Nucleotide segment (؉169)AAAA(؉172) constitutes an A-rich loop within human immunodeficiency virus type 1 (HIV-1) (HXB2D) RNA and is able to interact with the anticodon loop (33)/USUU(36) of primer tRNA 3 Lys. We have shown that the deletion of this A-rich loop resulted in diminished levels of infectivity and reduced synthesis of viral DNA in MT-2 cells and cord blood mononuclear cells. Endogenous reverse transcriptase (RT) assays revealed that the mutated viruses, termed HIV/del-A, generated fewer cDNA products than did wild-type virus, designated HIV/WT. We also employed in vitro RT assays with in vitro-synthesized viral RNA templates, recombinant HIV-1 RT(p66/51), and natural tRNA 3 Lys as primers to show that the mutated RNA templates, designated PBS/del-A, generated less minus-strand strong-stop DNA product than did the wild-type RNA template, designated PBS/WT. The initiation efficiency of reverse transcription from the mutated RNA template was significantly impaired compared with that from the wild-type RNA template when a single-base extension assay from the tRNA 3 Lys primer was employed. However, RT reactions performed with DNA oligonucleotides complementary to the primer binding site (PBS) as primers did not yield differences between the mutated PBS/del-A and wild-type RNA templates. Long-term culture of HIV/del-A in MT-2 cells resulted in the replacement of two G's at nucleotide positions 167 and 168 by two A's that possessed the same relationship to the 5 end of the PBS as did the wild-type A's at positions 171 and 172. In vitro RT assays performed with recombinant enzyme with tRNA 3 Lys as the primer showed that the RNA template thus generated, termed PBS/A2, yielded levels of minus-strand strong-stop DNA product similar to those yielded by the wild-type RNA template. Coincidentally, viruses containing A's at positions 167 and 168 were able to replicate with efficiencies similar to those of the wild-type viruses. Thus, the (؉169)AAAA(؉172) A-rich loop plays a key role in the synthesis of viral DNA.

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We have studied the relationship between the length of HIV-1 reverse transcriptase (RT)-mediated nucleotide polymerization and inhibitors of these reactions in cell-free RT assays performed in the presence of either of two dideoxynucleoside triphosphates (ddNTPs), i.e. AZTTP or 3TCTP, or nevirapine, a non-nucleoside RT inhibitor. These reactions employed a heterologous RNA template and three DNA oligonucleotide primers, i.e. pAR, dPR and PA, that yielded distinct full-length products of 65, 192 and 376 nt, respectively, in the absence of inhibitor. We now show that the extent of inhibition of RT activity was greatest with use of the PA primer, which normally yielded the longest reaction product, and that lesser degrees of inhibition were noted in the reactions that generated shorter products. For example, at a concentration of 5 microM AZTTP, the extent of inhibition was 75% with the PA primer but only 40% and <10% when reactions were primed by the dPR and pAR primers, respectively. Similar results were obtained when either a mutated form of HIV RT (i.e. M184V), associated with resistance to 3TC, was tested in the presence of 3TCTP or when RT derived from Moloney murine leukemia virus (M-MuLV) was tested in the presence of AZTTP.