IDENTIFICATION OF SEQUENCES DOWNSTREAM OF THE PRIMER BINDING-SITE THAT ARE IMPORTANT FOR EFFICIENT REPLICATION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1

Reverse transcription of retroviruses is initiated from an 18-nucleoti de (nt) primer binding site (PBS), located within the 5' region of vir al genomic RNA, to which the host cell-derived tRNA primer is annealed and also involves viral genomic sequences outside the PBS. We constru cted proviral DNA clones of human immunodeficiency virus (HIV) that ha d selective deletions of either a 7-nt segment found immediately downs tream of the PBS or an extended nontranslated 54-nt stretch located im mediately downstream of the PBS and containing the aforementioned 7-nt segment. Synthesis of minus-strand strong-stop DNA was assessed with MT-4 cells infected with viruses derived from COS-7 cells that had bee n transfected with these various constructs. We found that similar lev els of minus-strand strong-stop DNA as well as DNA produced after temp late switching were expressed in MT-4 cells infected with COS-7-derive d wild-type viruses or with viruses that had the 7-nt segment deleted. In contrast, significantly lower levels of viral DNA were detected in MT-4 cells after infection with viruses that had deletions of the 54- nt stretch. Furthermore, the molecular clone containing the 7-nt delet ion was able to replicate with wild-type kinetics, while that containi ng the 54-nt deletion displayed a significantly diminished capacity in this regard. Further deletion analysis showed that a 16-nt segment at the 3' end of this 54-nt segment was largely responsible for these ef fects. We also conducted studies to determine levels of viral mRNA in COS-7 cells that had been transfected with equivalent amounts of DNA d erived from either a wild-type HIV construct or our various deletion m utants. In the case of transfections performed with the 7-nt deletion mutant and wild-type HIV DNA, high levels of aral mRNA transcripts mer e detected, which was not the case for the 54 nt-deletion mutant. Howe ver, these various mRNAs possessed similar stabilities, as shown throu gh studies in which transcript formation was arrested by treatment of cells with actinomycin D. Thus, the 54-nt segment of 5' nontranslated RNA, located downstream of the PBS, is involved in efficient expressio n of each of viral DNA, mRNA, and infectious virus.