Alternative Coreceptor Requirements for Efficient CCR5- and CXCR4-Mediated HIV-1 Entry into Macrophages

Abstract

Macrophage tropism of human immunodeficiency virus type 1 (HIV-1) is distinct from coreceptor specificity of the viral envelope glycoproteins (Env), but the virus-cell interactions that contribute to efficient HIV-1 entry into macrophages, particularly via CXCR4, are not well understood. Here, we characterized a panel of HIV-1 Envs that use CCR5 (n ‫؍‬ 14) or CXCR4 (n ‫؍‬ 6) to enter monocyte-derived macrophages (MDM) with various degrees of efficiency. Our results show that efficient CCR5-mediated MDM entry by Env-pseudotyped reporter viruses is associated with increased tolerance of several mutations within the CCR5 N terminus. In contrast, efficient CXCR4-mediated MDM entry was associated with reduced tolerance of a large deletion within the CXCR4 N terminus. Env sequence analysis and structural modeling identified amino acid variants at positions 261 and 263 within the gp41-interactive region of gp120 and a variant at position 326 within the gp120 V3 loop that were associated with efficient CXCR4-mediated MDM entry. Mutagenesis studies showed that the gp41 interaction domain variants exert a significant but strain-specific influence on CXCR4-mediated MDM entry, suggesting that the structural integrity of the gp120-gp41 interface is important for efficient CXCR4-mediated MDM entry of certain HIV-1 strains. However, the presence of Ile326 in the gp120 V3 loop stem, which we show by molecular modeling is located at the gp120-coreceptor interface and predicted to interact with the CXCR4 N terminus, was found to be critical for efficient CXCR4-mediated MDM entry of divergent CXCR4-using Envs. Together, the results of our study provide novel insights into alternative mechanisms of Env-coreceptor engagement that are associated with efficient CCR5-and CXCR4-mediated HIV-1 entry into macrophages.The gp120 glycoproteins of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) initiate contact between the virus and the target cell (46). Viral attachment involves binding of gp120 to cellular CD4 and then to either CCR5 or CXCR4 as a coreceptor (reviewed in references 13 and 14). CD4 binding occurs with high affinity and triggers a conformational change in gp120 that exposes the coreceptor binding site. Current models of gp120 binding to coreceptor, supported recently by analysis of the crystal structure of CXCR4 (79), suggest that the crown of the V3 loop interacts principally with the coreceptor second extracellular loop (ECL2) region while the gp120 bridging sheet and the stem of the V3 loop interact with the coreceptor N terminus (7, 11,25,39). The interaction of CD4-bound gp120 with coreceptor induces additional conformational changes in gp120, which leads to a structural rearrangement in gp41 that enables fusion and virus entry (reviewed in reference 75).

Phenotypic Correlates of HIV-1 Macrophage Tropism

“…A number of Env substitutions have been suggested to contribute to macrophage tropism (24,25,27,(30)(31)(32)(33)(34)(35)(36)(37)(38)(39), but none of these suggested mutations can distinguish the paired M-tropic and T-tropic viruses used in our study. By including additional HIV-1 variants identified by intermediate CD4 usage, we observed that both MDM infection and low CD4 usage vary across a wide range.…”

“…Although it is widely observed that M-tropic viruses are able to enter more efficiently at low CD4 densities (14,19,(22)(23)(24)(25)(26) and are more sensitive to neutralization by soluble CD4 (sCD4) (27)(28)(29) than T-tropic viruses, few other characteristics are widely agreed upon. Similarly, several amino acid changes in the HIV-1 Env protein have been associated with macrophage tropism (24,25,27,(30)(31)(32)(33)(34)(35)(36)(37)(38)(39), but the changes are not consistent across different subjects when macrophage tropism is defined as a distinct set of evolutionary variants within that subject (40). Furthermore, viruses isolated from the brain are sometimes referred to as neurotropic but without a clear definition of what this means phenotypically other than being located in the central nervous system (CNS) at the time of isolation.…”

A common mechanism of clinical HIV-1 resistance to the CCR5 antagonist maraviroc despite divergent resistance levels and lack of common gp120 resistance mutations

“…Single round entry assays were conducted in NP2-CD4 cells expressing either wild type (WT) CCR5 or CCR5 containing various mutations in the N-terminal region using Env-pseudotyped luciferase reporter viruses, in the presence or absence of MVC (Figure 5). The levels of virus entry in cells expressing CCR5 mutants were expressed as percentages of that attained in cells expressing equivalent levels of WT CCR5, which was verified by flow cytometry as described previously [62,64,65] (data not shown). In the absence of MVC, both 17-Res and 24-Res Envs displayed significantly increased dependence on Tyr10, Tyr14 and Tyr15 residues in the CCR5 N-terminus, which are known to be sulfated in vivo , compared to their MVC-sensitive parental strains.…”

HIV-1 target cells in the CNS

“… a Musich et al (2011) b Cashin et al (2011) c Dunfee et al (2006) d Duenas-Decamp et al (2009) e Thomas et al (2007) f Duenas-Decamp et al (2008) g Dunfee et al (2007) h Ouyang et al (2014) …”