Human immunodeficiency virus (HIV) isolates with reduced sensitivity to zidovudine (3'-azido-3'-deoxythymidine, AZT) from individuals with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex were studied to determine the genetic basis of their resistance. Most were sequential isolates obtained at the initiation of and during therapy. Comparative nucleotide sequence analysis of the reverse transcriptase (RT) coding region from five pairs of sensitive and resistant isolates identified three predicted amino acid substitutions common to all the resistant strains (Asp67----Asn, Lys70----Arg, Thr215----Phe or Tyr) plus a fourth in three isolates (Lys219----Gln). Partially resistant isolates had combinations of these four changes. An infectious molecular clone constructed with these four mutations in RT yielded highly resistant HIV after transfection of T cells. The reproducible nature of these mutations should make it possible to develop rapid assays to predict zidovudine resistance by performing polymerase chain reaction amplification of nucleic acid from peripheral blood lymphocytes, thereby circumventing current lengthy HIV isolation and sensitivity testing.

The drug sensitivities of human immunodeficiency virus (HIV) isolates from a group of patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) who were receiving zidovudine (3'-azido-3'-deoythymidine, AZT) therapy were tested by means of a newly developed plaque assay in CD4+ HeLa cells. Fifty percent inhibitory dose (ID50) values of 18 isolates from untreated individuals ranged between 0.01 microM and 0.05 microM. In contrast, most isolates from patients who had received zidovudine for 6 months or more exhibited decreased sensitivity characterized by changes in ID50 or ID95 values (or both), with isolates from several patients (5/15) showing 100-fold increases in ID50. The latter isolates were also insensitive to 3'-azido-2',3'-dideoxyuridine; however, the isolates were still sensitive to 2',3'-dideoxycytidine, 2',3'-dideoxy-2',3'-didehydrothymidine, or phosphonoformate. It cannot be determined from this small sample of patients whether development of a less sensitive virus phenotype results in clinical resistance. Appearance of such variants was not associated with a consistent increase in viral p24 concentrations in patient plasma and did not herald any sudden deterioration in clinical status. More extensive studies are required to determine the clinical significance. Thus, it would be premature to alter any treatment protocols for HIV-infected individuals at present.

Resistant variants of human immunodeficiency virus type 1 (HIV-1) have been selected by limited passage in MT4 cells of both wild-type and 3'-azido-3'-deoxythymidine (AZT, zidovudine)-resistant strains with the nucleoside analogues (-)-2'-deoxy-3'-thiacytidine (3TC) and (-)-2'-deoxy-5-fluoro-3'-thiacytidine (FTC). Virus variants selected independently were crossresistant to both inhibitors. This rapid in vitro selection of resistant virus has not previously been seen with nucleoside analogues but is reminiscent of that observed with the nonnucleoside reverse transcriptase inhibitors. However, passage of wild-type virus with a combination of AZT and FTC appreciably delayed emergence of FTCresistant virus. DNA sequence analysis of the reverse transcriptase coding region from FTC-resistant virus revealed changes at codon 184 in the highly conserved Tyr, Met, Asp,

Human immunodeficiency virus (HIV) is the causative agent of AIDS (acquired immune deficiency syndrome) a disease which poses a serious challenge to modern medicine. If we are to conquer this disease we will need a protective vaccine or effective drugs able to block the life cycle of the virus. An early stage in the invasion of the host cell is the conversion of the RNA genome of the virus to a double-stranded DNA intermediate which subsequently becomes integrated into the host cell chromosome. The enzyme reverse transcriptase is crucial in this process and is thus an obvious chemotherapeutic target. In this study we have used site-directed mutagenesis of this enzyme expressed in Escherichia coli to reveal several important functional regions of the protein including putative components of the triphosphate binding site and pyrophosphate exchange sites.

It is recognized that high-level resistance to 3'-azido-3'-deoxythymidine (AZT, zidovudine, or Retrovir) is conferred by the presence of four mutations in the human immunodeficiency virus (HIV) reverse transcriptase [RT; deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase (RNA-directed), EC 2.7.7.49] coding sequence. However, a number of clinical isolates have been observed that exhibit high-level resistance but contain only three of the four identified mutations