Derivation and Characterization of a CD4-Independent, Non-CD4-Tropic Simian Immunodeficiency Virus

Abstract

CD4 tropism is conserved among all primate lentiviruses and likely contributes to viral pathogenesis by targeting cells that are critical for adaptive antiviral immune responses. Although CD4-independent variants of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) have been described that can utilize the coreceptor CCR5 or CXCR4 in the absence of CD4, these viruses typically retain their CD4 binding sites and still can interact with CD4. We describe the derivation of a novel CD4-independent variant of pathogenic SIVmac239, termed iMac239, that was used to derive an infectious R5-tropic SIV lacking a CD4 binding site. Of the seven mutations that differentiate iMac239 from wild-type SIVmac239, a single change (D178G) in the V1/V2 region was sufficient to confer CD4 independence in cell-cell fusion assays, although other mutations were required for replication competence. Like other CD4-independent viruses, iMac239 was highly neutralization sensitive, although mutations were identified that could confer CD4-independent infection without increasing its neutralization sensitivity. Strikingly, iMac239 retained the ability to replicate in cell lines and primary cells even when its CD4 binding site had been ablated by deletion of a highly conserved aspartic acid at position 385, which, for HIV-1, plays a critical role in CD4 binding. iMac239, with and without the D385 deletion, exhibited an expanded host range in primary rhesus peripheral blood mononuclear cells that included CCR5(+) CD8(+) T cells. As the first non-CD4-tropic SIV, iMac239-ΔD385 will afford the opportunity to directly assess the in vivo role of CD4 targeting on pathogenesis and host immune responses.

Importance: CD4 tropism is an invariant feature of primate lentiviruses and likely plays a key role in pathogenesis by focusing viral infection onto cells that mediate adaptive immune responses and in protecting virions attached to cells from neutralizing antibodies. Although CD4-independent viruses are well described for HIV and SIV, these viruses characteristically retain their CD4 binding site and can engage CD4 if available. We derived a novel CD4-independent, CCR5-tropic variant of the pathogenic molecular clone SIVmac239, termed iMac239. The genetic determinants of iMac239's CD4 independence provide new insights into mechanisms that underlie this phenotype. This virus remained replication competent even after its CD4 binding site had been ablated by mutagenesis. As the first truly non-CD4-tropic SIV, lacking the capacity to interact with CD4, iMac239 will provide the unique opportunity to evaluate SIV pathogenesis and host immune responses in the absence of the immunomodulatory effects of CD4(+) T cell targeting and infection.

FIG 1

Replication and fusion of CD4-independent variants of SIVmac239. (A) Replication of parental SIVmac239 and an uncloned CD4-independent viral swarm is shown in CD4⁺ SupT1/RhR5 cells (left) and CD4⁻ BC7/RhR5 cells (right), each of which stably expressed rhesus CCR5. RT, reverse transcriptase activity. (B) Fusion activity of SIVmac239 and four iMac239 env clones on QT6 cells using a cell-cell fusion assay. For each env gene, the level of CD4-independent fusion on rhesus CCR5 is shown as a percentage of fusion (luciferase activity) in the presence of rhesus CD4. Background fusion levels on cells expressing GFP only were subtracted. The data shown are the means of three experiments plus the standard errors of the means (SEM). (C) Growth curves in CD4-negative, BC7/RhR5 cells are shown for wild-type (WT) SIVmac239, the iMac239 viral swarm, and four recombinant SIVmac239-based viruses bearing the indicated iMac239 env clones. RT activity in culture supernatants was measured at the indicated time points. Results from a representative experiment are shown.

FIG 2

Determinants for iMac239 Env CD4 independence in cell-cell fusion and viral replication assays. (A) Fusion activity on rhesus CCR5 in the presence or absence of rhesus CD4 is shown for SIVmac239 Envs containing the indicated single mutations from iMac239. Data from 3 experiments plus SEM are shown as described for Fig. 1B. Two-way analysis of variance (ANOVA) with multiple comparisons was used to determine significance. (B) Replication of SIVmac239-based viruses bearing the indicated Envs is shown in CD4⁺ SupT1/RhR5 (left) and CD4⁻ BC7/RhR5 (right) cells. Four changes in gp120 are sufficient to confer CD4-independent replication. A virus with D178G alone was unable to replicate in either cell type but was rescued for replication in SupT1 by H224Q. RT activity was measured at the indicated time points. Results from a representative experiment are shown.

FIG 3

Neutralization sensitivity of Envs with iMac239 mutations. Viral pseudotypes containing the indicated Envs were preincubated with various dilutions of plasma, serum, or monoclonal antibodies prior to infection of TZM-bl cells. Reciprocal 50% inhibitory dilutions (ID₅₀) for plasma and serum are color coded (<100, green; 100 to 1,000, yellow; 1,000 to 100,000, orange; >100,000, red). Fifty percent inhibitory concentrations (IC₅₀) of monoclonal antibodies (mAb) are color coded (>2 μg/ml, green; 0.2 to 2 μg/ml, yellow; <0.01 to 0.2 μg/ml, red). (A) Neutralization of SIVmac239 and iMac239 relative to two viruses (SIVmac239 D178G and SIVmac239 D179G H224Q D337Y R427K), each containing the indicated mutations in gp120. (B) Neutralization of iMac239 and iMac239-ΔD385, containing a deletion of Asp-385 within the CD4 binding site. Gray-shaded areas indicate assays that were not tested.

FIG 4

Effect of the ΔD385 mutation in cell-cell fusion, viral replication, and neutralization assays. (A) Fusion activities of SIVmac239, SIVmac239-ΔD385, iMac239, and iMac239-ΔD385 on rhesus CCR5 only are shown for each Env as the percentage of fusion in the presence of rhesus CD4. Background was subtracted as described for Fig. 1B. The data shown are the means from four experiments plus SEM. (B) Replication of SIVmac239, SIVmac239-ΔD385, iMac239, and iMac239-ΔD385 viruses in CD4⁺ SupT1/RhR5 cells (left) and CD4⁻ BC7/RhR5 cells (right). RT activity in culture supernatants was measured at the indicated time points. Results from a representative experiment are shown. (C) Soluble CD4 (sCD4) neutralization of viral pseudotypes containing SIVmac239, SIVmac251.6, iMac239, and iMac239-ΔD385 Envs is shown on TZM-bl cells. Percent neutralization was calculated using luciferase activity normalized to infection in the absence of sCD4. Results from a representative experiment are shown.

FIG 5

Replication of SIVmac239 and iMac239 with and without the ΔD385 mutation in rhesus PBMCs. Replication of SIVmac239, SIVmac239-ΔD385, iMac239, and iMac239-ΔD385 in ConA/IL-2-stimulated rhesus PBMCs is shown. p27 Gag in culture supernatants was quantified by ELISA at the indicated time points. (A) Results from a representative experiment are shown. (B, left) For each virus, the percentage of total CD3⁺ T cells that are positive for p27 Gag are indicated. (Right) Flow cytometry cytograms from a representative experiment show the percentage of p27 Gag⁺ CD3⁺ T cells. (C, left) The percentage of p27 Gag⁺ CD3⁺ T cells that express CD4 and/or CD8 at peak infection is shown. (Right) Cytograms from a representative experiment show that for iMac239 and iMac239-ΔD385, a marked increase in p27 Gag is detectable in CD8⁺ T cells. One-way analysis of variance (ANOVA) was used to determine significance.

FIG 6

Use of alternative coreceptors by SIVmac239, iMac239, and iMac239-ΔD385. (A) Fusion activity of the indicated Envs on rhesus coreceptors in the presence (left) or absence (right) of rhesus CD4 was assessed in a cell-cell fusion assay. In each panel, luciferase activity was normalized to values for rhesus CCR5. Background fusion levels were subtracted prior to normalization. Data shown are the means from three experiments plus SEM. (B) Replication of SIVmac239, iMac239, and iMac239-ΔD385 viruses in CD4⁺ CCR5⁻ CEMx174 (left) and HUT-78 (right) cells. RT activity in culture supernatants was measured at the indicated time points. Results from a representative experiment are shown.