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. 1998 Nov;72(11):8650-8.
doi: 10.1128/JVI.72.11.8650-8658.1998.

Potential contributions of viral envelope and host genetic factors in a human immunodeficiency virus type 1-infected long-term survivor

K Grovit-Ferbas  1 , J Ferbas, V Gudeman, S Sadeghi, M B Goetz, J V Giorgi, I S Chen, W A O'Brien
Affiliations

Potential contributions of viral envelope and host genetic factors in a human immunodeficiency virus type 1-infected long-term survivor

K Grovit-Ferbas et al. J Virol. 1998 Nov.

Abstract

The lack of clinical progression in some individuals despite prolonged human immunodeficiency virus type 1 (HIV-1) infection may result from infection with less-pathogenic viral strains. To address this question, we examined the HIV-1 envelope protein from a donor with a low viral burden, stable CD4(+) T-lymphocyte counts, and little evidence of CD8(+) T-cell expansion, activation, or immune activity. To avoid potential changes in envelope function resulting from selection in vitro, envelope clones were constructed by using viral RNA isolated from uncultured peripheral blood mononuclear cells (PBMC). The data showed that recombinant viruses containing envelope sequences derived from RNA isolated from patient PBMC replicated poorly in primary CD4(+) T cells but demonstrated efficient growth in macrophages. The unusual phenotype of these viruses could not be explained solely by differential utilization of coreceptors since the chimeric viruses, as well as an uncloned isolate obtained from the same visit date, can utilize CCR5. In addition, the donor's own cells appeared resistant to infection with chimeric viruses containing autologous envelope sequences. Genotype analysis revealed that the donor was heterozygous for the previously described 32-bp deletion in CCR5 which may be linked with prolonged survival in HIV-1-infected individuals. These data suggest that the changes in envelope sequences confer properties of viral attenuation, which together with the CCR5 +/Delta32 genotype could account for the long-term survival of this patient.

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Figures

FIG. 1
FIG. 1
Virus isolated from CD4+ T lymphocytes of donor 6 grows slowly in culture relative to that of other HIV-infected individuals. CD4+ lymphocytes from HIV-infected donors were cocultured with a pool of allogeneic CD4+ T cells and sampled at the times indicated. CD4+ lymphocytes were selected onto AIS flasks coated with anti-CD4 antibodies. Thirty million PBMC were added to each flask, and after a 1-h incubation, the nonadherent cells were removed. Seven million allogeneic CD4+ lymphocytes activated with anti-CD3 antibody (45) were added to the adherent cell population, and the cultures were maintained in the presence of 5,000 U of interleukin-2 (Chiron)/ml for 21 to 28 days. Culture supernatants were sampled every 7 days, at which time fresh medium was added to the cultures. (A) CD4+ lymphocytes from eight HIV-infected individuals. Donors are as indicated on the figure. Donor 13, a rapid progressor to disease, is included for the purposes of comparison. (B) CD4+ lymphocytes from donor 6 were tested on four different occasions.
FIG. 2
FIG. 2
Viral envelope sequences of the clones are homogeneous. Plasmid DNA from full-length clones were prepared by using an anion-exchange resin membrane (Qiagen, Inc.) and were subjected to cycle sequencing with an automated DNA sequencer (Applied Biosystems, Inc.). Potential N-linked glycosylation sites are underlined. The numbering system is based on the sequence of gp120 from HIVJRFL. Dashes indicate that the corresponding sequences of the recombinant clones containing donor 6 envelope sequences are identical to those of HIVJRFL. Amino acid substitutions are indicated, and deletions are identified by a boldface dot. (A) V1 sequences. (B) V2 sequences. (C) V3 sequences.
FIG. 3
FIG. 3
Growth kinetics of recombinant viruses on allogeneic CD4+ T cells recapitulate growth patterns of the uncloned virus. Infections were performed in 96-well microtiter plates. CD4+ lymphocytes from three donors were selected onto individual, commercially available AIS flasks coated with anti-CD4 antibodies. Each flask was treated with anti-CD3 antibody and interleukin-2 for 5 days. A pool of the three donors was prepared at the end of the incubation period, and it was cryopreserved for future use. Cells were used for infection within 2 days after thaw. For infections, 2 × 105 cells were incubated with virus made by electroporation of PBMC for 2 h as described in Materials and Methods. Clones are as indicated. Data points represent the averages of two independent experiments.
FIG. 4
FIG. 4
Recombinant viruses are capable of replicating in M∅. Infections were performed in 96-well microtiter plates. For infections, 2 × 105 cells were incubated with virus made by electroporation of PBMC for 2 h as described in Materials and Methods. M∅ from three donors were prepared, and the cells were pooled and plated for infection on day 6. Infections were performed on 7-day-old M∅. The M∅ donors were different from the CD4+ T-cell donors in Fig. 3. Clones are as indicated. Data points represent the averages of at least two independent experiments. If error bars are drawn, data points represent the averages of three independent experiments.
FIG. 5
FIG. 5
Recombinant viruses use CCR5 for entry in a single round of infection. Coreceptor usage was determined after infection of HOS cells (gift of D. Littman) expressing CD4 and one of the following chemokine receptors: CCR1, CCR2, CCR3, CCR4, CCR5, GPR15 (BONZO), STRL33 (BOB), or CXCR4. In addition, the cells contain the GFP under the control of the HIV long terminal repeat. Cultures infected by HIV-1 chimeras resulted in the production of GFP 48 h postinfection. GFP expression was measured in paraformaldehyde-fixed (2%) samples by flow cytometry (Becton Dickinson; FACScan). GHOST cells were infected with 50 to 70 ng of recombinant virus and cultured for 2 days. (A) PBMC 7. (B) PBMC 14. (C) PBMC 17. Rows: 1, GHOST CD4 cells; 2, GHOST CD4 + CCR1 cells; 3, GHOST CD4 + CCR2b; 4, GHOST CD4 + CCR3; 5, GHOST CD4 + CCR4; 6, GHOST CD4 + CCR5; 7, GHOST CD4 + BOB; 8, GHOST CD4 + BONZO; 9, GHOST CD4 + CXCR4. In this assay, approximately 10 to 20% of the cells will be positive in a single round of infection. Infections performed with vesicular stomatitis virus protein-G pseudotyped envelope yielded an average of 10% positive cells in all nine cell lines (data not shown).
FIG. 6
FIG. 6
Cells from donor 6 are less susceptible to infection by virus containing autologous envelope sequences as well as heterologous envelope sequences. Infections were performed in 96-well microtiter plates. For infections, 2 × 105 cells were incubated with 50 pg of virus for 2 h. Allogeneic M∅ (B) and CD4+ T cells (A) were used as pools as described in the legends to Fig. 3 and 4. Infections were performed on 7-day-old M∅. The M∅ donors were different from the CD4+ T-cell donors. M∅ and CD4+ T cells from donor 6 were prepared according to the same methods. Clones are as indicated. Samples were tested for p24 production at day 14.
FIG. 7
FIG. 7
Donor 6 is CCR5 +/Δ32. Genomic DNA was extracted as described in Materials and Methods. One-tenth of the amplified product was run on a 4% NuSieve agarose gel in the presence of ethidium bromide. Lanes 1 to 3 contain control samples and are labeled accordingly. Lane 4 contains the sample from donor 6. A 172-bp band is indicative of the +/+ condition (lane 3), a 140-bp band is indicative of the Δ32/Δ32 condition (lane 2), and both bands indicate the heterozygous condition +/Δ32 (lanes 1 and 4).
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