Small molecule inhibition of HIV-1-induced MHC-I down-regulation identifies a temporally regulated switch in Nef action.

HIV-1 Nef triggers down-regulation of cell-surface MHC-I by assembling a Src family kinase (SFK)-ZAP-70/Syk-PI3K cascade. Here, we report that chemical disruption of the Nef-SFK interaction with the small molecule inhibitor 2c blocks assembly of the multi-kinase complex and represses HIV-1-mediated MHC-I down-regulation in primary CD4(+) T-cells. 2c did not interfere with the PACS-2-dependent trafficking of Nef required for the Nef-SFK interaction or the AP-1 and PACS-1-dependent sequestering of internalized MHC-I, suggesting the inhibitor specifically interfered with the Nef-SFK interaction required for triggering MHC-I down-regulation. Transport studies revealed Nef directs a highly regulated program to down-regulate MHC-I in primary CD4(+) T-cells. During the first two days after infection, Nef assembles the 2c-sensitive multi-kinase complex to trigger down-regulation of cell-surface MHC-I. By three days postinfection Nef switches to a stoichiometric mode that prevents surface delivery of newly synthesized MHC-I. Pharmacologic inhibition of the multi-kinase cascade prevents the Nef-dependent block in MHC-I transport, suggesting the signaling and stoichiometric modes are causally linked. Together, these studies resolve the seemingly controversial models that describe Nef-induced MHC-I down-regulation and provide new insights into the mechanism of Nef action.

Different in vivo effects of HIV-1 immunodominant epitope-specific cytotoxic T lymphocytes on selection of escape mutant viruses.

HIV-1 escape mutants are well known to be selected by immune pressure via HIV-1-specific cytotoxic T lymphocytes (CTLs) and neutralizing antibodies. The ability of the CTLs to suppress HIV-1 replication is assumed to be associated with the selection of escape mutants from the CTLs. Therefore, we first investigated the correlation between the ability of HLA-A*1101-restricted CTLs recognizing immunodominant epitopes in vitro and the selection of escape mutants. The result showed that there was no correlation between the ability of these CTLs to suppress HIV-1 replication in vitro and the appearance of escape mutants. The CTLs that had a strong ability to suppress HIV-1 replication in vitro but failed to select escape mutants expressed a higher level of PD-1 in vivo, whereas those that had a strong ability to suppress HIV-1 replication in vitro and selected escape mutants expressed a low level of PD-1. Ex vivo analysis of these CTLs revealed that the latter CTLs had a significantly stronger ability to recognize the epitope than the former ones. These results suggest that escape mutations are selected by HIV-1-specific CTLs that have a stronger ability to recognize HIV-1 in vivo but not in vitro.

Selection of escape mutation by Pol154-162-specific cytotoxic T cells among chronically HIV-1-infected HLA-B*5401-positive individuals.

Most escape mutations have been identified on cytotoxic T lymphocyte (CTL) epitopes presented by Caucasian or African human leukocyte antigen (HLA) class I alleles, whereas a limited number of studies have identified the escape mutations on epitopes presented by Asian alleles. HLA-B54 is a common HLA allele in Asian countries. We recently identified five HLA-B*5401-restricted HIV-1-specific CTL epitopes. We here investigated escape mutations in these CTL epitopes in Japanese HIV-1-infected individuals. The frequency of substitution from Glu (E) to Asp (D) at position 7 (FV9-7D) in the Pol 154-162 (FV9) epitope was significantly higher in HLA-B*5401(+) HIV-infected individuals than in HLA-B*5401(-) individuals, whereas substitutions that were significantly higher in HLA-B*5401(+) individuals than in HLA-B*5401(-) individuals were not found in the other four epitopes. FV9-specific CTLs showed reduced killing activity against target cells pulsed with the FV9-7D mutant peptide and failed to kill those infected with the FV9-7D mutant virus, strongly suggesting that FV9-7D is an escape mutant. Furthermore, longitudinal sequence analysis of the FV9 epitope in two HLA-B*5401(+) individuals revealed that the sequence had changed from the wild type to the FV9-7D during the clinical course. Taken together, these results indicate that the FV9-7D escape mutant had been selected by FV9-specific CTLs among chronically HIV-1-infected HLA-B*5401(+) individuals.

Clinical relevance of substitutions in the connection subdomain and RNase H domain of HIV-1 reverse transcriptase from a cohort of antiretroviral treatment-naïve patients.

Some mutations in the connection subdomain of the polymerase domain and in the RNase H domain of HIV-1 reverse transcriptase (RT) have been shown to contribute to resistance to RT inhibitors. However, the clinical relevance of such mutations is not well understood. To address this point we determined the prevalence of such mutations in a cohort of antiretroviral treatment-naïve patients (n=123) and assessed whether these substitutions are associated with drug resistance in vitro and in vivo. We report here significant differences in the prevalence of substitutions among subtype B, and non-subtype B HIV isolates. Specifically, the E312Q, G333E, G335D, V365I, A371V and A376S substitutions were present in 2-6% of subtype B, whereas the G335D and A371V substitutions were commonly observed in 69% and 75% of non-B HIV-1 isolates. We observed a significant decline in the viral loads of patients that were infected with HIV-1 carrying these substitutions and were subsequently treated with triple drug regimens, even in the case where zidovudine (AZT) was included in such regimens. We show here that, generally, such single substitutions at the connection subdomain or RNase H domain have no influence on drug susceptibility in vitro by themselves. Instead, they generally enhance AZT resistance in the presence of excision-enhancing mutations (EEMs, also known as thymidine analogue-associated mutations, TAMs). However, N348I, A376S and Q509L did confer varying amounts of nevirapine resistance by themselves, even in the absence of EEMs. Our studies indicate that several connection subdomain and RNase H domain substitutions typically act as pre-therapy polymorphisms.

Adaptation of HIV-1 to human leukocyte antigen class I.

The rapid and extensive spread of the human immunodeficiency virus (HIV) epidemic provides a rare opportunity to witness host-pathogen co-evolution involving humans. A focal point is the interaction between genes encoding human leukocyte antigen (HLA) and those encoding HIV proteins. HLA molecules present fragments (epitopes) of HIV proteins on the surface of infected cells to enable immune recognition and killing by CD8(+) T cells; particular HLA molecules, such as HLA-B*57, HLA-B*27 and HLA-B*51, are more likely to mediate successful control of HIV infection. Mutation within these epitopes can allow viral escape from CD8(+) T-cell recognition. Here we analysed viral sequences and HLA alleles from >2,800 subjects, drawn from 9 distinct study cohorts spanning 5 continents. Initial analysis of the HLA-B*51-restricted epitope, TAFTIPSI (reverse transcriptase residues 128-135), showed a strong correlation between the frequency of the escape mutation I135X and HLA-B*51 prevalence in the 9 study cohorts (P = 0.0001). Extending these analyses to incorporate other well-defined CD8(+) T-cell epitopes, including those restricted by HLA-B*57 and HLA-B*27, showed that the frequency of these epitope variants (n = 14) was consistently correlated with the prevalence of the restricting HLA allele in the different cohorts (together, P < 0.0001), demonstrating strong evidence of HIV adaptation to HLA at a population level. This process of viral adaptation may dismantle the well-established HLA associations with control of HIV infection that are linked to the availability of key epitopes, and highlights the challenge for a vaccine to keep pace with the changing immunological landscape presented by HIV.

Escape mutation selected by Gag28-36-specific cytotoxic T cells in HLA-A*2402-positive HIV-1-infected donors.

Gag-specific CTLs are known to have stronger ability to control HIV-1 replication than others that are protein-specific. Therefore, the analysis of Gag escape mutants is expected to clarify the mechanisms of immune control in HIV-1-infected donors. However, only a limited number of Gag escape mutants have been identified so far. A previous study suggested the possibility that Gag28-3R (KW9-3R) is an escape mutant from HLA-A*2402-restricted KW9-specific CTLs but did not show any evidence of it. Here we sought to demonstrate that KW9-3R is selected as escape mutant by KW9-specific CTLs. KW9-specific CTLs showed a remarkable reduction in recognition of target cells infected with the KW9-3R mutant. The sequence analysis of HIV-1 from 58 HIV-1-infected individuals showed that the frequency of the KW9-3R mutant was significantly higher in HLA-A*2402(+) individuals than in HLA-A*2402(-) individuals. Longitudinal analysis of an HLA-A*2402(+) individual with HIV-1 early infection showed that this escape mutant was selected over an approximately 2-year period. These results together indicate that Gag28-3R is an escape mutant selected by HLA-A*2402-restricted KW9-specific CTLs. Further analysis of this epitope will clarify the role of HIV-1-specific CTLs in the control of HIV-1 among the Japanese population, since 70% of them carry this allele.

Different abilities of escape mutant-specific cytotoxic T cells to suppress replication of escape mutant and wild-type human immunodeficiency virus type 1 in new hosts.

There is much evidence that in human immunodeficiency virus type 1 (HIV-1)-infected individuals, strong cytotoxic T lymphocyte (CTL)-mediated immune pressure results in the selection of HIV-1 mutants that have escaped from wild-type-specific CTLs. If escape mutant-specific CTLs are not elicited in new hosts sharing donor HLA molecules, the transmission of these mutants results in the accumulation of escape mutants in the population. However, whether escape mutant-specific CTLs are definitively not elicited in new hosts sharing donor HLA molecules still remains unclear. A previous study showed that a Y-to-F substitution at the second position (2F) of the Nef138-10 epitope is significantly detected in HLA-A*2402(+) hemophilic donors. Presently, we confirmed that this 2F mutant was an escape mutant by demonstrating strong and weak abilities of Nef138-10-specific CTL clones to suppress replication of the wild-type and 2F mutant viruses, respectively. We demonstrated the existence of the 2F-specific CTLs in three new hosts who had been primarily infected with the 2F mutant. The 2F-specific CTL clones suppressed the replication of both wild-type and mutant viruses. However, the abilities of these clones to suppress replication of the 2F virus were much weaker than those of wild-type-specific and the 2F-specific ones to suppress replication of the wild-type virus. These findings indicate that the 2F mutant is conserved in HIV-1-infected donors having HLA-A*2402, because the 2F-specific CTLs failed to completely suppress the 2F mutant replication and effectively prevented viral reversion in new hosts carrying HLA-A*2402.