The retrovirus matrix (MA) sequence of the Gag polyprotein has been shown to contain functions required for membrane targeting and binding during particle assembly and budding. Additional functions for MA have been proposed based on the existence of MA mutants in Rous sarcoma virus (RSV), murine leukemia virus, human immunodeficiency virus type 1, and human T-cell leukemia virus type 1 that lack infectivity even though they release particles of normal composition. Here we describe an RSV MA mutant with a surprising and previously unreported phenotype. In the mutant known as Myr1E, the small membrane-binding domain of the Src oncoprotein has been added as an N-terminal 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 capacity of this foreign sequence. The presence of myristic acid at the N terminus of the Myr1E Gag protein does not explain its replication defect, because 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]). Biochemical analyses of Myr1E particles reveal that they contain wild-type levels of the Gag cleavage products, Env glycoproteins, and reverse transcriptase activity when measured on an exogenous template. Genomic RNA incorporation appears to be mildly reduced compared to the wild-type level. Unexpectedly, RNA isolated from Myr1E particles is monomeric when analyzed on nondenaturing Northern blots. Importantly, the insertional mutation does not lie within previously identified dimer linkage sites. In spite of the dimerization defect, the genomic RNA from Myr1E particles serves efficiently as a template for reverse transcription as measured by an endogenous reverse transcriptase 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 formation or stabilization of RNA dimers or by the interference in such events by the mutant MA molecules. It is possible that Myr1E viruses package a single copy of viral RNA.The retrovirus Gag polyprotein directs the assembly and budding of virus particles from the plasma membrane of infected cells. Extensive functional mapping of several Gag proteins has led to the identification of three common assembly domains that are required for this activity. The N-terminal membrane-binding (M) domain directs Gag molecules from the cytoplasm to the inner leaflet of the plasma membrane (44,55,63) where aggregates of Gag proteins are formed through protein-protein interactions primarily involving the I (interaction) domains (4, 12, 22, 57). These interactions lead to the emergence of spherical particles that pinch off the membrane during the final step in the budding process, which is mediated by the L (late) assembly domain (27,45,58). Virus maturation occurs as the...

The genomic RNA of retroviruses exists within the virion as a noncovalently linked dimer. Previously, we identified a mutant of the viral matrix (MA) protein of Rous sarcoma virus that disrupts viral RNA dimerization. This mutant, Myr1E, is modified at the N terminus of MA by the addition of 10 amino acids from the Src protein, resulting in the production of particles containing monomeric RNA. Dimerization is reestablished by a single amino acid substitution that abolishes myristylation (Myr1E؊). To distinguish between cis and trans effects involving Myr1E, additional mutations were generated. In Myr1E.cc and Myr1E؊.cc, different nucleotides were utilized to encode the same protein as Myr1E and Myr1E؊, respectively. The alterations in RNA sequence did not change the properties of the viral mutants. Myr1E.ATG؊ was constructed so that translation began at the gag AUG, resulting in synthesis of the wild-type Gag protein but maintenance of the src RNA sequence. This mutant had normal infectivity and dimeric RNA, indicating that the src sequence did not prevent dimer formation. All of the src-containing RNA sequences formed dimers in vitro. Examination of MA-green fluorescent protein fusion proteins revealed that the wild-type and mutant MA proteins Myr1E.ATG؊, Myr1E؊, and Myr1E؊.cc had distinctly different patterns of subcellular localization compared with Myr1E and Myr1E.cc MA proteins. This finding suggests that proper localization of the MA protein may be required for RNA dimer formation and infectivity. Taken together, these results provide compelling evidence that the genomic RNA dimerization defect is due to a trans-acting effect of the mutant MA proteins.All retroviruses incorporate two identical copies of their RNA genome into each virion. The genomic RNA molecules are linked near their 5Ј ends by noncovalent interactions to form a stable structure possessing ordered secondary and tertiary structure. Although there are multiple contact points throughout the two parallel RNA molecules, the most stable linkage is called the dimer linkage structure. The dimer linkage structure can be visualized by electron microscopy and appears to be a region about 50 nucleotides (nt) in length near the 5Ј end of the genome (centered around nucleotide 511 in Rous sarcoma virus [RSV]) (1,20,23). Dimerization is required for infectivity, although precisely how it contributes to the replication cycle remains poorly understood. Dimerization is believed to facilitate recombination during reverse transcription by enabling close approximation of the viral RNA molecules, leading to increased genetic diversity and improved viral fitness (15,16,26). The dimeric RNA structure has also been implicated in inhibiting the translation of unspliced viral RNA so that genomic RNA is available for packaging; however, there is little experimental evidence in support of this idea (26).Because the RNA sequences that are important for dimerization overlap those required for RNA incorporation into virus particles, dimerization and packaging were postu...