In vitro transcription of Sonchus yellow net virus RNA by a virus-associated RNA-dependent RNA polymerase

<p/>The aim of the investigation presented in this thesis was to elucidate the nature of the RNA- dependent RNA polymerase, thought to be associated with <em>Sonchus</em> yellow net virus (SYNV), a rhabdovirus infecting plants. This research was initiated to shed light on the transcription activity in rhabdoviruses with similarities to rabies virus. It has been difficult to detect RNA polymerase activity in rabies virus particles in contrast to VSV with its highly active RNA- dependent RNA polymerase.<p/>The proteins of SYNV and their location in the virus particle are discussed in Chapter 2. We detected four major proteins, designated G, N, M1 and M2 in purified preparations of SYNV, which were separated on a 10% SDS-polyacrylamide gel. Minor proteins HMW1, HMW2, X and Y were usually also detected. To understand more about the interactions of SYNV and the host plant cell, it is important to determine which virus proteins are associated with the transcribing ribonucleoprotein (RNP) complex, which is infectious.<p/>We propose a model for the transcription of SYNV-RNA in which the single-stranded RNA with a negative polarity forms the transcribing RNP complex with HMW1 (RNAdependent RNA polymerase), N, X and M I proteins. The N protein-RNA complex serves as a template for the virus-associated RNA-dependent RNA polymerase. The X protein is assumed to be a form of the M1 protein which is phosphorylated to a different degree than M I. The M2 protein which forms a bridge between the G protein in the envelope and the RNP complex, may inhibit transcription.<p/>In Chapter 3 we showed, that an enzyme is associated with purified SYNV, which has the ability to catalyze the polymerization of ribonucleoside triphosphates.<p/>The RNA polymerase showed low activity after a 60 min incubation of purified SYNV. Prolonged incubation of purified SYNV considerably enhanced SYNV-specifiC [ <sup><font size="-1">3</font></SUP>H]UMP- incorporation. The results obtained by analysis of the protein composition of the reaction mixture during these prolonged incubations shows that the M1 and M2 proteins require modification before transcription could proceed.<p/>It is tempting to hypothesize that phosphorylation of one of the SYNV proteins, equivalent in function to the NS protein of VSV is necessary for efficient transcription of SYNV-RNA. In Chapter 3 we showed that the decrease in M1 protein corresponds to an increase in X protein. During chromatography of disrupted viruses on phosphocellulose columns, discussed in Chapter 4, the RNP complex was enriched in X protein when compared with the amount of X protein in purified preparations of SYNV. The lagtime in transcription was considerably reduced after eluting solubilized SYNV from a phosphocellulose column with KCI or K-acetate. The results of an experiment discussed in Chapter 5 has been interpreted as evidence that the X protein is analogous to the NS protein of VSV. The X protein is specifically inhibited by its own IgGs in an ALPA and this inhibition can be eliminated by incubating with anti-N serum which apparently mimicks the action of X by lifting the N protein from the template RNA, thereby activating the RNA polymerase. Kingsford and Emerson (1980) have shown that phosphorylation of the NS protein of VSV is necessary for transcription of VSV-RNA. This phosphorylation takes place in our SYNV system during incubation andactivates a protein. Changes in degree of phosphorylation is reflected in the increasing amounts of XI during the prolonged incubations as shown by SDS-polyacrylamide gel electrophoresis (Chapter 3). Van Beek <em>et al</em> . (1985) reported that the M 1 protein and sometimes a protein with a mol. wt. similar to the X protein of SYNV are phosphorylated in cowpea protoplasts infected with SYNV.<p/>Our results indicated an inhibitory role of the M2 protein. A regulatory role may be ascribed to the M2 protein, because we have shown in Chapter 3, that M2 protein is degraded to discrete products (M* and M**), concomitant with an increase in transcriptase activity <em>in vitro.</em> In experiments using phosphocellulose column purified RNP complexes the M2 protein of SYNV appeared to be modified to a more antigenic form and no longer inhibited transcription <em>in vitro.</em> Inhibition of transcription by the M protein of VSV has been reported by Carroll and Wagner (1979) and is now widely accepted for VSV. Pal <em>et al.</em> (1985a) recently showed that the exposure of only one epitope on the surface of the M protein of VSV may be important for the inhibition of <em>in vitro</em> transcription of VSV-RNA. In this thesis the importance of the M2 protein of SYNV in regulating transcription <em>in vitro</em> is stressed. The regulatory role of the M protein of VSV in <em>in vivo</em> and <em>in vitro</em> transcription was further investigated by Rosen <em>et al.</em> (1983). The M protein of VSV can be cleaved <em>in vivo</em> and <em>in vitro</em> by a protease to a discrete product (M'). The M' protein could be involved in regulating the transcription of VSV in a positive manner, while the M protein inhibits transcription. Whether the products of M2 had any role in the transcription of SYNV-RNA was not studied.<p/>To establish that the HMW1 protein of SYNV is the RNA-dependent RNA polymerase associated with SYNV, an ALPA was used in which transcribing RNP complexes are immobilized on nitrocellulose via an IgG bridge between SDS-denatured, electrophoretically separated SYNV proteins and the RNP complex. The polymerase activity is detected by incubating the nitrocellulose filters in a transcription mixture containing the ribonucleoside triphosphates. The newly synthesized RNA is then precipitated onto the nitrocellulose and visualized by autoradiography. Using this approach we provided evidence that the HMW 1 protein of SYNV exhibits polymerase activity.<p/>To prove that the <em>in</em><em>vitro</em> synthesized RNA was specific for SYNV, a direct and an indirect approach was used. Dotblot hybridizations demonstrated that SYNV-RNA was transcribed into complementary RNA. In a coupled <em>in vitro</em> transcription-translation experiment it was shown that the SYNV-associated polymerase was capable of elongating RNA into fulllength mRNAs, which were translated into the major SYNV proteins in a wheat germ cell-free system.<p/>It proved difficult to visualize the product RNAs on denaturing gels. Many bands were found (not shown), indicating random initiation or premature termination of the polymerase on the SYNV template. Perrault and McLear (1984) have demonstrated that the VSV RNA polymerase often aborts the transcription of VSV-RNA <em>in</em><em>vitro.</em> It is of interest to note that Thornton <em>et al.</em> (1984) reported that the M protein of VSV can abolish the transcriptionof the leader RNA whereas increasing amounts of fragments with 11 to 14 nucleotides transcribed from the 3' end of the N-mRNA gene are found. This has been explained by a M protein-mediated inhibition at the level of leader RNA synthesis and elongation.<p/>One of the major problems in studying plant virus replication is the occurrence of a host- encoded RNA-dependent RNA polymerase in healthy plants which is usually enhanced upon infection of the plant (Dorssers, 1983; Dorssers <em>et al.,</em> 1982 and 1983; and Hall <em>et al.,</em> 1982). The results obtained with the phosphocellulose-purified RNP particle of SYNV did not indicate the presence of contaminating host polymerases in that fraction, but rather in fractions eluting at lower salt concentrations, which is in agreement with the results obtained by Mouches <em>et al.</em> (1984), who eluted the host RNA polymerase at low salt concentrations, while the turnip yellow mosaic virus-induced RNA polymerase elutes at higher salt concentrations. Furthermore the polymerase of rhabdoviruses requires an N protein-RNA complex as template and it is inconceivable that a host plant RNA-dependent RNA polymerase would also accept this template. We conclude therefore that a host RNA-dependent RNA polymerase did not play a significant role in our investigations concerning the <em>in vitro</em> transcription of SYNV-RNA.<p/>In conclusion, we have provided evidence that the HMW1 protein of SYNV in possible combination with the M1 protein, phosphorylated to a different degree, can transcribe protein-RNA complexes <em>in vitro</em> when the inhibitory M2 protein has been modified