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Journal of Virology, June 2008, p. 5584-5593, Vol. 82, No. 11
0022-538X/08/$08.00+0     doi:10.1128/JVI.02676-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Coreceptor Tropism Can Be Influenced by Amino Acid Substitutions in the gp41 Transmembrane Subunit of Human Immunodeficiency Virus Type 1 Envelope Protein

Wei Huang,^* Jonathan Toma, Signe Fransen, Eric Stawiski, Jacqueline D. Reeves, Jeannette M. Whitcomb, Neil Parkin, and Christos J. Petropoulos

Monogram Biosciences, South San Francisco, California

Received 17 December 2007/ Accepted 11 March 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Many studies have demonstrated that the third variable region (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is a major determinant of coreceptor tropism. Other regions in the surface gp120 subunit of Env can modulate coreceptor tropism in a manner that is not fully understood. In this study, we evaluated the effect of env determinants outside of V3 on coreceptor usage through the analysis of (i) patient-derived env clones that differ in coreceptor tropism, (ii) chimeric env sequences, and (iii) site-directed mutants. The introduction of distinct V3 sequences from CXCR4-using clones into an R5-tropic env backbone conferred the inefficient use of CXCR4 in some but not all cases. Conversely, in many cases, X4- and dual-tropic env backbones containing the V3 sequences of R5-tropic clones retained the ability to use CXCR4, suggesting that sequences outside of the V3 regions of these CXCR4-using clones were responsible for CXCR4 use. The determinants of CXCR4 use in a set of dual-tropic env sequences with V3 sequences identical to those of R5-tropic clones mapped to the gp41 transmembrane (TM) subunit. In one case, a single-amino-acid substitution in the fusion peptide of TM was able to confer CXCR4 use; however, TM substitutions associated with CXCR4 use varied among different env sequences. These results demonstrate that sequences in TM can modulate coreceptor specificity and that env sequences other than that of V3 may facilitate efficient CXCR4-mediated entry. We hypothesize that the latter plays an important role in the transition from CCR5 to CXCR4 coreceptor use.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Human immunodeficiency virus type 1 (HIV-1) entry is mediated by a heterotrimeric envelope (Env) glycoprotein comprised of two subunits, the gp120 surface (SU) subunit, which binds CD4 and coreceptors, and the gp41 transmembrane (TM) subunit, which mediates membrane fusion (16). The major coreceptors for HIV-1 infection in vivo are CCR5 and CXCR4 (2). Based on coreceptor usage in vitro, the majority of HIV-1 variants can be classified as those that use CCR5 (R5 tropic), those that can use CCR5 and CXCR4 (R5X4 or dual tropic), and, much less frequently, those that use CXCR4 (X4 tropic). R5-tropic variants predominate in acute and early infections (2). Individuals that are homozygous for a defective ccr5 gene (32 ccr5) essentially are protected from HIV-1 infection, while individuals heterozygous for the 32 ccr5 mutation have slower disease progression (11, 30, 37, 43, 58). CXCR4-using (either X4-tropic or dual-tropic) variants tend to emerge in late disease and are present in approximately 50% of infected patients with advanced disease (2, 9, 31). CXCR4-using viruses are associated with more rapid CD4⁺ T-cell losses than R5-tropic viruses (2, 4, 13, 31, 32, 55), as well as having a poorer prognosis for survival (10, 27). The mechanism(s) underlying the emergence of CXCR4-using variants is not well defined. CCR5 is present on cells of the monocyte/macrophage lineage and primary T cells, whereas CXCR4 is more widely expressed, including various CD4⁺ T-cell subsets, macrophages, and other cell lines (7). It has been suggested that transitions from CCR5 use to CXCR4 use allow the virus to replicate in expanded target cell populations.

In the multiple-step process of HIV-1 Env-mediated entry, the binding of SU to the CD4 receptor creates and/or exposes a coreceptor binding site (16). The engagement of SU with a coreceptor eventually triggers conformational changes in the TM that lead to the fusion of the virus and host cell membranes (16). Previous studies have demonstrated that the V3 loop of SU contains strong determinants of coreceptor specificity and tropism (8, 22, 24, 25, 34, 45, 46, 48, 49, 53, 56). Increased numbers of basic residues and fewer potential N-linked glycosylation sites in V3 have been associated with CXCR4 use, and certain substitutions in the V3 loop can be used to estimate coreceptor tropism (12, 18, 21, 26, 38). The exchange of V3 loop sequences between some X4- and R5-tropic env sequences has provided evidence that V3 can be necessary and sufficient for the determination of coreceptor tropism (25). In addition to V3, other regions in SU have been implicated in CXCR4 usage, including the V1/V2 region (5, 29, 33, 35, 36). However, the determinants of coreceptor usage in env regions outside of the V3 loop remain poorly defined and may be context dependent.

HIV-1 populations in plasma are comprised of closely related but not identical variants, and these variants can display dramatic differences in coreceptor preference. The analysis of env function and sequence in these variants can facilitate the identification of genetic determinants of coreceptor tropism. In this study, we explored these differences to assess the impact of V3 and non-V3 regions on coreceptor usage by interchanging V3 regions between closely related CXCR4-using and R5-tropic clones. We observed that patient env sequences may share identical V3 loop sequences but differ in coreceptor tropism (i.e., R5 or dual tropic). The determinants of CXCR4 use by dual-tropic clones with R5-like V3 sequences mapped to TM, providing a dramatic demonstration that regions outside of the coreceptor binding site and outside of SU can contribute to coreceptor use.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Isolation of env clones from patient plasma. Full-length env genes (gp160) were amplified by reverse transcription-PCR from the plasma of an individual infected with subtype B HIV-1. env genes were cloned into an expression vector, generating a patient env expression vector library as previously described (54). Twenty-two functional env clones were selected from the env expression vector library based on their ability to infect U87 cells expressing CD4 and either the CCR5 or CXCR4 coreceptor.

Sequence analysis of patient HIV-1 env clones. The nucleotide sequences of patient-derived env clones were determined using conventional dideoxy chain-terminator chemistry (ABI, Foster City, CA). Phylogenetic analysis was performed using neighbor-joining methods as implemented in MEGA, version 3.1 (28); topology support was estimated with a 1,000-replicate bootstrap resampling of the data.

Construction of env chimeras and site-directed mutants. The megaprimer method of site-directed mutagenesis (44) was used to construct a series of chimeric env sequences from parental R5-, X4-, and dual-tropic clones selected from the patient's viral population. Chimeric clones were generated by exchanging V3 loop sequences from clones of a particular tropism with those of another. To map determinants of CXCR4 use located outside of the V3 region, chimeric env sequences were generated by exchanging SU and TM regions, as well as different regions within TM. Single and multiple substitutions at positions 515, 529, and/or 607 were introduced into the TM region of an R5-tropic clone by site-directed mutagenesis. The entire gp160 env sequence of all chimeric and mutant env genes was confirmed to ensure that only the desired substitutions were present.

Coreceptor tropism and sensitivity to entry inhibitors. The coreceptor tropism of patient env clones, chimeras, and site-directed mutants was determined using the Trofile HIV coreceptor tropism assay (Monogram Biosciences) (54). Briefly, pseudotyped HIV-1 viruses were generated by the cotransfection of HEK293 cells with various patient virus-derived env expression vectors and a proviral vector containing a luciferase reporter gene in place of env. Coreceptor tropism was evaluated by the parallel infection of U87 cells expressing CD4 and either CXCR4 or CCR5. Coreceptor specificity was determined by inhibiting infection with CXCR4 or CCR5 inhibitors. Drug susceptibility also was evaluated by using the PhenoSense HIV Entry assay (Monogram Biosciences) (52). The susceptibility of pseudotyped viruses to a fusion inhibitor (enfuvirtide [ENF]; Roche/Trimeris), a CCR5 antagonist (Merck), and a CXCR4 antagonist (AMD3100; AnorMed) was evaluated using serial dilutions of the test inhibitor.

Cell-cell fusion assay. For the cell-cell fusion assay, HEK293 effector cells were transfected with patient-derived env expression vectors and an env-defective HIV genomic vector. After 2 days, effector cells were washed and cocultured overnight with CEM 5.25.Luc4.M7 target cells (provided by Nathan Landau, Smilow Research Center, New York University School of Medicine), which express the CD4 receptor and CCR5 and CXCR4 coreceptors and contain tat-inducible luciferase and green fluorescent protein reporter genes under the control of the HIV-1 long terminal repeat. The nonnucleoside reverse transcriptase inhibitor efavirenz (0.25 µM) was added to cocultured cells to prevent target cell infection. env-mediated cell-cell fusion was measured by assaying luciferase activity in cell lysates and by the microscopic visualization of green fluorescent protein expression in the coculture.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
R5- and dual-tropic env clones can have identical V3 loop sequences. Coreceptor usage, viral infectivity (luciferase activity), and V3 amino acid sequences of 22 clones derived from a single patient are listed in Table 1. Eight clones were R5 tropic, one clone was X4 tropic, and the remaining 13 clones were dual tropic. All R5-tropic clones had identical V3 amino acid sequences. The V3 sequence of the X4-tropic clone differed from the V3 sequences of R5-tropic clones at nine amino acid positions, including a deletion at position 16 and eight amino acid substitutions (i.e., N7S, S11G, T13L, M14V, G17T, A19R, G24R, E25N, numbered from the start of the V3 loop). The 13 dual-tropic clones were classified as dual R tropic (dual-R) (n = 5) or dual X tropic (dual-X) (n = 8) based on their V3 amino acid sequences and their ability to infect CXCR4 and CCR5 target cells, as previously described (23) (Table 1). Specifically, the five dual-R clones had V3 loop sequences that were identical to the V3 sequences of the eight R5-tropic clones, while the eight dual-X clones had V3 sequences that closely resembled the V3 sequence of the X4-tropic clone (i.e., T13H, G17H, A19R, G24K, E25N, and the position 16 deletion); clone 11D contained an additional R3G substitution. In general, the five dual-R clones exhibited much lower infectivity as determined by luciferase activity (in relative light units [RLU]) on CXCR4-expressing cells (10² to 10⁴ RLU) than dual-X or X4-tropic clones (10⁴ to 10⁵ RLU). Dual-R clones were equivalent to the R5-tropic clones in their ability to infect CCR5-expressing cells (10⁵ to 10⁶ RLU), while dual-X clones displayed lower infectivity (10³ to 10⁴ RLU) on CCR5 target cells (Table 1). In contrast to the phenotype determinations, all 22 clones were designated R5-tropic based on the 11KR/25KR rule (12, 18, 21) and position-specific scoring matrix (26) genotypic predictive algorithms (data not shown). Taken together, these data suggest that a sequence(s) outside of the V3 loop in the five dual-R clones is responsible for the ability to enter cells via the CXCR4 coreceptor.

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TABLE 1. Coreceptor tropism and V3 sequences of env clones

Dual-R clones are genetically distinct from dual-X and X4-tropic clones. A phylogenetic analysis of full-length (gp160) sequences of the 22 env clones was performed (Fig. 1). The alignment of these sequences with representative env sequences from group M HIV-1 subtypes indicated that all clones in this study were contiguous subtype B sequences (data not shown). Overall, the sequences of dual-R clones clustered with the R5-tropic clones and were genetically distinct from dual-X and X4-tropic clones in a neighbor-joining tree. These results are consistent with our previous observations for subtype D viruses (23). Among the five dual-R clones, clone 86d exhibited the highest infectivity in CXCR4-expressing cells, was genetically more distant from other dual-R and R5-tropic clones, and was closer to dual-X and X4-tropic clones in a neighbor-joining tree. This observation suggested that clone 86d has some env sequence features in common with dual-X and X4-tropic clones outside of the V3 loop, which may confer more efficient CXCR4 use.

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FIG. 1. X4-tropic and dual-X clones are genetically distinct from R5-tropic and dual-R clones. A neighbor-joining tree was generated using MEGA (28) from an alignment of gp160 nucleotide sequences of molecular clones derived from the patient virus population. The numbers at the nodes are bootstrap values greater than 50% determined from 1,000 replicate trials. Different colors indicate the coreceptor tropism of individual clones. R5-tropic clones are shown in red, dual-R clones are shown in light blue, dual-X clones are shown in purple, and the X4-tropic clone is shown in green. The clones designated by an asterisk were selected for additional analysis.

env sequences outside of V3 influence coreceptor tropism. To better understand the impact of various regions of env on coreceptor usage, we selected 6 of the 22 clones for further investigation based upon coreceptor tropism, V3 sequences, and phylogenetics: an R5-tropic clone (10R), the X4-tropic clone (42X), two dual-X clones (11D and 88D), and two dual-R clones (21d and 86d). Differences in SU (excluding V3) and TM amino acid sequences among these six clones are shown in Table 2. Compared to the amino acid sequence of the R5-tropic clone 10R, the X4-tropic clone 42X had three additional positively charged residues (lysine or arginine) in SU. All six clones contained identical patterns of potential N-linked glycosylation sites in gp160, except for the X4-tropic clone 42X, which lacked one N-linked glycosylation site immediately upstream of the V3 loop due to an N295K substitution. The same clone also contained a single-amino-acid insertion in the V1 region. Compared to the sequence of the R5-tropic clone 10R, the CXCR4-using clones contained 11 to 30 amino acid differences outside of the V3 region. The dual-R clone with a weak ability to infect CXCR4-expressing cells (21d) contained the least number of substitutions (n = 11), while a dual-R clone with higher infectivity in CXCR4-expressing cells (86d) had a similar number of substitutions (n = 24) compared to those of the two dual-X clones, 11D and 88D (n = 17 and 21, respectively). The X4-tropic clone (42X) had the most substitutions (n = 30). Similar numbers of amino acid substitutions were observed in both SU and TM for two dual-R clones, while SU of the dual-X and X4-tropic clones contained more amino acid substitutions than TM. The dual-R clone 86d had six amino acid residues in SU and seven amino acid residues in TM, which are identical to those of the dual-X clone(s) but different from that of R5-tropic clones (Table 2).

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TABLE 2. Comparison of amino acid differences of selected env clones with different coreceptor tropism (V3 excluded)

To evaluate the impact of V3 and non-V3 env regions on CXCR4 use, we constructed a panel of chimeric env clones by exchanging the V3 regions between the R5-tropic clone 10R and three other CXCR4-using clones that had different V3 sequences: 11D, 88D, and 42X. The infectivity of these chimeric and parental clones in CCR5- and CXCR4-expressing cells, as well as the amino acid sequences of their V3 loops, is shown in Fig. 2. The three chimeras that contained the V3 region from R5-tropic clone 10R in the context of either dual-X or X4-tropic env backbones retained some level of CXCR4-mediated entry, although the ability to infect CXCR4-positive cells was reduced compared to that of the parental CXCR4-using envelopes. These data indicate that non-V3 determinants of CXCR4 use were present in the parental CXCR4-using clones 11D, 88D, and 42X. Each of these chimeras was able to infect CCR5-expressing cells well compared to the R5-tropic clone 10R (Fig. 2).

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FIG. 2. V3 and non-V3 regions contribute to Env-mediated virus entry. (A) The infectivity of pseudotyped viruses bearing parental and chimeric env sequences was determined by measuring luciferase production (in RLU) after the inoculation of U87/CD4 target cells expressing CCR5 (gray bars) or CXCR4 (black bars). The sources of V3 and the env backbones for each chimeric env are indicated at the bottom. nd, no detectable infectivity. (B) V3 loop amino acid sequences of parental and chimeric env clones.

Three chimeras contained V3 sequences from either dual-X (11D and 88D) or X4-tropic (42X) clones in the context of an R5-tropic (10R) env backbone. Two of these chimeras with the V3 loop from 88D or 42X were able to use CXCR4, albeit much less efficiently than the parental clones 88D and 42X. The chimera containing the V3 loop of the dual-X clone 11D in the backbone of the R5 clone 10R was not functional. The V3 loops of 11D and 88D differed by a single amino acid, R3G. Overall, the infectivity of chimeras with X4-like V3 sequences (from dual-X or X4-tropic clones) on an R5-tropic env backbone was lower on both CCR5 and CXCR4 target cells than on the corresponding CXCR4-using parental clones (Fig. 2).

env chimeras with X4-like V3 sequences in dual-R backbones (21d or 86d) had improved infectivity on both CXCR4- and CCR5-expressing cells compared to that of paired chimeras bearing the R5-tropic backbone from clone 10R (Fig. 3). The chimeras constructed on the 86d backbone displayed higher infectivity than those on the 21d backbone, consistent with the infectivity of the parental clones 86d and 21d, respectively. In comparison to the parental R5-tropic (10R) and dual-R (21d and 86d) clones, all chimeras with X4-like V3 sequences exhibited increased infectivity in CXCR4-expressing cells but dramatically reduced infectivity in CCR5-expressing cells (Fig. 3).

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FIG. 3. Chimeric env sequences with a dual-R env backbone exhibit higher infectivity than those with an R5-tropic env backbone. (A) Infectivity as measured by luciferase production (in RLU) on U87/CD4 target cells expressing CCR5 (gray bars) or CXCR4 (black bars). The sources of V3 and the env backbones for each chimeric env are indicated at the bottom of the figure. (B) V3 loop amino acid sequences of parental and chimeric env clones.

Taken together, these data indicate that determinants in both V3 and other regions of env are required for the efficient use of CXCR4.

Determinants of CXCR4 use in dual-R clones reside in TM. To identify the specific determinants of CXCR4 use in dual-R clones, we further analyzed the dual-R clones 21d and 86d in conjunction with the R5-tropic clone 10R. Amino acid sequences of clones 21d and 86d differed from the R5-tropic clone 10R at 11 and 24 residues, respectively (Table 2). Clone 21d had six amino acid substitutions in SU and five substitutions in TM. Clone 86d had 12 amino acid substitutions in SU and 12 substitutions in TM. To localize the domains required for the CXCR4-using phenotype, we first created chimeric env sequences by exchanging the sequences of the SU and TM subunits between the dual-R and R5-tropic clones. Unexpectedly, chimeras containing the SU sequence from the R5-tropic clone (10R) and TM sequences from the dual-R clones (21d or 86d) exhibited the dual-R phenotype, whereas chimeras that contained the SU sequence from dual-R clones (21d or 86d) and TM sequence from the R5-tropic clone (10R) had an R5-tropic phenotype (Fig. 4A and B). These results indicate that the determinants responsible for conferring CXCR4 use to the dual-R clones 21d and 86d are located in TM rather than SU. To confirm this observation, we also generated chimeric env sequences that contained the TM sequence from either clone 21d or 86d and the SU sequence from the unrelated R5-tropic strain JRCSF. Both chimeras were able to infect CCR5 and CXCR4 target cells (Fig. 4C), further confirming that determinants of CXCR4 use in the dual-R clones 21d and 86d are located in TM. Similar results were obtained using SF162, another R5-tropic strain (data not shown).

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FIG. 4. Determinants of CXCR4 use reside in the TM region of dual-R clones. (A) SU/TM chimeric env sequences constructed from R5-tropic clone 10R and dual-R clone 86d. (B) SU/TM chimeric env sequences constructed from R5-tropic clone 10R and dual-R clone 21d. (C) Chimeric env sequences derived from the TM region of dual-R clone 86d or 21d and SU of the R5-tropic strain JRCSF. Infectivity as measured by luciferase production (in RLU) on U87/CD4 target cells expressing CCR5 (gray bars) or CXCR4 (black bars) is shown.

Fine mapping of TM determinants of CXCR4 use in dual-R clones. We next attempted to define the specific substitutions within the TM subunit of dual-R clones that were responsible for CXCR4 use. Four chimeric env genes were made, each containing different TM regions of clone 86d in the backbone of clone 10R (Table 3). Chimera 86d.1 contained a portion of the TM extracellular domain (fusion peptide to the HR2 domain) with three substitutions (G515V, A539V, and D607A). Chimera 86d.2 contained the entire extracellular domain of the 86d TM protein (fusion peptide to the transmembrane domain) with seven amino acid substitutions (G515V, A539V, D607A, T644N, D648E, E654D, and M687I). Chimera 86d.4 contained the cytoplasmic domain of 86d and had five substitutions (G746E, C767S, H787R, W790R, and I812V). Chimera 86d.3 contained the cytoplasmic domain of 86d along with the HR2 and TM regions, including four additional substitutions (T644N, D648E, E654D, and M687I). Chimeras 86d.1 and 86d.2 exhibited a dual-tropic phenotype, while chimeras 86d.3 and 86d.4 both were R5 tropic, thus localizing the determinants of dual tropism to the extracellular domain of the 86d TM protein. The infectivity of chimera 86d.1, which had the least number of substitutions in TM, also was less than that of either the 10R/86d chimera bearing the complete TM of 86d or the 86d parental clone, indicating that additional substitutions in TM and SU enhance infection on CXCR4 or CCR5 cells.

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TABLE 3. Comparison of amino acids exchanged in chimeras constructed between clone 10R and clone 86d

To determine which of the three substitutions (G515V, A539V, or D607A) contributed to CXCR4 use in the 86d.1 chimera, each was introduced separately into the backbone of the R5-tropic env clone 10R by site-directed mutagenesis (Fig. 5). The G515V mutation alone was able to confer the dual-tropic phenotype, whereas env sequences containing either an A539V or D607A mutation remained R5 tropic. A valine at amino acid position 515 also was present in the dual-X clones 11D and 88D (Table 2) as well as in other dual-X clones, but it was not in R5-tropic and other dual-R clones listed in Table 1 (data not shown). The G515V substitution was absent from the dual-R clone 21d; however, this clone contained five other substitutions in TM compared to the sequence of clone 10R (positions T529A, A539V, C767S, H787R, and I792S). Therefore, additional TM chimeras were generated between clone 21d and clone 10R (Table 4). Chimera 21d.1, with two substitutions (T529A and A539V) located in the fusion peptide and the region between the fusion peptide and HR1, and chimera 21d.2, with three substitutions (C767S, H787R, and I792S) in the cytoplasmic tail of clone 21d, were unable to efficiently infect CXCR4-positive target cells but retained infectivity on CCR5 target cells. Based on these results, it appears that substitutions in both the fusion peptide and cytoplasmic tail of TM are required for detectable CXCR4 use by the dual-R clone 21d.

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FIG. 5. Fine mapping of the determinants of CXCR4 use within TM. Infectivity mediated by env genes containing single mutations (G515V, A539V, and D607A) or the triple mutant (G515V/A539V/D607A) in the backbone of the R5-tropic clone 10R was measured in U87 CD4 target cells expressing CCR5 (gray bars) or CXCR4 (black bars).

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TABLE 4. Comparison of amino acids exchanged in chimeras constructed between clone 10R and clone 21d

Altered fusogenicity and ENF susceptibility of dual-R env sequences. Based upon the critical role that TM plays in cell-virus membrane fusion (6, 51), we investigated whether dual-R clones with determinants of CXCR4 use in TM exhibited altered fusion activity compared to that of closely related R5-tropic env clones. The fusion activities of R5-tropic clone 10R, dual-R clones 21d and 86d, SU/TM chimeric env sequences, and the G515V mutant clone are summarized in Table 5. Compared to that of the R5-tropic clone 10R, the dual-R clone 86d and the chimeric env with 86d TM sequences exhibited 7- to 10-fold higher levels of membrane fusion. No differences in fusion were observed with the chimeric env-containing 86d SU sequences or the G515V mutation. Similarly, enhanced fusion was not observed for dual-R clone 21d and related chimeric env clones.

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TABLE 5. Effect of TM determinants of CXCR4 use on cell-cell fusion and susceptibility to entry inhibitors

Since sequence changes in TM can alter susceptibility to the fusion inhibitor ENF and since ENF resistance mutations usually are found in the HR1 domain of TM (19, 20, 42, 50), we were interested in determining whether the presence of TM determinants of CXCR4 use influenced the susceptibility to ENF (Table 5). Despite identical HR1 sequences, dual-R clones 86d and 21d both exhibited four- to fivefold-reduced susceptibility to ENF compared to that of the R5-tropic clone 10R. The chimeric env with 21d TM sequences also exhibited reduced susceptibility to ENF, but the chimera with the 21d SU sequence did not. The 86d chimeras and the G515V mutant also did not exhibit differences in ENF susceptibility. The data support findings that sequence changes outside of HR1 or TM can influence ENF susceptibility (14, 15, 20, 39, 41). The dual-R clones (86d and 21d) and respective SU/TM chimeric env sequences were as susceptible to a CCR5 inhibitor as the R5-tropic clone 10R. The G515V mutant was slightly more susceptible to CCR5 and CXCR4 inhibitors than the parental R5-tropic clone 10R and dual-R clone 86d.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this study, we evaluated the contribution of HIV-1 V3 and non-V3 env regions to coreceptor utilization by analyzing individual env sequences isolated from a single patient's plasma viral population that exhibited dual/mixed tropism. Dual-tropic variants with V3 sequences that were identical to those of R5-tropic variants were observed, indicating that determinants of CXCR4 use were present outside of the V3 region. This is consistent with observations that we recently reported for subtype D HIV-1 (23). The study of these subtype D env sequences and previous observations (35, 36) led us to hypothesize that mutations in V3 that could allow efficient CXCR4 use are highly detrimental to the infectivity (fitness) of CCR5-using strains, resulting in a high genetic barrier for evolution from R5 to X4 tropism in the absence of compensatory changes elsewhere in env. We further hypothesized that dual-R variants, which have R5-like V3 sequences that can use CXCR4, albeit inefficiently, represent a possible evolutionary intermediate between R5-tropic variants and dual-X or X4-tropic variants and provide a context that is better suited to tolerate V3 mutations that confer more efficient CXCR4 utilization. In this study, we tested this hypothesis by exchanging the V3 regions of subtype B R5-tropic and CXCR4-using env clones to evaluate the impact of V3 and non-V3 env regions on coreceptor tropism. Chimeric env clones with R5-like V3 sequences in the context of dual-X and X4-tropic env backbones utilized CCR5 efficiently while retaining their ability to use CXCR4. Conversely, chimeric env clones with X4-like V3 sequences in the context of an R5-tropic env backbone utilized both CXCR4 and CCR5 inefficiently. These observations are consistent with our proposed hypothesis and demonstrate that the determinants of CXCR4 use can reside outside of V3 and improve the efficiency of CXCR4 entry. Our results extend the previously reported findings that mutations in other regions of SU, such as V1/V2, can play a role during coreceptor switching (35, 36).

The determinants of coreceptor tropism have been mapped to sequences in V3 and other regions of HIV-1 SU. The SU subunit of env generally is considered important for CD4 binding, coreceptor binding, and specificity, while the TM subunit facilitates membrane fusion. The identification of determinants in TM as a contributor to CXCR4 use highlights the complexity of determinants of coreceptor tropism and env function in general. Consistent with our observations here, a recent study demonstrated that mutations within a region of the avian sarcoma/leukosis virus TM protein, corresponding to the HR1 domain of HIV-1 TM, resulted in expanded cellular tropism (1). Hence, in these two distinct retroviruses, mutations in the TM protein subunit of the Env protein can influence coreceptor-mediated tropism. In addition to coreceptor utilization, several studies have shown that sequence changes in the TM subunit can affect other functions of SU. In two different studies, we and others recently identified that amino acid substitutions in the TM subunit of patient-derived env sequences can affect the interaction between Env and CD4 and alter the sensitivity of viruses to soluble CD4 (3, 47). Furthermore, truncations of the TM cytoplasmic tail and ENF resistance mutations in HR1 have been reported to change SU protein conformation and alter the kinetics of infectivity and membrane fusion (17, 40). In this study, the degree to which alterations in TM affect ENF susceptibility or membrane fusion appears to be variable and likely is dependent on contextual effects of the particular Env backbone. Specifically how the determinants of CXCR4 use in TM identified in this study are able to change the nature of SU interactions with coreceptors is unknown, but it could involve indirect conformational changes in SU that facilitate CXCR4 coreceptor binding or direct modifications in TM that alter CXCR4 utilization.

We have shown that mutations in the fusion peptide and cytoplasmic tail of TM contribute to CXCR4 use by the dual-R clone 21d, while a single G515V mutation present in the fusion peptide of the dual-R clone 86d was sufficient to confer CXCR4 use to the R5-tropic clone 10R. Among 372 different patient-derived sequences for which the coreceptor tropism data are in LANL/GenBank, G515V is present in five viruses, all of which use CXCR4. Other substitutions at this position include T, A, I, and G. There is no evidence for an association between these substitutions and specific coreceptor usage.

The existence of dual-tropic variants with differential CCR5 and CXCR4 use may have implications for therapy with coreceptor inhibitors. It has been reported that some dual-tropic variants are able to use both CCR5 and CXCR4 to infect macrophages but are largely restricted to CXCR4-mediated entry in primary lymphocytes (57). These dual-tropic variants may use CXCR4, not CCR5, for the infection of T cells in vivo when both coreceptors are available. Using samples from patients enrolled in a clinical trial of the CXCR4 antagonist AMD3100, we recently demonstrated that AMD3100 was able to suppress the replication of some dual-X variants that use CXCR4 efficiently but not dual-R variants that use CXCR4 poorly (S. Fransen, G. Bridger, J. Whitcomb, J. Toma, E. Stawiski, N. T. Parkin, C. Petropoulos, and W. Huang, unpublished data). This suggests that differential coreceptor use by dual-tropic variants affects clinical responses to coreceptor inhibitors.

 
We are grateful to the Monogram Biosciences Clinical Reference Laboratory for the performance of tropism assays. We thank Frederick M. Hecht and Steve Deeks (San Francisco General Hospital, UCSF) for providing the patient plasma sample, Merck Research Laboratory for providing the CCR5 inhibitor, AnorMED for providing the CXCR4 inhibitor, AMD3100, and Trimeris for providing ENF. We also are grateful to Peter Hughes and Ellen Paxinos (Monogram Biosciences) for critical reviews of the manuscript and Cynthia Sedik for editorial assistance.

This work was supported in part by NIH-NIAID SBIR grant R44-AI048990.

 
* Corresponding author. Mailing address: Monogram Biosciences, 345 Oyster Point Blvd., South San Francisco, CA 94080. Phone: (650) 866-7429. Fax: (650) 624-4132. E-mail: whuang{at}monogrambio.com

Published ahead of print on 19 March 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Virology, June 2008, p. 5584-5593, Vol. 82, No. 11
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