Independent replication and expression of B-component RNA of cowpea mosaic virus

SUMMARYCowpea protoplasts were inoculated with different combinations of the four nucleoprotein components of alfalfa mosaic virus (A1MV), and the synthesis of viral particles, coat protein and RNA was analysed. Synthesis of viral particles and coat protein was detectable by polyacrylamide gel electrophoresis only when the inoculum contained the components B, M and Tb, accommodating RNAs 1, 2 and 3 respectively.Upon inoculation with B + M + Tb, the synthesis of viral RNA was detectable by annealing of radiolabelled RNA from protoplasts to virus-specific double-stranded RNA. On inoculation with B + Tb or M ÷ Tb no significant amount of viral RNA was produced. After inoculation with B + M, however, the production of virus-specifiC RNA was about 40 % of that induced by complete infection, indicating that genetic information encoded by RNAs 1 and 2 is involved in viral RNA replication. As a mixture of the genomic RNAs did not induce the synthesis of virus-specific RNA, it is concluded that coat protein is required for the RNA synthesis induced by the mixture of B and M nucleoproteins.The observation that no coat protein is produced upon inoculation with B + M + Ta components indicates that the replicase activity induced by B + M does not replicate RNA 4, the messenger for coat protein present in Ta particles.

Section: Discussionmentioning

confidence: 99%

Localization of Genetic Information Involved in the Replication of Alfalfa Mosaic Virus

Nassuth

Alblas

Bol

1981

“…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). Using infectious transcripts derived from cDNA clones of the M RNA it has been shown that both 58K/48K movement and the capsid proteins are required for cellto-cell movement .…”

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

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.

“…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).…”

mentioning

confidence: 99%

“…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.…”

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

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).