EQUINE INFECTIOUS-ANEMIA VIRUS UTILIZES A YXXL MOTIF WITHIN THE LATE ASSEMBLY DOMAIN OF THE GAG P9 PROTEIN

We have previously demonstrated that the Gag p9 protein of equine infe ctious anemia virus (EIAV) is functionally homologous with Rous sarcom a virus (RSV) p2b and human immunodeficiency virus type 1 (HIV-1) p6 i n providing a critical late assembly function in RSV Gag-mediated budd ing from transfected COS-1 cells (L. J. Parent et al., J. Virol. 69:54 55-5460, 1995). In light of the absence of amino acid sequence homolog y between EIAV p9 and the functional homologs of RSV and HIV-1, we hav e now designed an EIAV Gag-mediated budding assay to define the late a ssembly (L) domain peptide sequences contained in the EIAV p9 protein. The results of these particle budding assays revealed that expression of EIAV Gag polyprotein in COS-1 cells yielded extracellular Gag part icles with a characteristic density of 1.18 g/ml, while expression of EIAV Gag polyprotein lacking p9 resulted in a severe reduction in the release of extracellular Gag particles. The defect in EIAV Gag polypro tein particle assembly could be corrected by substituting either the R SV p2b or HIV-1 p6 protein for EIAV p9. These observations demonstrate d that the L domains of EIAV, HIV-1, and RSV were interchangeable in m ediating assembly of EIAV Gag particles in the COS-1 cell budding assa y. To localize the L domain of EIAV p9, we next assayed the effects of deletions and site-specific mutations in the p9 protein on its abilit y to mediate budding of EIAV Gag particles. Analyses of EIAV Gag const ructs with progressive N-terminal or C-terminal deletions of the p9 pr otein identified a minimum sequence of 11 amino acids (21)L(22)Y(23)P( 24)D(25)L(26)S(27)E(28)I(29)K(30)) capable of providing the late assem bly function. Alanine scanning studies of this L-domain sequence demon strated that mutations of residues Y-23, P-24, and L-26 abrogated the p9 late budding function; mutations of other residues in the p9 L doma in did not substantially affect the level of EIAV Gag particle assembl y. These data indicate that the L domain in EIAV p9 utilizes a YXXL mo th which we hypothesize may interact with cellular proteins to facilit ate virus particle budding from infected cells.