Multiple functions of the 32K and 60K proteins in cowpea mosaic virus RNA replication

Cowpea mosaic virus (CPMV) is the type member of the comoviridae , a group of 14 different plant viruses that have a divided genome consisting of two plus-strand RNAs. These RNAs, designated B-RNA and M-RNA, have a small protein, VPg, attached to the 5'-end and a poly(A) tail at the 3'-end and are separately packaged into icosahedral particles of 28 nm in diameter. Nucleotide sequence analysis has revealed that each RNA contains one large open reading frame. Upon infection the RNAs are translated into large polyproteins that are subsequently processed into several stable intermediate and final cleavage products.The B-RNA and its encoded enzyme activities constitute an autonomous RNA replicon, since the B- RNA can replicate independently of M-RNA in isolated plant cells. However, B-RNA is dependent on M-RNA for cell-to-cell movement in intact plants. The development of full-length cDNA clones, of B- and M-RNA from which infectious RNA transcripts can be derived, has made it possible to study the mechanism of viral gene expression in more detail. By introducing specific mutations in B cDNA clones, several functional domains in the B-polyprotein were identified, but the understanding of the activity of each individual B-RNA encoded protein in the replicative machinery is still incomplete. At the start of the research described in this thesis the B-RNA encoded 110K, which consists of the 24K protein and the 97K core polymerase protein, has been shown to represent the viral RNA-dependent RNA-polymerase. The 60K protein has been proposed to function as a precursor for VPg, that probably has a role as a primer in the initiation of viral RNA replication. Furthermore, the 60K protein is thought to function in anchoring the viral RNA replication complex to membranes, known to be the site of viral RNA replication. Processing of the B-polyprotein is accomplished by the B-RNA encoded 24K proteinase, cleaving the viral proteins at specific Gln/Gly, Gln/Ser and Gln/Met sites. However, for efficient trans processing of the Gin/Met site in the M-polyprotein also the B-RNA encoded 32K protein is required.The studies described in this thesis were concentrated on elucidating the role of the 32K and 60K proteins in the viral replication process. The work presented in chapter 2 of this thesis was directed towards the role of the 32K protein in the polyprotein processing. By employing an in vitro transcription/translation system, to express specifically modified cDNA clones, it was shown that the 32K protein regulates both M- and B-polyprotein processing, by interacting with the 58K domain of the 170K and 84K precursors of the 24K proteinase, thereby modulating the cleavage activity and specificity of the 24K proteinase. In chapters 3 and 4 processing of several VPg precursors has been examined. This study showed that in vitro processing of the 170K protein can occur via three alternative pathways to generate 112K, 84K and 60K putative VPg precursor proteins. The 60K protein was found to be stable in this in vitro system, whereas the 112K and 84K proteins were processed and might function as a VPg precursor. Using a transient expression system, the 112K protein was evidently shown to function as a direct VPg precursor in cowpea protoplasts (chapter 4).A study on the biochemical properties of the B-RNA encoded 60K and 84K proteins is described in chapters 5 and 6 of this thesis. A covalent affinity labelling assay was exploited and the 60K and 84K protein were shown to specifically bind ATP, possibly at a ribonucleoside triphosphate binding motif (NTBM) located in the 58K domain of these proteins (chapter 5). In chapter 6 the effect of mutations that were introduced in the coding region of the NTBM is described. With this study an essential role in viral RNA replication could be attributed to the NTBM