Nef is an HIV-encoded accessory protein that enhances pathogenicity by down-regulating major histocompatibility class I (MHC-I) expression to evade killing by cytotoxic T lymphocytes (CTLs). A potent Nef inhibitor that restores MHC-I is needed to promote immune-mediated clearance of HIV-infected cells. We discovered that the plecomacrolide family of natural products restored MHC-I to the surface of Nef-expressing primary cells with variable potency. Concanamycin A (CMA) counteracted Nef at subnanomolar concentrations that did not interfere with lysosomal acidification or degradation and were nontoxic in primary cell cultures. CMA specifically reversed Nef-mediated down-regulation of MHC-I, but not CD4, and cells treated with CMA showed reduced formation of the Nef:MHC-I:AP-1 complex required for MHC-I down-regulation. CMA restored expression of diverse allotypes of MHC-I in Nef-expressing cells and inhibited Nef alleles from divergent clades of HIV and simian immunodeficiency virus, including from primary patient isolates. Lastly, we found that restoration of MHC-I in HIV-infected cells was accompanied by enhanced CTL-mediated clearance of infected cells comparable to genetic deletion of Nef. Thus, we propose CMA as a lead compound for therapeutic inhibition of Nef to enhance immune-mediated clearance of HIV-infected cells.

Host proteins are essential for HIV entry and replication and can be important nonviral therapeutic targets. Large-scale RNA interference (RNAi)-based screens have identified nearly a thousand candidate host factors, but there is little agreement among studies and few factors have been validated. Here we demonstrate that a genome-wide CRISPR-based screen identifies host factors in a physiologically relevant cell system. We identify five factors, including the HIV co-receptors CD4 and CCR5, that are required for HIV infection yet are dispensable for cellular proliferation and viability. Tyrosylprotein sulfotransferase 2 (TPST2) and solute carrier family 35 member B2 (SLC35B2) function in a common pathway to sulfate CCR5 on extracellular tyrosine residues, facilitating CCR5 recognition by the HIV envelope. Activated leukocyte cell adhesion molecule (ALCAM) mediates cell aggregation, which is required for cell-to-cell HIV transmission. We validated these pathways in primary human CD4⁺ T cells through Cas9-mediated knockout and antibody blockade. Our findings indicate that HIV infection and replication rely on a limited set of host-dispensable genes and suggest that these pathways can be studied for therapeutic intervention.

Immune control of human immunodeficiency virus type 1 (HIV) infection is typically associated with effective Gag-specific CD8⁺ T-cell responses. We here focus on HLA-B*14, which protects against HIV disease progression, but the immunodominant HLA-B*14-restricted anti-HIV response is Env specific (ERYLKDQQL, HLA-B*14-EL9). A subdominant HLA-B*14-restricted response targets Gag (DRYFKTLRA, HLA-B*14-DA9). Using HLA-B*14/peptide-saporin-conjugated tetramers, we show that HLA-B*14-EL9 is substantially more potent at inhibiting viral replication than HLA-B*14-DA9. HLA-B*14-EL9 also has significantly higher functional avidity (P < 0.0001) and drives stronger selection pressure on the virus than HLA-B*14-DA9. However, these differences were HLA-B*14 subtype specific, applying only to HLA-B*14:02 and not to HLA-B*14:01. Furthermore, the HLA-B*14-associated protection against HIV disease progression is significantly greater for HLA-B*14:02 than for HLA-B*14:01, consistent with the superior antiviral efficacy of the HLA-B*14-EL9 response. Thus, although Gag-specific CD8⁺ T-cell responses may usually have greater anti-HIV efficacy, factors independent of protein specificity, including functional avidity of individual responses, are also critically important to immune control of HIV.IMPORTANCE In HIV infection, although cytotoxic T lymphocytes (CTL) play a potentially critical role in eradication of viral reservoirs, the features that constitute an effective response remain poorly defined. We focus on HLA-B*14, unique among HLAs associated with control of HIV in that the dominant CTL response is Env specific, not Gag specific. We demonstrate that Env-specific HLA-B*14-restricted activity is substantially more efficacious than the subdominant HLA-B*14-restricted Gag response. Env immunodominance over Gag and strong Env-mediated selection pressure on HIV are observed only in subjects expressing HLA-B*14:02, and not HLA-B*14:01. This reflects the increased functional avidity of the Env response over Gag, substantially more marked for HLA-B*14:02. Finally, we show that HLA-B*14:02 is significantly more strongly associated with viremic control than HLA-B*14:01. These findings indicate that, although Gag-specific CTL may usually have greater anti-HIV efficacy than Env responses, factors independent of protein specificity, including functional avidity, may carry greater weight in mediating effective control of HIV.

Some HIV-1-infected patients develop broad and potent HIV-1 neutralizing antibodies (bNAbs) that when passively transferred to mice or macaques can treat or prevent infection. However, bNAbs typically fail to neutralize coexisting autologous viruses due to antibody-mediated selection against sensitive viral strains. We describe an HIV-1 controller expressing HLA-B57*01 and HLA-B27*05 who maintained low viral loads for 30 years after infection and developed broad and potent serologic activity against HIV-1. Neutralization was attributed to three different bNAbs targeting nonoverlapping sites on the HIV-1 envelope trimer (Env). One of the three, BG18, an antibody directed against the glycan-V3 portion of Env, is the most potent member of this class reported to date and, as revealed by crystallography and electron microscopy, recognizes HIV-1 Env in a manner that is distinct from other bNAbs in this class. Single-genome sequencing of HIV-1 from serum samples obtained over a period of 9 years showed a diverse group of circulating viruses, 88.5% (31 of 35) of which remained sensitive to at least one of the temporally coincident autologous bNAbs and the individual's serum. Thus, bNAb-sensitive strains of HIV-1 coexist with potent neutralizing antibodies that target the virus and may contribute to control in this individual. When administered as a mix, the three bNAbs controlled viremia in HIV-1_YU2-infected humanized mice. Our finding suggests that combinations of bNAbs may contribute to control of HIV-1 infection.