The mapping of HIV-1 gpl60 epitopes required for interleukin-1 and tumor necrosis factor a production in glial cells

Abstract

The pathology in central nervous system (CNS) AIDS suggests that direct infection with HIV-1 is not required for changes in glia and neurons. Induction of a variety of pathological responses in vitro in rodent brain cultures also suggests that CD4 is not the receptor for HIV-1 in the brain, given that human and rodent CD4 are not homologous. This implies that the epitopes on HIV-1 which bind glia and activate them are novel, non-CD4-binding domains. We have therefore mapped the envelope (env) regions required for production in rat glial cultures of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFα) which we hypothesize are important in CNS AIDS. Serially truncated deletion mutants from the gp120/gp41 carboxy terminus representing folded, glycosylated recombinant env proteins were expressed in HeLa cells via a vaccinia virus vector. These proteins, linear gp120/gp41 peptides, as well as polyclonal and monoclonal antibodies reactive to defined regions of gp120/gp41 were used to map the epitopes involved in production of IL-1 and TNFα. Compared to HeLa cell and wild-type vaccinia virus controls, the vaccinia recombinant env protein gp160 containing cleaved gp120 and gp41 induced both IL-1 and TNFa. If gp160 was not cleaved into gp120 and gp41, IL-1 but not TNFa induction was reduced. Peak production of TNFa by gp120/gp41 was at 4 h while IL-1 production was still significantly elevated at 44 h at the highest concentrations of env protein. Using the truncation deletions, the V3 loop of gp120 appeared to be critical for IL-1. Glycosylation and folding of V3 is probably important in IL-1 induction since a V3 peptide was not as active. While removal of glycosylated, folded V4 and C4 regions had no effect on IL-1, linear peptides in the region from the V4 loop to the C4 domain were strong inducers of IL-1. Non-glycosylated, linear V4 loop peptide induced more IL-1 than the V4 in protein generated in HeLa cells, suggesting that glycosylation and/or conformational structures sequester V4 inducer epitopes. Using the truncation deletions, the carboxy terminus region (V4-C5) of gp120 as well as gp41 were shown to be critical for TNFa production. Peptides representing linear epitopes in the V3 loop, C5 domain of gp120, and the ectodomain of gp41 were all strong inducers of TNFα; a protein representing almost the entire gp41 was the strongest inducer of TNFα. This suggests both conformational and linear determinants induce TNFα. Glycosylated, folded VI-3 and C1-3, exclusive of other gp120 domains, did not induce TNFα production. These data were further confirmed by antibody inhibition of env regions. Cytokine induction by env proteins was transcriptionally regulated, since messenger RNA levels were increased as determined by Northern blot analysis. In summary, virus infection is not required and gp160 epitopes, other than the classical CD4 binding domains, are involved in induction of IL-1 and TNFa in glial cell cultures. These data suggest that HIV-1 env proteins in their native form, as well as those that are partially denatured (linearized or non-glycosylated) could induce two proinflammatory cytokines which are possibly important in CNS AIDS.