Identification of the human immunodeficiency virus reverse transcriptase residues that contribute to the activity of diverse nonnucleoside inhibitors.

The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is potently inhibited by a structurally diverse group of nonnucleoside compounds. These include pyridinone derivatives, tetrahydroimadazo[4,5,1-j,k][1,4]-benzodiazepin-2(1H)-one and -thione, and BI-RG-587 (nevirapine). The compounds act noncompetitively, by an unknown mechanism, with respect to template-primer and nucleotide substrates. Despite a high degree of similarity between the HIV-1 and HIV-2 RTs, the HIV-2 enzyme is totally insensitive to these inhibitors. Using a novel method for joining DNA sequences, we have exploited this difference between the two enzymes to identify the regions of the RT that contribute to the compounds' inhibitory activities. The relative in vitro sensitivities of HIV-1/HIV-2 chimeric and site-specific mutant enzymes were determined. Sensitivity to inhibition was largely, though not exclusively, dependent upon the RT region defined by amino acid residues 176 to 190, with specific contributions by residues 181 and 188. The region defined by residues 101 to 106 was found to functionally interact with the domain from 155 to 217. In addition, the functional equivalence of the three inhibitor groups was shown.

Peptide substrate cleavage specificity of the human cytomegalovirus protease.

The human cytomegalovirus UL80 gene encodes an 80-kDa precursor polyprotein whose N-terminal 256-amino acid domain is a protease. This enzyme cleaves a specific peptide bond that results in its own release from the precursor, as well as a peptide bond near the C terminus of the viral assembly protein. The latter cleavage is apparently required for encapsidation of the viral genomic DNA and maturation of the viral capsid. A series of peptide substrates, representing the assembly protein cleavage site, was used to study the enzyme's substrate requirements and specificity. It was found that efficient cleavage minimally required the amino acid residues spanning the P4 to P4' positions. Substitution at any of these residues adversely affected the reaction. Conservation of the hydrophobic residues at P3 and P4 was essential. In addition, cleavage of a peptide representing the protease domain release site was reduced almost 100-fold relative to cleavage of the assembly protein maturation site peptide substrate.

Comprehensive mutant enzyme and viral variant assessment of human immunodeficiency virus type 1 reverse transcriptase resistance to nonnucleoside inhibitors.

The nonnucleoside reverse transcriptase (RT) inhibitors comprise a class of structurally diverse compounds that are functionally related and specific for the human immunodeficiency virus type 1 RT. Viral variants resistant to these compounds arise readily in cell culture and in treated, infected human. Therefore, the eventual clinical usefulness of the nonnucleoside inhibitors will rely on a thorough understanding of the genetic and biochemical bases for resistance. A study was performed to assess the effects of substitutions at each RT amino acid residue that influences the enzyme's susceptibility to the various nonnucleoside compounds. Single substitutions were introduced into both purified enzyme and virus. The resulting patterns of resistance were markedly distinct for each of the tested inhibitors. For instance, a > 50-fold loss of enzyme susceptibility to BI-RG-587 was engendered by any of four individual substitutions, while the same level of relative resistance to the pyridinone derivatives was mediated only by substitution at residue 181. Similarly, substitution at residue 181. Similarly, substitution at residue 106 had a noted effect on virus resistance to BI-RG-587 but not to the pyridinones. The opposite effect was mediated by a substitution at residue 179. Such knowledge of nonucleoside inhibitor resistance profiles may help in understanding the basis for resistant virus selection during clinical studies of these compounds.

Functional analysis of HIV-1 reverse transcriptase amino acids involved in resistance to multiple nonnucleoside inhibitors.

Several novel, structurally distinct classes of specific human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) nonnucleoside inhibitors have been described recently. These include the pyridinone derivatives L-697,639, L-697,661, and L-696,229 as well as BI-RG-587 and the tetrahydroimidazo[4,5,1-j,k]-benzodiazepin-2(1H)-one and -thione compounds. Previous studies have implicated involvement of the RT amino acid residues at positions 103, 181, and 188 in the activity of the compounds. Accordingly, HIV-1 RT mutants containing a series of amino acid substitutions at these positions were constructed. The relative resistance of purified mutant enzymes to each of the inhibitors was assessed. This analysis established the functional equivalence of the three inhibitor classes and provided evidence for the interaction of the 103 site with the 181/188 region. Amino acid substitutions at these positions were also found to influence RT sensitivity to inhibition by phosphonoformate, thereby suggesting a close association between this pyrophosphate analog's binding site in RT and the binding site of the nonnucleoside inhibitors. In addition, aromatic stacking of the amino acid side groups at residues 181 and 188 was suggested to be required for inhibitor activity.

Susceptibilities of human immunodeficiency virus type 1 enzyme and viral variants expressing multiple resistance-engendering amino acid substitutions to reserve transcriptase inhibitors.

To evaluate the potential that multiply resistant human immunodeficiency virus type 1 variants may arise during combination nucleoside and nonnucleoside reverse transcriptase inhibitor therapy, we constructed a series of mutant reverse transcriptase enzymes and viruses that coexpressed various combinations of resistance-associated amino acid substitutions. Substitutions at residues 100 (Leu-->Ile) and 181 (Tyr-->Cys), which mediate resistance to the nonnucleosides, suppressed resistance to 3'-azido-3'-deoxythymidine (AZT) when coexpressed with AZT-specific substitutions. However, a number of viral variants that exhibited significantly reduced susceptibilities to both classes of inhibitors were constructed.