Virtues of being faithful: can we limit the genetic variation in human immunodeficiency virus?

Human immunodeficiency virus (HIV) infections are characterized by a high degree of viral variation. The genetic variation is thought to be a combined effect of a high error rate of reverse transcriptase (RT), viral genomic recombination, the selection forces of the human immune system, the requirement for growth in multiple cell types during pathogenesis, and persistent immune activation associated with HIV disease. This hypermutability gives the virus an ability to escape mechanisms of innate immune surveillance and therapeutic interventions. Indeed, HIV variants that are resistant to drugs that antagonize both the HIV protease and RT enzymes are well described. Furthermore, there are seemingly no procedures to restrict this disarming property of HIV to mutate rapidly. Recently we have shown that some of the drug-resistant RTs display an increased in vitro polymerase fidelity. The question is whether this finding will stimulate new approaches that will not only help the immune system to deal with the virus more efficiently but also to reduce or delay resistance to various classes of anti-HIV drugs. The pros and cons of this concept and the influence of viral replication rates and viral fitness on HIV variability are discussed.

The influence of 3TC-resistance mutations E89G and M184V in the human immunodeficiency virus reverse transcriptase on mispair extension efficiency.

Two nucleoside analog resistance mutations in HIV-1 reverse transcriptase (RT), E89G and M184V, were previously shown to increase the dNTP insertion fidelity of HIV-1 RT. However, forward mutation assays using a lacZ alpha reporter gene have revealed a lack of impact on the overall error rate of these variants. In an effort to investigate the basis for this discrepancy, we have examined whether the increases in misinsertion fidelity observed for E89G and M184V RTs are accompanied by an increase in mispair extension fidelity. The relative efficiencies with which the wild type, E89G, M184V and M184V/E89G HIV-1 RTs extend model template-primer duplexes containing 3'-OH terminal mismatches were measured. The calculated efficiencies of mispair extension ( f ext) were, in general, not significantly decreased from the wild type HIV-1 RT. In fact, the efficiency of extension from one of the mispaired primer-template duplexes was significantly increased for two of the mutants tested. These results suggest that amino acid substitutions that increase the fidelity of dNTP insertion do not necessarily increase misextension fidelity, and that the decreased misextension fidelity may counterbalance the increases in misinsertion fidelity observed for E89G and M184V RTs.

The influence of 3TC resistance mutation M184I on the fidelity and error specificity of human immunodeficiency virus type 1 reverse transcriptase.

A common target for therapies against human immuno-deficiency virus type 1 (HIV-1) is the viral reverse transcriptase (RT). Treatment with the widely used nucleoside analog (-)-2', 3'-deoxy-3'-thiacytidine (3TC) leads to the development of resistance-conferring mutations at residue M184 within the YMDD motif of RT. First, variants of HIV with the M184I substitution appear transiently, followed by viruses containing the M184V substitution, which persist and become the dominant variant for the duration of therapy. In the three-dimensional crystal structure of HIV-1 RT complexed with double-stranded DNA, the M184 residue lies in the vicinity of the primer terminus, near the incoming dNTP substrate. Recent studies have shown that 3TC resistance mutations, including M184I, increase the nucleotide insertion and mispair extension fidelity. Therefore, we have examined the effects of the M184I mutation on the overall polymerase fidelity of HIV-1 RT via an M13-based forward mutation assay. We found the overall error rate of the M184I variant of HIV-1 RT to be 1.7 x 10(-5) per nucleotide. This represents a 4-fold increase in fidelity over wild-type HIV-1Hxb2RT (7.0 x 10(-5) per nucleotide) and a 2.5-fold increase in fidelity over the M184V variant (4.3 x 10(-5) per nucleotide). Of the nucleoside analog resistance mutations studied using the forward assay, the M184I variant has shown the greatest increase in fidelity observed to date. Interestingly, the M184I variant RT displays significantly altered error specificity, both in terms of error rate at specific sites and in the overall ratio of substitution to frameshift mutations in the entire target.

Increased misincorporation fidelity observed for nucleoside analog resistance mutations M184V and E89G in human immunodeficiency virus type 1 reverse transcriptase does not correlate with the overall error rate measured in vitro.

Nucleoside analog-resistant variants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) that displayed higher in vitro polymerase fidelity were previously identified via nucleotide insertion and mispair extension assays. To evaluate the contribution of increased nucleotide insertion and primer extension fidelities on the overall error rate of HIV-1 RT, we have measured the impact of two such mutations, E89G and M184V, on DNA copying fidelity in an M13 phage-based forward mutation assay. Using this assay, we observed mutation frequencies of 8.60 x 10(-3), 6.26 x 10(-3), 5.53 x 10(-3), and 12.30 x 10(-3) for wild-type, E89G, M184V, and double-mutant E89G/M184V HIV-1 RTs, respectively. Therefore, the overall polymerase fidelities of wild-type, E89G, M184V, and E89G/M184V HIV-1 RTs are similar (less than twofold differences) for DNA-dependent DNA synthesis. Thus, rather large increases in fidelity of deoxynucleoside triphosphate insertion and mispair extension observed previously appear not to influence the overall error rate of these mutants. However, a qualitative analysis of the mutations induced revealed significant differences in the mutational spectra between the wild-type and mutant enzymes.

Increased polymerase fidelity of E89G, a nucleoside analog-resistant variant of human immunodeficiency virus type 1 reverse transcriptase.

Nucleoside analog resistance in human immunodeficiency virus type 1 results from mutations in reverse transcriptase (RT) that allow the enzyme to discriminate against such analogs. To evaluate the possible impact of such mutations on the ability of human immunodeficiency virus RT to selectively incorporate Watson-Crick base-paired deoxynucleotide triphosphates (dNTPs) over incorrectly paired dNTPs, we have measured the fidelity of dNTP insertion by the E89G variant of RT in in vitro reaction mixtures containing synthetic template primers. The E89G RT was previously shown to be resistant to several ddNTPs and to phosphonoformic acid. Our results show that the mutant enzyme displays a lower level of efficiency of misinsertion than did the wild-type RT for every mispair tested (ranging from 2- to 17-fold.

Use of chimeric human immunodeficiency virus types 1 and 2 reverse transcriptases for structure-function analysis and for mapping susceptibility to nonnucleoside inhibitors.

The human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2) reverse transcriptases (RTs) are evolutionary related. To study the effect of homologous sequence replacements on polymerase function and to map the determinants of the lack of susceptibility of HIV-2 RT to nonnucleoside drugs, a series of chimeric HIV-1/HIV-2 RTs were constructed. Analysis of the chimeric RTs showed that wild-type levels of RNA-dependent DNA polymerase activity were retained when both finger and palm subdomains were exchanged as a unit between the two parental RTs. Analysis of enzymatically active chimeras for inhibition by the thiobenzimidazolone derivative TIBO R82150 showed that a segment of HIV-2 RT at 212-250, when placed in the HIV-1 RT context, conferred a 40-fold decrease in susceptibility to TIBO R82150. Site-directed mutagenesis of this segment found Tyr227 to be a key residue in this segment for the natural resistance of HIV-2 RT to TIBO R82150.

Enhanced fidelity of 3TC-selected mutant HIV-1 reverse transcriptase.

Monotherapy with (-)2',3'-dideoxy-3'-thiacytidine (3TC) leads to the appearance of a drug-resistant variant of human immunodeficiency virus-type 1 (HIV-1) with the methionine-184 --> valine (M184V) substitution in the reverse transcriptase (RT). Despite resulting drug resistance, treatment for more than 48 weeks is associated with a lower plasma viral burden than that at baseline. Studies to investigate this apparent contradiction revealed the following. (i) Titers of HIV-neutralizing antibodies remained stable in 3TC-treated individuals in contrast to rapid declines in those treated with azidothymidine (AZT). (ii) Unlike wild-type HIV, growth of M184V HIV in cell culture in the presence of d4T, AZT, Nevirapine, Delavirdine, or Saquinavir did not select for variants displaying drug resistance. (iii) There was an increase in fidelity of nucleotide insertion by the M184V mutant compared with wild-type enzyme.