein (P2). In vitro both polyproteins are proteolytically processed by the RNAl-encoded 24 kDa chymotrypsin-like cysteine proteinase (Margis & Pinck, 1992;Margis et al., 1991). Polyprotein P1 is cleaved at various sites: Cys415/Ala 416 (Margis et al., 1994), Cys1217/Ser 121s, Gly124X/Glu ~242 , and Arg1461/Gly 1402 (Ritzenthaler et al., 1991) to release, from the N terminus to the C terminus, a 46 kDa protein, an 88 kDa putative helicase, the VPg, the 24 kDa proteinase and a 94 kDa putative viral RNA polymerase. Upon in vitro translation, polyprotein P2 is converted into three final products: a 28 kDa N-terminal protein (P28), a 38 kDa protein (P38) and the C-terminal 56 kDa coat protein (CP) (Fig. 1 a). The cleavages occur at the Cys257/Ala 258 and Arg6°5/Gly 6°6 sites between P28/P38 and P38/CP respectively Serghini et al., 1990 Biologically active full-length transcripts of RNA2 (clone pACN) and RNAI (clone pMV) have been obtained (Viry et al., 1993) which allowed us to demonstrate that GFLV RNA1 can autoreplicate in protoplasts and thus encodes the viral replication functions. In contrast RNA2, which requires for its replication the functions encoded by RNA1, is needed for virus spread in plants and thus encodes functions necessary for viral movement (Viry et al., 1993). By analogy with related comoviruses, it has been proposed that the protein adjacent to the capsid protein cont...

Section: P41fllcontrasting

confidence: 96%

Section: Total Proteinsmentioning

confidence: 99%

Grapevine fanleaf nepovirus P38 putative movement protein is not transiently expressed and is a stable final maturation product in vivo

Ritzenthaler

Pinck

1995

“…It is therefore possible that the 58K and 48K proteins have different cellular locations and different functions. Evidence for this has been presented by Wellink et al (1987), Holness et al (1989) andRezelman et al (1989), who found that the 48K protein is present in both cytoplasmic and membrane fractions of protoplasts, whereas the 58K protein is found only in the cytoplasmic fraction. It is also interesting that the 48K protein is the only non-structural C P M V protein detectable in the culture medium (Wellink et al, 1987).…”

Section: Discussionmentioning

confidence: 85%

“…The regions encoding these proteins are located on the M RNA of the bipartite genome. This M RNA is translated into two overlapping polyproteins of 105K and 95K (Pelham, 1979;Vos et al, 1984;Rezelman et al, 1989), which are proteolytically processed to give the overlapping 58K and 48K nonstructural proteins and, via a 60K precursor, the 37K and 23K coat proteins . The B RNA encodes all the functions necessary for RNA replication (Goldbach et al, 1980;Eggen & van Kammen, 1988) and is able to replicate independently of the M RNA in cowpea protoplasts (Rezelman et al, 1982).…”

Section: Introductionmentioning

confidence: 99%

Tubular structures involved in movement of cowpea mosaic virus are also formed in infected cowpea protoplasts

Lent¹,

Storms²,

Meer³

et al. 1991

Self Cite

166

104

In cowpea plant cells infected with cowpea mosaic virus, tubular structures containing virus particles are formed in the plasmodesmata between adjacent cells; these structures are supposedly involved in cell-to-cell spread of the virus. Here we show that similar tubular structures are also formed in cowpea protoplasts, from which the cell wall and plasmodesmata are absent. Between 12 and 21 h post-inoculation, tubule formation starts in the periphery of the protoplast at the level of the plasma membrane. Upon assembly, the viruscontaining tubule is enveloped by the plasma membrane and extends into the culture medium. This suggests that the tubule has functional polarity and makes it likely that a tubule 'grows' into a neighbouring cell in vivo. On average, 75 % of infected protoplasts were shown to possess tubular structures extending from their surface. The tubule wall was 3 to 4 nm thick and they were up to 20 gm in length, as shown by fluorescent light microscopy and negative staining electron microscopy. By analogy to infected plant cells, both the viral 58K/48K movement and capsid proteins were located in these tubules, as determined by immunofluorescent staining and immunogold labelling using specific antisera against these proteins. These results demonstrate that the formation of tubules is not necessarily dependent on the presence of plasmodesmata or the cell wall, and that they are composed, at least in part, of virus-encoded components.

“…1). Rezelman et aL (1989) showed recently that these proteins are also produced in CPMV-infected cowpea protoplasts.…”

mentioning

confidence: 99%

Evidence for the Involvement of the 58K and 48K Proteins in the Intercellular Movement of Cowpea Mosaic Virus

Lent¹,

Wellink

Goldbach³

1990

Self Cite

136

Infection of cowpea cells with cowpea mosaic virus (CPMV) is accompanied by the appearance of tubular structures containing virus-like particles which protrude from or penetrate the cell wall. Immunogold labelling of sections of infected cells using antisera against a CPMV M RNA translation products, and Protein A -g o l d , showed that the 58K and/or 48K tentative transport proteins of CPMV were located in or on these tubular structures. Furthermore, these proteins were detected in small electron-dense areas near the tail-end of the tubules. The possible function of these structures in virus movement from cell to cell is discussed.The bipartite genome of cowpea mosaic virus (CPMV) consists of two positive single-stranded RNA molecules (B and M RNA) that each contain only one large open reading frame and are translated into polyproteins which are cleaved by a B RNA-encoded protease into functional proteins (Goldbach & van Kammen, 1985;Vos et al., 1988). B RNA encodes all the functions necessary for replication of the RNAs (Goldbach et al., 1980;Eggen & van Kammen, 1988) and is able to replicate independently of the M RNA in cowpea protoplasts (Rezelman et al., 1982). The M RNA is translated in vitro into two overlapping polyproteins of Mr 105K and 95K (Vos et al., 1984), which are processed proteolytically to give the overlapping 58K and 48K non-structural proteins and, via a 60K precursor, the 37K and 23K coat proteins and Fig. 1). Rezelman et aL (1989) showed recently that these proteins are also produced in CPMV-infected cowpea protoplasts.Although the B RNA can replicate independently of M RNA in protoplasts, M RNA is essential for infection of plants (Rezelman et al., 1982). Using infectious transcripts of the M RNA, WeUink & van Kammen (1989) showed that mutations and deletions in the coding regions of both 58K/48K proteins and the capsid proteins prevented systemic infection in cowpea plants. These results suggest that both the 58K/48K proteins and the coat proteins of CPMV are indispensable for cell-tocell movement of the virus and also that CPMV is transported as particles and not as naked RNA.To study the possible involvement of M RNA products in virus movement we detected these products in sections of infected cells by the binding of specific antisera to antigens as shown by its reaction with Protein A-gold.To achieve a near-simultaneous infection of the cells in secondary leaves of cowpea plants (Vigna unguiculata cv. California Blackeye), plants were given a differential temperature treatment (DTT) after inoculation of the primary leaves with 1 mg/ml of purified CPMV in 0.01 M-phosphate buffer pH 7.0 (Dawson & Schlegel, 1976).