Two viraj proteins involved in the proteolytic processing of the cowpea mosaic virus polyproteins

Abstract

A series of specific deletion mutants derived from a full-length cDNA clone of cowpea mosaic virus (CPMV) B RNA was constructed with the aim to study the role of viral proteins in the proteolytic processing of the primary translation products. For the same purpose cDNA clones were constructed having sequences derived from both M and B RNA of CPMV. In vitro transcripts prepared from these clones with T7 RNA polymerase, were efficiently translated in rabbit reticulocyte lysates. The translation products obtained were processed in the lysate by specific proteolytic cleavages into smaller products, which made it possible to study subsequently the effect of the various mutations on this process. The results obtained indicate that the B RNA-encoded 24K polypeptide represents a protease responsible for all cleavages in the polyproteins produced by both CPMV B and M RNA. For efficient cleavage of the glutamine-methionine site in the M RNA encoded polyprotein the presence of a second B RNA encoded protein, the 32K polypeptide, is essential, although the 32K polypeptide itself does not have proteolytic activity. A number of cleavage-site mutants were constructed in which the coding sequence for the glutamine-glycine cleavage site between the two capsid proteins was changed. Subsequent in vitro transcription and translation of these cleavage site mutants show that a correct dipeptide sequence is a prerequisite for efficient cleavage but that the folding of the polypeptide chain also plays an important role in the formation of a cleavage site.

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

“…The 58K and/or 48K proteins were also found in small electron-dense areas close to the end of the tubules, but not in the large electron-dense areas which are made up of non-structural B R N A products (Wellink et al, 1988). These small electron-dense areas associated with tubules may be the s i t e where virus-containing tubules are formed.…”

“…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).…”

Expression of Cowpea Mosaic Virus M RNA in Cowpea Protoplasts

“…Therefore M RNA multiplication seems to be essential for the detection of primary translation products. It is probably also important that the viral protease responsible for processing the polyproteins is present at much lower amounts than at later stages of infection (Vos et al, 1988). At these later times it was possible to detect the polyproteins only when ZnC12 was used to inhibit the viral protease (Wellink et al, 1987).…”

“…The expression of the two positive-stranded RNAs of cowpea mosaic virus (CPMV) has been studied extensively both in vitro and in vivo (Goldbach & van Kammen, 1985;Wellink et al, 1987;Vos et aL, 1988). Both RNAs are translated into polyproteins that are cleaved by a B RNA-encoded protease into functional proteins (Vos et aL, 1988).…”

“…Both RNAs are translated into polyproteins that are cleaved by a B RNA-encoded protease into functional proteins (Vos et aL, 1988). Remarkably, M RNA is translated in vitro into two polyproteins (Mr 105K and 95K) with overlapping amino acid sequences, as a result of translation initiating at different AUG codons (Vos et al, 1984).…”