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. 2002 Jul;76(14):6966-73.
doi: 10.1128/jvi.76.14.6966-6973.2002.

Coreceptor phenotype of natural human immunodeficiency virus with nef deleted evolves in vivo, leading to increased virulence

Andreas Jekle  1 , Birgit Schramm, Prerana Jayakumar, Verena Trautner, Dominique Schols, Erik De Clercq, John Mills, Suzanne M Crowe, Mark A Goldsmith
Affiliations

Coreceptor phenotype of natural human immunodeficiency virus with nef deleted evolves in vivo, leading to increased virulence

Andreas Jekle et al. J Virol. 2002 Jul.

Abstract

The Sydney Blood Bank Cohort is a group of patients with slowly progressive infection by a human immunodeficiency virus strain containing spontaneous deletions within the nef long terminal repeat region. In 1999, 18 years after the initial infection, one of the members (D36) developed AIDS. In this work, we used an ex vivo human lymphoid cell culture system to analyze two viral isolates obtained from this patient, one prior to the onset of AIDS in 1995 and one after disease progression in 1999. Both D36 isolates were less potent in depleting CD4(+) T cells than a reference dualtropic, nef-bearing viral isolate. However, the 1999 isolate was measurably more cytotoxic to CD4(+) T cells than the 1995 isolate. Interestingly, although both isolates were nearly equally potent in depleting CCR5(+) CD4(+) T cells, the cytotoxic effect of the 1999 isolate toward CCR5(-) CD4(+) T cells was significantly higher. Furthermore, GHOST cell infection assays and blocking experiments with the CXCR4 inhibitor AMD3100 showed that the later D36 1999 isolate could infect both CCR5(+) and CCR5(-) CXCR4(+) cells efficiently, while infection by the 1995 isolate was nearly completely restricted to CCR5(+) cells. Sequence analysis of the V1/V2 and V3 regions of the viral envelope protein gp120 revealed that the more efficient CXCR4 usage of the later isolate might be caused by an additional potential N-glycosylation site in the V1/V2 loop. In conclusion, these data show that an in vivo evolution of the tropism of this nef-deleted strain toward an X4 phenotype was associated with a higher cytopathic potential and progression to AIDS.

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Figures

FIG. 1.
FIG. 1.
D36/99 isolate depletes CD4+ T cells more efficiently and replicates faster than D36/95 in ex vivo human lymphoid cell cultures. (A) CD4 depletion induced by infection with viral strains D36/99, D36/95, 7/86, 1/85, NL4-3, and 49-5 was assessed by FACS analysis at the indicated time points. Shown are the mean relative CD4/CD8 ratios (n = 3) with standard error of the mean (SEM) of a representative experiment from among experiments with six different donor tissues. (B) Viral replication was monitored in the same infections by assessing the accumulation of p24 in the culture medium at days 3, 6, 9, and 12 after infection with an anti-p24 ELISA. Shown are the mean values (n = 3) with SEM of a representative experiment from among experiments with six different donor tissues.
FIG. 2.
FIG. 2.
D36/99 induces high levels of apoptosis among CD4+ T cells in human lymphoid cell cultures. Apoptosis in CD4+ T cells induced by infection with viral strains D36/99, D36/95, 7/86, 1/85, NL4-3, and 49-5 was measured at the indicated time points independently by annexin V binding (A), activation of caspase-3 (B), and depolarization of the mitochondrial membrane potential (C) by FACS analysis. Shown are the mean values (n = 3) with SEM of a representative experiment from among experiments with six different donor tissues.
FIG. 3.
FIG. 3.
D36/99 induces depletion and apoptosis of both CCR5+ and CCR5− CD4+ T cells in human ex vivo lymphoid cell cultures. CD4 depletion induced by infection with viral strains D36/99, D36/95, 7/86, and 1/85 within the CCR5+ (A) and CCR5− (B) subsets of CD4+ T cells was analyzed at the indicated time points by multiparameter FACS analysis. Shown are the mean relative CD4/CD8 ratios (n = 3) with SEM of a representative experiment from among experiments with six different donor tissues. In the same infections, apoptosis was measured in the CCR5+ (C) and CCR5− (D) CD4+ T-cell subsets by annexin V binding. Shown are the mean values (n = 3) with SEM of a representative experiment from among experiments with six different donor tissues.
FIG. 4.
FIG. 4.
D36/99 can use CXCR4 as a coreceptor more efficiently than D36/95. (A) GHOST-R5 (open bars) and GHOST-X4 cells (solid bars) were infected with D36/99, D36/95, NL4-3, and 49-5 at an MOI of 0.1 and cultured for 72 h. Infected cells were identified by FACS analysis. Shown are the mean values (n = 3) with SEM of a representative experiment from among three independent experiments. (B) CXCR4+ CCR5+ HeLa CD4 cells were pretreated for 12 h with the indicated concentrations of AMD3100 and infected with D36/99, D36/95, NL4-3, and 49-5 at an MOI of 0.01. Viral replication was measured 72 h after infection by assessing the concentration of p24 in the culture medium. Shown are the mean values (n = 3) with SEM of a representative experiment from among three independent experiments.
FIG. 5.
FIG. 5.
D36/99 depletes and induces apoptosis in both the CCR5+ and CCR5− subsets of CD4+ T cells in human ex vivo lymphoid cultures. Human ex vivo lymphoid cell cultures were pretreated with AMD3100 (250 nM) for 12 h and infected with D36/95 and D36/99. AMD3100 was replenished in the cultures at each medium change. Depletion of total CD4+ T cells (A) and of the CCR5+ and CCR5− subsets of CD4+ T cells (C) as well as apoptosis of total CD4+ T cells (B) were measured by FACS analysis 12 days after infection. Shown are the mean values (n = 3) with SEM of a representative experiment from among experiments with four different donor tissues.
FIG. 6.
FIG. 6.
V1/V2 loop of D36/99 contains an additional potential N-glycosylation site. The top panel shows the gp120 V1/V2 and the bottom panel shows the gp120 V3 loop sequence of both D36 isolates. Potential N-glycosylation sites are printed in bold, and changes between the D36/95 and the D36/99 isolates are highlighted by a box. Amino acid numbers are based on the D36/95 sequence.
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