A 700-bp region corresponding to the second constant region through the fifth variable region of the envelope gene (C2 to V5) was used to evaluate the diversity of HIV-1 proviral DNA sequences in five patients at various stages of disease progression. Sequences derived from each patient belonged to envelope sequence subtype B, and each set clustered as monophyletic groups compared with those from other patients in this study (see Fig. ) and the positive control sequences used in our experiments. Furthermore, no closely matched sequences were found in our local database or published databases of HIV sequences (33
). Hence, there was no evidence of sample mix-up or contamination.
FIG. 2 Neighbor-joining phylogenetic tree of proviral sequences from the blood and semen of five patients. Triangles represent sequences from NSMC; squares represent sequences from PBMC. Open symbols are from direct sequencing of molecular endpoints from PCR; (more ...)
The JO sample patient had clinical AIDS but CD4+
T-cell counts of 500 at the time of sampling. Initially, 10 plasmid subclones from his PBMC (JO-B series in Fig. ) and 4 from his NSMC (JO-S series) were sequenced. The diversity among the NSMC-derived clones was so low (mean, 0.6%; range, 0.3 to 0.8%; Fig. a) that we were concerned that provirus resampling had occurred and the divergence noted was due to Taq
polymerase misincorporation during PCR (33
) rather than representing viral quasispecies diversity in these cells. Hence, to rule out resampling, sequences from 11 molecular endpoints from his NSMC (JO-SE) were also derived. From this sampling, clonal virus populations were evident, as indicated by the bimodal distribution of divergence values between pairs of sequences in this population (Fig. b). Phylogenetic tree analysis revealed that 10 semen-derived variants formed a tightly clustered group of sequences distinct from that found in his PBMC (Fig. ). This cluster consisted exclusively of variants expected to be of the SI phenotype, based on positively charged amino acids encoded at positions 11 and/or 25 of the V3 loop (19
), while all other variants from his blood and semen were predicted to be NSI (Fig. and ). The fact that this cluster was formed from endpoint-derived sequences (e.g., each was derived from a single proviral template) as well as from multiple sequences from plasmids derived from a separate PCR indicates that its generally narrow diversity was a true reflection of that found in the semen proviral DNA. Additional, smaller tight clusters (e.g., JO-SE11, -SE12, and -SE22) were detected in the blood and semen. A comparison of the divergences measured between all pairwise comparisons of sequences revealed a bimodal distribution in each compartment, with the groups being most divergent in the semen (Fig. a and b).
FIG. 1 Distribution of divergences between pairwise comparisons of sequences. Provirus populations in the semen or PBMC were in each case sampled by PCR on multiple templates followed by cloning and sequencing of multiple plasmids (“clones”) (more ...)
FIG. 3 Deduced amino acid alignment over the C2-to-V5 region of HIV-1 env. For each patient, sequences are compared to one sequence from the PBMC. Dots are placed at positions at which individual sequences match that of the reference sequence. Dashes were introduced (more ...)
FIG. 4 Distribution of divergences between pairwise comparisons of sequences. Proviral sequences from semen or PBMC derived by either endpoint dilution, cloning, or a combination of the two methods were grouped for analysis of compartments in each patient. For (more ...)
To evaluate the requirement for endpoint dilution to provide accurate measures of viral population diversity, we determined sequences from both plasmid subclones from the PE sample AIDS patient’s PBMC (PE-B; n = 13) and NSMC (PE-S; n = 8), as well as from molecular endpoints from both compartments (sequences derived from PBMC endpoints are designated PE-BE [n = 9]; those from NSMC endpoints are designated PE-SE [n = 7]). The distribution of sequences was similar with either the molecular endpoint- or clone-derived variants (Fig. ). Three clusters were noted in the phylogenetic analysis (Fig. ): a tight cluster consisting of 16 variants from the semen and 1 from the blood, a more diverse cluster consisting of 8 variants found only in the blood, and a third loose cluster made up of variants from both compartments. As with the JO sample patient, viruses with the SI signature mutations were found as a cluster. However, in contrast to the JO sample patient, the PBMC-specific cluster was the one with the predicted SI phenotype, while all other variants from his blood and semen were predicted to be NSI.
Fifteen HIV plasmid subclones from NSMC proviral DNA (613-S) and eight subclones from PBMC (613-P) from asymptomatic patient 613 were generated and sequenced (Fig. ). Again, a bimodal distribution of divergences was evident in the virus populations, and again this was most pronounced in the semen proviral population (Fig. ). Proviral load was not quantitated in these (and patient 064) specimens, and yet the divergence noted between the two- and three-member clusters was above the level expected from Taq
polymerase error alone (33
). Hence, a fair estimate of quasispecies diversity was likely to have been obtained. Despite the fact that this patient was asymptomatic at the time of sampling, V3 loop sequences suggestive of the SI phenotype were evident in his proviruses. In contrast to the two patients above, however, the SI variants were distributed in both PBMC and NSMC populations (Fig. and ). Again, the SI-like cluster was phylogenetically distinct, forming the upper cluster of sequences with 93.5% bootstrap support in Fig. .
Nine HIV plasmid subclones from NSMC proviral DNA (064-S) and 12 subclones from PBMC (064-B) from asymptomatic patient 064 were generated and sequenced (Fig. and ). In this instance, however, no clear bimodal distribution was evident in the semen or blood (Fig. ). This patient was asymptomatic at sampling, and no mutations suggestive of the SI phenotype were found. No tight clustering of clonal outgrowths was noticed in the phylogenetic tree, and the pattern of variant representation was similar in the blood and semen.
The MA sample patient (22
) had been infected with HIV for approximately 7 years prior to sampling; he has remained generally asymptomatic with peripheral blood CD4+
T-cell counts in the range of 300 to 500/mm3
throughout the course of his infection. Fifteen PBMC variants sequenced after plasmid subcloning (MA-B300 series) sorted into two diverse sequence clusters, with two outlying variants (MA-B311 and -B312). His proviral load in the NSMC fraction of semen was quite low. Thus, to avoid resampling viral templates (34
), all 14 sequences derived from the NSMC were obtained by endpoint dilution and then PCR and direct sequencing (MA-SE series). Again, no clear bimodal distribution was evident in the semen or blood (Fig. ). Phylogenetic analysis revealed a distributed pattern of variant representation, with little evidence of substantial clonal outgrowths and no clustering in one or the other tissue evident (Fig. ). The deduced amino acid sequences of the V3 loop of each virus suggested an NSI phenotype (Fig. ). Thus, no distinct differences were found between provirus populations in his blood and semen.