Sequencing of the reverse transcriptase (RT) region of 26 human immunodeficiency virus type 1 (HIV-1) isolates from eight patients treated with 3'-azido-3'-deoxythymidine (AZT) revealed a mutation at codon 210 from TTG (leucine) to TGG (tryptophan) exclusively in association with resistance to AZT. The mutation Trp-210 was observed in 15 of the 20 isolates phenotypically resistant to AZT, being more commonly observed than resistance-associated mutations at codons 67, 70, and 219. Trp-210 was never observed before the emergence of resistance-associated mutations Leu-41 and Tyr-215, and in a sequential series of five isolates from one patient the order of emergence of mutations was found to be Tyr-215, Leu-41, and then Trp-210. Trp-210 was also found in association with the Leu-41, Asn-67, Arg-70, and Tyr-215 resistance genotype. To define the role of Trp-210 in AZT resistance, molecular HIV-1 clones were constructed with various combinations of RT mutations at codons 41, 67, 70, 210, and 215 and tested for susceptibility to AZT. In clones with polymerase genes derived either from HXB2-D or clinical isolates, Trp-210 alone did not increase AZT resistance, whereas in conjunction with Leu-41 and Tyr-215, Trp-210 contributed to high-level resistance (50% inhibitory concentration of >1 microM). In HXB2-D, Trp-210 with Tyr-215 generated a virus with resistance comparable to one with Leu-41, Tyr-215, and Trp-210. Inserting Trp-210 into the genetic context of mutations at codons 41, 67, 70, and 215 further enhanced resistance from a 50% inhibitory concentration of 1.44 microM to 8.41 microM. Molecular modeling of the tertiary structure of HIV-1 RT revealed that the distance between the side chains of Trp-210 (in helix alphaF) and Tyr-215 (in strand beta11a) approximated 4 A (1 A = 0.1 nm), sufficiently close to result in significant energetic interaction between these two aromatic side chains. In conclusion, Trp-210 contributes significantly to phenotypic AZT resistance of HIV-1 by augmenting resistance at least three- to sixfold in the context of two resistant genotypes, and its effect may require an interaction with an aromatic amino acid at position 215.

A blood donor infected with human immunodeficiency virus-type 1 (HIV-1) and a cohort of six blood or blood product recipients infected from this donor remain free of HIV-1-related disease with stable and normal CD4 lymphocyte counts 10 to 14 years after infection. HIV-1 sequences from either virus isolates or patient peripheral blood mononuclear cells had similar deletions in the nef gene and in the region of overlap of nef and the U3 region of the long terminal repeat (LTR). Full-length sequencing of one isolate genome and amplification of selected HIV-1 genome regions from other cohort members revealed no other abnormalities of obvious functional significance. These data show that survival after HIV infection can be determined by the HIV genome and support the importance of nef or the U3 region of the LTR in determining the pathogenicity of HIV-1.

Foscarnet (PFA) is a pyrophosphate analogue antiviral active against human immunodeficiency virus (HIV-1) and herpesviruses, Strains of HIV-1 resistant to PFA have mutations in the HIV-1 reverse transcriptase (RT), We examined the influence of PFA resistance mutations, in different genetic backgrounds, on HIV-1 replication competency in both replication kinetics and growth competition assays. In replication kinetics assays, the recombinant strains HX89K, HX92I, and HX156A (encoding RT mutations E89K, L92I, and S156A, respectively, in the HXB2-D genetic background) replicated to lower titers than the wild-type parent in the absence of drug, and the degree of replication impairment increased as PFA resistance increased. PFA-resistant strains LAI 92I and LAI 156A (encoding RT mutations L92I and S156A, respectively) were replication impaired in comparison to the wild-type parent LAI to a similar degree as observed for strains in the HXB2-D background. In growth competition assays with wild-type LAI, strains LAI 92I and LAI 156A had relative fitness values of 0.5 and 0.8, respectively. These results show that the RT mutations E89K, L92I and S156A, observed in PFA-resistant strains selected in cell culture, reduce replication competence. Furthermore, these data show a correlation of increasing PFA resistance and decreasing replication competence mediated by single amino acid substitutions in the RT.