Sequence analysis of envelope genes amplified from patient brain and LN tissue samples.
We first sequenced the variable V1V2 loops of each envelope clone obtained to identify individual genotypes and to omit replicate clones from further study. A total of 19 distinct V1V2 genotypes were identified from the five patients (Table ). V1V2 sequences were distinct for each patient. NA118 and NA420 sequences are also closely related with previously published envelope sequences from these individuals (17
). For NA420, brain and LN V1V2 sequences were clearly distinct genotypes consistent with tissue-specific evolution. For NA118, brain and LN V1V2 sequences revealed distinct genotypes; however, specific compartmentalization was less clear, as previously reported for this individual (17
V1V2 loop amino acid sequences of patient envelopes
V3 loop charge.
The overall positive charge of the V3 loop has been used as an important indicator of HIV-1 tropism as either syncytium inducing or non-syncytium inducing (12
). Syncytium-inducing and non-syncytium-inducing phenotypes are now known to represent viruses that use CXCR4 or are predominantly CCR5 using, respectively (2
). All V3 loop sequences carried an overall charge of 3 or 4, consistent with a CCR5-using phenotype (Table ).
V3 loop amino acid sequences of patient envelopes
Infectivity and coreceptor use of pseudotype viruses carrying LN- and brain-derived envelopes.
Pseudotype viruses were prepared by cotransfecting 293T cells with env+ pSVIIIenv together with env− pNL43. Pseudotype viruses were harvested after 48 h, and their infectivity was analyzed for the GHOST and NP2/CD4 series of coreceptor-expressing cell lines. Infectivity data for envelope-positive pseudotype viruses are shown in Table . Thirteen of the 19 distinct envelope genotypes yielded pseudotypes with infectivity for both GHOST/CCR5 and NP2/CD4/CCR5 cells. Infectivity of these pseudotypes was comparable to pseudotypes carrying well-characterized HIV-1 envelopes, e.g., SF162. Some envelopes (NA20 B76, NA353 B13, NA118 B18, NA118 B44, NA118 LN9, and NA420 B3) failed to yield infectious pseudotype viruses, while others consistently yielded pseudotypes with lower levels of infectivity (NA118 B45, NA176 B72, NA420 LN85, and NA420 LN40). Three distinct phenotypes of coreceptor use were observed. First, some envelopes, e.g., NA420 brain envelopes B33, B42, and B13, were mainly restricted for CCR5 use and unable to infect cells expressing other coreceptors. Second, the majority of functional envelopes consistently used CCR3 in addition to CCR5 but did not use other coreceptors. Third, a single envelope genotype from NA20 (B59) showed a broader use of coreceptors, including CCR3, CCR8, GPR1, GPR15, CXCR6, and CCR5 (Table ).
Summary of macrophage syncytia induction by patient and control envelopes
Cell-cell fusion induced by patient envelopes.
The capacity of the different patient-derived envelopes to induce cell-cell fusion was evaluated. 293T cells were cotransfected with the pNL43 env− plasmid together with each patient env+ pSVIIIenv construct. After overnight incubation, the transfected 293T cells were trypsinized and cocultivated with different CD4+, coreceptor-positive cell lines (the GHOST and NP2/CD4 series of coreceptor-expressing cell lines). After incubations between 4 h and overnight, cocultivations were fixed and stained as described in Materials and Methods. Typical spreading syncytia were observed in cocultivations (Fig. ). Syncytium induction assays confirmed the coreceptor use detected by infectivity assays described above. For example, envelopes that conferred infection of NP2/CD4/CCR3 also induced cell-cell fusion with this cell line (data not shown). The broad coreceptor use by NA20 envelope B59 was also confirmed by induction of syncytia via CCR5, CCR3, CCR8, GPR1, and very weakly via CXCR4 (but not via APJ) on NP2/CD4 cells and via GPR15 and CXCR6 on GHOST cells (Fig. ). Cell-cell fusion was not detected for any other patient envelope via CXCR4 expressed either on NP2/CD4 or on GHOST cells. However, these CXCR4+ cells supported extensive syncytium formation when cocultivated with 293T cells expressing the X4 envelope of NL43 (data not shown).
Coreceptor use of NA20 B59 envelope evaluated by cell-cell fusion assays. NA20 B59-induced syncytia in GHOST and NP2/CD4 cells expressing a range of different coreceptors but not in the parental cell lines lacking coreceptors.
Patient envelopes that failed to yield infectious pseudotypes also failed to induce cell-cell fusion. This result suggests that these envelopes carry a defect(s) that eliminates their capacity to function in these assays, as reported for other envelopes PCR amplified from patient tissue (28
). This conclusion is supported by the NA20 B76 envelope, where a single premature stop codon, identified in the extracellular domain of gp41 (data not shown), precluded the production of a full-length functional envelope. Repair of this stop codon restored full function to the NA20 B76 envelope. The repaired NA20 B76 was shown to be an R3R5 envelope similar to the majority of patient envelopes described here but distinct from the broadly tropic B59 envelope also from NA20 (Table ).
Two patient envelopes, NA420 LN40 and NA420 LN85, consistently yielded pseudotypes that carried only low-level infectivity for GHOST/CCR5 and NP2/CD4/CCR5 cells. Interestingly, these envelopes were fully functional for cell-cell fusion and induced syncytia formation on both GHOST/CCR5 and NP2/CD4/CCR5 cells. Moreover, both low-infectivity envelopes conferred cell-cell fusion in NP2/CD4/CCR3 cells, confirming an R5R3 phenotype. Both NA420 LN40 and NA420 LN85 envelopes contained mutations in the gp41 cytoplasmic domain, mutations that severely reduced their incorporation onto budding virions (data not shown). These envelopes were also restored to full infectivity by preparing chimeric constructs that contained gp120 sequences from either NA420 LN40 or NA420 LN85 and gp41 sequence from NA420 B33.
Infection and cell-cell fusion of primary macrophage cultures.
Both Gorry's UK1-br brain isolate (16
) and Shieh's microglia-passaged BORI variant (39
) were reported to be more infectious and fusigenic for microglial cells and macrophages. We therefore compared the capacity of the patient-derived envelopes to confer infectivity and syncytium induction in primary macrophage cultures. Patient env+
pseudotypes with high infectivity titers on CD4+
cell lines also infected primary macrophages (Fig. ). Ratios of patient env+
pseudotype infectivity for macrophages compared to those for GHOST/CCR5 are shown in Fig. . These results indicate that four brain envelopes infected primary macrophages efficiently, while other patient envelopes conferred less-efficient or no infection. We also tested envelopes of several well-characterized HIV-1 strains including macrophage-tropic (SF162 and AD8) and brain- or cerebrospinal fluid-derived (YU2, JRFL, and JRCSF) viruses. These envelopes were cloned from infectious molecular clones into pSVIIIenv. Four of these (YU2, JRFL, SF162, and AD8) conferred infection of primary macrophage cultures. However, at least two of the brain-derived patient envelopes (NA20 B59 and NA353 B27) conferred even higher macrophage-GHOST/CCR5 infectivity ratios, indicating that these envelopes carry an enhanced macrophage tropism. Both R5 and R5R3 patient envelopes segregated with macrophage-tropic and non-macrophage-tropic envelopes, and thus no obvious association was observed between CCR3 use and macrophage infection.
FIG. 2. Infection of primary macrophages by patient LN and brain envelopes. (A) Infectivity titers for patient envelope-positive pseudotype viruses titrated on primary macrophages from different donors and on GHOST/CCR5 cells. Arrows indicate that infection is (more ...)
NA118 B12, LN27, and LN33 envelopes failed to confer macrophage infection, even though they conferred high infectivity for GHOST/CCR5. The three NA420 brain envelopes conferred macrophage infection with NA420 B33 infecting most efficiently of the three. In contrast, both repaired NA420 LN envelopes failed to confer any macrophage infection. Taken together, these results show that all LN-derived envelopes were non-macrophage tropic, while most brain-derived envelopes (all except NA118 B12) conferred macrophage infection (Fig. ).
We next evaluated the capacity of patient envelopes to induce cell-cell fusion in primary macrophage cultures. These fusion assays were carried out by cocultivation of primary macrophage cultures with 293T cells cotransfected with patient env+ pSVIIIenv and env− pNL43 vectors as described in Materials and Methods. A minority of envelopes induced very large and spreading macrophage syncytia containing numerous nuclei (Fig. ). The same four patient envelopes that efficiently infected macrophages consistently induced large spreading syncytia over 2 to 3 days of coculture. The extent of macrophage syncytia induction was donor dependent. Of the well-characterized control envelopes (YU2, JRCSF, JRFL, SF162, and AD8), only AD8 conferred levels of macrophage syncytia similar to the patient brain envelopes described above. Macrophage syncytium induction is summarized in Table .
FIG. 3. Syncytium induction in primary macrophages by patient brain-derived envelopes. Large spreading syncytia induced by patient brain envelopes NA176 B93 and NA353 B27 are shown. Note that the syncytia shown contain hundreds of nuclei (top panels). No syncytia (more ...)
Summary of macrophage syncytia induction by patient and control envelopes
Brain-derived HIV-1 envelopes are not CD4 independent.
Several studies have correlated SIV macrophage tropism with the capacity to infect CCR5+
cell lines in vitro in the absence of CD4 (24
). We therefore tested whether pseudotype viruses carrying patient envelopes could infect CD4−
NP2/CCR5 cells. We also tested a primary HIV-2 strain (ALI) that was previously shown to infect CCR5+
cells in the absence of CD4 (33
). In contrast to HIV-2 ALI, none of the brain or LN envelopes conferred any infection of CD4−
NP2/CCR5 cells (data not shown), even though many envelopes conferred high levels of infection on NP2/CD4/CCR5 cells (Table ). It was previously shown that pretreatment of HIV-2 with soluble CD4 induced infection of CD4−
). However, soluble CD4 (1 μg/ml) had no effect on the capacity of patient envelopes to confer infection or cell-cell fusion of CD4−
NP2/CCR5 cells (data not shown). Together, these observations show that all the patient envelopes investigated here, whether from LN or brain, were completely dependent on cell surface CD4 for infection and cell-cell fusion.
Some brain envelopes can infect cells expressing low levels of CD4 and/or CCR5.
Although, none of the patient envelopes studied here were CD4 independent, it was possible that brain envelopes required less cell surface CD4 to trigger infection as described by Gorry et al. (16
) and Martin et al. (23
). We therefore tested the capacity of pseudotype viruses carrying brain and LN envelopes to infect HeLa cells expressing either high or low levels of CD4 and three different levels of CCR5 (30
). Platt et al. reported that the high CD4 clones express approximately 4 × 105
molecules/cell and the low CD4 clones express approximately 104
receptors/cell. Low, medium, and high CCR5 clones were reported to express approximately 2 × 103
, and 105
molecules/cell, respectively. Immunostaining and fluorescence-activated cell sorter analysis confirmed that these different clones expressed low or high CD4 and low, medium, or high CCR5 amounts on their cell surfaces.
All envelopes tested were capable of infecting HeLa cells expressing high levels of CD4 and different levels of CCR5 (Fig. ). Infection of HeLa/HiCD4 cells with the lowest CCR5 was within 1 log of maximum infectivity recorded in the presence of the highest CCR5 levels. In contrast, infectivity recorded on HeLa cells with low levels of CD4 (HeLa/LowCD4) showed that only particular envelopes were able to infect these cells efficiently (Fig. ). These included the four brain-derived envelopes that scored highest for macrophage fusion and infection. Infection by patient NA118 envelopes (B12, LN27, and LN33) was severely affected by CD4 expression. NA420 brain and LN envelopes clearly segregated into separate phenotypes with NA420 B33, B13, and B42 envelopes conferring infection of HeLa/LowCD4 relatively efficiently, while infection by the repaired NA420 LN envelopes was below the level of detection on cells expressing low levels of CD4. JRCSF showed a CD4 requirement similar to those of the NA118 and NA420 LN envelopes. Several of the control macrophage-tropic HIV-1 envelopes (AD8 as well as brain tissue-derived YU2 and JRFL) also infected in the presence of low CD4 and low CCR5. These envelopes were as efficient on the HeLa/LowCD4/CCR5 cultures as were patient brain envelopes NA20 B59 and NA353 B27.
FIG. 4. Infection of cells expressing low levels of CD4 and/or CCR5 by patient envelopes. Infection of HeLa/HiCD4 cells expressing high levels of CD4 and three different amounts of CCR5 (top panels). Infection of HeLa/LowCD4 expressing low levels of CD4 and three (more ...)
Dejucq et al. previously reported that particular R5 HIV-1 isolates were able to infect the CD4+
T-cell line MOLT 4. Although these cells express high levels of CD4, CCR5 expression was undetectable by flow cytometry and only weakly detectable by reverse transcription-PCR (7
). Nevertheless, MOLT 4 infection by R5 isolates was completely inhibited by CCR5-specific ligands (7
). Here, we tested whether the patient envelopes could infect or induce cell-cell fusion of MOLT 4 cells. The four macrophage-tropic brain-derived envelopes conferred infection of MOLT 4 cells (data not shown) and were able to induce the formation of large syncytia (Fig. ). Syncytium formation by NA20 B59 and by NA176 B93 was completely blocked by TAK779 and unaffected by a range of ligands for other potential coreceptors, including AMD3100, the ligand for CXCR4 (data not shown). Several other patient envelopes induced low levels of small syncytia. Of the control envelopes, only AD8 and YU2 consistently induced low levels of MOLT 4 syncytia.
FIG. 5. Cell-to-cell fusion induced by patient envelopes in MOLT 4 cells that express very low levels of CCR5. 293T cells expressing different patient envelopes were cocultivated with MOLT 4 cells. The presence of syncytia was assessed by light microscopy after (more ...)
Together these results show that four brain-derived envelopes were capable of exploiting low levels of CD4 and/or CCR5 for infection and cell-cell fusion of macrophages and T cells.
Sensitivity of patient envelopes to inhibition by ligands to CD4 and CCR5.
We next tested whether the distinct phenotypes of the patient envelopes impacted on their sensitivity to inhibition by reagents that blocked either CD4 or CCR5. We first tested the blockade of CCR5 by TAK779. GHOST/CCR5 cells were treated with twofold dilutions of TAK779 for 1 h before challenging with 100 to 200 FFU of patient envelope-positive pseudotype viruses as described in Materials and Methods. Inhibition was assessed as 90% inhibitory concentrations (IC90s) (Fig. ). A range of IC90s was evident and no clear association of TAK779 sensitivity and envelope phenotype could be seen (Fig. ). IC90s were also obtained for envelope-positive pseudotype inhibition by Q4120 (Fig. ). Q4120 is a monoclonal antibody that recognizes the N-terminal domain of CD4 and blocks gp120 binding and infectivity. In contrast to TAK779, Q4120 IC90s showed a strong correlation with patient envelope phenotype. The four brain-derived envelopes that showed enhanced fusigenicity and tropism for macrophages and MOLT 4 cells required the highest doses of Q4120 for inhibition. These results are consistent with an increase in envelope affinity for CD4, conferring an adaptation to infect cells expressing low levels of CD4. Of note, all four LN envelopes clustered along with JRCSF as the most sensitive to Q4120 inhibition. These results are consistent with the hypothesis that envelopes that evolve a higher affinity for CD4 will have an increased tropism for cells expressing low levels of CD4 and/or CCR5, including both macrophages and T cells.
FIG. 6. Sensitivity of patient envelopes to CCR5 and CD4 ligands. (A) TAK779 (CCR5 inhibitor) IC090 0 inhibitory doses. (B) Q4120 (anti-CD4 domain 1) IC090 0 inhibitory doses. Black symbols represent macrophage-tropic patient envelopes. Gray symbols are other (more ...)