JOURNAL OF VIROLOGY, June 2008, p. 5584–5593 0022-538X/08/$08.00⫹0 doi:10.1128/JVI.02676-07 Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 11
Coreceptor Tropism Can Be Influenced by Amino Acid Substitutions in the gp41 Transmembrane Subunit of Human Immunodeficiency Virus Type 1 Envelope Protein䌤 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
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 CXCR4mediated entry. We hypothesize that the latter plays an important role in the transition from CCR5 to CXCR4 coreceptor use.
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 Nlinked 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 R5tropic 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.
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
* 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:
[email protected]. 䌤 Published ahead of print on 19 March 2008. 5584
VOL. 82, 2008
CHANGES IN gp41 CAN AFFECT CORECEPTOR TROPISM
5585
TABLE 1. Coreceptor tropism and V3 sequences of env clones Luciferase activity (RLU)
env clone name
CCR5 cells
CXCR4⫹ cells
55R 2R 8R 5R 25R 15R 79R 10Rc 13d 23d 22d 21dc 86dc 87D 16D 73D 12D 67D 88Dc 56D 11Dc 42Xc
158,594 1,082,926 273,662 145,655 735,520 293,016 1,898,438 1,149,479 214,029 705,068 1,985,072 1,278,284 3,790,005 3,145 19,281 27,100 16,830 8,862 8,456 7,114 11,893 71
60 69 67 74 144 145 101 102 798 805 841 1,426 63,759 130,982 176,477 329,832 349,224 413,493 931,105 963,659 27,700 328,864
⫹
Tropisma
V3 loop amino acid sequenceb
R5 R5 R5 R5 R5 R5 R5 R5 Dual-R Dual-R Dual-R Dual-R Dual-R Dual-X Dual-X Dual-X Dual-X Dual-X Dual-X Dual-X Dual-X X4
CTRPGNNTRRSITMGPGRAFYTTGEIIGDIRKAHC ................................... ................................... ................................... ................................... ................................... ................................... ................................... ................................... ................................... ................................... ................................... ................................... ............H..-H.R....KN.......... ............H..-H.R....KN.......... ............H..-H.R....KN.......... ............H..-H.R....KN.......... ............H..-H.R....KN.......... ............H..-H.R....KN.......... ............H..-H.R....KN.......... ..G.........H..-H.R....KN.......... ......S...G.LV.-T.R....RN..........
a Determined using the Trofile assay; dual-R clones have the same V3 sequences as R5-tropic clones, while dual-X clones have V3 sequences that resemble those of the X4-tropic clone (23). b For the V3 amino acid sequences, dots represent amino acids identical to the first sequence, and dashes indicate gaps. c Clones chosen for chimeric env analysis.
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 R5tropic 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. MATERIALS AND METHODS 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 chainterminator 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 M) 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.
RESULTS 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
5586
HUANG ET AL.
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. Dual-R clones are genetically distinct from dual-X and X4tropic 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. 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
J. VIROL.
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.
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). 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
VOL. 82, 2008
CHANGES IN gp41 CAN AFFECT CORECEPTOR TROPISM
5587
Clone (tropism) 3 4
R5-tropic V M consensus 10R (R5) G 21d (dual-R) 86d (dual-R) 11D (dual-X) 88D (dual-X) 42X (X4) T
C1
T
A
L
I
N
S
N
S R
V1
G
E
E
G
F
L L I
V2
I
V
gp120
N
D
A
T
Q
R
N
K
T
V V V V K
E
T
I
C3
I
R R R
E
G
P
S S S S
V4
N
K
K
E E E
G
V V V
C5
T
A
V
A
Amino acid according to env region and amino acid positiona for:
C2
E
K
D
A A
A
FP
T
N N
D
E E
M
I I
FP-HR1
E
D D
gp41
S
H
R
H
C
HR1HR2
G
E E
E
TABLE 2. Comparison of amino acid differences of selected env clones with different coreceptor tropism (V3 excluded)
K
E
I
S
HR2
W
R S
I
V
V
A
T
I
CT
F
V
TM*
L
16 37 84 92 121 141 146 148 152 175 184 279 281 287 290 295 337 348 361 389 403 405 406 485 515 529 539 607 644 648 654 687 746 767 787 790 792 812 815 817 818
N
K K K K
a Amino acid identity at the indicated position is shown (according to HXB2 numbering). The amino acids that are identical to the R5-tropic clone consensus are not shown. FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; FP-HR1, region between FP and HR1; HR1-HR2, region between HR1 and HR2; TM*, transmembrane domain in gp41; and CT, cytoplasmic tail.
CXCR4-mediated entry, although the ability to infect CXCR4positive 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). 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 CXCR4and 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). 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
5588
HUANG ET AL.
J. VIROL.
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.
86d are located in TM. Similar results were obtained using SF162, another R5-tropic strain (data not 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
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.
VOL. 82, 2008
CHANGES IN gp41 CAN AFFECT CORECEPTOR TROPISM
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.
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
5589
(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. 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 dualtropic 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. Altered fusogenicity and ENF susceptibility of dual-R env sequences. Based upon the critical role that TM plays in cellvirus 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 R5tropic 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 7to 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. 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 mu-
5590
HUANG ET AL.
J. VIROL.
TABLE 3. Comparison of amino acids exchanged in chimeras constructed between clone 10R and clone 86d Luciferase activity (RLU) of:
Amino acid according to domain and position in TMa Clone or construct
Clone 10R Chimera 86d.1 Chimera 86d.2 Chimera 86d.3 Chimera 86d.4 10RSU ⫹ 86dTMb
FP
FPHR1
HR1HR2
515
539
607
644
648
654
687
746
767
787
790
812
G V V G G V
A V V A A V
D A A D D A
T T N N T N
D D E E D E
E E D D E D
M M I I M I
G G G E E E
C C C S S S
H H H R R R
W W W R R R
I I I V V V
HR2
TM*
Tropism
CT
R5 Dual Dual R5 R5 Dual
CCR5⫹ cells
CXCR4⫹ cells
1,036,805 480,215 792,017 891,001 1,821,581 1,676,485
103 7,277 11,428 88 122 31,395
a Boldface indicates amino acid residues derived from clone 86d. FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; FP-HR1, region between FP and HR1; HR1-HR2, region between HR1 and HR2; TM*, transmembrane domain in gp41; and CT, cytoplasmic tail. b Chimeric env contains SU from clone 10R and TM from clone 86d.
tant 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. DISCUSSION 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 CCR5using 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 X4tropic 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
TABLE 4. Comparison of amino acids exchanged in chimeras constructed between clone 10R and clone 21d
Clone or construct
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).
Clone 10R Chimera 21d.1 Chimera 21d.2 10SU ⫹ 21TMb
Amino acid according to domain and position in TMa
Luciferase activity (RLU) for: Tropism
FP
FPHR1
CT
529
539
767 787 792
T A T A
A V A V
C C S S
H H R R
I I S S
R5 R5 R5 Dual
CCR5⫹ cells
CXCR4⫹ cells
1,036,805 435,581 1,755,181 1,106,515
103 94 101 1,575
a Boldface indicates amino acid residues derived from clone 21d. FP, fusion peptide; HR1, heptad repeat 1; FP-HR1, region between FP and HR1; and CT, cytoplasmic tail. b Chimeric env contains SU from clone 10R and TM from clone 21d.
VOL. 82, 2008
CHANGES IN gp41 CAN AFFECT CORECEPTOR TROPISM
5591
TABLE 5. Effect of TM determinants of CXCR4 use on cell-cell fusion and susceptibility to entry inhibitors IC50a (ng/ml)
Luciferase activity (RLU) of: env construct
10R 21d 86d 10SU ⫹ 21SU ⫹ 10SU ⫹ 86SU ⫹ G515V a
21TM 10TM 86TM 10TM
SU
10R 21d 86d 10R 21d 10R 86d 10R
TM
10R 21d 86d 21d 10R 86d 10R 10R
Tropism
R5 Dual-R Dual-R Dual-R R5 Dual-R R5 Dual-R
CCR5⫹ cells
CXCR4⫹ cells
1,036,805 1,278,284 3,790,005 1,106,515 435,581 1,676,485 645,526 716,490
103 1,426 63,759 1,575 94 31,395 105 5,449
Fusion (RLU)
846 251 6,163 552 188 8,248 616 689
ENF
CCR5 inhibitor
CXCR4 inhibitor
47 272 205 155 32 62 54 25
4.4 3.8 3.6 2.6 4.4 3.4 5.0 1.7
NA NA 5.6 NA NA 2.2 NA 1.2
IC50, 50% inhibitory concentration; NA, unreportable IC50 data due to undetectable or low-level infectivity on CXCR4 target cells.
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. ACKNOWLEDGMENTS 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 R44AI048990. REFERENCES 1. Amberg, S. M., R. C. Netter, G. Simmons, and P. Bates. 2006. Expanded tropism and altered activation of a retroviral glycoprotein resistant to an entry inhibitor peptide. J. Virol. 80:353–359. 2. Berger, E. A., P. M. Murphy, and J. M. Farber. 1999. Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu. Rev. Immunol. 17:657–700. 3. Blish, C. A., M. A. Nguyen, and J. Overbaugh. 2008. Enhancing exposure of HIV-1 neutralization epitopes through mutations in gp41. PLoS Med. 5:e9. 4. Brumme, Z. L., J. Goodrich, H. B. Mayer, C. J. Brumme, B. M. Henrick, B. Wynhoven, J. J. Asselin, P. K. Cheung, R. S. Hogg, J. S. Montaner, and P. R. Harrigan. 2005. Molecular and clinical epidemiology of CXCR4-using HIV-1 in a large population of antiretroviral-naive individuals. J. Infect. Dis. 192:466–474. 5. Carrillo, A., and L. Ratner. 1996. Human immunodeficiency virus type 1 tropism for T-lymphoid cell lines: role of the V3 loop and C4 envelope determinants. J. Virol. 70:1301–1309. 6. Chan, D. C., and P. S. Kim. 1998. HIV entry and its inhibition. Cell 93:681–684. 7. Clapham, P. R., J. D. Reeves, G. Simmons, N. Dejucq, S. Hibbitts, and A. McKnight. 1999. HIV coreceptors, cell tropism and inhibition by chemokine receptor ligands. Mol. Membr. Biol. 16:49–55. 8. Cocchi, F., A. L. DeVico, A. Garzino-Demo, A. Cara, R. C. Gallo, and P. Lusso. 1996. The V3 domain of the HIV-1 gp120 envelope glycoprotein is critical for chemokine-mediated blockade of infection. Nat. Med. 2:1244– 1247. 9. Connor, R. I., K. E. Sheridan, D. Ceradini, S. Choe, and N. R. Landau. 1997. Change in coreceptor use coreceptor use correlates with disease progression in HIV-1-infected individuals. J. Exp. Med. 185:621–628. 10. Daar, E. S., K. L. Kesler, C. J. Petropoulos, W. Huang, M. Bates, A. E. Lail, E. P. Coakley, E. D. Gomperts, and S. M. Donfield. 2007. Baseline HIV type 1 coreceptor tropism predicts disease progression. Clin. Infect. Dis. 45:643–649. 11. Dean, M., M. Carrington, C. Winkler, G. A. Huttley, M. W. Smith, R. Allikmets, J. J. Goedert, S. P. Buchbinder, E. Vittinghoff, E. Gomperts, S. Donfield, D. Vlahov, R. Kaslow, A. Saah, C. Rinaldo, R. Detels, S. J. O’Brien, et al. 1996. Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Science 273:1856–1862.
5592
HUANG ET AL.
12. De Jong, J. J., A. De Ronde, W. Keulen, M. Tersmette, and J. Goudsmit. 1992. Minimal requirements for the human immunodeficiency virus type 1 V3 domain to support the syncytium-inducing phenotype: analysis by single amino acid substitution. J. Virol. 66:6777–6780. 13. Demarest, J., T. Bonny, C. Vavaro, C. LaBranche, K. Kitrinos, C. McDanal, S. Sparks, S. Chavers, S. Castillo, D. Elrick, D. McCarty, J. Whitcomb, W. Huang, C. Petropoulos, and S. Piscitelli. 2004. HIV-1 co-receptor tropism in treatment naı¨ve and experienced subjects, abstr. H-1136. 44th Intersci. Conf. Antimicrob. Agents Chemother. American Society for Microbiology, Washington, DC. 14. Derdeyn, C. A., J. M. Decker, J. N. Sfakianos, X. Wu, W. A. O’Brien, L. Ratner, J. C. Kappes, G. M. Shaw, and E. Hunter. 2000. Sensitivity of human immunodeficiency virus type 1 to the fusion inhibitor T-20 is modulated by coreceptor specificity defined by the V3 loop of gp120. J. Virol. 74:8358–8367. 15. Derdeyn, C. A., J. M. Decker, J. N. Sfakianos, Z. Zhang, W. A. O’Brien, L. Ratner, G. M. Shaw, and E. Hunter. 2001. Sensitivity of human immunodeficiency virus type 1 to fusion inhibitors targeted to the gp41 first heptad repeat involves distinct regions of gp41 and is consistently modulated by gp120 interactions with the coreceptor. J. Virol. 75:8605–8614. 16. Doms, R. W., and J. P. Moore. 2000. HIV-1 membrane fusion: targets of opportunity. J. Cell Biol. 151:F9–F14. 17. Edwards, T. G., S. Wyss, J. D. Reeves, S. Zolla-Pazner, J. A. Hoxie, R. W. Doms, and F. Baribaud. 2002. Truncation of the cytoplasmic domain induces exposure of conserved regions in the ectodomain of human immunodeficiency virus type 1 envelope protein. J. Virol. 76:2683–2691. 18. Fouchier, R. A., M. Brouwer, S. M. Broersen, and H. Schuitemaker. 1995. Simple determination of human immunodeficiency virus type 1 syncytiuminducing V3 genotype by PCR. J. Clin. Microbiol. 33:906–911. 19. Greenberg, M. L., and N. Cammack. 2004. Resistance to enfuvirtide, the first HIV fusion inhibitor. J. Antimicrob. Chemother. 54:333–340. 20. Heil, M. L., J. M. Decker, J. N. Sfakianos, G. M. Shaw, E. Hunter, and C. A. Derdeyn. 2004. Determinants of human immunodeficiency virus type 1 baseline susceptibility to the fusion inhibitors enfuvirtide and T-649 reside outside the peptide interaction site. J. Virol. 78:7582–7589. 21. Hoffman, N. G., F. Seillier-Moiseiwitsch, J. Ahn, J. M. Walker, and R. Swanstrom. 2002. Variability in the human immunodeficiency virus type 1 gp120 Env protein linked to phenotype-associated changes in the V3 loop. J. Virol. 76:3852–3864. 22. Hoffman, T. L., and R. W. Doms. 1999. HIV-1 envelope determinants for cell tropism and chemokine receptor use. Mol. Membr. Biol. 16:57–65. 23. Huang, W., S. H. Eshleman, J. Toma, S. Fransen, E. Stawiski, E. E. Paxinos, J. M. Whitcomb, A. M. Young, D. Donnell, F. Mmiro, P. Musoke, L. A. Guay, J. B. Jackson, N. T. Parkin, and C. J. Petropoulos. 2007. Coreceptor tropism in human immunodeficiency virus type 1 subtype D: high prevalence of CXCR4 tropism and heterogeneous composition of viral populations. J. Virol. 81:7885–7893. 24. Hung, C. S., N. Vander Heyden, and L. Ratner. 1999. Analysis of the critical domain in the V3 loop of human immunodeficiency virus type 1 gp120 involved in CCR5 utilization. J. Virol. 73:8216–8226. 25. Hwang, S. S., T. J. Boyle, H. K. Lyerly, and B. R. Cullen. 1991. Identification of the envelope V3 loop as the primary determinant of cell tropism in HIV-1. Science 253:71–74. 26. Jensen, M. A., F. S. Li, A. B. van ’t Wout, D. C. Nickle, D. Shriner, H. X. He, S. McLaughlin, R. Shankarappa, J. B. Margolick, and J. I. Mullins. 2003. Improved coreceptor usage prediction and genotypic monitoring of R5to-X4 transition by motif analysis of human immunodeficiency virus type 1 env V3 loop sequences. J. Virol. 77:13376–13388. 27. Koot, M., I. P. Keet, A. H. Vos, R. E. de Goede, M. T. Roos, R. A. Coutinho, F. Miedema, P. T. Schellekens, and M. Tersmette. 1993. Prognostic value of HIV-1 syncytium-inducing phenotype for rate of CD4⫹ cell depletion and progression to AIDS. Ann. Intern. Med. 118:681–688. 28. Kumar, S., K. Tamura, and M. Nei. 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief. Bioinform. 5:150–163. 29. Labrosse, B., C. Treboute, A. Brelot, and M. Alizon. 2001. Cooperation of the V1/V2 and V3 domains of human immunodeficiency virus type 1 gp120 for interaction with the CXCR4 receptor. J. Virol. 75:5457–5464. 30. Liu, R., W. A. Paxton, S. Choe, D. Ceradini, S. R. Martin, R. Horuk, M. E. MacDonald, H. Stuhlmann, R. A. Koup, and N. R. Landau. 1996. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiplyexposed individuals to HIV-1 infection. Cell 86:367–377. 31. Melby, T., M. Despirito, R. Demasi, G. Heilek-Snyder, M. L. Greenberg, and N. Graham. 2006. HIV-1 coreceptor use in triple-class treatment-experienced patients: baseline prevalence, correlates, and relationship to enfuvirtide response. J. Infect. Dis. 194:238–246. 32. Moyle, G. J., A. Wildfire, S. Mandalia, H. Mayer, J. Goodrich, J. Whitcomb, and B. G. Gazzard. 2005. Epidemiology and predictive factors for chemokine receptor use in HIV-1 infection. J. Infect. Dis. 191:866–872. 33. Nabatov, A. A., G. Pollakis, T. Linnemann, A. Kliphius, M. I. Chalaby, and W. A. Paxton. 2004. Intrapatient alterations in the human immunodeficiency virus type 1 gp120 V1V2 and V3 regions differentially modulate coreceptor
J. VIROL.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44. 45.
46.
47.
48.
49.
50.
51.
52.
53.
usage, virus inhibition by CC/CXC chemokines, soluble CD4, and the b12 and 2G12 monoclonal antibodies. J. Virol. 78:524–530. O’Brien, W. A., Y. Koyanagi, A. Namazie, J. Q. Zhao, A. Diagne, K. Idler, J. A. Zack, and I. S. Chen. 1990. HIV-1 tropism for mononuclear phagocytes can be determined by regions of gp120 outside the CD4-binding domain. Nature 348:69–73. Pastore, C., R. Nedellec, A. Ramos, O. Hartley, J. L. Miamidian, J. D. Reeves, and D. E. Mosier. 2007. Conserved changes in envelope function during human immunodeficiency virus type 1 coreceptor switching. J. Virol. 81:8165–8179. Pastore, C., R. Nedellec, A. Ramos, S. Pontow, L. Ratner, and D. E. Mosier. 2006. Human immunodeficiency virus type 1 coreceptor switching: V1/V2 gain-of-fitness mutations compensate for V3 loss-of-fitness mutations. J. Virol. 80:750–758. Paxton, W. A., S. R. Martin, D. Tse, T. R. O’Brien, J. Skurnick, N. L. VanDevanter, N. Padian, J. F. Braun, D. P. Kotler, S. M. Wolinsky, and R. A. Koup. 1996. Relative resistance to HIV-1 infection of CD4 lymphocytes from persons who remain uninfected despite multiple high-risk sexual exposure. Nat. Med. 2:412–417. Pollakis, G., S. Kang, A. Kliphuis, M. I. Chalaby, J. Goudsmit, and W. A. Paxton. 2001. N-linked glycosylation of the HIV type-1 gp120 envelope glycoprotein as a major determinant of CCR5 and CXCR4 coreceptor utilization. J. Biol. Chem. 276:13433–13441. Reeves, J. D., S. A. Gallo, N. Ahmad, J. L. Miamidian, P. E. Harvey, M. Sharron, S. Pohlmann, J. N. Sfakianos, C. A. Derdeyn, R. Blumenthal, E. Hunter, and R. W. Doms. 2002. Sensitivity of HIV-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics. Proc. Natl. Acad. Sci. USA 99:16249–16254. Reeves, J. D., F. H. Lee, J. L. Miamidian, C. B. Jabara, M. M. Juntilla, and R. W. Doms. 2005. Enfuvirtide resistance mutations: impact on human immunodeficiency virus envelope function, entry inhibitor sensitivity, and virus neutralization. J. Virol. 79:4991–4999. Reeves, J. D., J. L. Miamidian, M. J. Biscone, F. H. Lee, N. Ahmad, T. C. Pierson, and R. W. Doms. 2004. Impact of mutations in the coreceptor binding site on human immunodeficiency virus type 1 fusion, infection, and entry inhibitor sensitivity. J. Virol. 78:5476–5485. Rimsky, L. T., D. C. Shugars, and T. J. Matthews. 1998. Determinants of human immunodeficiency virus type 1 resistance to gp41-derived inhibitory peptides. J. Virol. 72:986–993. Samson, M., F. Libert, B. J. Doranz, J. Rucker, C. Liesnard, C. M. Farber, S. Saragosti, C. Lapoumeroulie, J. Cognaux, C. Forceille, G. Muyldermans, C. Verhofstede, G. Burtonboy, M. Georges, T. Imai, S. Rana, Y. Yi, R. J. Smyth, R. G. Collman, R. W. Doms, G. Vassart, and M. Parmentier. 1996. Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature 382:722–725. Sarkar, G., and S. S. Sommer. 1990. The “megaprimer” method of sitedirected mutagenesis. BioTechniques 8:404–407. Shioda, T., J. A. Levy, and C. Cheng-Mayer. 1991. Macrophage and T cell-line tropisms of HIV-1 are determined by specific regions of the envelope gp120 gene. Nature 349:167–169. Speck, R. F., K. Wehrly, E. J. Platt, R. E. Atchison, I. F. Charo, D. Kabat, B. Chesebro, and M. A. Goldsmith. 1997. Selective employment of chemokine receptors as human immunodeficiency virus type 1 coreceptors determined by individual amino acids within the envelope V3 loop. J. Virol. 71:7136–7139. Toma, J., S. Fransen, T. Wrin, J. Whitcomb, N. Parkin, C. Petropoulos, and W. Huang. 2008. The gp41 transmembrane subunit of HIV-1 envelope influences CD4 receptor-dependent virus entry. 15th Conf. Retrovir. Opportun. Infect., abstr. 251. Trkola, A., T. Dragic, J. Arthos, J. M. Binley, W. C. Olson, G. P. Allaway, C. Cheng-Mayer, J. Robinson, P. J. Maddon, and J. P. Moore. 1996. CD4dependent, antibody-sensitive interactions between HIV-1 and its co-receptor CCR-5. Nature 384:184–187. Trujillo, J. R., W. K. Wang, T. H. Lee, and M. Essex. 1996. Identification of the envelope V3 loop as a determinant of a CD4-negative neuronal cell tropism for HIV-1. Virology 217:613–617. Wei, X., J. M. Decker, H. Liu, Z. Zhang, R. B. Arani, J. M. Kilby, M. S. Saag, X. Wu, G. M. Shaw, and J. C. Kappes. 2002. Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob. Agents Chemother. 46:1896–1905. Weissenhorn, W., A. Dessen, S. C. Harrison, J. J. Skehel, and D. C. Wiley. 1997. Atomic structure of the ectodomain from HIV-1 gp41. Nature 387: 426–430. Westby, M., C. Smith-Burchnell, J. Mori, M. Lewis, M. Mosley, M. Stockdale, P. Dorr, G. Ciaramella, and M. Perros. 2007. Reduced maximal inhibition in phenotypic susceptibility assays indicates that viral strains resistant to the CCR5 antagonist maraviroc utilize inhibitor-bound receptor for entry. J. Virol. 81:2359–2371. Westervelt, P., D. B. Trowbridge, L. G. Epstein, B. M. Blumberg, Y. Li, B. H. Hahn, G. M. Shaw, R. W. Price, and L. Ratner. 1992. Macrophage tropism determinants of human immunodeficiency virus type 1 in vivo. J. Virol. 66:2577–2582.
VOL. 82, 2008
CHANGES IN gp41 CAN AFFECT CORECEPTOR TROPISM
54. Whitcomb, J. M., W. Huang, S. Fransen, K. Limoli, J. Toma, T. Wrin, C. Chappey, L. D. Kiss, E. E. Paxinos, and C. J. Petropoulos. 2007. Development and characterization of a novel single-cycle recombinant-virus assay to determine human immunodeficiency virus type 1 coreceptor tropism. Antimicrob. Agents Chemother. 51:566–575. 55. Wilkin, T. J., Z. Su, D. R. Kuritzkes, M. Hughes, C. Flexner, R. Gross, E. Coakley, W. Greaves, C. Godfrey, P. R. Skolnik, J. Timpone, B. Rodriguez, and R. M. Gulick. 2007. HIV type 1 chemokine coreceptor use among antiretroviral-experienced patients screened for a clinical trial of a CCR5 inhibitor: AIDS Clinical Trial Group A5211. Clin. Infect. Dis. 44:591–595. 56. Wu, L., N. P. Gerard, R. Wyatt, H. Choe, C. Parolin, N. Ruffing, A. Borsetti, A. A. Cardoso, E. Desjardin, W. Newman, C. Gerard, and J. Sodroski. 1996.
5593
CD4-induced interaction of primary HIV-1 gp120 glycoproteins with the chemokine receptor CCR-5. Nature 384:179–183. 57. Yi, Y., F. Shaheen, and R. G. Collman. 2005. Preferential use of CXCR4 by R5X4 human immunodeficiency virus type 1 isolates for infection of primary lymphocytes. J. Virol. 79:1480–1486. 58. Zimmerman, P. A., A. Buckler-White, G. Alkhatib, T. Spalding, J. Kubofcik, C. Combadiere, D. Weissman, O. Cohen, A. Rubbert, G. Lam, M. Vaccarezza, P. E. Kennedy, V. Kumaraswami, J. V. Giorgi, R. Detels, J. Hunter, M. Chopek, E. A. Berger, A. S. Fauci, T. B. Nutman, and P. M. Murphy. 1997. Inherited resistance to HIV-1 conferred by an inactivating mutation in CC chemokine receptor 5: studies in populations with contrasting clinical phenotypes, defined racial background, and quantified risk. Mol. Med. 3: 23–36.
