Evidence for a cytopathogenicity determinant in HIV-1 Vpr

Abstract

HIV-1 is cytopathic for CD4(+) T lymphocytes in vitro and this property of HIV-1 is generally considered to account for some of its in vivo cytopathogenicity. Thus, the extent of lymphocyte depletion correlates with the level of viremia whereas low levels of viral replication are typically associated with stable lymphocyte levels and asymptomatic infection such as is observed in non-progressors. Here, we describe a non-progressor who did not fit this general pattern in that CD4(+) T lymphocyte homeostasis was maintained in the face of high-level viral replication. Biological viral isolates from this patient replicated in primary lymphocytes without inducing cytopathicity. Because this phenotype is reminiscent of Vpr-deleted viruses, we examined the contribution of the Vpr gene to the viral phenotype. Vpr alleles derived from this patient contained both premature stop codons and an unusual Q3R polymorphism. Insertion of patient-derived Vpr alleles or a Q3R substitution into a cytopathic HIV-1 clone resulted in a marked impairment of cytopathicity without affecting viral replication efficiency. The effect of Vpr on cytopathicity was unrelated to reported activities of Vpr including virion association, interaction with uracil DNA glycosylase, G(2) arrest, or enhancement of macrophage infection but correlated with the ability of Vpr to induce host cell apoptosis. This study suggests the presence of a determinant of in vivo cytopathogenicity within HIV-1 Vpr and further indicates that viral replication can be uncoupled from cytopathicity in vitro and in vivo.

Figure 1

Patient characteristics and impact of Vpr mutations on viral cytopathicity. (A) Plasma viral RNA and CD4 lymphocyte profiles in patient P1016 with asymptomatic infection. Time intervals are shown relative to initiation of Nevirapine monotherapy. (B) Vpr truncation in virus isolates established from P1016 immediately before initiation of therapy. (C) In vitro cytopathic phenotype of wild-type and accessory gene deletion mutants of HIV-1.

Figure 2

Cytopathic activity of Vpr alleles derived from plasma viral RNA. (A) Frequency of Vpr alleles containing premature stop codons as assessed by oligonucleotide probe-specific hybridization. Bacterial colonies transformed with wild-type, Vpr_stop and patient-derived Vpr alleles were hybridized with probes recognizing wild-type or truncated Vpr sequences. Hybridization conditions were adjusted to promote specific binding of each probe to its cognate sequence. One of four clones hybridizing specifically to the Vpr stop probe (clone 0) and four hybridizing specifically to the wild-type Vpr probe (clones I-IV) were sequenced (B). Consensus Vpr sequences were derived from 101 clade B Vpr sequences listed in the Los Alamos HIV-1 Sequence Database. (C and D) Replication and cytopathicity of X4-tropic (C) and R5-tropic (D) HIV-1 LAI variants containing wild-type and patient-derived Vpr alleles.

Figure 3

In vitro activities of patient-derived Vpr alleles. (A) Analysis of relative levels of cell-associated and virion-associated Vpr proteins. (B) Schematic of HIV-1 LAI/ADA, an R5-tropic variant of HIV-1 LAI that contains envelope sequences spanning the V3 region of HIV-1_ADA. (C) Replication profiles of LAI/ADA variants containing LAI Vpr and patient Vpr alleles in monocyte-derived macrophages. (D) Interaction of Vpr with uracil DNA glycosylase. (E) Induction of G₂ arrest. G₁ and G₂ profiles for 293T cells expressing wild-type and patient-derived Vpr clones was determined specifically for productively infected cells.

Figure 4

Influence of the Q3R polymorphism on cytopathic and apoptotic activities of Vpr. (A) Cell viability and viral replication in CD8 depleted CD4⁺ T lymphocytes infected with wild-type HIV-1 LAI and an LAI variant containing a Q3R Vpr mutation. (B) Apoptotic activity of HIV-1 LAI/ADA variants containing LAI Vpr or a patient-derived Vpr allele (clone I). (C) Effect of amino acid substitutions on apoptotic activity of Vpr. The indicated amino acid substitutions were inserted within either LAI Vpr or a patient-derived vpr allele (clone I). The Q3R polymorphism in the patient-derived Vpr allele (clone I) was mutated to a Q3 consensus sequence (clone I R3Q). W54R and R90K substitutions impair UDG-association and G₂ arrest activities of Vpr, respectively. For ease of comparison, apoptotic activity was normalized to values obtained for the parental LAI Vpr.