JOURNAL OF VIROLOGY, Aug. 2003, p. 9069–9073 0022-538X/03/$08.00⫹0 DOI: 10.1128/JVI.77.16.9069–9073.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.
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NOTES Role of the Human Immunodeficiency Virus Type 1 Envelope Gene in Viral Fitness Hector R. Rangel,1 Jan Weber,1 Bikram Chakraborty,1 Arantxa Gutierrez,2 Michael L. Marotta,1 Muneer Mirza,1 Patti Kiser,1 Miguel A. Martinez,2 Jose A. Este,2 and Miguel E. Quin ˜ones-Mateu1,3* Department of Virology, Lerner Research Institute, Cleveland Clinic Foundation,1 and Center for AIDS Research, Case Western Reserve University,3 Cleveland, Ohio, and Laboratori de Retrovirologia, Fundacio irsiCaixa, Hospital Universitari Germans Trias i Pujol, 08916 Badalona, Spain2 Received 21 April 2003/Accepted 30 May 2003
A human host offers a variety of microenvironments to the infecting human immunodeficiency virus type 1 (HIV-1), resulting in various selective pressures, most of them directed against the envelope (env) gene. Therefore, it seems evident that the replicative capacity of the virus is largely related to viral entry. In this study we have used growth competition experiments and TaqMan real-time PCR detection to measure the fitness of subtype B HIV-1 primary isolates and autologous env-recombinant viruses in order to analyze the contribution of wild-type env sequences to overall HIV-1 fitness. A significant correlation was observed between fitness values obtained for wild-type HIV-1 isolates and those for the corresponding env-recombinant viruses (r ⴝ 0.93; P ⴝ 0.002). Our results suggest that the env gene, which is linked to a myriad of viral characteristics (e.g., entry into the host cell, transmission, coreceptor usage, and tropism), plays a major role in fitness of wild-type HIV-1. In addition, this new recombinant assay may be useful for measuring the contribution of HIV-1 env to fitness in viruses resistant to novel antiretroviral entry inhibitors. the replication capacity of wild-type (wt) subtype B HIV-1 strains and the way in which host cell entry seems to define ex vivo HIV-1 fitness in the absence of any unusual alterations affecting other steps of the HIV-1 life cycle (e.g., deletions on the HIV-1 nef gene [17] and the presence of drug resistance mutations in the pol gene [23]). HIV-1 isolates and env-pseudotyped viruses from eight subtype B HIV-1 strains with different biophenotypes (i.e., syncytium-inducing [SI] or non-syncytium-inducing [NSI] and CCR5-tropic [R5], CXCR4-tropic [X4], or dual-tropic [R5/X4] viruses) were analyzed (Table 1). Two HIV-1 primary isolates harboring similar env genes but with distinct patterns of drug resistance mutations in the pol genes (F96 and F98) were obtained from an HIV-1-infected individual treated at the Hospital Universitari Germans Trias i Pujol in Badalona, Spain (7). Two HIV-1 primary isolates that became resistant to the CXCR4 antagonist AMD3100 and their parental strains (i.e., CI-1, CI-1⫹, CI-2, and CI-2⫹) were obtained from a previous study (11). Finally, two SI X4 HIV-1 isolates (laboratory-adapted strain HIV-1B-HXB2 and primary isolate HIV1B-92USO76) were obtained from the AIDS Research and Reference Reagent Program. This collection of viruses covers a broad genotypic and phenotypic selection (i.e., wt strains, multidrug-resistant variants, and phylogenetically related viruses with different coreceptor usage patterns), which allowed us to analyze the contribution of the HIV-1 env gene to viral fitness. Recombinant viruses carrying env genes corresponding to those of these eight HIV-1 strains were constructed as previously described (6) (Fig. 1A). Briefly, A3.01/CCR5-F7 cells
Human immunodeficiency virus type 1 (HIV-1) fitness, the replicative adaptation of the virus to its environment (10), is the result of the interaction of a multitude of viral and host factors (reviewed in references 22 and 23). Among the viral factors, many biological processes in the HIV-1 life cycle (e.g., cell entry, genome replication, protein synthesis and processing, and particle assembly and release from cells) may affect viral fitness. It is evident, however, that the envelope (env) gene plays a major role in the competitive ability of the virus. For example, the env gene is associated with viral transmission (13, 15, 28) and host cell tropism (4, 14) and is the main target of the host immune response (19, 27, 31). Consequently, many studies have evaluated its direct contribution to viral replication and HIV-1 pathogenesis (2, 4, 5, 12, 19, 24, 28). In addition, a whole new generation of antiretroviral drugs is being developed with the env gene as a primary target (e.g., HIV entry inhibitors that involve viral env glycoproteins and their cellular receptors) (8, 21). A recent study showed preliminary evidence that the efficiency of host cell entry may be the factor with the greatest impact on HIV-1 fitness in the absence of drug selective pressure (3). In this study, we have used growth competition experiments and TaqMan real-time PCR to measure fitness of both HIV-1 isolates and autologous env-recombinant viruses. Our results reveal the impact of the env gene on * Corresponding author. Mailing address: Cleveland Clinic Foundation, Lerner Research Institute, Department of Virology/NN10, 9500 Euclid Ave., Cleveland, OH 44195. Phone: (216) 444-2515. Fax: (216) 444-2998. E-mail:
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TABLE 1. HIV-1 isolates used to evaluate the role of env in viral fitness Fitnessc (%) of: Virusa
Drug resistance phenotypeb
Biophenotype
HIV-1 isolates
env recombinants
F96 F98 CI-1 CI-1⫹ CI-2 CI-2⫹ HXB2 92US076
wt PR and RT resistant wt AMD3100 resistant wt AMD3100 resistant wt wt
SI, R5/X4 SI, R5/X4 SI, R5/X4 SI, R5/X4 SI, X4 NSI, R5 SI, X4 SI, X4
96 25 86 55 79 49 94 100
81 105 74 47 60 34 105 97
a HIV-1 isolates F96 and F98 were obtained from a single patient in February 1996 and April 1998, respectively (7). CD4 counts and viral loads at these time points were as follows: F96, 10 cells/l and 891,205 HIV RNA copies/ml; F98, 388 cells/l and 66,070 HIV RNA copies/ml. HIV-1 clinical isolates CI-1⫹ and CI-2⫹ were obtained after in vitro selection with AMD3100 (11). CI-1 and CI-2 correspond to parental HIV-1 strains from the same individuals. HXB2 and 92US076 correspond to laboratory strain HIV-1B-HXB2 and primary isolate HIV1B-92US076, respectively. b wt indicates an absence of mutations in the pol gene associated with drug resistance. After a history of antiretroviral treatment that included zidovudine, didanosine, lamivudine, stavudine, nevirapine, indinavir, ritonavir, and saquinavir, the F98 HIV-1 isolate showed multiple protease (PR) (10I, 48V, 54V, 63P, 71V, 77I, 82A, and 90M) and reverse transcriptase (RT) (41L, 67N, 181C, 184V, 190A, 215Y, and 219E) drug resistance mutations (30) (http://www.iasusa.org). c Viral fitness values are from the averages of two relative fitness values corresponding to the competitions of each HIV-1 isolate with two HIV-1 control strains (see text for details) and are calculated relative to the fitness of the wild-type HIV-192US076 virus control (100%).
(obtained from Q. Sattentau through the Centralised Facility for AIDS Reagents, Medical Research Council) were transfected by electroporation with a mixture of the env-defective HIVHXB2 plasmid pJJ5 (9) and the corresponding PCR-amplified HIV-1 env fragment. PCR amplification of the complete gp160-encoding sequence (the region from 5580 to 8586 of the HIV-1HXB2 genome) was performed by nested PCR by using the following external primers: Rec2F, 5⬘-GATAAAGCCAC CTTTGCCTAGT-3⬘ (nucleotide [nt] position 5514), and env2, 5⬘-TTCTAGGTCTCGAGATACTGCT-3⬘ (nt position 8889). The following primers were used for the second PCR: Rec1F, 5⬘-AAGGGCCACAGAGGGAGCCATA-3⬘ (nt position 5580), and E270R, 5⬘-GCGTCCCAGAAGTTCCACAA-3⬘ (nt position 8566). Before transfection, the pJJ5 plasmid was digested with NcoI and BamHI at positions 5675 and 8475 of the HIV1HXB2 genome. Open plasmid and PCR products were coprecipitated and resuspended in water. After transfection, infectious viruses were recovered from the supernatants of cell cultures and stored at ⫺80°C until use. All viral stocks (i.e., HIV-1 isolates and env-recombinant viruses) were propagated in phytohemagglutinin-stimulated, interleukin-2-treated peripheral blood mononuclear cells (PBMC) as previously described (24). The tissue culture dose for 50% infectivity was determined for each isolate in triplicate with serially diluted supernatants from each viral propagation. Reverse transcriptase activity (29) in culture supernatants, on day 8 of culture, was used to calculate the tissue culture dose for 50% infectivity by using the Reed and Muench method (26). The MT-2 assay (28) was used to analyze the viral phenotype (i.e., SI or NSI). Coreceptor usage was determined by using viral stocks to infect U87 cells expressing CD4 and either CCR5 or CXCR4 chemokine receptors as previously described (3) (Table 1).
FIG. 1. Schematic representation of the construction of HIV-1 recombinant viruses, growth competition experiments, and TaqMan real-time PCR detection. (A) Patient PBMC samples were used to (i) isolate HIV-1 and (ii) PCR amplify and clone the whole gp160-encoding region of the env gene to construct HIV-1 env-recombinant viruses (see Materials and Methods for details). (B) Individual dual infections with a subtype B query HIV-1 isolate and one control strain (HIV-1A-92UG029 or HIV-1AE-CMU06) were performed at a multiplicity of infection (MOI) of 0.01 infectious U/cell. Wells I and III correspond to positive controls for the query and control viruses, respectively. (C) Three sets of subtype-specific primers and probes were designed to quantify the proportion of both HIV-1 variants in the dual infection. Subtype B-specific primers Bgag-S and Bgag-AS3 and probe pBgagROX were used to PCR amplify and recognize a conserved region within the subtype B HIV-1 gag gene. Similarly, subtype A-specific primers A2env-S and A2env-AS2 plus probe pA2env-FAM and CRF01_AE-specific primers E1env-S and E1env-AS, together with probe pE1env-FAM, were used to PCR amplify and recognize conserved regions in the HIV-1 env genes of clade A and the circulating recombinant form CRF01_AE, respectively (30).
Finally, nucleotide sequence analysis of the complete gp160encoding region of the env gene was used to verify the identity of all viral stocks (i.e., HIV-1 isolates and env-recombinant viruses) as previously described (24). To estimate ex vivo HIV-1 fitness, growth competition experiments were carried out as previously described (30). Briefly, each subtype B HIV-1 primary isolate or env-recombinant virus competed against two different non-subtype B HIV-1 control strains (HIV-1A-92UG029 and HIV-1AE-CMU06)
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FIG. 2. Fitness of HIV-1B-92US076 and the autologous env-recombinant virus. (A) Growth competition experiments with the HIV-1B-92US076 isolate and each one of the control viruses (HIV-1A-92UG029 and HIV-1AE-CMU06) in PBMC, with TaqMan real-time PCR to measure viral production. Similar experiments were performed by using the B-92US076 env-recombinant virus and both non-subtype B control viruses. TaqMan real-time PCR amplification plots and charts showing proportions of virus production are indicated. (B) Relative fitness relationships between query viruses (i.e., HIV-1 isolates or env recombinants) and non-subtype B HIV-1 controls. Numbers in parentheses under B-92US076 correspond to the relative fitness value in each condition (24). Arrow directions indicate fitness differences (positive or negative) between both viral strains (24). For example, the HIV-1B-92US076 isolate is 9.2-fold more fit than HIV-1AE-CMU06. Numbers in black boxes correspond to the fitness values relative to the that of the wt HIV-192US076 control (100%) as described in Table 1.
in a 1:1 initial proportion with a multiplicity of infection of 0.01 infectious U/cell (Fig. 1B). One milliliter of these viral mixtures was incubated with 106 PBMC for 2 h at 37°C and 5% CO2. Subsequently, the cells were washed three times with 1⫻ phosphate-buffered saline and then resuspended in culture medium (106/ml). Cells were washed and fed with medium after 4 days. Supernatants and cells were harvested at day 8, resuspended in dimethyl sulfoxide-fetal bovine serum, and stored at ⫺80°C for subsequent analysis. To determine viral fitness, the final ratio of the two viruses produced from each growth competition experiment was determined by TaqMan real-time PCR and compared to viral production from the monoinfections as previously described (30). Three sets of subtype-specific primers and probes were designed (Fig. 1C). These sets of primers and probes allowed subtype-specific PCR amplification and hybridization so that cross-hybridization between subtype B, A, and AE probes did not occur (30). A relative fitness value for each virus in the competition was estimated by using the production of each individual HIV-1 strain in the dual infection (24, 25). A total relative fitness value was calculated as the average of the two relative fitness values corresponding to the competition between each subtype B HIV-1 isolate or recombinant virus and each of the nonsubtype B HIV-1 control strains. The total relative fitness values were then compared and expressed as percentages
of the fitness of a wt subtype B HIV-1 primary isolate (HIV-1B-92US076, with a fitness value set at 100%) (25, 30). Despite differences in viral phenotypes and coreceptor usage patterns, our growth competition and real-time PCR method was able to accurately determine fitness of both HIV-1 isolates and env-recombinant viruses (Table 1). For example, when the B-92US076 env-recombinant virus competed in PBMC against both HIV-1 controls, followed by real-time PCR detection, a relative fitness value similar to that observed with the HIV-1 isolate was obtained (1.45 and 1.62 for the env recombinant and the HIV-1 isolate, respectively, corresponding to 97 and 100% of the fitness of the HIV-1 control) (Fig. 2). When the rest of the viruses were analyzed, a strong, statistically significant correlation was observed between the fitness values calculated for wt HIV-1 isolates and those for the env-recombinant viruses (r ⫽ 0.86; P ⫽ 0.01; Pearson product moment) (Fig. 3), suggesting that the env gene may be driving viral fitness in wt HIV-1 strains. It is important to note that the multidrug-resistant F98 isolate and the corresponding env-recombinant virus were not included in this correlation. This highly mutated HIV-1 isolate showed impairment in fitness, which was not evident when its env gene was introduced into a wt HIV-1 backbone (Fig. 3A). A recent study analyzed the fitness levels of both wt F96 and multidrug-resistant F98 viruses by using HIV-1 isolates and recombinant viruses carrying
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FIG. 3. Contribution of the env gene to HIV-1 fitness. (A) Relative fitness of wt subtype B HIV-1 isolates and autologous env-recombinant viruses (constructed in an HIV-1HXB2 backbone). Values are percentages of the fitness of the wt HIV-1B-92US076 control (100%). (B) Correlation between relative fitness values measured for HIV-1 isolates and those for the corresponding env-recombinant viruses in an HIV-1HXB2 background. The multidrug-resistant F98 HIV-1 isolate and the corresponding env-recombinant virus were not included in this analysis (see text for details). The Pearson product moment correlation coefficient was used to determine the strength of association or correlation between ex vivo HIV-1 fitness values of HIV-1 isolates and those of env-recombinant viruses.
the protease gene, the reverse transcriptase gene, and the 3⬘ end of the gag gene (30). Fitness values of the wt F96 viruses were similar to that of the HIV-1 control (i.e., viral isolate, 96%; pol recombinant, 102%). Analyses of the drug-resistant F98 HIV-1 isolate and the autologous pol-recombinant virus showed a comparable reduction in fitness (i.e., viral isolate, 25%; pol recombinant, 19%) (30). Thus, a considerable decrease in replication capacity due to selection and accumulation of drug-resistant mutations in the pol gene seems to have overcome the effect of other viral genomic regions (e.g., the env gene) on the overall ex vivo fitness of the F98 virus. A previous study showed that the addition of the CXCR4 antagonist AMD3100 to PBMC infected with R5/X4 HIV-1 isolates resulted in the selection of R5 HIV-1 variants or AMD3100-resistant viruses without a change in coreceptor usage (11). Here we determined the fitness of four HIV-1 primary isolates (i.e., CI-1, CI-1⫹, CI-2, and CI-2⫹) and the corresponding env-recombinant viruses (Table 1 and Fig. 3). In agreement with results in a previous report (1), both AMD3100-resistant viruses, regardless of coreceptor usage, showed a decrease in fitness compared with their parental strains. Interestingly, fitness values of all four env-recombinant viruses (i.e., wt parental and AMD3100 resistant) mimic the values of the autologous HIV-1 isolates (Fig. 3). Finally, a slight reduction in fitness values was observed for most of the env-recombinant viruses compared to those of their parental HIV-1 isolates (Table 1; Fig. 3). Although this may stress the importance of relative harmony between different viral genomic regions in order to maintain optimal viral fitness, it may also be a consequence of the env recombination procedure. Nevertheless, this phenomenon did not affect the correlation between fitness values determined with HIV-1 isolates and env-recombinant viruses. Numerous studies have addressed HIV-1 fitness and the potential effects on viral load, resistance to protease inhibitors
(PI) and/or reverse transcriptase inhibitors (RTI), and disease progression (22, 23). However, great effort is being devoted to the development of new drugs that may inhibit the entry of HIV-1 into susceptible cells (8, 21). In addition, current clinical trials involve treatment of HIV-infected individuals with combinations of PI, RTI, and viral entry inhibitors (e.g., enfuvirtide, formerly T-20) (18). Therefore, studies to estimate fitness of HIV-1 with genes for potential multidrug resistance (i.e., pol and env) must be designed. The growth competition and real-time PCR assay has been demonstrated to be useful in analyzing fitness of wt (3, 24) and drug-resistant HIV-1 isolates and pol-recombinant viruses (1, 25, 30). In this case, our ex vivo assay may be useful for analyzing fitness of both wt HIV-1 isolates and isolates resistant to PI, RTI, and/or entry inhibitors. These studies are currently under way in our laboratory. In this study, we have analyzed the fitness of different subtype B HIV-1 isolates with distinct phenotypes and coreceptor usage patterns, showing a statistically significant correlation with the fitness of the corresponding autologous env-recombinant viruses. Although HIV-1 fitness is the result of many biological processes in the virus life cycle (i.e., cell entry, reverse transcription, integration, gene expression, and virion assembly and release from cells), our results suggest that early events in the life cycle of wt HIV-1 isolates (e.g., viral entry) may make a major contribution to overall viral fitness. Recent studies have proposed that differences in fitness levels among HIV-1 subtypes may map to the env gene, perhaps having an impact on disease progression as well as transmission, evolution, and diversification of HIV-1 in different regions of the world (1, 3, 16, 20, 23). Further studies on the role of the wt HIV-1 env gene in viral fitness will help us to understand its effects on viral tropism, replication, and/or persistence in a variety of microenvironments within the human host, in addition to contributing to the development of novel antiretroviral treatments.
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H.R.R. and J.W. contributed equally to the experiments described in this work. Research performed at the Cleveland Clinic Foundation (M.E.Q.M.) was supported by research grants from the National Heart, Lung, and Blood Institute, NIH (5-KO1-HL67610-03), and the Center for AIDS Research (AI36219) at Case Western Reserve University. Research at the Fundacio irsiCaixa was supported by research grants from la marato ´ de TV3 (FIS 01/0067-02 and Red Tematica Cooperativa de Investigacion en Sida) and from Fundacio ´n para la Investigacio ´n y la prevencio ´n del SIDA en Espan ˜a (FIPSE 36293/02 and 36207/01 [M.A.M.] and BFM-2000-1382 and FIS01/1116 [J.A.E.]).
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17. 18.
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