Abstract
CD4+ T cell depletion and immune activation are hallmarks of HIV infection. Despite extensive studies, the mechanisms underlying immune modulation remain elusive. HIV-1 Nef protein is secreted in exosomes from infected cells and is abundant in the plasma of HIV+ individuals. Exosomal Nef (exNef) was also shown to induce apoptosis in bystander CD4+ T cells. We hypothesized that exNef contributes to HIV pathogenesis. A HIV-1 NL4-3 virus containing alanine substitutions in the secretion modification region (SMR; amino acids 66 to 70; HIVNefsmr5a) was developed. Nef protein containing this modified SMR was shown to be deficient in exNef secretion in nef-transfected cells. Using both HIV-1 NL4-3 wild type (HIVwt) and HIVNefsmr5a, correlates of pathogenesis were evaluated in cell-lines, human peripheral blood mononuclear cells, and humanized NOD-RAG1-/- IL2r-/- double mutant (NRG) mice. Disruption of the SMR did not affect viral replication or exNef secretion from infected cell cultures as compared with nef-transfected cells. However, T cell apoptosis was reduced in HIVNefsmr5a infected cell cultures and CD4+ T cell depletion was reduced in the spleen and peripheral blood of similarly infected NRG mice. Inflammatory cytokine release was also decreased in the sera of HIVNefsmr5a infected mice relative to HIVwt infected controls. These findings demonstrate the importance of Nef and the SMR motif in HIV pathogenesis and suggest a potential role for exNef in HIV-driven immune modulation.
Highlights
Chronic immune activation and CD4+ T-cell depletion are hallmarks of HIV infection
While the levels of p24 released from HIV-1 NL4-3 wild type (HIVwt) infected A3.01 cells started to increase by day 3, peaking by day 7, there was a slight delay in p24 release from cells infected with HIVNefsmr5a as well as HIVdsNef, with peak p24 release observed at day 9 (Figure 2A)
One report in the nonhuman primate model of HIV showed that secreted SIV Nef was able to affect hematopoiesis [29], the authors did not test whether Nef was or was not released in exosomes
Summary
Chronic immune activation has been well established as one of the strongest predictors of disease progression [19]. The source of such sustained immune activation has been ascribed to several factors that often use overlapping mechanisms including: (i) continuous plasmacytoid dendritic cell production of Type I IFN [10]; (ii) direct activation via the effect of viral proteins such as Tat, Nef and gp120 [11]; and (iii) bacterial translocation [5]. We submit that additional mechanisms underlie immune activation/immune dysregulation One such candidate is the secretion of exosomes containing the HIV accessory protein Nef. Nef is a 27-35 kDa protein that is expressed early in the replication cycle of HIV-1 [12]. Several studies have revealed Nef to be a determinant of disease progression in both HIV and SIV infection: (i) Mice and rats transgenic for HIV-1 nef develop immunodeficiencies [14,15,16,17,18,19,20,21,22]; (ii) the clinical course and both acute and chronic viral loads were markedly attenuated or abolished in both humans and rhesus macaques infected with viral strains that harbor deletions in nef [23,24,25,26,27]
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