trans-acting inhibition of genomic RNA dimerization by Rous sarcoma virus matrix mutants

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 mu tant, Myr1E, is modified at the N terminus of MA by the addition of 10 amin o acids from the Src protein, resulting in the production of particles cont aining 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 t he same protein as Myr1E and Myr1E-, respectively, The alterations in RNA s equence did not change the properties of the viral mutants. Myr1E.ATG- was constructed so that translation began at the gag AUG, resulting in synthesi s of the wild-type Gag protein but maintenance of the src RNA sequence, Thi s mutant had normal infectivity and dimeric RNA, indicating that the src se quence did not prevent dimer formation. All of the src-containing RNA seque nces formed dimers in vitro. Examination of MA-green fluorescent protein fu sion proteins revealed that the wild-type and mutant MA proteins Myr1E.ATG- , Myr1E-, and Myr1E-,cc had distinctly different patterns of subcellular lo calization compared with Myr1E and Myr1E.cc MA proteins. This finding sugge sts that proper localization of the MA protein may be required for RNA dime r formation and infectivity, Taken together, these results provide compelli ng evidence that the genomic RNA dimerization defect is due to a trans-acti ng effect of the mutant MA proteins.