Genotypic and Mechanistic Characterization of Subtype-Specific HIV Adaptation to Host Cellular Immunity.

The extent to which viral genetic context influences HIV adaptation to human leukocyte antigen (HLA) class I-restricted immune pressures remains incompletely understood. The Ugandan HIV epidemic, where major pandemic group M subtypes A1 and D cocirculate in a single host population, provides an opportunity to investigate this question. We characterized plasma HIV RNA gag, pol, and nef sequences, along with host HLA genotypes, in 464 antiretroviral-naive individuals chronically infected with HIV subtype A1 or D. Using phylogenetically informed statistical approaches, we identified HLA-associated polymorphisms and formally compared their strengths of selection between viral subtypes. A substantial number (32%) of HLA-associated polymorphisms identified in subtype A1 and/or D had previously been reported in subtype B, C, and/or circulating recombinant form 01_AE (CRF01_AE), confirming the shared nature of many HLA-driven escape pathways regardless of viral genetic context. Nevertheless, 34% of the identified HLA-associated polymorphisms were significantly differentially selected between subtypes A1 and D. Experimental investigation of select examples of subtype-specific escape revealed distinct underlying mechanisms with important implications for vaccine design: whereas some were attributable to subtype-specific sequence variation that influenced epitope-HLA binding, others were attributable to differential mutational barriers to immune escape. Overall, our results confirm that HIV genetic context is a key modulator of viral adaptation to host cellular immunity and highlight the power of combined bioinformatic and mechanistic studies, paired with knowledge of epitope immunogenicity, to identify appropriate viral regions for inclusion in subtype-specific and universal HIV vaccine strategies.IMPORTANCE The identification of HIV polymorphisms reproducibly selected under pressure by specific HLA alleles and the elucidation of their impact on viral function can help identify immunogenic viral regions where immune escape incurs a fitness cost. However, our knowledge of HLA-driven escape pathways and their functional costs is largely limited to HIV subtype B and, to a lesser extent, subtype C. Our study represents the first characterization of HLA-driven adaptation pathways in HIV subtypes A1 and D, which dominate in East Africa, and the first statistically rigorous characterization of differential HLA-driven escape across viral subtypes. The results support a considerable impact of viral genetic context on HIV adaptation to host HLA, where HIV subtype-specific sequence variation influences both epitope-HLA binding and the fitness costs of escape. Integrated bioinformatic and mechanistic characterization of these and other instances of differential escape could aid rational cytotoxic T-lymphocyte-based vaccine immunogen selection for both subtype-specific and universal HIV vaccines.

HIV-1 Nef-induced cardiotoxicity through dysregulation of autophagy.

Cardiovascular disease is a leading cause of co-morbidity in HIV-1 positive patients, even those in whom plasma virus levels are well-controlled. The pathogenic mechanism of HIV-1-associated cardiomyopathy is unknown, but has been presumed to be mediated indirectly, owing to the absence of productive HIV-1 replication in cardiomyocytes. We sought to investigate the effect of the HIV-1 auxiliary protein, Nef, which is suspected of extracellular release by infected CD4+ T cells on protein quality control and autophagy in cardiomyocytes. After detection of Nef in the serum of HIV-1 positive patients and the accumulation of this protein in human and primate heart tissue from HIV-1/SIV-infected cells we employed cell and molecular biology approaches to investigate the effect of Nef on cardiomyocyte-homeostasis by concentrating on protein quality control (PQC) pathway and autophagy. We found that HIV-1 Nef-mediated inhibition of autophagy flux leads to cytotoxicity and death of cardiomyocytes. Nef compromises autophagy at the maturation stage of autophagosomes by interacting with Beclin 1/Rab7 and dysregulating TFEB localization and cellular lysosome content. These effects were reversed by rapamycin treatment. Our results indicate that HIV-1 Nef-mediated inhibition of cellular PQC is one possible mechanism involved in the development of HIV-associated cardiomyopathy.

Nef inhibits glucose uptake in adipocytes and contributes to insulin resistance in human immunodeficiency virus type I infection.

Human immunodeficiency virus (HIV) infection is associated with insulin resistance. HIV type 1 Nef downregulates cell surface protein expression, alters signal transduction, and interacts with the cytoskeleton and proteins involved in actin polymerization. These functions are required for glucose uptake by insulin-stimulated adipocytes. We sought to determine whether Nef alters adipocyte glucose homeostasis. Using radiolabeled glucose, we found that adipocytes exposed to recombinant Nef took in 42% less glucose after insulin stimulation than did control cells. This reduction resulted from a Nef-dependent inhibition of glucose transporter 4 (GLUT4) trafficking, as assessed by means of immunofluorescence microscopy. Immunoblot analysis revealed a decrease in phosphorylation of signal transducing proteins after Nef treatment, and fluorescence microscopy showed a dramatic alteration in cortical actin organization. We conclude that Nef interferes with insulin-stimulated processes in adipocytes. We have identified HIV Nef, which is detectable and antigenic in serum samples from HIV-infected people, as a novel contributor to the development of insulin resistance.

Effect of multiple intravenous injections of butaphosphan and cyanocobalamin on the metabolism of periparturient dairy cows.

Numerous adjunct therapeutic agents have been investigated for the treatment or control of fat mobilization syndrome in periparturient dairy cows. The aim of this study was to determine the effects of multiple i.v. injections of 10% butaphosphan and 0.005% cyanocobalamin combination (Catosal, Bayer Animal Health, Leverkusen, Germany) between 1 and 2 wk antepartum (a.p.) on the metabolism and health of dairy cows. Forty-five late-gestation Holstein-Friesian cows (second pregnancy) were allocated randomly to 1 of 3 groups with 15 cows/group: group C6 (6 daily i.v. injections of butaphosphan at 10 mg/kg of body weight (BW) and cyanocobalamin at 5 microg/kg of BW in the last 2 wk of gestation); group C3 (3 daily i.v. injections of butaphosphan at 10 mg/kg of BW and cyanocobalamin at 5 microg/kg of BW in the last week of gestation); and group C0 (equivolume daily i.v. injections of 0.9% NaCl solution). Serum biochemical analysis was performed on jugular venous blood samples that were periodically obtained a.p. and postpartum (p.p.). Health status and milk production were monitored p.p. Serum cyanocobalamin concentration increased in groups C6 and C3 p.p. Multiple daily i.v. injections of Catosal before parturition increased p.p. glucose availability, as evaluated by p.p. serum glucose concentration, and decreased peripheral fat mobilization and ketone body formation, as evaluated by p.p. serum nonesterified fatty acid and beta-OH butyrate concentrations. The number of puerperal infections in the first 5 d after calving was decreased in group C6, relative to group C0. We conclude that multiple injections of Catosal during the close-up period have a beneficial effect on the metabolism of periparturient dairy cows. Our results are consistent with the hypothesis that high-producing dairy cows in early lactation may have a relative or actual deficiency of cyanocobalamin.

Circulating Nef induces dyslipidemia in simian immunodeficiency virus-infected macaques by suppressing cholesterol efflux.

Human immunodeficiency virus (HIV) infection and subsequent antiretroviral therapy have been associated with an increased incidence of dyslipidemia and cardiovascular disease and has been shown to suppress cholesterol efflux from virus-infected macrophages by inducing Nef-dependent down-regulation of adenosine triphosphate-binding cassette transporter A1 (ABCA1). Here, the simian immunodeficiency virus (SIV)-infected macaque model was used to examine the consequences and mechanisms involved. SIV infection drove a significant remodeling of high-density lipoprotein profiles, suggesting that systemic inhibition of the ABCA1-dependent reverse cholesterol transport pathway occurred. The ABCA1 cholesterol transporter was significantly down-regulated in the livers of the SIV-infected macaques, and the viral protein Nef could be detected in the livers as well as in the plasma of infected animals. Extracellular myristoylated HIV Nef inhibited cholesterol efflux from macrophages and hepatocytes. Moreover, serum samples from SIV-infected macaques also suppressed cholesterol efflux in a Nef-dependent fashion. These results indicate that SIV infection is a significant contributor to primary dyslipidemia, likely through the ability of Nef to suppress ABCA1-dependent reverse cholesterol transport.

Functional adaptation of Nef to the immune milieu of HIV-1 infection in vivo.

Nef-mediated down-regulation of MHC class I (MHC-I) molecules on HIV-1-infected cells has been proposed to enhance viral persistence through evasion of host CTLs. This conclusion is based largely on demonstrations that Nef from laboratory HIV-1 strains reduces the susceptibility of infected cells to CTL killing in vitro. However, the function and role of Nef-mediated MHC-I down-regulation in vivo have not been well described. To approach this issue, nef quasispecies from chronically HIV-1-infected individuals were cloned into recombinant reporter viruses and tested for their ability to down-regulate MHC-I molecules from the surface of infected cells. The level of function varied widely between individuals, and although comparison to the immunologic parameters of blood CD4(+) T lymphocyte count and breadth of the HIV-1-specific CTL response showed positive correlations, no significant correlation was found in comparison to plasma viremia. The ability of in vivo-derived Nef to down-regulate MHC-I predicted the resistance of HIV-1 to suppression by CTL. Taken together, these data demonstrate the functionality of Nef to down-regulate MHC-I in vivo during stable chronic infection, and suggest that this function is maintained by the need of HIV-1 to cope with the antiviral CTL response.