The Gag domains required for avian retroviral RNA encapsidation determinedby using two independent assays

The Rous sarcoma virus (RSV) Gag precursor polyprotein is the only viral pr otein which is necessary fur specific packaging of genomic RNA. To map doma ins within Gag which are important for packaging, we constructed a series o f Gag mutations in conjunction with a protease (PR) active-site point mutat ion in a full-length viral construct. We found that deletion of either the matrix (MA), the capsid (CA), or the protease (PR) domain did not abrogate packaging, although the MA domain is likely to be required for proper assem bly. A previously characterized deletion of both Cys-His motifs in RSV nucl eocapsid protein (NC) reduced both the efficiency of particle release and s pecific RNA packaging by 6- to 10-fold, consistent with previous observatio ns that the NC Cys-His motifs played a role in assembly and RNA packaging. Most strikingly, when amino acid changes mt Arg 549 and 551 immediately dow nstream of the distal NC Cys-His box were made, RNA packaging was reduced b y more than 25-fold with no defect in particle release, demonstrating the i mportance of this basic amino acid region in packaging. We also used the ye ast three-hybrid system to study avian retroviral RNA-Gag interactions. Usi ng this assay, we found that the interactions of the minimal packaging regi on (M psi) with Gag are of high affinity and specificity. Using a number of M psi and Gag mutants, we have found a clear correlation between a reporte r gene activation in a yeast three-hybrid binding system and an in vivo pac kaging assay. Our results showed that the binding assay provides a rapid ge netic assay of both RNA and protein components for specific encapsidation.