The human immunodeficiency virus type 1 (HIV-1) early gene product Nef is a multifunctional protein that alters numerous pathways of T-cell function, including endocytosis, signal transduction, vesicular trafficking, and immune modulation, and is a major determinant of pathogenesis. Individual Nef functions include PAK-2 activation, CD4 downregulation, major histocompatibility complex (MHC) class I downregulation, and enhancement of viral particle infectivity. How Nef accomplishes its multiple tasks presents a difficult problem of mechanistic analysis because of the complications associated with multiple, overlapping functional domains in the context of significant sequence variability. To address these issues we determined the conservation of each Nef residue based on 1,643 subtype B Nef sequences. Mutational analysis based on conservative substitutions and Nef sequence data allowed us to search for amino acids on the surface of Nef that are specifically required for PAK-2 activation. We found residues 85, 89, and 191 to be highly significant determinants for Nef's PAK-2 activation function but functionally unlinked to CD4 and MHC class I downregulation or enhancement of infectivity. These residues are not conserved across HIV-1 subtypes but are confined to separate sets of surface elements within a subtype. Thus, L85/H89/F191 and F85/F89/R191 are dominant in subtype B and subtype E or C, respectively. Our results provide support for developing subtype-specific interventions in HIV-1 disease.

No abstract

We previously reported that inhibition of endosomal/lysosomal function can dramatically enhance human immunodeficiency virus type 1 (HIV-1) infectivity, suggesting that under these conditions productive HIV-1 infection can occur via the endocytic pathway. Here we further examined this effect with bafilomycin A1 (BFLA-1) and show that this enhancement of infectivity extends to all HIV-1 isolates tested regardless of coreceptor usage. However, isolate-specific differences were observed in the magnitude of the effect. This was particularly evident in the case of the weakly infectious HIV-1 SF2 , for which we observed the greatest enhancement. Using reciprocal chimeric viruses, we were able to determine that both the disproportionate increase in the infectivity of HIV-1 SF2 in response to BFLA-1 and its weak infectivity in the absence of BFLA-1 mapped to its envelope gene. Further, we found HIV-1 SF2 to have lower fusion activity and to be 12-fold more sensitive to the fusion inhibitor T-20 than HIV-1 NL4-3 . Proteasomal inhibitors also enhance HIV-1 infectivity, and we report that the combination of a lysosomal and a proteasomal inhibitor greatly enhanced infectivity of all isolates tested. Again, HIV-1 SF2 was unique in exhibiting a synergistic 400-fold increase in infectivity. We also determined that inhibition of proteasomal function increased the infectivity of HIV-1 pseudotyped with vesicular stomatitis virus G protein. The evidence presented here highlights the important role of the lysosomes/ proteasomes in the destruction of infectious HIV-1 SF2 and could have implications for the development of novel antiviral agents that might take advantage of these innate defenses.

SIV infection of natural host species such as sooty mangabeys results in high viral replication without clinical signs of simian AIDS. Studying such infections is useful for identifying immunologic parameters that lead to AIDS in HIV-infected patients. Here we have demonstrated that acute, SIV-induced CD4(+) T cell depletion in sooty mangabeys does not result in immune dysfunction and progression to simian AIDS and that a population of CD3(+)CD4(-)CD8(-) T cells (double-negative T cells) partially compensates for CD4(+) T cell function in these animals. Passaging plasma from an SIV-infected sooty mangabey with very few CD4(+) T cells to SIV-negative animals resulted in rapid loss of CD4(+) T cells. Nonetheless, all sooty mangabeys generated SIV-specific antibody and T cell responses and maintained normal levels of plasma lipopolysaccharide. Moreover, all CD4-low sooty mangabeys elicited a de novo immune response following influenza vaccination. Such preserved immune responses as well as the low levels of immune activation observed in these animals were associated with the presence of double-negative T cells capable of producing Th1, Th2, and Th17 cytokines. These studies indicate that SIV-infected sooty mangabeys do not appear to rely entirely on CD4(+) T cells to maintain immunity and identify double-negative T cells as a potential subset of cells capable of performing CD4(+) T cell-like helper functions upon SIV-induced CD4(+) T cell depletion in this species.

The continual threat posed by newly emerging influenza virus strains is demonstrated by the recent outbreak of H5N1 influenza virus in Hong Kong. Currently, immunization against influenza virus infection is fairly adequate, but it is imperative that improved vaccines are developed that can protect against a variety of strains and be generated rapidly. Since humoral immunity is ineffective against serologically distinct viruses, one strategy would be to develop vaccines that emphasize cellular immunity. Here we report the successful protection of C57BL/6 mice from a lethal A/HK/156/97 (HK156) infection by immunizing first with an H9N2 isolate, A/Quail/HK/G1/97 (QHKG1), that harbours internal genes 98 % homologous to HK156. This strategy also protected mice that are deficient in antibody production, indicating that the immunity is T-cell-mediated. In the course of these studies, we generated a highly pathogenic H5N1 reassortant which implicated NP and PB2 as having an important contribution to pathogenesis when present with a highly cleavable H5. These results provide the first demonstration that protective cell-mediated immunity can be established against the highly virulent HK156 virus and have important implications for the development of novel strategies for the prevention and treatment of HK156 infection and the design of future influenza vaccines.