Human immunodeficiency virus type 1 (HIV‐1) variants with resistance mutations in the reverse transcriptase (RT) gene appear during drug therapy with the nucleoside analogue 2′,3′‐dideoxy‐3′‐thiacytidine (3TC). These resistance mutations alter the methionine (Met) residue of the conserved YMDD motif, which is part of the catalytic core of the RT enzyme. Isoleucine (Ile) variants are initially observed, followed by the appearance and eventual outgrowth of viruses encoding valine (Val). Similar replication kinetics were measured for wild‐type and 3TC‐resistant HIV‐1 viruses in tissue culture infections of a T cell line, but we measured reduced polymerase activity for the two mutant RT enzymes compared with the wild‐type enzyme (Ile = 43% and Val = 67%). Gel analysis of the reverse transcription products revealed that both 3TC‐resistant RT mutants produce significantly shorter cDNA molecules than the wild‐type enzyme [Met (wt)>Val>Ile], indicating that 3TC‐resistant RT polymerases are less processive enzymes. Interestingly, these enzyme defects were more pronounced under limiting dNTP concentrations and we therefore assayed virus replication in primary cells that contain relatively low dNTP levels. Under these conditions, we measured significantly reduced replication kinetics for the 3TC‐resistant HIV‐1 variants [Met (wt)>Val>Ile]. If the level of virus replication can be similarly reduced in 3TC‐treated patients that develop drug‐resistant HIV‐1 variants, this may be of considerable clinical benefit.

The third variable domain (V3) of the human immunodeficiency virus type 1 external envelope contains determinants of cell tropism, cytopathicity, and infectivity and elicits antibodies able to block infectivity in vitro and in vivo. Our study encompassed point-mutational analysis of HXB-2 viruses containing patient-derived V3 regions and expressing a non-syncytium-inducing, low-replicating phenotype in T-cell line SupTl. The mutation within V3 of a serine at position 306 into an also naturally occurring arginine (S to R) required an additional, naturally occurring mutation at position 320 (aspartate to glutamine, D to Q) or 324 (aspartate to asparagine, D to N) for ful expression of the syncytium-inducing, high-replicating (SI) phenotype. The naturally occurring mutation of an aspartate into an arginine at position 320 (D to R) was sufficient for production of the SI phenotype. This study proves that introduction of a positively charged amino acid at position 306 or 320, previously shown to be strongly associated with the SI phenotype in field isolates (R. A. M.

No abstract

Chimeric human immunodeficiency virus type 1 (HIV-1) molecular clones differing only in the envelope V3 region were constructed. The V3 regions were derived from two HIV-1 isolates with a non-syncytium-inducing, non-T-cell-tropic phenotype and from four HIV-1 isolates with a syncytium-inducing, T-cell-tropic phenotype. When assayed in SupT1 cells, the two chimeric viruses with a V3 region derived from the non-syncytium-inducing isolates did not induce syncytia and showed a low level of replication. The four chimeric viruses with a V3 region derived from the syncytium-inducing isolates did induce syncytia and replicated efficiently in SupT1 cells. In A3.01 cells, which do not support syncytium formation, the V3 loop affected replication similarly. Upon prolonged culture in SupT1 cells, the phenotype of a non-syncytium-inducing, low-replicating chimeric HIV-1 converted into a syncytium-inducing, high-replicating phenotype. Mutations within the usually conserved GPGR tip of the loop, which were shown to be responsible for the conversion into the syncytium-inducing, high-replicating phenotype, had occurred. In vitro mutagenesis showed that coupled changes of amino acids at both sides of the tip of the V3 loop were able to convert the viral phenotype from non-syncytium-inducing, low replicating into syncytium inducing, high replicating. Our data show that the V3 loop is involved in both syncytium forming and replicative capacity of HIV-1.

The relative fitness of viral variants has previously been defined as the slope of the logarithmic ratio of the genotype or phenotype frequencies in time plots of pairwise competition experiments. Developing mathematical models for such experiments by employing the conventional coefficient of selection s, we demonstrate that this logarithmic ratio gives the fitness difference, rather than the relative fitness. This fitness difference remains proportional to the actual replication rate realized in the particular experimental setup and hence cannot be extrapolated to other situations. Conversely, the conventional relative fitness (1 ؉ s) should be more generic. We develop an approach to compute the generic relative fitness in conventional competition experiments. This involves an estimation of the total viral replication during the experiment and requires an estimate of the average lifetime of productively infected cells. The novel approach is illustrated by estimating the relative fitness, i.e., the relative replication rate, of a set of zidovudine-resistant human immunodeficiency virus type 1 variants. A tool for calculating the relative fitness from observed changes in viral load and genotype (or phenotype) frequencies is publically available on the website at http://www-binf.bio.uu.nl/ ϳ rdb/fitness.html.Differences in the in vitro replication rate (or fitness) between viral variants can be estimated experimentally by pairwise competition experiments in tissue culture. The outcome of such an experiment is typically depicted in a logarithmic time plot of the ratio of the genotype or phenotype frequencies (7). On a logarithmic scale the ratio tends to change linearly in time, and the rate of change (i.e., the slope of the line) has previously been defined as the relative fitness (7). According to population genetics theory, the relative fitness (1 ϩ s) of a variant represents its relative contribution to the next generation. The parameter s is defined as the coefficient of selection. The intertwined concepts of relative fitness (1 ϩ s) and selection coefficient s are traditionally employed in systems with discrete generations. They are equally valid for populations growing continuously, however, when time is scaled with respect to the generation time (11).Developing conventional population genetics models for pairwise competition experiments, we show that the abovementioned slope in a logarithmic time plot provides the absolute fitness difference between the two variants rather than the generic relative fitness (1 ϩ s) of one with respect to the other. As the fitness difference remains proportional to the replication rate realized in the particular experimental setup, viral strains having similar selection coefficients s may have large fitness differences. On the other hand, variants differing markedly in the selection coefficients will yield almost horizontal lines in logarithmic time plots when the realized replication rate in the experiment is sufficiently low. This has indeed caused confusion in the literatu...

No abstract

We report the DNA sequence of a previously cloned Mycobacterium bovis BCG gene encoding an immunogenic 64-kilodalton protein. This protein, MbaA, was purified from overproducing Escherichia coli K-12 cells, and the presence of antibodies to MbaA in human sera was determined by an enzyme-linked immunosorbent assay. In about 80% of serum samples from tuberculosis patients and in about 60% of samples from BCG-vaccinated individuals, significant levels of anti-MbaA antibodies were found. Surprisingly, in about 30% of the control serum samples obtained from children, anti-MbaA antibodies were also observed. Guinea pigs sensitized with M. bovis BCG or MbaA showed a delayed-type hypersensitivity reaction after challenge with purified MbaA, supporting the previously observed strong reactivity of human T-cell clones with this, for mycobacteria, common antigen.

The third variable domain (V3) of the envelope gene of human immunodeficiency virus type 1 contains a major neutralization epitope and determinants of syncytium-inducing (SI) capacity and replication rate (reviewed by J. P. Moore and P. L. Nara, AIDS Suppi. 2:S21-S33, 1991). Sequences were generated from DNA of samples taken 3 months apart over a period of 24 and 30 months from peripheral blood mononuclear cells (PBMC) of two individuals, both before and after cocultivation with uninfected donor PBMC. The isolated virus shifted from the non-syncytium-inducing (NSI) phenotype to the SI phenotype during the study period. This shift was associated with distinct changes in the V3 domain in both patients. The association of the phenotype shift with the V3 sequence changes was confirmed by construction of viruses with chimeric V3 loops. The shift from NSIto SI-associated V3 variants was also seen in the uncultured PBMC of both patients, but not until 3 and 9 months after the detection of SI virus in culture. In the samples of uncultured PBMC DNA, several subgroups of sequences were found, indicating that the process of evolution may not be gradual and that several distinct populations can coexist. The paucity of intermediate sequences indicated that strong selection pressure was exerted on this part of the envelope. The early emergence of disease-associated SI variants in cultured material indicates that virus culture may have relevance for the in vivo situation.