In Vitro Evidence That the Coat Protein of Alfalfa Mosaic Virus Plays a Direct Role in the Regulation of Plus and Minus RNA Synthesis: Implications for the Life Cycle of Alfalfa Mosaic Virus

Abstract

The coat protein of alfalfa mosaic virus has both structural and regulating functions. The latter is evident from the fact that the genomic RNAs of the virus, although they are of messenger polarity, cannot start an infection cycle in the absence of cost protein. The reason could be that the coat protein is needed for viral RNA synthesis. Indeed, the coat protein has been found in tight association with the viral RNA polymerase (R. Quadt et al., 1991, Virology 182, 309-315). To investigate the role of the coat protein, if any, in viral RNA synthesis, we have isolated that viral RNA polymerase (RNA-dependent RNA polymerase, RdRp) from mock-inoculated tobacco plants transformed with cDNAs 1 and 2, known as P12 plants (P. E. M. Taschner et al., 1991, Virology 181, 687-693), which express the nonstructural proteins P1 and P2. Such an enzyme (called M-RdRp) will contain the viral subunits P1 and P2 but not the coat protein. As a comparison we also isolated the RdRp from virion-inoculated P12 plants (C-RdRp). This enzyme will contain the coat protein. We found that both M-RdRp and C-RdRp could synthesize minus RNA, showing that coat protein is not needed for minus-strand synthesis. In contrast, minus-strand synthesis by both enzymes was inhibited by coat protein. Plus-strand synthesis was unaffected by coat protein in the case of C-RdRp, but strongly stimulated by coat protein in the case of M-RdRp. These data might explain why infected cells, which do not produce coat protein, display a very low accumulation of viral plus-strand RNA. They also give a possible explanation for the noninfectious character of the genomes of alfalfa mosaic virus and ilarviruses in the absence of coat protein. The fact that an active enzyme could be isolated from the same membrane fraction in infected and noninfected P12 plants shows that coat protein is not needed for assembly and targeting of the viral RNA polymerase.

Alfalfa mosaic virus and ilarviruses: involvement of coat protein in multiple steps of the replication cycle.

“…RdRp isolated from mock-infected P12 plants does not contain CP but specifically transcribed AMV plus-strand RNAs in vitro into minus-strand copies (de Graaff & Jaspars, 1995). Addition of CP to this assay mixture inhibited minus-strand RNA synthesis.…”

“…In the absence of CP, the RdRp extracted from mock-infected P12 plants did not recognize AMV minus-strand RNA 3 as a template, but after addition of CP to the assay mixture plus-strand RNA 4 was synthesized (de Graaff & Jaspars, 1995). Also, when CP was added to membrane-bound AMV replication complexes that contain an endogenous minus-strand template, an increase in the accumulation of plus-strand RNA was observed.…”

The 3' untranslated region of alfalfa mosaic virus RNA3 contains a core promoter for minus-strand RNA synthesis and an enhancer element.

“…[α-$#P]UTP was incorporated during the synthesis of the minusstrand RNA in vitro. The products were treated with S1 nuclease and subsequently analysed by electrophoresis on agarose gels and autoradiography, all essentially as previously described (Houwing & Jaspars, 1986 ;de Graaff et al, 1995). Fig.…”

The 42-kDa coat protein of Andean potato mottle virus acts as a transcriptional activator in yeast

“…In the case of alfalfa mosaic virus, the coat protein is necessary for viral RNA synthesis and initiation of the infection cycle (15). Indeed, the coat protein was found to be tightly associated with the viral RNA polymerase, which was purified by immunoaffinity chromatography with antibodies raised against the coat protein (30).…”

“…Synthetic transcripts or purified genomic RNAs become infectious and able to replicate only upon the addition of few coat protein molecules. This phenomenon is known as genome activation and indicates that coat proteins have also a func-tional role in viral replication (15,16).…”