THE BOVINE LEUKEMIA-VIRUS ENCAPSIDATION SIGNAL IS DISCONTINUOUS AND EXTENDS INTO THE 5' END OF THE GAG GENE

In order to define bovine leukemia virus (BLV) sequences required for efficient vector replication, a series of mutations were made in a BLV vector. Testing the replication efficiency of the vectors with a help er virus and helper plasmids allowed for separation of the mutant vect ors into three groups. The replication efficiency of the first group w as reduced hy a factor of 7; these mutants contained deletions in the 5' end of the gag gene. The second group of mutants had replication re duced by a factor of 50 and had deletions including the 5' untranslate d leader region. The third group of mutants replicated at levels compa rable to those of the parental vector and contained deletions of the 3 ' end of the gag gene, the pol gene, and the env gene. Analysis of cyt oplasmic and virion RNA levels indicated that vector RNA expression wa s not affected but that the vector RNA encapsidation was less efficien t for group 1 and group 2 mutants. Additional mutations revealed two r egions important for RNA encapsidation, The first region is a 132-nucl eotide-base sequence within the gag gene (nucleotides 1015 to 1147 of the proviral DNA) and facilitates efficient RNA encapsidation in the p resence of the second region. The second region includes a 147-nucleot ide-base sequence downstream of the primer binding site (nucleotide 55 1) and near the gag gene start codon (nucleotide 698; gag begins at nu cleotide 628) and is essential for RNA encapsidation, We conclude that the encapsidation signal is discontinuous; a primary signal, essentia l for RNA encapsidation, is largely in the untranslated leader region between the primer binding site and near the gag start codon. A second ary signal, which facilitates efficient RNA encapsidation, is in a 132 -nucleotide-base region within the 5' end of the gag gene.