Distinct Molecular Pathways to X4 Tropism for a V3-Truncated Human Immunodeficiency Virus Type 1 Lead to Differential Coreceptor Interactions and Sensitivity to a CXCR4 Antagonist

Abstract

During the course of infection, transmitted HIV-1 isolates that initially use CCR5 can acquire the ability to use CXCR4, which is associated with an accelerated progression to AIDS. Although this coreceptor switch is often associated with mutations in the stem of the viral envelope (Env) V3 loop, domains outside V3 can also play a role, and the underlying mechanisms and structural basis for how X4 tropism is acquired remain unknown. In this study we used a V3 truncated R5-tropic Env as a starting point to derive two X4-tropic Envs, termed ⌬V3-X4A.c5 and ⌬V3-X4B.c7, which took distinct molecular pathways for this change. The ⌬V3-X4A.c5 Env clone acquired a 7-amino-acid insertion in V3 that included three positively charged residues, reestablishing an interaction with the CXCR4 extracellular loops (ECLs) and rendering it highly susceptible to the CXCR4 antagonist AMD3100. In contrast, the ⌬V3-X4B.c7 Env maintained the V3 truncation but acquired mutations outside V3 that were critical for X4 tropism. In contrast to ⌬V3-X4A.c5, ⌬V3-X4B.c7 showed increased dependence on the CXCR4 N terminus (NT) and was completely resistant to AMD3100. These results indicate that HIV-1 X4 coreceptor switching can involve (i) V3 loop mutations that establish interactions with the CXCR4 ECLs, and/or (ii) mutations outside V3 that enhance interactions with the CXCR4 NT. The cooperative contributions of CXCR4 NT and ECL interactions with gp120 in acquiring X4 tropism likely impart flexibility on pathways for viral evolution and suggest novel approaches to isolate these interactions for drug discovery.For human immunodeficiency virus type I (HIV-1) to enter a target cell, the gp120 subunit of the viral envelope glycoprotein (Env) must engage CD4 and a coreceptor on the cell surface. Although numerous coreceptors have been identified in vitro, the two most important coreceptors in vivo are the CCR5 (3,11,19,22,24) and CXCR4 (27) chemokine receptors. HIV-1 variants that can use only CCR5 (R5 viruses) are critical for HIV-1 transmission and predominate during the early stages of infection (86,90). The importance of CCR5 for HIV-1 transmission is underscored by the fact that individuals bearing a homozygous 32-bp deletion in the CCR5 gene (ccr5-⌬32) are largely resistant to HIV-1 infection (15, 49, 84). Although R5 viruses typically persist into late disease stages, viruses that can use CXCR4, either alone (X4 viruses) or in addition to CCR5 (R5X4 viruses), emerge in approximately 50% of individuals infected with subtype B or D viruses (12, 39, 44). Although not required for disease progression, the appearance of X4 and/or R5X4 viruses is associated with a more rapid depletion of CD4 ϩ cells in peripheral blood and faster progression to AIDS (12,44,77,86). However, it remains unclear whether these viruses are a cause or a consequence of accelerated CD4 ϩ T cell decline (57). The emergence of CXCR4-using viruses has also complicated the use of smallmolecule CCR5 antagonists as anti-HIV-therapeutics as these compounds can select for the outgrow...

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

“…Human SupT1 and BC7 cell lines (29) were transfected with a lentiviral vector to express rhesus CCR5 (RhCCR5) (70). SupT1/RhCCR5, BC7/RhCCR5, CEMx174, and HUT-78 cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 2 mM penicillin-streptomycin (RPMI-complete).…”

Correlation of the Virological Response to Short-Term Maraviroc Monotherapy with Standard and Deep-Sequencing-Based Genotypic Tropism Prediction Methods

“…However, the virological response observed in other patients, especially patient 29, points to a failure of the genotypic algorithm predictions as the more plausible explanation. The discordance rates between the virological response and genotypic algorithm prediction could be explained by the use of only the V3 loop sequence in the modeling of these algorithms, because other variable regions of the HIV envelope, and even gp41, can be involved in HIV tropism (3,14,16,27). Furthermore, although the discordance rates were similar for comparing UDS and standard genotypic methods, they did not occur in the same set of patients.…”

Interaction of the HIV-1 gp120 Viral Protein V3 Loop with Bacterial Lipopolysaccharide

“…We preincubated gp120 and LPS before adding to the cells. gp120 otherwise first interacts with CD4 on the cell surface, and the V3 loop interacts with a coreceptor (3)(4)(5). A shift in the flow cytometric analysis of gp120 binding to the surface of H9 cells (Fig.…”

“…Deletion of the V3 loop renders the envelope unable to elicit neutralizing antibodies (6) and renders the virus noninfective (8), thereby identifying the role of the V3 loop in the of HIV-1 fusion. However, some mutants have been reported in which varying V3 truncations have nevertheless enabled infectious virus (5).…”

“…gp120 is composed of an inner, outer, and connecting domain and contains five surfaceexposed mobile loops (V1 to V5) (2). The early stage of viral entry involves binding of gp120 to the CD4 cell receptor causing a conformational change within gp120, resulting in the exposure of the bridging sheet and V3 loop, allowing it to bind to a chemokine coreceptor, either CCR5 or CXCR4, which is required for viral entry (3)(4)(5). The V3 loop thus represents one of the principal neutralizing determinants of HIV-1 (6).…”