Role of the SH3-ligand domain of simian immunodeficiency virus Nef in interaction with Nef-associated kinase and simian AIDS in rhesus macaques

Abstract

The nef gene of the human and simian immunodeficiency viruses (HIV and SIV) is dispensable for viral replication in T-cell lines; however, it is essential for high virus loads and progression to simian AIDS (SAIDS) in SIV-infected adult rhesus macaques. Nef proteins from HIV type 1 (HIV-1), HIV-2, and SIV contain a proline-Xaa-Xaa-proline (PxxP) motif. The region of Nef with this motif is similar to the Src homology region 3 (SH3) ligand domain found in many cell signaling proteins. In virus-infected lymphoid cells, Nef interacts with a cellular serine/threonine kinase, designated Nef-associated kinase (NAK). In this study, analysis of viral clones containing point mutations in the nef gene of the pathogenic clone SIVmac239 revealed that several strictly conserved residues in the PxxP region were essential for Nef-NAK interaction. The results of this analysis of Nef mutations in in vitro kinase assays indicated that the PxxP region in SIV Nef was strikingly similar to the consensus sequence for SH3 ligand domains possessing the minus orientation. To test the significance of the PxxP motif of Nef for viral pathogenesis, each proline was mutated to an alanine to produce the viral clone SIVmac239-P104A/P107A. This clone, expressing Nef that does not associate with NAK, was inoculated into seven juvenile rhesus macaques. In vitro kinase assays were performed on virus recovered from each animal; the ability of Nef to associate with NAK was restored in five of these animals as early as 8 weeks after infection. Analysis of nef genes from these viruses revealed patterns of genotypic reversion in the mutated PxxP motif. These revertant genotypes, which included a second-site suppressor mutation, restored the ability of Nef to interact with NAK. Additionally, the proportion of revertant viruses increased progressively during the course of infection in these animals, and two of these animals developed fatal SAIDS. Taken together, these results demonstrated that in vivo selection for the ability of SIV Nef to associate with NAK was correlated with the induction of SAIDS. Accordingly, these studies implicate a role for the conserved SH3 ligand domain for Nef function in virally induced immunodeficiency.

FIG. 1

In vitro kinase assay of SIVmac239 Nef mutants for Nef association with NAK and activation of PAK. (A) The conserved SH3 ligand domains from HIV-1 and SIV Nef are represented by amino acid sequences from the HIV-1-NL4-3 and SIVmac239 clones. SIV Nef mutants that affected NAK association and PAK activation are denoted by asterisks. For all mutants, the codon for the prototype amino acid was changed to the codon for alanine. (B and C) In vitro kinase assays were performed on anti-SIV Nef (B) and anti-PAK-1 (C) immunoprecipitates from extracts of uninfected CEMx174 cells and CEMx174 cells chronically infected with SIVmac239nef+, SIVmac239-RR/LL, SIVmac239-  P¹⁰⁴A/ P¹⁰⁷A, SIVmac239-R¹⁰³A, SIVmac239-P¹⁰⁴A, SIVmac239-K¹⁰⁵A, SIVmac239- V¹⁰⁶A, SIVmac239-P¹⁰⁷A, SIVmac239-L¹⁰⁸A, SIVmac239-R¹⁰⁹A, SIVmac239-F¹²²A, and SIVmac239Δnef. A total of 10⁷ cells were analyzed per lane. (D) The level of Nef expression in cell lines infected with prototype and mutant viruses was determined by immunoblot analysis with the anti-SIV Nef monoclonal antibody 17.2. The lanes are the same as in panels B and C. Immunoprecipitates were electrophoresed on a 12% polyacrylamide gel under denaturing conditions and transferred to PVDF membranes. Phosphorylation of proteins was visualized by autoradiography. Kinase and immunoblot assays were performed as described in Materials and Methods.

FIG. 2

Virus load measured by bDNA assay in plasma from infected macaques (expressed as viral RNA copies per milliliter of plasma). Solid lines indicate animals that were euthanized within the study, and the stars show the time at which each animal was euthanized for necropsy. Dashed lines indicate animals that were alive at the conclusion of the study. The lower limit of detection for the bDNA assay is 10,000 copies of viral RNA per ml. (A) Virus load in plasma in seven macaques infected by the intravenous route with the mutant SIVmac239-P¹⁰⁴A/P¹⁰⁷A. (B) Virus load in plasma in control macaques infected with virus containing full-length nef, SIVmac239nef+.

FIG. 3

CD4 cell count in peripheral blood of infected macaques. Lymphocyte subsets in peripheral blood were analyzed by flow cytometry, in a FACScan (Becton Dickinson, Mountain View, Calif.) with marker antibodies recognizing CD4 T cells (OKT4), CD8 T cells (Leu2A), CD2 T cells (Leu5b), and CD19 B cells (Leu16). Solid lines indicate animals that were euthanized within the study, and the stars show the time at which each animal was euthanized for necropsy. Dashed lines indicate animals that were alive at the conclusion of the study. (A) CD4 cell count in seven macaques infected with SIVmac239-P¹⁰⁴A/P¹⁰⁷A. (B) CD4 cell count in control macaques infected with virus containing full-length nef, SIVmac239nef+.

FIG. 4

Phenotypic reversions of the SIVmac239-P¹⁰⁴A/P¹⁰⁷A mutant in macaques. In vitro kinase assays were performed on anti-Nef immunoprecipitates from uninfected CEMx174 cells and from CEMx174 cells infected with prototype virus and viruses recovered from several macaques infected with the mutant virus. (A) Control kinase assays were done on anti-SIV Nef immunoprecipitates from extracts of uninfected CEMx174 cells and on extracts of CEMx174 cells chronically infected with SIVmac239nef+ or SIVmac239-P¹⁰⁴A/P¹⁰⁷A. In vitro kinase assay results are shown for anti-Nef immunoprecipitates from extracts of CEMx174 cells acutely infected with virus isolated from Mmu 25905 at weeks 20 (20wk) and 30, with virus from Mmu 27659 at weeks 8 and 24, and with virus from Mmu 27626 at week 24 postinoculation. (B) In vitro kinase assay performed on virus isolated from Mmu 27626 and Mmu 27659 at 42 weeks (42wk) postinoculation, analyzed in the same manner as in panel A with controls: in vitro kinase analysis of anti-SIV Nef immunoprecipitates from extracts of uninfected CEMx174 cells and CEMx174 cells chronically infected with SIVmac239nef+. (C) Association of Nef with NAK in the mutant virus, SIVmac239-K¹⁰⁵R/P¹⁰⁷A; the mutation in nef in this virus was constructed on the basis of the second-site revertant sequence in virus isolated from Mmu 27659 (Table 2). Results of in vitro kinase analysis of anti-SIV Nef immunoprecipitates from uninfected CEMx174 cells and from CEMx174 cells chronically infected with SIVmac239nef+ and SIVmac239-K¹⁰⁵R/P¹⁰⁷A are shown. A total of 10⁷ cells were analyzed per lane. The kinase assays were performed as described in Materials and Methods. Immunoprecipitates were electrophoresed on a 12% polyacrylamide gel under denaturing conditions, and phosphorylation of proteins was visualized by autoradiography.