HIV-1 Resistance to CCR5 Antagonists Associated with Highly Efficient Use of CCR5 and Altered Tropism on Primary CD4 ⁺ T Cells

Abstract

We previously reported on a panel of HIV-1 clade B envelope (Env) proteins isolated from a patient treated with the CCR5 antagonist aplaviroc (APL) that were drug resistant. These Envs used the APL-bound conformation of CCR5, were cross resistant to other small-molecule CCR5 antagonists, and were isolated from the patient's pretreatment viral quasispecies as well as after therapy. We analyzed viral and host determinants of resistance and their effects on viral tropism on primary CD4 ؉ T cells. The V3 loop contained residues essential for viral resistance to APL, while additional mutations in gp120 and gp41 modulated the magnitude of drug resistance. However, these mutations were context dependent, being unable to confer resistance when introduced into a heterologous virus. The resistant virus displayed altered binding between gp120 and CCR5 such that the virus became critically dependent on the N terminus of CCR5 in the presence of APL. In addition, the drug-resistant Envs studied here utilized CCR5 very efficiently: robust virus infection occurred even when very low levels of CCR5 were expressed. However, recognition of drug-bound CCR5 was less efficient, resulting in a Entry of human immunodeficiency virus (HIV) into target cells is a complex, multistep process that is initiated by interactions between the viral envelope (Env) protein gp120 and the host cell receptor CD4, which trigger conformational changes in gp120 that form and orient the coreceptor binding site (9,24). Upon binding to coreceptor, which is either CCR5 or CXCR4 for primary HIV isolates, Env undergoes further conformational changes resulting in insertion of the gp41 fusion peptide into the host cell membrane and gp41-mediated membrane fusion (8,15,26). Targeting stages of the HIV entry process with antiretroviral drugs is a productive method of inhibiting HIV replication, as demonstrated by the potent antiviral effects of small-molecule CCR5 antagonists and fusion inhibitors (23,35,49). As with other antiretroviral drugs, HIV can develop resistance to entry inhibitors, and a detailed understanding of viral and host determinants of resistance will be critical to the optimal clinical use of these agents.The coreceptor binding site that is induced by CD4 engagement consists of noncontiguous regions in the bridging sheet and V3 loop of gp120 (4,18,42,43,50). Interactions between gp120 and CCR5 occur in at least two distinct areas: (i) the bridging sheet and the stem of the V3 loop interact with sul-

HIV-1 Clinical Isolates Resistant to CCR5 Antagonists Exhibit Delayed Entry Kinetics That Are Corrected in the Presence of Drug

“…Late fusion events occur at similar rates in different viral isolates, so it is likely that the differences we observed in entry kinetics reflect changes in the kinetics of binding to CD4, CCR5, or both (29). Increased coreceptor affinity accelerates viral fusion; because CCR5 antagonist-resistant HIV-1 uses the drug-bound CCR5, the V3 loop mutations that confer resistance must increase gp120 affinity for the antagonist-coreceptor complex (36,40,44,54,59). A clonal analysis of env sequences from Sub07 and Sub57, however, demonstrated that even after the virus acquired the capacity to use the drug-coreceptor complex, resistance mutations continued to accumulate.…”

HIV-1 Resistance to Maraviroc Conferred by a CD4 Binding Site Mutation in the Envelope Glycoprotein gp120

“…HIV-1 resistance to such inhibitors is likely to entail unique escape mechanisms given that a host receptor, not a viral enzyme, is the drug target. Potential pathways of resistance to these inhibitors include coreceptor switching to CXCR4-using viruses (16), increased affinity and binding to CD4 and/or CCR5 (17,18), use of inhibitorbound conformations of CCR5 (19,20), and increased kinetics of membrane fusion (21). Although outgrowth of CXCR4-using virus remains a concern for the therapeutic administration of CCR5 antagonists and is why patients are screened for X4-tropic virus prior to starting a maraviroc regimen, de novo mutations altering coreceptor tropism do not appear to be the preferential pathway for resistance (22,23).…”

HIV-1 Pathogenesis: The Virus

“…Because of down regulation of CD4 by HIV-1, cells actively producing virus are seen as CD4/CD8 double-negative T cells (Kaiser et al 2007). In cell culture HIV-1 infects activated cells with much greater efficiency than quiescent cells (Korin and Zack 1998), with central and effector memory cells as the primary targets (Pfaff et al 2010). However, in vivo there appears to be a type of CD4 þ T cell that does not express surface activation markers but supports significant levels of infection, particularly in the gut mucosa (Veazey et al 2000;Brenchley et al 2004b;Li et al 2005;Mattapallil et al 2005;Mehandru et al 2007).…”