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Phylogenetic analysis indicated that env sequences (C1-V2 region) for all clones from each patient clustered together along with the consensus sequence obtained for bulk envelope sequences amplified directly from plasma by RT-PCR (Fig. 1). Viruses with both R5 and X4 tropism (see below) were isolated from 4 patients. For patients 1 and 2, sequences for viruses with R5 tropism appeared to be phylogenetically distinct from those of viruses with X4 (patient 2) or dual (patient 1) tropism, and the sequence diversity among viruses with similar tropism was lower than that of the entire viral population. In contrast, for patients 4 and 5, envelope sequences in the V1-V2 region for viruses with R5 tropism were not segregated from those of viruses with X4 (patient 4) or dual (patient 5) tropism. For patient 2, the consensus env sequence for plasma viruses clustered with the sequences of viruses with X4 tropism at month 26, but clustered with the sequences of viruses with R5 tropism at month 34.
Figure 1. Phylogenetic tree of envelope sequences of clonal viruses. Shown is the neighbor-joining phylogenetic tree for nucleotide sequences coding the region of env encompassing C1 to V2 (corresponding to nucleotides 6440 – 6809 of HXB2) of the 70 clonal viruses evaluated in the study (open circles, strict R5 tropism, solid black circles, strict X4 tropism; solid gray circles, dual tropism), the consensus sequence of the same region for viral RNA amplified by RT-PCR from an aliquot of the plasma sample used to generate the clonal viruses (stars), and the sequence of the laboratory strain pNL4-3. Bootstrap values are also indicated. For patient 2, the consensus sequence of plasma viruses grouped with clonal viruses with X4 tropism at M26, and with clonal viruses with R5 tropism at M34.
The nucleotide diversity of env sequences extending from V1 to the middle of V4, calculated by the method of Tajima-Nei, ranged from 0.018 – 0.060, values typical of those obtained for sequences amplified from plasma by RT-PCR. The greatest diversity was observed for patient 3, despite that all viruses from this patient showed strict R5 tropism.
To explore the functional capacities of Env proteins expressed by these clonal viruses, we generated recombinant reporter viruses in which the env sequence (gp120 + the extracellular domain of gp41) was derived from the different clonal viruses, and evaluated the ability of these luciferase-expressing viruses to infect U373 cells stably expressing CD4 and either CCR5 or CXCR4 co-receptors. All of the 70 recombinant viruses were infectious (Fig. 2). Viruses with strict R5 tropism were identified in all patients (n = 53), but clones with R5X4 tropism (n = 5) and/or strict X4 tropism (n = 12) were also identified in 4 of the 5 patients studied. The infectivity of dual-tropic viruses tended to be similar in the U373-R5 and U373-X4 target cells (compare gray symbols in Figs. 2A and 2B). Without exception, the infectivity of clones with R5 tropism was at least 5-fold lower than the infectivity of recombinant viruses carrying the Env from the laboratory-adapted strain NL-AD8 [mean infectivity in U373-R5 cells: 22524 (RLU/sec)/(ng p24/ml)]. The infectivity of some clones with X4 tropism was equivalent to that observed for recombinant viruses carrying the Env from pNL4-3 [mean infectivity in U373-X4 cells: 2650 (RLU/sec)/(ng p24/ml)].
Figure 2. Infectivity and co-receptor usage of envelope proteins expressed by clonal viruses. Recombinant reporter viruses were generated in which the env sequence (gp120 + the extracellular domain of gp41) was derived from clonal viruses isolated from plasma of patients chronically infected with HIV-1. The ability of these viruses, which express Renilla luciferase in the place of Nef, to infect U373 cells stably expressing CD4 and either CCR5 (panel A) or CXCR4 (panel B) co-receptors was measured by evaluating luciferase expression in target cells 44 hours after infection. For patient 2, clonal viruses were obtained from plasma samples obtained 8 months apart (26 and 34 months after initial diagnosis). The ability of all recombinant viruses to infect the two cell types was evaluated, but results are shown only for viruses that induced significant luciferase activity in the indicated target cell type [>2 (RLU/sec)/(ng p24/ml)]. Viruses could be classified as having strict R5 tropism (open symbols) strict X4 tropism (solid black symbols) and dual tropism (sold gray symbols). Results for viruses expressing env sequences amplified by RT-PCR from patient plasma is also shown (stars). Each symbol is the mean of at least 3 independent experiments; the mean coefficient of variation for these results is as follows: U373-R5 cells, 42% (range 3 – 83%); U373-X4 cells, 50% (range 10 – 78%). For each patient, significant differences were found comparing the viruses with the highest and lowest infectivity (p < 0.05 – 0.001 by t-test).
Considerable variability was observed in the infectivity of viruses carrying different env sequences from the same patient. When U373-R5 cells were used as targets, the difference in infectivity between the most and least infectious viruses from each of the 5 patients averaged 1.0 ± 0.26 log10 (range 0.8 – 1.3 log10 difference). Some inter-patient variability in infectivity was also observed. When considered as a group, no significant differences in the infectivity of clones carrying Env proteins from patients 1–4 were observed, but the infectivity of clones from each of these patients was significantly greater than that of clones from patient 5 (p < 0.05 for all comparisons).
The intracellular portion of gp41 is known to interact with Gag. To avoid potential incompatibilities between these viral proteins [43,44], we initially evaluated the infectivity of recombinant viruses in which both the intracellular portion of gp41 and Gag were derived from the pNL4-3 viral strain. Changes in the intracellular domain of gp41, however, have also been reported to influence Env function [45-48], and it was important to evaluate the possibility that incompatibilities between the extracellular domains of Env in some of the viruses and the intracellular domain of gp41 from pNL4-3 contributed to the wide range of infectivities observed. To do so, we compared the infectivity of selected recombinant viruses in which the intracellular domain of gp41 was derived either from pNL4-3 or from the primary viral isolate. As shown in Fig. 3, the infectivities of the two constructs were strongly correlated (Spearman r = 0.86; p < 0.0001). Viruses that demonstrated relatively poor infectivity when the intracellular domain of gp41 was derived from pNL4-3 did not show improved infectivity when the homologous intracellular domain of gp41 was used (e.g., the viruses with Env from patient 5 and the least infectious virus from patient 4). Indeed, the use of the homologous intracellular domain of gp41 led to a moderate loss in infectivity for the viruses from patient 4 and some viruses from patient 5, consistent with the possibility that incompatibilities existed in these cases between the gp41 sequences and the gag protein from pNL4-3.
Figure 3. The effect of the origin of the intracellular portion of gp41 on infectivity. For selected clonal viruses isolated from patient 3 (inverted triangles), patient 4 (triangles), patient 5 (squares) or from the laboratory-adapted strain NL-AD8 (diamond), two types of recombinant reporter viruses were generated, one in which gp120 + only the extracellular domain of gp41 was derived from the clonal virus (pNL4-3-ΔectoENV-lucR, abscissa), and one in which gp120 and all of gp41 was derived from the clonal virus (pNL4-3-ΔENV-lucR vector, ordinate). The ability of these viruses to infect U373-R5 cells was compared by evaluating luciferase expression in the target cells 44 hours after infection. The infectivity of each pair of viruses was evaluated in three independent experiments, and each symbol represents the mean of these determinations. The dotted line is the line of identity. The correlation coefficient (Spearman) for the data shown is 0.86 (p < 0.0001).
For all 70 recombinant viruses, the amount of luciferase activity resulting from infection of U373 cells was greater than that seen after infection of MT4-R5 cells. For each patient, a significant correlation was observed between the infectivity of viruses with R5-exclusive tropism for U373-R5 and MT4-R5 target cells (p < 0.05 for all comparisons). As was observed when U373 cells were used as targets, viruses carrying different env sequences from the same patient showed considerable variability in infectivity when MT4-R5 cells were infected (Fig. 4A). The difference in infectivity between the most and least infectious R5 viruses from each of the 5 patients averaged 1.2 ± 0.31 log10 (range 0.8 – 1.5 log10 difference). As indicated above, the infectivity of viruses from patient 5 were strikingly lower than that of viruses from other patients when U373-R5 cells were used as targets. This difference was less striking when MT4-R5 cells were infected (compare Figs. 2A and 4A), although the infectivity of R5 viruses from patient 5 remained significantly lower than that of R5 viruses from patients 2 and 3 (p < 0.05 for both comparisons).
Figure 4. Infectivity of recombinant viruses carrying primary Env sequences in MT4-R5 cells. (A) Recombinant reporter viruses were generated as described in Fig. 1 legend, and the ability of these viruses to infect MT4-R5 cells was measured by evaluating luciferase expression in the target cells 44 hours after infection. For patient 2, clonal viruses were obtained from plasma samples obtained 8 months apart (26 and 34 months after initial diagnosis). The tropism of the recombinant viruses, as defined by their ability to infect U373-R5 and U373-X4 cells is shown: strict R5, open symbols; dual, solid gray symbols; strict X4, solid black symbols. Results for viruses expressing env sequences amplified by RT-PCR from patient plasma is also shown (stars). Each symbol is the mean of at least 3 independent experiments; the mean coefficient of variation for these results is 37% (range 1 – 78%). For each patient, significant differences were found comparing the viruses with the highest and lowest infectivity (p < 0.05 – 0.005 by t-test). (B) For each recombinant virus, the infectivity [(RLU/sec)/(ng p24/ml)] observed using U373 target cells and MT4-R5 target cells is expressed as a ratio.
Interestingly, considerable variability was observed when luciferase activity obtained following infection of the two cell types was expressed as a ratio (Fig. 4B). This ratio did not correlate with the infectivity of the viruses for U373 cells (Spearman r = 0.10, p = 0.49), but only viruses with relatively low infectivity for MT4-R5 cells [i.e., <110 (RLU/sec)/(ng p24/ml)] had values for this ratio that were greater than 20 (Fig. 5). The level of expression of both CD4 and CCR5 was approximately two-fold higher on U373-R5 cells than on MT4-R5 cells (Fig. 6). Thus, a possible explanation for these observations is that the infectivity of viruses for which a high ratio was observed are particularly sensitive to the levels of expression of CD4 and/or co-receptor, although other differences between U373-R5 cells and MT4-R5 cells may also influence the ability of viruses carrying different envelopes to infect these cell types.
Figure 5. Relationship between the infectivity of recombinant viruses bearing envelope proteins from plasma viruses for MT4-R5 cells and U373-R5 cells. Recombinant reporter viruses were generated as described in Fig. 1 legend, and the ability of these viruses to infect MT4-R5 cells and U373-R5 cells was measured by evaluating luciferase expression in the target cells 44 hours after infection. For each of the 53 viruses with strict R5 tropism, the infectivity ratio (infectivity for U373-R5 cells/infectivity for MT4-R5 cells) is expressed as a function of the infectivity for MT4-R5 cells [(RLU/sec)/(ng p24/ml)]. A significant inverse correlation between these parameters was observed (Spearman r = -0.64, p < 0.0001).
Figure 6. Expression of CD4 and CCR5 on U373-R5 cells and MT4-R5 cells. Cells were resuspended in PBS containing 20% human serum and incubated with directly-conjugated monoclonal antibodies or isotype-matched control antibodies conjugated with the same flurochrome. (A) CD4 expression on U373-R5 cells (thin solid line) and MT4-R5 cells (heavy solid line) detected using FITC-labelled mouse anti-human CD4 monoclonal antibody (clone RPA-T4). Binding of an isotype-matched control antibody conjugated with FITC is shown in the corresponding dashed lines. (B) CCR5 expression on U373-R5 cells (thin solid line) and MT4-R5 cells (heavy solid line) detected using phycoerythrin-conjugated mouse anti-human CCR5 monoclonal antibody (clone 2D7). Binding of an isotype-matched control antibody conjugated with phycoerythrin to these cells is shown in the corresponding dashed lines.
It is noteworthy that the proportion of viruses with a relatively high infectivity ratio was different in different patients. The infectivity ratios of clones from patients 1, 3 and 4 were significantly greater than that of patient 2 (p < 0.001, p < 0.001 and p < 0.05, respectively), and the infectivity ratio of clones from patient 3 was also significantly greater than that of patient 5 (p < 0.05).
In parallel with studies evaluating the infectivity of recombinant viruses carrying env sequences derived from clonal viruses, we evaluated the infectivity of recombinant viruses expressing env sequences amplified by RT-PCR from viral RNA extracted from the same plasma specimen from which the clonal viruses had been derived. The infectivity of viruses carrying plasma-derived env sequences for U373-R5 cells, although detectable in all cases except patient 2, was generally low, and was less than that of the clonal viruses from the same patient, usually by a substantial margin (Fig. 2A). The infectivity of viruses expressing plasma-derived env sequences for U373-X4 cells was detectable in only two samples (Fig. 2B). In both of these cases (patient 2, M26 and patient 4), clonal viruses with X4 exclusive tropism had been identified. The failure to detect infectivity of viruses carrying plasma-derived env sequences in other samples from which clonal viruses with strict X4 or dual tropism were identified may reflect the somewhat lower infectivity of the viruses with X4 tropism (e.g., patient 2, M34 and patient 5) and/or a lower proportion of viruses with X4 tropism in the sample (e.g., patient 1). As was observed for viruses carrying env sequences derived from clonal viruses, the infectivity of viruses carrying plasma-derived env sequences for MT4-R5 cells was usually reduced compared to that observed for U373 cells (Fig. 4A). Indeed, for patients 1 and 5, low level infectivity was detected toward U373-R5 cells, but infectivity was below detection when MT4-R5 cells were targeted.
Because the infectivity of clonal viruses carrying different env sequences from a given patient varied over a wide range, these viruses were useful in exploring the possible relationship between infectivity and sensitivity to inhibition by entry inhibitors. To do so, clonal viruses with R5-tropism that exhibited a spectrum of infectivities towards U373-R5 cells and/or MT4-R5 cells were selected from 4 of the patients (Figs. 7A and 7B). For each of these 20 viruses, the IC50s were determined on U373-R5 target cells for two entry inhibitors (enfuvirtide and TAK-799), soluble CD4, and neutralizing monoclonal antibodies recognizing either gp120 (2G12, 48d) or gp41 (2F5). No significant correlations were observed between the IC50s of these six inhibitors and the infectivity of the clonal viruses for either U373-R5 cells or MT4-R5 cells (data not shown, p > 0.09 for all Spearman correlations).
Figure 7. Sensitivity of recombinant viruses carrying primary envelope sequences to entry inhibitors. Clonal viruses with R5-tropism exhibiting a spectrum of infectivities towards U373-R5 cells (panel A) and/or MT4-R5 cells (panel B) were selected from among those obtained from patients 1–4. For each of these 20 viruses, the IC50s were determined on U373-R5 cells for soluble CD4 (C), enfuvirtide (E), TAK-799 (G), and neutralizing monoclonal antibodies 2F5 (D), 2G12 (F), and 48d (H). Each symbol represents the mean of three independent determinations. For all inhibitors except TAK-799, significant patient-specific differences in IC50 were observed using the Kruskal-Wallis test. Brackets indicate significant pairwise differences in post-test comparisons performed using Dunn's multiple comparison test (* p < 0.05; ** p < 0.01). For patient 2, clonal viruses were obtained from plasma samples obtained at both 26 months (solid symbols) and 34 months (open symbols) after initial diagnosis.
Viruses from different patients did, however, demonstrate differential sensitivity to these entry inhibitors, independent of infectivity (Fig. 7). Thus, significant differences in the median sensitivity to entry inhibitors by clonal viruses from different patients was observed by ANOVA for all entry inhibitors except TAK-799 (p values for Kruskal-Wallis test: p < 0.001 – 0.05), and for each of these inhibitors, significant differences in were also identified in pair-wise comparisons of IC50s for viruses from different patients (Fig. 7).
Nucleotide sequences for env extending from the signal peptide to mid C4 region were available for all clones. The PSSM score developed by Jensen et al.  correctly distinguished all clones with R5-exclusive tropism and X4-exclusive tropism, even though viruses from two of the patients were non-B subtypes. Three of the 6 Env proteins with dual tropism, however, were predicted to exhibit R5-exclusive tropism (one clone each from patients 1, 2 and 5). The amino acid sequence of the V3 region of these dual-tropic clones from patients 1 and 2 differed at 6 and 1 positions, respectively, compared to that of the most similar R5-tropic Env identified in that patient, and these differences may explain the change in tropism. The V3 region of the misidentified dual-tropic envelope sequence from patient 5, however, was identical to that of other Env with R5-exclusive tropism, indicating that sequences outside V3 influenced the tropism of this Env.
In general, viruses with strict R5-tropism from the same individual expressed a relatively small number of haplotypes within a given variable (V) region. For example, the number of distinct haplotypes identified for the V3 region ranged from 1 (patient 1) to 4 (patient 2). Significant differences in the infectivity of R5-tropic viruses as a function of haplotype were observed for the variable regions 2 and 3 (V2 and V3) of patient 2 (p < 0.02 and p < 0.03 respectively using the Kruskal-Wallis test). As shown in Fig. 8, the viruses from this patient expressing V3 region haplotypes 2 and 3 were significantly less infectious than those expressing haplotype 1 (p < 0.01 using the Mann-Whitney test), and viruses expressing the V2 region haplotype 4 were less infectious than those expressing the V2 region haplotypes 1 – 3 (p < 0.001). Most of the viruses expressing the V2 haplotype associated with low infectivity (haplotype 4) also expressed V3 haplotypes associated with low infectivity. However, one of the viruses expressed this V2 haplotype in association with the V3 haplotype 1, which was usually associated with good infectivity (red arrow in Fig. 8A). The infectivity of this virus was, nevertheless, low (red arrow in Fig. 8B), suggesting that expression of the V2 haplotype 4 was a major determinant for low infectivity in this patient. It is noteworthy that clones from patient 2 expressing the V2 haplotype 4 were obtained only from the plasma sample obtained at month 34, and 9/13 clones with R5-tropism isolated at this time point expressed this V2 haplotype.
Figure 8. Expression of V2 and V3 region haplotypes in Env proteins of clonal viruses from patient 2 and viral infectivity. (A) The haplotypes in the V2 region (left) and V3 region (right) of the Env proteins expressed by the 17 clonal viruses from patient 2 with strict R5 tropism are shown. The consensus amino sequence is shown on the top line, and only amio acids differing from the consensus sequence are shown for each clone. For each variable region, sequences that are identical or that differ by a single amino acid substitution not identified in another sequence are highlighted by the same color, and these haplotypes are also identifed by numbers adjacent to the brackets. The red arrow indicates the envelope expressing the V2 region haplotype 4 associated with the V3 region haplotype 1 (see text). (B) The infectivity of recombinant viruses expressing these Env proteins using U373-R5 cells is shown. For each of the variable regions, the color of the symbols corresponds to that of the V region haplotype expressed by that virus. The red arrow indicates the infectivity of the clone expressing the V2 region haplotype 4 associated with the V3 region haplotype 1.
No significant associations between infectivity and haplotype were observed for the variable regions expressed by the other patients, and no association between infectivity and the haplotypes in the constant regions were identified. One factor that could confound such analyses is the presence of deleterious mutations elsewhere in the envelope sequence. In this regard, we found that four of the 53 viruses with strict R5 tropism had V3 sequences containing a single amino acid polymorphism not seen in any other sequence from that patient. The recombinant viruses carrying 3 of these unique V3 polymorphisms had the lowest infectivity for U373-R5 cells of any virus evaluated from that sample.
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