Phenotype and envelope gene diversity of nef-deleted HIV-1 isolated from long-term survivors infected from a single source
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The Sydney blood bank cohort (SBBC) of long-term survivors consists of multiple individuals infected with attenuated, nef-deleted variants of human immunodeficiency virus type 1 (HIV-1) acquired from a single source. Long-term prospective studies have demonstrated that the SBBC now comprises slow progressors (SP) as well as long-term nonprogressors (LTNP). Convergent evolution of nef sequences in SBBC SP and LTNP indicates the in vivo pathogenicity of HIV-1 in SBBC members is dictated by factors other than nef. To better understand mechanisms underlying the pathogenicity of nef-deleted HIV-1, we examined the phenotype and env sequence diversity of sequentially isolated viruses (n = 2) from 3 SBBC members.
The viruses characterized here were isolated from two SP spanning a three or six year period during progressive HIV-1 infection (subjects D36 and C98, respectively) and from a LTNP spanning a two year period during asymptomatic, nonprogressive infection (subject C18). Both isolates from D36 were R5X4 phenotype and, compared to control HIV-1 strains, replicated to low levels in peripheral blood mononuclear cells (PBMC). In contrast, both isolates from C98 and C18 were CCR5-restricted. Both viruses isolated from C98 replicated to barely detectable levels in PBMC, whereas both viruses isolated from C18 replicated to low levels, similar to those isolated from D36. Analysis of env by V1V2 and V3 heteroduplex tracking assay, V1V2 length polymorphisms, sequencing and phylogenetic analysis showed distinct intra- and inter-patient env evolution.
Independent evolution of env despite convergent evolution of nef may contribute to the in vivo pathogenicity of nef-deleted HIV-1 in SBBC members, which may not necessarily be associated with changes in replication capacity or viral coreceptor specificity.
KeywordsCoreceptor Usage Sydney Blood Bank Cohort D36L Clone R5X4 Phenotype
The Sydney blood bank cohort (SBBC) of long-term survivors (LTS) consists of multiple individuals who became infected with an attenuated strain of HIV-1 via contaminated blood products from a common blood donor between 1981 and 1984 [1, 2, 3]. Viral attenuation has been attributed to gross deletions in the nef and nef/long-terminal repeat (LTR) overlapping regions of the HIV-1 genome. Despite being infected from a single source, long-term prospective studies on SBBC members demonstrated that the cohort now consists of subjects with slow disease progression (SP), as well as individuals who remain true long-term nonprogressors (LTNP) and antiretroviral therapy-naive with stable CD4 counts and low or undetectable HIV-1 RNA levels . Three SBBC members (one SP and two LTNP) have since died from causes unrelated to HIV-1 infection [3, 4]. Although the cohort members had differing clinical courses, a comprehensive longitudinal analysis of nef/LTR sequences in the SBBC donor and four of the transfusion recipients demonstrated a convergent pattern of nef sequence evolution, characterized by progressive sequence deletions evolving toward a minimal nef/LTR structure that retains only the key sequence elements that are required for viral replication . Thus, HIV-1 pathogenicity in SBBC members is dictated by viral and/or host determinants other than those that impose a unidirectional selection pressure on the nef/LTR region of the HIV-1 genome.
The HIV-1 env gene, which encodes the viral envelope glycoproteins (Env) is a significant viral determinant in HIV-1 pathogenesis [reviewed in [5, 6, 7]]. HIV-1 Env initiates viral entry via binding to CD4 and subsequently to a coreceptor, either CCR5 [8, 9, 10, 11, 12] or CXCR4 . CCR5-using (R5) HIV-1 strains predominate at early, asymptomatic stages of infection. In 40–50% of infected adults, progression of HIV-1 infection is accompanied by a switch in coreceptor specificity to HIV-1 variants able to use CXCR4 or both CCR5 and CXCR4 for entry (X4 or R5X4 strains, respectively) [14, 15]. A switch in the specificity of HIV-1 Env from R5 to X4 or R5X4 is considered an indicator of poor prognosis, partly because it increases the number of CD4+ cells that are susceptible to cytolytic infection by HIV-1, and is associated with rapid progression of HIV-1 infection. R5 HIV-1 variants are present exclusively in the remaining 50–60% of infected individuals who progress to AIDS, without switching coreceptor specificity [16, 17], and exert pathogenic effects that contribute to HIV-1 progression via mechanisms that remain poorly understood . Thus, changes in HIV-1 env that affect viral tropism are important for progression of HIV-1 infection.
Analysis of inter- and intra-host evolution of env sequence has provided important insights relevant for HIV-1 transmission and progression. While several reports showed an inverse relationship between the rate and extent of viral diversification and progression of HIV-1 infection [18, 19, 20, 21, 22, 23, 24, 25], other studies demonstrated that disease progression is associated with increasing rates of viral diversity [26, 27, 28]. A later study made significant headway in reconciling these conflicting studies by identifying 3 distinct phases of HIV-1 env sequence diversity and divergence during the asymptomatic period preceding the development of AIDS ; an early phase of variable duration with linear increases (approximately 1% per year) in both viral divergence and diversity; an intermediate phase characterized by a continued increase in viral divergence but with a stabilisation or decline in viral diversity; and a late phase characterized by a stabilisation of viral divergence and a continued stability or decline in viral diversity. The emergence of X4 HIV-1 variants often coincided with transition between the early and intermediate phases. More recent studies identified convergent sequence evolution in env during the early phase toward a common ancestral sequence , suggesting that HIV-1 recovers certain ancestral features early in HIV-1 infection that most likely serve to restore viral fitness. However, other studies examining HIV-1 progression in individuals harbouring only R5 variants showed an increase in viral diversity in viral isolates obtained from patients with AIDS compared to isolates from asymptomatic individuals , raising the possibility that selection pressures driving HIV-1 evolution may be distinct in patients who maintain R5 viral variants compared to those who experience a coreceptor switch.
While the viral determinants underlying the pathogenicity of nef-deleted HIV-1 strains harbored by SBBC members are presently unknown, several lines of evidence support the hypothesis that evolution of HIV-1 env contributes to disease progression in this cohort; 1) compartmentalized evolution of HIV-1 V3 env sequence in cerebrospinal fluid (CSF) of the SBBC donor was shown to contribute to the development of HIV-associated dementia (HIVD) ; 2) enhanced cell killing in ex vivo human tissue cultures by HIV-1 isolates from the same SBBC subject was predicted to result from more efficient coreceptor usage ; and 3) increased Env-mediated fusion was shown to increase the in vivo pathogenicity of nef-deleted simian immunodeficiency virus (SIV) .
To better understand the role of HIV-1 env in the pathogenesis of nef-deleted HIV-1 strains harbored by SBBC members, we examined the phenotype and env sequence diversity of sequential viruses isolated from 3 SBBC members. Isolates from the SBBC "donor" (subject D36; SP) were R5X4 phenotype and replicated to low levels in peripheral blood mononuclear cells (PBMC). In contrast, isolates from 2 SBBC "recipients" (subjects C98 and C18; SP and LTNP, respectively) were CCR5-restricted with variable replication kinetics. Analysis of env by V1V2 and V3 heteroduplex tracking assay, V1V2 length polymorphisms, sequencing and phylogenetic analysis showed distinct intra- and inter-patient env evolution. Thus, independent evolution of env despite convergent evolution of nef may contribute to the in vivo pathogenicity of nef-deleted HIV-1 in SBBC members, which may not necessarily be associated with changes in replication capacity or viral coreceptor specificity.
Results and discussion
Subjects and laboratory studies
Date of infection
Date of blood sample
CD4+ T-cell count (cells/μl)a
Viral load (RNA copies/ml)b
Status of HIV-1 progressiond
ABC, AZT, NVP (1/1999–9/2004)
ABC, NVP, 3TC (9/2004-present)
d4T, NVP, IND (11/1999-death)
HIV-1 isolated from PBMC on 2 consecutive occasions was used in this study (Table 1). The time between isolations ranged from 2 to 6 years. For the purpose of this report, the initial isolates are referred to as "early" isolates, and the subsequent isolates are referred to as "late" isolates (designated "E" and "L", respectively).
V1V2 and V3 HTA analysis
Changes in the dominant viral quasispecies may serve to augment HIV-1 pathogenicity in vivo without increasing replication capacity or changing coreceptor preference in vitro . Therefore, to determine whether distinct viral variants are present in early and late D36, C18 and C98 viruses, Env V1V2 and V3 HTA analyses were conducted.
The V3 heteroduplex patterns also demonstrated distinct viral variants using either probe (Fig. 4). However, the resolution of V3 heteroduplexes was more readily achieved using the NL4-3 probe. V3 HTAs demonstrated 4 major variants in C18L that were distinct from a single variant present in C18E; 2 major variants in C98L that were distinct from a single major variant present in C98E; and 2 major variants in D36L that were distinct from 2 major variants present in D36E. Similar to results of the V1V2 HTAs (Fig. 3), the V3 heteroduplexes also appeared to be distinct between subjects. Together, the results of the V1V2 and V3 HTAs suggest significant inter- and intra-patient evolution of HIV-1 Env. In contrast to convergent sequence evolution previously reported for HIV-1 nef in the study subjects , the V1V2 and V3 HTA results suggest independent evolution of HIV-1 Env.
V1V2 length polymorphism analysis
These results suggest that significant evolution of V1V2 Env occurred in each of the study subjects, an interpretation supported also by results of the V1V2 HTA analysis (Fig. 3). That C18E and C98E viruses contained dominant variants with identical V1V2 nt length raises the possibility that these 2 subjects once harboured Env variants with some shared features. However, the increase in number of V1V2 length polymorphisms in D36L and C18L viruses compared to D36E and C18E viruses, respectively, the shift in dominant V1V2 length polymorphism in C98 viruses, and the lack of overlap between V1V2 length variants detected in D36L, C98L and C18L viruses suggests divergent evolution of HIV-1 Env in these SBBC study subjects. In contrast to previous studies [23, 42], long-term survival of HIV-1 infection in these subjects was not associated with increased V1V2 nt length. Furthermore, significant increases in V1V2 nt length diversity were observed in late viruses from a SP (D36) and a LTNP (C18) compared to respective early viruses, and no increase in V1V2 nt length diversity was observed in late virus from a SP (C98); this suggests that divergent evolution of HIV-1 Env in the study subjects was neither necessary nor sufficient for disease progression.
The base of the V1V2 stem contains a highly conserved potential N-linked glycosylation site in the CNTS sequence of NL4-3 (Fig. 7A), which is present in all but seven of 208 clade B HIV-1 Env sequences screened from the Los Alamos National Laboratory HIV Database . One of 3 D36E clones and 2/3 D36L clones lacked a potential N-linked glycosylation site at this position (Fig. 7A). Similarly, the glycosylation site at this position was lacking in 1/3 C98E and 1/3 C98L clones. In contrast, the glycosylation site at the V1V2 stem was conserved among all C18E and C18L clones, and in contrast to D36 and C98 clones there was a high degree of sequence homology in this region to that of NL4-3. Elimination of a glycosylation site at this position is sufficient for CD4-independent infection by HIV-1 ADA, achieved by altering the position of the V1V2 loops and exposing the coreceptor binding site in gp120 [49, 50, 51, 52]. Thus, alterations in glycosylation at the V1V2 stem may serve to enhance receptor binding, which could contribute to HIV-1 pathogenicity at later stages of HIV-1 infection. To this end, it is interesting to note that Env clones lacking this glycosylation site were present only in SBBC slow progressors (D36 and C98), whereas the glycosylation site was present in all Envs from the LTNP (C18). Further sequence analysis of a greater number of Env clones is required to determine the significance of this sequence change in SBBC SPs and LTNPs. In addition, further studies to biologically characterize these Envs are required to determine whether SBBC Envs exhibit functional changes that could potentially contribute to HIV-1 pathogenicity.
In this study, we analyzed the phenotype and Env sequences of HIV-1 present in 3 SBBC members who were slow progressors or long-term nonprogressors. Early and late viruses from D36 were R5X4 whereas viruses isolated from C98 and C18 remained CCR5-restricted, indicating that a coreceptor switch was neither necessary nor sufficient for disease progression in these subjects. Replication capacity of these viruses in PBMC ranged from rapid to barely detectable and was not associated with disease progression. Although SBBC subjects had evidence of convergent evolution of nef sequence , analysis of Env diversity by V1V2 and V3 HTA, V1V2 length polymorphism assay, and maximum likelihood phylogeny suggest that Env sequence evolution was divergent in SP and LTNP subjects. Our results suggest that evolution toward a pathogenic Env phenotype may occur in long-term survivors infected with nef-deleted HIV-1, which is not necessarily associated with changes in replication capacity or coreceptor usage, or degree of Env sequence diversity.
Isolation of HIV-1
HIV-1 was isolated from patient's PBMC by coculture with selected PBMC according to published methods . Briefly, 2 × 106 patient PBMC were mixed with 10 × 106 PHA-activated PBMC from 2 normal uninfected donors, and cocultured for 28 days in RPMI-1640 medium containing 10% (vol/vol) fetal calf serum (FCS) and 20 U/ml interleukin-2 (IL-2). Fifty percent media changes were performed twice weekly. Five million PHA-activated PBMC from a different donor were added at every second media change. Supernatants were tested for reverse transcriptase (RT) activity using [33P]dTTP incorporation as described previously . Supernatants testing positive for RT activity were filtered through 0.45 μm filters and stored at -80°C.
HIV-1 replication kinetics
Five hundred thousand PHA-activated PBMC were infected in 48-well tissue culture plates by incubation with 1 × 106 [33P] cpm RT units of virus supernatant in a volume of 250 μl for 3 h at 37°C, as described previously [32, 54]. Virus was then removed, and PBMC were washed 3 times with phosphate-buffered saline (PBS) and cultured in RPMI-1640 medium containing 10% (vol/vol) FCS and 20 U/ml IL-2 for 27 days. Fifty percent medium changes were performed twice weekly, and supernatants were tested for HIV-1 replication by RT assays on days 1, 7, 14, 21 and 28 post-infection.
Coreceptor usage by primary HIV-1 isolates was determined using Cf2-Luc cells expressing CD4 alone, or expressing CD4 together with CCR5 or CXCR4, as described previously [32, 36, 54, 55]. Briefly, Cf2-Luc cells were transfected with 10 μg of plasmid pcDNA3-CD4 and 20 μg of plasmid pcDNA3 containing CCR5 or CXCR4 using the calcium phosphate method, and infected 48 h later by incubation with 1 × 106 [33P] cpm RT units of HIV-1 in the presence of 2 μg of Polybrene (Sigma) per ml. After overnight infection, virus was removed and the cells were cultured for an additional 48 h prior to lysis in 200 μl of cell lysis buffer (Promega, Madison, Wis.). Cell lysates were cleared by centrifugation, and assayed for luciferase activity (Promega) according to the manufacturer's protocol.
V1V2 and V3 HTA
The V1V2 probes were generated by PCR amplification of a 282 bp fragment of the HIV-1 ADA or NL4-3 Env using primers SK122 (5'-CAAGCCTAAAGCCATGTGTA-3'; corresponding to nucleotide positions 6561 to 6580 of NL4-3) and SK123 (5'-TAATGTATGGGAATTGGCTCAA-3'; corresponding to nucleotide positions 6822 to 6843 of NL4-3). The V3 probes were generated by PCR amplification of a 239 bp fragment of the HIV-1 ADA or NL4-3 Env using primers V3c (5'-CCATAATAGTACAGCTGAATG-3'; corresponding to nucleotide positions 7062 to 7081 of NL4-3) and V3d (5'-ATTTCTGGGTCCCCTCCTGAGGATTG-3'; corresponding to nucleotide positions 7276 to 7301 of NL4-3). Labelling was achieved by incorporation of α- [32P]-dCTP in the PCR, which consisted of an initial denaturation at 95°C for 5 min, followed by 25 cycles of 95°C for 30 sec, 52°C for 1 min, and 72°C for 2 min followed by a final extension step of 72°C for 7 min. Unincorporated nucleotides were removed using a QIAquick spin column (Qiagen). The V1V2 and V3 Env target DNA sequences were generated from genomic DNA of PBMC infected with each primary HIV-1 isolate by PCR using primers SK122/SK123 and V3c/V3d, respectively. Genomic DNA of PBMC infected with HIV-1 ADA, NL4-3 or 89.6 was used as controls. PCR reactions proceeded as described above, except that radiolabelled dCTP was not included. Amplimers were purified using a QIAquick spin column (Qiagen). Heteroduplex reactions were performed as described previously  with the following modifications. The reactions consisted of 1× annealing buffer [1 M NaCl, 100 mM Tris-HCL (pH 7.5), 20 mM EDTA], 5 μl unlabelled V1V2 or V3 target PCR product, and 2.5 μl labelled V1V2 or V3 probe. The reactions were denatured at 95°C for 4 min and then allowed to anneal on wet ice for 5 min. The heteroduplexes were separated in 5.5% (wt/vol) polyacrylamide gels in 1× Tris-borate-EDTA buffer, and were visualized by autoradiography of dried HTA gels.
V1V2 length polymorphism analysis
V1V2 length polymorphisms in HIV-1 Env were quantified using a fluorescent-based assay that has been described in detail previously . This technique measures HIV-1 sequence diversity by taking advantage of frequent length polymorphisms that occur within the V1V2 region of HIV-1 Env, and has the sensitivity to detect a single nucleotide deletion or insertion. Briefly, the V1V2 region of HIV-1 Env was amplified from genomic DNA of PBMC infected with each primary HIV-1 isolate by nested PCR using outer primers V12-51 and V12-52, and inner primers V12-50 and V12-53, as described previously . The V12-50 primer used in the second round PCR was labelled with a fluorophore, 6-carboxy-fluorescien, at the 5' end (PE Biosystems). PCR amplified, fluorescently labelled products were purified using QIAquick spin columns (Qiagen), separated in 6% (wt/vol) denaturing polyacrylamide gels using an automated sequencer (ABI PRISM 377; PE Biosystems) and analysed using GeneScan software (PE Biosystems). Peaks with areas <10% of the total peak area were considered not significant, as described previously . The fraction of sequences in the viral quasispecies with a given nucleotide length was calculated from GeneScan data and plotted against nucleotide length, as described previously .
Env cloning, sequencing and phylogenetic analysis
A 2.1 kb fragment of HIV-1 Env (corresponding to nucleotide positions 6332 to 8452 in NL4-3) was amplified from genomic DNA of PBMC infected with each primary HIV-1 isolate by nested PCR using outer primers env 1A and env 1M , and inner primers env KpnI and env BamHI , as described previously , and cloned into pGEM-T Easy (Promega). The V1 to V3 region of 2 to 3 independent Envs cloned from each primary HIV-1 isolate was sequenced using a SequiTherm EXCEL II DNA sequencing kit (Epicenter Technologies, Madison, WI) and a model 4000L LI-COR DNA sequencer (LI-COR, Lincoln, NE). Nucleotide sequences were aligned using CLUSTALW and corrected by hand. Phylogeny was estimated by a maximum likelihood algorithm (DNAml) with a transition/transversion ratio of 2.0, empirical base frequencies, and a randomised input order of sequences. Bootstrap values were calculated from 100 resamplings of the same alignment using Seqboot.
Nucleotide sequence accession numbers
The V1 to V3 Env nucleotide sequences reported here have been assigned GenBank accession numbers DQ665223 to DQ665240.
We thank J. Sodroski and B. Etemad-Moghadam for providing Cf2-Luc cells, and J. Sodroski for providing CD4 and coreceptor plasmids. This study was supported, in part, by a grant from the National Health and Medical Research Council of Australia (NHMRC) to PRG (251520), a grant from the American Foundation for AIDS Research (amfAR) to DAM (106669), and grants from the National Institutes of Health to PRG (AI054207) and DG (NS37277). LG and JS are recipients of NHMRC Dora Lush Biomedical Research Scholarships. PRG is the recipient of an NHMRC R. Douglas Wright Biomedical Career Development Award.
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