In 2005, we reported a case of dual-tropic multidrug-resistant (MDR) HIV-1 infection associated with rapid disease progression.1 In general, MDR viruses are considered less fit and less pathogenic than drug-sensitive wild-type viruses because of the fitness costs conferred by the emergence of drug-resistance–associated mutations.2–5 Along similar lines, MDR viruses are believed to be less transmissible than wild-type viruses.6,7 Nonetheless, this individual (MDR-1) was infected with an MDR virus and progressed to AIDS within as few as 4 and up to 20 months postinfection. Subsequently, we reported that select biologically clonal isolates and a bulk viral isolate demonstrated very high replication rates, syncytium-forming activity, and enhanced cytopathicity in vitro,1 suggesting that viral factors were at least in part responsible for the observed rapid disease progression.
To study the potential viral determinants of the observed phenotype, we developed a strategy to generate biologically relevant, full-length molecular clones. We constructed 3 infectious molecular clones from this case: pMDR-1a, an R5-tropic variant; pMDR-1b, a dual-tropic variant; and pMDR-1c, a second dual-tropic variant with very rapid replication kinetics. In addition, we constructed a less cytopathic MDR virus clone, pMDR-5a, an R5-tropic variant with slow replication kinetics and multidrug resistance isolated from a patient (MDR-5) with slower disease progression. To determine the gene(s) responsible for the enhanced replication fitness of the MDR variant, despite the presence of drug-resistance mutations, we constructed chimeric viruses of a rapidly replicating and cytopathic clone, pMDR-1c, with a drug-sensitive, wild-type pNL4-38 and with a slower replicating pMDR-5a. We characterized these chimeras and performed growth competition assays to understand determinants underlying the replication advantage of the MDR virus variants isolated from the HIV-1–infected individual with rapid disease progression.
Patients Infected With MDR HIV-1 Associated With Rapid and Slow Disease Progression
Individuals infected with HIV-1 were identified and evaluated by the Clinical Program of the Aaron Diamond AIDS Research Center and gave written informed consent for study under a Rockefeller University Hospital Institutional Review Board–approved observational protocol. The rapidly progressing individual (MDR-1) from whom pMDR-1 clones were constructed has been well described.1 By contrast, subject MDR-5 presented in April 2006, approximately 10 months postinfection, was found to be infected with MDR HIV-1 with triple-class resistance (Table 1). He exhibited slower disease progression when compared with subject MDR-1 (Table 2) and elected to initiate therapy approximately 3.5 years after infection. He was treated with fixed-dose combination tenofovir disoproxil fumarate/emtricitabine, etravirine, ritonavir-enhanced darunavir, and raltegravir, and an excellent response to therapy was observed (Table 2).
Construction of Molecular Clones
A strategy to construct relevant molecular clones was developed (see Figure S1, Supplemental Digital Content, https://links.lww.com/QAI/A627). The supernatant of the primary end point culture, ie, a biologically clonal isolate, was inoculated onto PHA-stimulated, HIV-1 uninfected peripheral blood mononuclear cells (PBMCs), from which genomic DNA was purified and used for the molecular cloning. A complete LTR fragment was amplified and cloned into a vector (designated as pLTR). Then, the remaining HIV-1 genome with the length of ∼9.1 kb was amplified and cloned into a corresponding pLTR. Resulted clones with full-length HIV-1 genome were screened for the same infectivity and replication rates as of their parental isolates using TZM-bl cell9 and PBMC cultures. The detailed procedure is described in Supplemental Digital Content Text 1 (https://links.lww.com/QAI/A627).
Sequencing reactions were performed by Genewiz (South Plainfield, NJ). Multiple bidirectional sequences were aligned to obtain a full-length genome sequence using MegAlign within Lasegene V6 (DNASTAR, Madison, WI). The sequences of clones were analyzed by RIP10 for HIV-1 subtype determination and recombination detection, and N-Glycosite11 for putative N-glycosylation sites in envelope (http://www.hiv.lanl.gov). Drug-resistance mutations were detected by using Stanford University HIV Drug Resistance Database (http://hivdb.stanford.edu).12 For phylogenic analysis, the full-length genome sequences were aligned with subtype reference sequences using ClustralW, and the phylogenic tree was constructed by Maximum Likelihood Method in MEGA5.13
Construction of Envelope-Chimeras
We divided the HIV-1 genome into 2 parts: the envelope (gp160) gene and the remaining backbone genome, and constructed envelope-chimeras (env-chimera). Chimeras were designated as follows; the backbone strain followed by the gene replaced and its origin in parentheses. For example, pNL4-3(envMDR-1c) indicates a chimeric virus with the backbone of pNL4-3 and the env gene derived from pMDR-1c. Note that the envelope gene (6225-8795, in HXB2) also codes a part of vpu (6225–6310), the second exons of tat (8379–8469), rev (8379–8653), and rev response element. The detailed procedures for constructing each env-chimera are indicated in Supplemental Digital Content Text 1 (https://links.lww.com/QAI/A627).
Replication Kinetics Assay
Viruses and chimeric viruses were produced on transfection of 293T cells with plasmid DNA, and their infectivity was measured by end point dilution culture with PHA-stimulated, uninfected donor-derived PBMC as described earlier.14 To determine the replication kinetics, 1000 TCID50 of each virus was used to infect 2 × 106 PHA-stimulated PBMC, unless otherwise indicated. Cultures were washed after 6 hours, and then maintained for 7–8 days. The supernatant was collected daily for p24 antigen determinations with Coulter HIV-1 p24 antigen assay kit (Beckman-Coulter, Jersey City, NJ). At the end of culture, infected PBMCs were stained for CD3, CD4, and CD8, and the CD4+/CD8+T cell ratio was used as an indicator of the cytopathicity of each virus.14
Growth Competition Assay
The growth competition assay is a sensitive, quantitative, and reliable assay that provides a relative fitness value.15–17 Growth competition cultures between 2 viruses were performed in 2 wells; 1 well with PBMC from 1 donor and the other with PBMC from a second donor to ensure that results were not dependent on a unique source of PBMC. The same 2 sources of PBMC were used for all competition experiments. Combinations of 2 viruses, each at 1000 TCID50, were inoculated to 2 × 106 PHA-stimulated PBMC in 1.5 mL of RPMI1640/10%FCS supplemented with 20 U/mL of IL-2 (Roche Applied Science, Indianapolis, IN). On day 1, infected PBMCs were washed twice by low-speed centrifugation and resuspended in prewarmed new culture media. The culture was kept until day 4, when 50 μL of the supernatant was transferred to a newly prepared PHA-stimulated PBMC culture. The competition culture was maintained until day 16 with the virus mixture passaged every 4 days. Culture supernatants were harvested every 2 days for viral RNA concentration determinations.
Virion-RNA copy numbers of 2 viruses in the culture supernatant were determined by the gene-specific real-time polymerase chain reaction assays, which targeted either V1 region of envelope gene or nef gene. The detailed procedures are described in the Supplemental Digital Content (see Text 1, https://links.lww.com/QAI/A627). The ratios of HIV-1 RNA copy numbers of virus 1 over virus 2 were converted into natural log (ln) and used to determine the best-fit slope with 95% confidence intervals using Prism 4 (GraphPad Software, Inc., La Jolla, CA). This slope was defined as “log relative fitness”18 and used for the calculation of w2/w1 = eslope, where “w1” and “w2” were net growth rates (or the fitness) for virus 1 and virus 2, respectively. To compare fitness among different viruses, we determined fitness value relative to a reference, wild-type virus clone pNL4-3. Thus, a relative fitness value indicates the change in the ratio of copy numbers of a virus over NL4-3 in each day of the culture. Relative fitness was also defined for HIV-1 subgenomes, where we assumed that the relative fitness of a whole virus is a product of the relative fitness of subgenomes.
Construction of Infectious Molecular Clones
According to our strategy (see Figure S1, Supplemental Digital Content, https://links.lww.com/QAI/A627), we have constructed 3, full-length infectious molecular clones, pMDR-1a, -1b, and -1c from the MDR HIV-1 isolates (MDR-1a, -1b, -1c),14 and 1 infectious molecular clone, pMDR-5a, from the less cytopathic MDR HIV-1 isolate (MDR-5a) (Table 2; see Figure S2A&B, Supplemental Digital Content, https://links.lww.com/QAI/A627). The replication kinetics and cytopathicity of the molecular clones were equivalent to their parental isolates (Fig. 1A–E). Coreceptor tropisms of the molecular clones were identical to their parental isolates14 (Table 1, data not shown).
Sequence Characteristics of MDR-1 Clones
The nucleotide sequence alignment and the construction of a phylogenic tree of the molecular clones along with subtype reference sequences indicated that all 3 MDR-1 clones were of subtype B (see Figure S3, Supplemental Digital Content, https://links.lww.com/QAI/A627), and their mean sequence distance was 0.8%, which is consistent with a minimum genetic diversity shortly after transmission (83 days).
All open reading frames are intact in the clones constructed. Some of their genetic characteristics are indicated in Table 1. Drug-resistant mutations of the molecular clones are identical with their parental isolates,14 except for pMDR-1b, which was derived from 1 of 2 variants within the isolate, MDR-1b. Accordingly, pMDR-1b carries the same protease (PR) mutations as of the original isolate MDR-1b, but only M41L and T215D in the reverse transcriptase gene. All MDR-1 clones shared N348I in RT, which confers a low-level reduction in susceptibility to zidovudine and nevirapine.19 Deduced Gag amino acid sequences revealed changes at the cleavage sites including A431V at p7/p120,21 and P453L at p1/p622 in all 3 clones. Mutations within matrix protein including R76K and Y79F are reported to reduce the susceptibility to PI and improve viral replication fitness.23
The differences in deduced amino acid sequences in gp120 were primarily within the variable regions. In the V3 loop, pMDR-1a had a glycine, whereas both pMDR-1b and -1c had an arginine at position 11, being consistent with their coreceptor usage.24 In gp41, the N651F/I mutation was observed within the HR-2 region and has been reported to be associated with enhanced fusion and increased viral pathogenicity.25 Enhanced fitness has been thought to be associated with increased positive charges in gp120.26 However, the net charge of gp120 did not directly correlate with the fitness values among these envelopes (Table 1).
Construction of Envelope-Chimeras
We used pMDR-1c, a molecular clone with robust replication dynamics, dual tropism, and enhanced cytopathicity, to construct env-chimeras with wild-type virus, pNL4-3, and a less cytopathic MDR variant, pMDR-5a (Fig. 2A; see Supplemental Digital Content, Text 1, https://links.lww.com/QAI/A627). The env-chimeras were all infectious, except for 1 chimera, pMDR-1c(envMDR-5a), which showed limited infectivity in TZM-bl cell culture (data not shown) but no infectivity in PBMC culture (Fig. 2B).
We determined the ratio of the infectious titer in PBMC culture (TCID50/ml) over the p24 concentration (pg/mL) in each virus stock (Fig. 2C). TCID50/p24 ratios of the original virus clones, pMDR-1c, pNL4-3, and pMDR-5a were 0.93, 0.66, and 0.36, respectively. TCID50/p24 was reduced in the NL4-3 env-chimera, pMDR-1c(envNL4-3), by 0.19-fold (0.18), whereas enhanced in 2 MDR-1c env-chimeras pNL4-3(envMDR-1c) by 2.4-fold (1.61) and pMDR-5a(envMDR-1c) by 2.8-fold (1.01) (Fig. 2C; see Table S2, Supplemental Digital Content, https://links.lww.com/QAI/A627). These results suggested that infectivity per virion in the respective viral stock was in large part determined by envelope, and that the envelope of pMDR-1c gave the highest infectivity/p24 ratio when inserted into all 3 viral backbones. Note that all virus stocks were produced, stored, and titrated for infectivity simultaneously, thereby controlling experimental conditions and minimizing procedure-induced variation in determining infectious titers.
Growth Competition Assays
To determine the relative fitness of viruses/chimeras and their subgenomes, we set up 8 different growth competition cultures with various combinations of paired viruses (Fig. 3A). Changes in the fractional viral RNA copy numbers (%) over days of culture are shown for each competition culture. Apparently, pMDR-1c grew much faster than pNL4-3 (#1) or pMDR-5a (#2). The env-chimera, pNL4-3(envMDR-1c), grew faster than pNL4-3 (#3) or pMDR-1c (#4), whereas the env-chimera, pMDR-5a(envMDR-1c), grew faster than pMDR-5a (#5) but slower than MDR-1c (#6). In contrast, the env-chimera, pMDR-1c(envNL4-3), grew slower than pMDR-1c (#7) or pNL4-3 (#8). These results suggested that the fitness of the envelope of pMDR-1c is higher than the envelope of pNL4-3 (#3, #7) or the envelope of pMDR-5a (#5). The fitness of backbone of pMDR-1c is lower than the backbone of pNL4-3 (#4, #8), but higher than the backbone of pMDR-5a (#6).
Estimating Relative Fitness of Envelope and Backbone Genome in pMDR-1c and pMDR-5a
For the quantitative comparison, the relative fitness value for each virus was estimated as described in the Methods section (see Tables S3 and S4A, Supplemental Digital Content, https://links.lww.com/QAI/A627). Relative fitness value was high in pMDR-1 [1.62 with 95% confidence intervals (1.42, 1.85)] and low in pMDR-5a [0.82 (0.69, 0.97)] as compared with the reference virus, pNL4-3 (1.00). For the env-chimeras, relative fitness value was very high in pNL4-3(envMDR-1c) [2.17 (1.94, 2.43)], low in pMDR-1c(envNL4-3) [0.56 (0.39, 0.81)], or equivalent to pNL4-3 in pMDR-5a(envMDR-1c) [1.06 (0.94, 1.19)] (Fig. 3A).
To determine the contribution of subgenomes to fitness, we estimated relative fitness values for envelope and backbone in each virus (see Table S4B, Supplemental Digital Content, https://links.lww.com/QAI/A627). The relative fitness of pMDR-1c envelope was calculated as high as 2.29 (2.05, 2.56), whereas the relative fitness of its backbone was 0.56 (0.39, 0.81). Similarly, the relative fitness of pMDR-5a envelope was as high as 1.64 (1.42, 1.90), and the relative fitness of its backbone was as low as 0.49 (0.46, 0.53) (Fig. 3C). Given the reduced fitness of the backbone of pMDR-1c, which is composed of all genes except for envelope gene, the envelope is considered as the major determinant of the enhanced fitness of the MDR virus. Similarly, the envelope of pMDR-5a contributed to the fitness of the virus, yet the fitness of pMDR-5a did not exceed the fitness of pNL4-3.
We used viruses derived from 2 patients with contrasting clinical profiles both infected with MDR HIV-1 to probe the issue of viral fitness in the context of multidrug resistance. Critical to our studies were the construction of biologically relevant molecular clones of the MDR HIV variants. By selecting biologically clonal isolates that are reflecting the growth characteristics of bulk isolates, and subsequently demonstrating comparable replication characteristics of molecular clones to their source clonal isolates, we believe our findings to be relevant. In our experiments, we separated the HIV-1 genome into 2 parts: the envelope and the remaining backbone. By using env-chimeras and growth competition assay, we could estimate the relative fitness of each segment independently. This allowed us to determine the contribution of each subgenome to the overall fitness of the virus. The backbone genomes of 2 MDR viruses had reduced fitness, likely because of the presence of the resistant conferring mutations that in turn affect the activity of RT and protease. The relative fitness of the backbone of pMDR-5a was lower than that of pMDR-1c, perhaps because of its mutations, M184V and T215Y, which are known to exert a substantial impact on viral fitness.27 In contrast, the envelope of pMDR-1c was found to have a relative fitness of 229% as compared with the envelope of pNL4-3.
We believe this compensated for the reduced fitness of the backbone genome, calculated to be 56%, and actually resulted in an increase in the overall fitness of the complete virus to 162%, which far exceeded that of the reference laboratory strain, pNL4-3. Thus, it would seem that the enhancing effect of the envelope of the cytopathic virus, pMDR-1c, more than compensates for the fitness loss conferred by the emergence of drug-resistance mutations in constitutive enzyme-coding regions. This is in contrast with the less cytopathic MDR virus, pMDR-5a, in which an enhanced fitness of the envelope (164%) compensated for the reduced fitness of the backbone genome (49%), but the overall fitness of the virus (82%) did not exceed the wild-type virus. Given the dramatic differences in clinical presentation seen between the 2 cases, this observation does support the contention that viral factors may play a significant role in the course of HIV-1 infection in vivo.
Note that we constructed env-chimeras by replacing the entire gp160 region, which also encodes the second exons of tat and rev, 3'half of vpu and the rev response element (Fig. 2A), thus it is conceivable that the observed enhanced fitness may result from these gene products rather than envelope. However, we believe the following observations support the major role of the envelope protein in conferring increased fitness. First, the TCID50/p24 ratio in the transfection-derived virus stock of pMDR-1c or 2 chimeras with MDR-1c envelope was 2- to 5-fold higher than the virus/chimeras with NL4-3 envelope or MDR-5a envelope (Fig. 2C). The ratio of TCID50 over p24 in virus stock or “infectivity per particle” is a reflection of the initial steps of infection. This ratio has been used by others as a measurement of env function.28 Although tat, rev, and vpu have critical roles in the viral life cycle, their activity resides more in levels of virus production as opposed to these initial steps in the viral life cycle.28–31 Second, the virus production on transfection of pMDR-1c plasmid DNA was not different from that of pNL4-3 (289 ng/mL vs. 292 ng/mL), which does not support a significant effect of tat, rev, or vpu. Nonetheless, further studies are needed to conclusively prove that the envelope protein is conferring the observed replication advantage. This could be accomplished with the use of site-directed mutagenesis of the env-coding region.
The viral envelope is known to play crucial roles during the early steps of viral life cycle: (1) a surface protein, gp120 mediates viral binding to the receptors, the CD4 molecule and coreceptor, CCR5 or CXCR4, or other receptors, and (2) a transmembrane protein, gp41 mediates membrane fusion/semifusion between virion and target cell, or the infected cell and uninfected cell.31–33 Thus, envelope is critical in viral entry and determines the infectivity, cell tropism, and cytopathicity.34–36 The comparison of envelope amino acid sequences among 3 clones, especially with pMDR-1a (R5, intermediate growing virus) indicated the difference within V3 loop, which explains cell tropism as described earlier. There are amino acid differences between less cytopathic pMDR-1a and cytopathic pMDR-1b and -1c in gp41, especially within HR-2 region. Given the critical role of gp41 in membrane fusion activity, these changes may be associated with the enhanced pathogenicity in vivo.25 However, at this time, we have yet to determine which functions of the envelope are the most critical for the enhanced fitness and cytopathicity we have observed. Measuring the affinity of the envelope to the CD4 molecule and its coreceptors, as well as determining its membrane fusion activity may provide some insights into the mechanisms whereby the envelope confers fitness advantage.
We compared the effects of the envelope on the backbone genome in 2 MDR HIV-1 variants to determine whether the observations seen in pMDR-1c were unique. Although pMDR-5a was constructed from a patient with slow disease progression, a similar profile was observed (Fig. 3C). The backbone genome was defective compared with NL4-3; however, this was compensated by a fitter envelope, although the degree of fitness enhancement was less than that observed for pMDR-1c. This suggests that MDR viruses may use envelope as a compensatory mechanism for the reduced fitness. This is consistent with our earlier study,37 in which the replication capacity of MDR viruses was higher than that of drug-sensitive wild-type viruses when measured by GHOST cell assay, a single-cycle infectivity assay where the viral input was normalized by p24, suggesting that a higher TCID50/p24 ratio may have been responsible for the observed increased fitness in vitro. The fitness compensation by envelope may not be unique to MDR viruses. For example, Das et al38 reported that the reduced viral replication with impaired translation could be rescued by adaptive mutations in env.
How did this MDR virus with enhanced fitness arise? We speculate that in the treated patient failing therapy, the MDR virus first compensated for reduced fitness by the selection for secondary mutations within the targeted molecules,39 ie, protease and RT proteins, and by the mutations within Gag cleavage sites, and perhaps by the mutations within MA protein as well, with these changes being maintained by drug pressure. However, further compensation occurred by the changes in the envelope glycoprotein through stepwise mutations. On transmission, this MDR virus started reverting toward wild type or recombined with a co-infected wild-type virus and gained further fitness. In fact, pMDR-1c had lost M184V, T215Y, and L210W, which were detected in the earliest sample as a mixture.1 Thus, the enhanced fitness of RT, together with the pre-existed enhanced fitness of the envelope, could have generated a highly pathogenic virus. Additionally, although not defined here, it is likely that host factors may also contribute to viral evolution in vivo and the emergence of HIV-1 viral variants.
A number of studies have concluded that MDR viruses demonstrate impaired replication capacity or fitness. However, this conclusion was based on the determination of RC values of a recombinant virus derived from the patient pol gene products being inserted into a cassette. As we have demonstrated in this study, drug-resistant virus may use envelope, an area outside of the product of the pol gene, to compensate for apparent reduced fitness. Thus, we believe that full understanding of the fitness and cytopathicity of MDR viruses requires study of the intact virus, and not chimeric viruses with only portions of the viral genome included.
In summary, we have demonstrated that the envelope is a major determinant of enhanced fitness of a cytopathic MDR virus isolated from an HIV-1–infected patient with rapid progression. Furthermore, in the 2 viruses studied, the enhanced fitness conferred by the envelope compensated for the reduced fitness conferred by the backbone genome of MDR viruses. Further studies are needed to understand whether this phenomenon is restricted to the 2 viruses studied here, or perhaps is a more global mechanism of viral evolution to compensate for deleterious mutations that arise as a consequence of drug resistance that reduces fitness in the absence of drug-selective pressure.
The authors thank Mark Muesing for helpful discussion; Kristina Rodriguez, Brandi Davis, Amir Figueroa, Aliza Lloyd, and Leslie St. Bernard for technical assistance; Wendy Chen for preparation of Figures and Tables. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: TZM-bl cells from Dr. John C. Kappes, Dr. Xiaoyun Wu, and Tranzyme Inc.; pNL4-3 from Dr. Malcolm Martin; p89.6 from Dr. Ronald G. Collman; pJRCSF from Dr. Irvin SY Chen and Dr. Yoshio Koyanagi.
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