Molecular cloning and expression of full-length DNA copies of the genomic RNAs of cowpea mosaic virus.

The experiments described in this thesis were designed to unravel various aspects of the mechanism of gene expression of cowpea mosaic virus (CPMV). For this purpose full-length DNA copies of both genomic RNAs of CPMV were constructed. Using powerful <u>in</u><u>vitro</u> transcription systems RNA transcripts closely resembling the viral RNAs were prepared from these clones, which were efficiently translated <u>in</u><u>vitro</u> and were able to infect cowpea protoplasts. Mutations were introduced in specific regions in the cDNA clones using methods for site-direced mutagenesis. Investigation of the effect of these mutations on viral gene expression has increased our insight in the multiplication cycle of CPMV.Most of the mutagenized cDNA clones described in this thesis were designed to study the proteolytic processing of the CPMV polyproteins. The proteolytic processing of CPMV lends itself very well to be studied <u>in</u><u>vitro</u> because it reliably mimicks the processing <u>in</u><u>vivo</u> upon translation of the viral RNAs in rabbit reticulocyte lysates (Pelham, 1978; Franssen <u>et</u><u>al</u> ., 1984). The results obtained here clearly indicate that the B RNA encoded 24K polypeptides is the proteinase involved in all cleavage reactions in the CPMV polyproteins. The question remains however how to processing of the CPMV polyproteins is regulated <u>in</u><u>vivo</u> . Some cleavage reactions which occur slowly <u>in</u><u>vitro</u> occur so rapidly <u>in</u><u>vivo</u> , that the precursor polypeptides cannot be detected. In the proteolytic processing of the M RNA encoded polyproteins. the 32K polypeptide also plays a role. because in its absence the glutamine-methionine site is cleaved very oorly by the 24K proteinase (chapter 6). Furthermore it is not clear whether the 24K polypeptide is proteolytically active in infected cells in the form one or mroe of the precursor polypeptides: the 170K, 110K and 84K polypeptides or as a free protein. It may therefore be expected that the proteoytic cleavages <u>in</u><u>vivo</u> are strictly regulated depending on the different processes in the life cycle of the virus, like RNA multiplication and virus assembly.The only way to gain more insight in the regulation of the proteolytic cleavage reactions is to study the expression of specifically modified cDNA clones <u>in</u><u>vivo</u> . In view of that the expression of RNA transcripts of fulllength cDNA clones in cowpea protoplasts is very promising. The expression of such transcripts in protoplasts was successful, but their infectivity in comparison to viral RNA was rather low and therefore the usefulness of these transcripts at this moment is limited. Besides the infection of whole cowpea plants could not be achieved. It is obvious that the infectivity of the RNA transcripts needs to be improved to increase their applicability. The difference in infectivity between viral RNA and the <u>in</u><u>vitro</u> synthesized RNA sent originate from the different properties of the RNA molecules. Compared to viral RNA the <u>in</u><u>vitro</u> synthesized RNAs lack the genome-linked protein VPg and have two additional nucleotides at the 5' end and 4 to 5 extra nucleotides to the 3' end of the RNA. The <u>in</u><u>vitro</u> synthesized RNA transcripts of full-length cDNA clones of tobacco mosaic virus (TMV) and bromo mosaic virus (BMV) have a relatively high level of infectivity (Damson <u>et</u><u>al</u> ., 1986; Meshi <u>et</u><u>al</u> ., 1986; Ahlquist <u>et</u><u>al</u> ., 1984), but these transcripts have exactly the same 5' end as the viral RNAs; i.e. a cap structure. Using one of the currently known <u>in</u><u>vitro</u> transcription systems it seems difficult to synthesize <u>in</u><u>vitro</u> transcripts from DNA copies of CPMV RNA, which have exactly the same 5' end as the viral RNAs. It may be hoped that the infectivity of the transcripts will increase if the number of extra nucleotides at the 5' end is reduced, but the synthesis of such transcripts will most likely be less efficient (Kang and Nu, 1987; Dunn and Studier, 1983). It in also possible that the extra nucleotides at the 3' end affect the infectivity, although in case of other viruses for which the <u>in</u><u>vitro</u> transcription of cDNA clones has been reported sofar a limited number of extra nucleotides added to the 3' end did not significantly influence infectivity (Ahlquist <u>et</u><u>al</u> ., 1984: Van der Werf <u>et</u><u>al</u> ., 1986; Dasmahapatra <u>et</u><u>al</u> ., 1986; Meshi <u>et</u><u>al</u> ., 1986; Dawson <u>et</u><u>al</u> ., 1986). It should be possible to construct cDNA clones, which allow the <u>in</u><u>vitro</u> synthesis of RNA transcripts terminating in a poly(A) tail without extra nucleotides at the 3' end using a restriction enzym for linearization of the template DNA, which restricts the DNA upstream of its recogntion sequence like e.g. BspM1.The possibilities for investigating the various aspects of the multiplication cycle of CPMV and of virus-host interaction will increase significantly when RNA transcripts can be synthesized which are much more infectious than the present ones. Since B RNA replicates independently in cowpea protoplasts because it encodes all proteins necessary for replication of the viral RNAs and proteolytic processing of the polyproteins. various mutations can be introduced in M RNA transcripts without affecting the multiplication and expression of B RNA. The infection of cowpea protoplasts with B RNA transcripts together with specifically modified K RNA transcripts may lead to a better understanding of the proteolytic processing of the M RNA encoded polyproteins <u>in</u><u>vivo</u> and to the elucidation of sequences involved in viral RNA replication.<TT>