Nef Involvement in Lymphocyte Depletion
Faculty Involved:
Description:
There is a body of literature that provides circumstantial evidence suggesting that the Nef protein is a major determinant of HIV and SIV pathogenesis. Primates and humans infected with nef-deleted viral strains have delayed, or no pathogenicity ({977}, {980}, {978}, {4465}, {2270}). Mice and rats expressing Nef as a transgene develop symptoms similar to those observed in human AIDS patients ({2816}, {2350}, {2349}, {2348}, {4735}, {2347}, {4536}, {4472}, {968}). Nef protein has been linked with the induction of apoptosis ({3242}, {3244}, {3245}). Our results show:
- Soluble HIV-1, -2, and SIV Nef proteins, alone, induce apoptosis in CD4+ T lymphocytes (James et al., 2004 {5060}).
- Nef proteins cytotoxic activity is mediated through the chemokine receptor CXCR4 ({5060}, Huang et al., 2004, {5298}).
- The apoptotic motifs in the HIV-1 Nef protein have been localized in two 10-amino acid regions (Huang et al., 2004, ({5298}), and in SIV Nef the motif has been localized to the C-terminal amino acids (preliminary data).
- In tissue culture, Nef protein is secreted into the extracellular environment (James et al., 2004, {5060} Huang et al., 2004, {5298}; unpublished evidence).
- HIV-1 Nef protein has been identified in patient plasma samples, and shown to be apoptotic (unpublished evidence).
- Nef is secreted in vesicles that appear to be exosomes (manuscripts submitted or in preparation).
- We have developed an in vivo mouse model for Nef induced lymphocyte depletion (Huang et al., submitted).
- The HIV-1 Nef apoptotic motif was observed to induce thymic apoptosis in our model (Huang et al., submitted).
- Lymphocytic depletion in the peripheral blood of Nef-treated mice was also observed, which was found to be blocked only by induction of a humoral response to the Nef apoptotic epitope (Huang et al., submitted). These observations are consistent with soluble Nef-induced changes including apoptosis driving some portion of HIV pathogenesis. We hypothesize that soluble, extracellular Nef, through the apoptotic motifs, which is not neutralized by the host immune response, drives CD4+ T-cell depletion in vivo.
As presented, the accumulated evidence suggests that soluble Nef-induced effects, including apoptosis, are responsible for at least some portion of HIV pathogenesis. Ultimately, it is envisioned that this work could lead to development of therapeutics, (e.g., vaccine) targeting the Nef motifs, that would antagonize pathogenesis, and prolong, or possibly halt progression towards AIDS. A full patent has been filed on the potential of this concept.
One major ongoing branch of this work involves a collaborative pilot study with the UC Davis Primate Center to examine our hypothesis in primates and an in vivo primate pathogenesis model.
Our Relevant Publications:
5060. James et al., 2004
5298. Huang et al., 2004
Patent:
"Modulating vaccine against HIV-1 Nef protein induced lymphocyte depletion." Filed 01/07/05. USSN 11/030,315. This invention relates to the use of identified apoptotic motif sequences in HIV Nef as targets for a vaccine that would block Nef apoptotic motif driven pathogenesis. Full Patent Application.
Examination of Nef-induced exosome secretion
Faculty Involved:
Description:
It has been demonstrated by our group and others that Nef protein can be secreted from transfected cells ({5448}, {5060}). Expression of Nef is known to cause membrane perturbations in the cell and induce the accumulation of multivesicular bodies ({5256}, {5259}). Our current results suggest that the secreted Nef is in the form of vesicular bodies (submitted publication). It has also been demonstrated that at least some of the Nef protein expressed within the cell ends up on the cell surface and can be cytocidal for human CD4+ cells ({3244}, {5253}, {5254}). Therefore, it is not unlikely that at least some of the Nef protein present in vesicular bodies is present on the surface where it can exert an effect on bystander cells. Our published data support this conclusion as Nef vesicles can indeed induce apoptosis through the CXCR4 receptor when applied to cultured cells ({5060}). Further, our current results confirm this conclusion (submitted publication). This phenomenon also appears to be relevant to the situation in vivo during an HIV-1 infection. Nef has been reported to be present in the sera of infected patients at a level of approximately 10ng/ml of serum ({3245}). We have found similar results in our own labs (submitted publication). Our current results also show that Nef vesicles can be taken up by (a) cell lines through a Nef-dependent, but non-CXCR4 mechanism, as well as by (b) virion particles. A body of literature has already shown that soluble Nef protein induces a variety of changes in the gene expression pattern of a number of cell types. Thus, the Nef-containing exosomes could either (a) induce apoptosis in certain cell types or, (b) they could be taken up by those cells causing the changes reported in the literature, or (c) they could be taken up by defective viral particles allowing some to become infectious.
The literature and our results suggest that the Nef protein directly induces vesicle secretion. Thus, we have speculated that there are Nef sequences that drive secretion, and mapped these sequences (manuscript in preparation). We have identified three sequence motifs on the HIV-1 Nef protein that regulate Nef-induced secretion. Two of those motifs have been previously identified on the Nef protein to be involved in other Nef-induced activities (membrane interaction, MHC-I downregulation). The third sequence is a newly identified regulatory sequence on the HIV-1 Nef protein. We have also shown that these three sequences are highly conserved across HIV-1, HIV-2 and SIV; with the new motif almost 100% conserved suggesting the importance of these sequences. . In our overall model for Nef pathogenesis (developed as part of the lymphocyte depletion project), secretion of Nef is important in driving lymphocyte depletion leading to AIDS pathogenesis. Based on our model, this region should be highly conserved across clades and types (HIV-1, HIV-2, and SIV). We are in discussion with the Emory Center for AIDS Research (of which we are members) to do a pilot study testing the relevance of Nef-induced secretion to in vivo SIV pathogenesis in macaques.
Nef Involvement in HIVAN
Faculty Involved:
Description:
HIV-associated nephropathy (HIVAN) is a kidney disease affecting predominantly African Americans (AA) who can progress to end stage renal failure. The exact cause of the disease is unknown. We found that endothelin-1 (ET-1), implicated in renal and cardiovascular disease, was increased in plasma from both HIVAN and African American HIV+ patients and was associated with a single nucleotide polymorphism, Lys198Asn, in the endothelin-1 gene (p=0.001). In addition, preproendothelin-1 (ppET-1) and endothelin converting enzyme-1 (ECE-1) mRNA expressions were up-regulated in HIVAN and AA HIV + patients. ppET-1 is proteolytically cleaved into big ET-1 which is then converted to the active ET-1 peptide by ECE-1. An increase in either ppET-1 or ECE-1 could lead to increased ET-1 levels observed in HIVAN patients. Renal biopsies from HIVAN patients stained positive for endothelin-1 indicating the presence of the peptide in the kidneys of these patients.
Peripheral blood derived macrophages from HIVAN patients stimulated with HIV Nef for 4 hours significantly increased ECE-1 mRNA when compared to the Nef-induced ECE-1 mRNA by cells from healthy donors and other HIV + patients (P<0.03 and P<0.01 respectively)(fig 3). In addition, Nef treatment significantly increased ECE-1 mRNA in macrophages from AA HIV + donors when compared to cells from healthy donors and other HIV + donors (P<0.006 and P<0.001 respectively). HIV gp120 did not induce any detectable ECE-1 mRNA from any of the donor's cells. Further, we found that macrophages from HIVAN and AA HIV+ patients treated with Nef or LPS displayed significantly higher expression of ppET-1 mRNA when compared to cells from healthy donors or other HIV + patients macrophages treated with either Nef or LPS (P<0.01 for both)( fig 4). HIV Nef treatment induced a significantly higher amount of ppET-1 mRNA from HIVAN donors when compared to other HIV+ patients (5.6 copies/106 copies 18S rRNA and 1.0 copies, respectively; P<0.05). No significant differences were observed in ppET-1 expression when cells were treated with HIV-1 gp120.
Thus, this evidence suggests (i) ET-1 plays a role in the pathogenesis of HIV-associated nephropathy, and (ii) an endothelin-1 polymorphism may predispose HIV infected African Americans to the development of renal disease. Further, and most significantly to the relationship between these projects, (iii) the evidence points to HIV Nef protein as the stimulus of increased expression of ppET-1 and ECE-1 in a subpopulation of African-Americans. As mentioned above, an increase in either ppET-1 or ECE-1 could lead to increased ET-1 levels observed in HIVAN patients. And increased ET-1 has already been shown to be implicated in renal and cardiovascular disease in non-HIV infected patients.