RNA-dependent RNA polymerases from cowpea mosaic virus-infected cowpea leaves

The aim of the research described in this thesis was the purification and identification of the RNA-dependent RNA polymerase engaged in replicating viral RNA in cowpea mosaic virus (CPMV)- infected cowpea leaves.Previously, an RNA-dependent RNA polymerase produced upon infection of Vigna unguiculata plants with CPMV, was partially purified (Zabel, 1978). This enzyme was thought to be responsible for the replication of viral RNA and believed to be encoded, at least in part, by the virus genome.In chapter III, it is shown that this enzyme is actually a host- encoded enzyme that occurs in small amounts in uninfected cowpea leaves and is greatly enhanced upon infection with CPMV. The host-encoded RNA- dependent RNA polymerase was extensively purified by DEAE-Sepharose, poly(U)-Sepharose and phosphocellulose column chromatography. Using antiserum directed against the purified host-encoded enzyme and a novel antibody-linked polymerase assay (ALPA), the host-enzyme was found to consist of a monomeric polypeptide with a molecular weight of 130,000 dalton (chapter IV).Since it was uncertain whether the purified host-encoded RNA polymerase plays any role in viral RNA replication, we reinvestigated the membranebound RNA-dependent RNA polymerase activity associated with endogenous template and known to be capable of synthesizing viral RNAs in vitro. Detailed analysis of the RNAs synthesized in vitro by RNA- dependent RNA polymerase activity associated with the crude membrane fraction of CPMV-infected leaves revealed the existence of two functionally different enzyme-template complexes (chapter V). One of the activities constitutes the CPMV RNA replication complex engaged in the production of full-length (+)-type viral RNA. The other activity occurs both in uninfected and infected leaves and transcribes endogenous plant RNAs into small RNAs of negative polarity. The occurrence of two distinct RNA-synthesizing activities in infected leaves is supported by their respective behaviour upon extracting the crude membrane fraction with a Mg 2+ -deficient buffer. Whereas the host-enzyme-template complex is solubilized, the viral RNA replication complex remains membranebound.A further analysis as to the role of the host-enzyme in viral RNA replication is presented in chapter VI. Using an antiserum directed against the purified host-encoded RNA polymerase to identify the 130K polypeptide on protein blots, it was confirmed that the amount of host- enzyme is greatly enhanced in leaves upon invasion of the virus. In contrast, no increase of 130K polypeptide occurs in isolated cowpea protoplasts infected with CPMV, indicating that the increase of host-enzyme represents a tissue- specific response to virus inoculation. Furthermore, the host-encoded 130K RNA polymerase is not found associated with the CM RNA replication complex. Thus, it is concluded that the RNA-dependent RNA polymerase of cowpea does not participate in CPU RNA replication.The purification procedure and the analysis of the protein composition of the viral replication complex is described in chapter VII. After solubilization from the membranes with a nonionic detergent and extensive purification by chromatography on Sepharose 2B and glycerol gradient centrifugation, an RNA replication complex preparation is obtained that is still capable of elongating nascent RNA chains in vitro. The purified replication complex appeared to contain three polypeptides, as revealed by SDS-polyacrylamide gel electrophoresis and silver staining. One of these polypeptides was found to be identical to the CPU B-RNA encoded 110K polypeptide. Since the hostencoded RNA-dependent RNA polymerase of cowpea is not involved in viral RNA replication, it is tempting to speculate that the 110K viral protein, by analogy to bacterial- and animal RNA viruses, is the core polymerase engaged in elongating CM RNAs in the viral RNA replication complex. Further experiments are required to verify this conclusion.The data presented in this thesis dispute the hypothesis that plant virus RNA replication might be mediated by a host-encoded RNA-dependent RNA polymerase. Contradictory to this hypothesis, CM RNA replication does not require the host-encoded RNA polymerase of cowpea but uses a different polymerase, probably of viral origin. This virus-specific RNA polymerase is present in very low amounts in infected tissue in comparison with the hostenzyme. and has previously been overlooked during purification of the viral replicase (Dorssers et al. , 1982). Only by employing an assay which keeps track of the synthesis of viral RNA, we have been able to distinguish between these RNA-dependent RNA polymerase activities.In other host plants inoculated with different viruses, a virus- specific RNA polymerase may similarly be present in low amounts in the particulate fraction of the leaves in addition to an enhanced amount of host-encoded RNA-dependent RNA polymerase. It seems conceivable, that in some cases where the purification of a plant viral replicase has been attempted without success, as for example with M, AM and M, the virus- specific RNA polymerase has been overlooked because of improper assay conditions applied during isolation and the very small amount of viral replicase relative to the hostencoded RNA-dependent RNA polymerase. It seems almost imperative in purifying plant viral RNA replicases to use test methods which keep control on the ability of the enzyme to produce full-length viral RNAs. In particular, this should apply when the purification of a viral RNA replication complex turns out to be impossible because the endogenous template is rapidly lost (Zaitlin et al. , 1973). By using a proper assay, a BMV replicase preparation from BMV-infected barley displaying a high degree of template-specificity has been obtained (Bujarski et al. , 1982; Miller and Hall, 1983).In those cases where it is not possible to isolate intact viral RNA replication complexes, an appropriate primer complexed to the template may be useful to test for the presence of a different (viral ?) core polymerase. Thus, the first step in viral RNA replication - i.e. specific template recognition and initiation of RNA synthesis for which specific factors are required that are often readily lost - is bypassed and the core polymerase is allowed to start transcription by elongating the primer. This approach has proven to be successful in identifying the core polymerase of several picornaviruses (Flanegan and Baltimore, 1977; Van Dijke and Flanegan, 1980; van Dijke et at., 1982; Baron and Baltimore, 1982; Lowe and Brown, 1981) and it is rather surprising that it has not yet been used in the study of plant viral RNA replication.Concerning CPMV RNA replication, the mechanism of initiation of CPMV RNA synthesis is not yet understood. The occurrence of a protein covalently linked to the 5'-end of the virion RNAs suggest that VPg might be of vital importance for the initiation of CPMV RNA synthesis. Evidence to support this hypothesis may be obtained by determining the nature of the 5'-termini of RNA replication intermediates. De novo initiation of CPMV RNA synthesis invitro has not been detected sofar, not even in the viral RNA replication complex still being an intergral part of the cellular membranes. The lacking of de novo initiation of CPMV RNA synthesis, suggests that some factor(s) required for initiation are already lost or inactivated upon the first fractionation of CPMV-infected leaves.A CPMV RNA-specific binding activity has previously been detected by using a nitrocellulose filter-binding assay (Zabel et al. , 1979). This specific binding activity was found in preparations devoid of the CPU RNA replication complex and can therefore not be ascribed to functions of the viral replication complex. However, this binding activity, which should be identified further, may well be involved in initiation of CPMV RNA synthesis.In conclusion, the replication of CPMV RNA may be described by the following model, taking into account the well established features of the process. (i) Infecting CPMV B-RNA directs the synthesis of a large B-RNA-specific polyprotein which is subsequently processed. Some mature protein then induces the expansion of cellular membranes. (ii) Proteolytic processing of the B-RNA-encoded precursors seems to occur in association with membranes, as virtually all 170K polypeptides are found in the particulate fraction (chapter VII, fig. 4A). (iii) The CPMV RNA replication complex is assembled using the viral RNA, the putative 110K core polymerase and probably other virus-encoded and host proteins, and is integrated in the cellular membranes to start RNA synthesis. Since the B-RNA-encoded proteins do not seem to be typical membrane proteins - in majority, they are solubilized by extraction with a Mg 2+ -deficient buffer - host proteins should play an important role in the compartimentalization of viral RNA synthesis. (iv) Initiation of viral RNA synthesis may involve VPg to be donated by either the 60K VPg-precursor or the 112K VPg-containing polypeptide. The latter seems to be largely overlapping with the putative 110K core polymerase and may be converted into the active core polymerase upon donating VPg.