RNA dimerization defect in a Rous sarcoma virus matrix mutant

The retrovirus matrix (MA) sequence of the Gag polyprotein has been shown t o contain functions required for membrane targeting and binding during part icle assembly and budding, Additional functions for MA have been proposed b ased on the existence of MA mutants in Rous sarcoma virus (RSV), murine leu kemia virus, human immunodeficiency virus type 1, and human T-cell leukemia virus type 1 that lack infectivity even though they release particles of n ormal composition. Here we describe an RSV MA mutant with a surprising and previously unreported phenotype. In the mutant known as Myr1E, the small me mbrane-binding domain of the Src oncoprotein has been added as an N-termina l extension of Gag, While Myr1E is not infectious, full infectivity can be reestablished by a single amino acid substitution in the Src sequence (G2E) , which eliminates the addition of myristic acid and the membrane-binding c apacity of this foreign sequence. The presence of myristic acid at the N te rminus of the Myr1E Gag protein does not explain its replication defect, be cause other myristylated derivatives of RSV Gag are fully infectious (e.g., Myr2 [C. R. Erdie and J, W Wills, J. Virol. 64:5204-5208, 1990]). Biochemi cal analyses of Myr1E particles reveal that they contain wild-type levels o f the Gag cleavage products, Env glycoproteins, and reverse transcriptase a ctivity when measured on an exogenous template. Genomic RNA incorporation a ppears to be mildly reduced compared to the mild-type level. Unexpectedly, RNA isolated from Myr1E particles is monomeric when analyzed on nondenaturi ng Northern blots. Importantly, the insertional mutation does not lie withi n previously identified dimer linkage sites. In spite of the dimerization d efect, the genomic RNA from Mgr1E particles serves efficiently as a templat e for reverse transcription as measured by an endogenous reverse transcript ase assay. In marked contrast, after infection of avian cells, the products of reverse transcription are nearly undetectable. These findings might be explained either by the loss of a normal function of MA needed in the forma tion or stabilization of RNA dimers or by the interference in such events b y the mutant MA molecules, It is possible that Myr1E viruses package a sing le copy of viral RNA.