Coat protein stimulates replication complexes of alfalfa mosaic virus to produce virion RNAs in vitro

Abstract

Viral replication complexes (RCs) were gradient-purified from cowpea mesophyll protoplasts 21 h after inoculation with alfalfa mosaic virus. These membranous structures incorporate [ 32P]UMP into double- and single-stranded RNAs in the absence of added template. When coat protein is added prior to the reaction the incorporation in both RNA fractions is stimulated several times. Part of the single-stranded product RNAs are released from the RCs. The stimulation of incorporation in high molecular mass RNAs by coat protein can be mimicked only to a certain extent by addition of a ribonuclease inhibitor or of an excess of viral RNA prior to the reaction. This shows that the coat protein is not only protecting the product of the RCs against degradation by ribonuclease, but that it is stimulating the synthesis and release of viral RNAs from RCs as well. This leads to the hypothesis that with alfalfa mosaic virus some coat protein is necessary for the release of messenger RNA from the RC. The hypothesis explains why the viral genome RNAs, although they are of messenger polarity, cannot start a replication cycle in the absence of coat protein: RCs containing the parental RNAs could be formed but no amplication of them could take place since no messenger RNAs needed for the production of viral polymerase proteins would be released into the cytoplasm.