In vivo analysis of Nef's role in HIV-1 replication, systemic T cell activation and CD4(+) T cell loss.

BACKGROUND: Nef is a multifunctional HIV-1 protein critical for progression to AIDS. Humans infected with nef(-) HIV-1 have greatly delayed or no disease consequences. We have contrasted nef(-) and nef(+) infection of BLT humanized mice to better characterize Nef's pathogenic effects. RESULTS: Mice were inoculated with CCR5-tropic HIV-1JRCSF (JRCSF) or JRCSF with an irreversibly inactivated nef (JRCSFNefdd). In peripheral blood (PB), JRCSF exhibited high levels of viral RNA (peak viral loads of 4.71 × 10(6) ± 1.23 × 10(6) copies/ml) and a progressive, 75% loss of CD4(+) T cells over 17 weeks. Similar losses were observed in CD4(+) T cells from bone marrow, spleen, lymph node, lung and liver but thymocytes were not significantly decreased. JRCSFNefdd also had high peak viral loads (2.31 × 10(6) ± 1.67 × 10(6)) but induced no loss of PB CD4(+) T cells. In organs, JRCSFNefdd produced small, but significant, reductions in CD4(+) T cell levels and did not affect the level of thymocytes. Uninfected mice have low levels of HLA-DR(+)CD38(+)CD8(+) T cells in blood (1-2%). Six weeks post inoculation, JRCSF infection resulted in significantly elevated levels of activated CD8(+) T cells (6.37 ± 1.07%). T cell activation coincided with PB CD4(+) T cell loss which suggests a common Nef-dependent mechanism. At 12 weeks, in JRCSF infected animals PB T cell activation sharply increased to 19.7 ± 2.9% then subsided to 5.4 ± 1.4% at 14 weeks. HLA-DR(+)CD38(+)CD8(+) T cell levels in JRCSFNefdd infected mice did not rise above 1-2% despite sustained high levels of viremia. Interestingly, we also noted that in mice engrafted with human tissue expressing a putative protective HLA-B allele (B42:01), JRCSFNefdd exhibited a substantial (200-fold) reduced viral load compared to JRCSF. CONCLUSIONS: Nef expression was necessary for both systemic T cell activation and substantial CD4(+) T cell loss from blood and tissues. JRCSFNefdd infection did not activate CD8(+) T cells or reduce the level of CD4(+) T cells in blood but did result in a small Nef-independent decrease in CD4(+) T cells in organs. These observations strongly support the conclusion that viral pathogenicity is mostly driven by Nef. We also observed for the first time substantial host-specific suppression of HIV-1 replication in a small animal infection model.

In vivo analysis of highly conserved Nef activities in HIV-1 replication and pathogenesis.

BACKGROUND: The HIV-1 accessory protein, Nef, is decisive for progression to AIDS. In vitro characterization of the protein has described many Nef activities of unknown in vivo significance including CD4 downregulation and a number of activities that depend on Nef interacting with host SH3 domain proteins. Here, we use the BLT humanized mouse model of HIV-1 infection to assess their impact on viral replication and pathogenesis and the selection pressure to restore these activities using enforced in vivo evolution. RESULTS: We followed the evolution of HIV-1LAI (LAI) with a frame-shifted nef (LAINeffs) during infection of BLT mice. LAINeffs was rapidly replaced in blood by virus with short deletions in nef that restored the open reading frame (LAINeffs∆-1 and LAINeffs∆-13). Subsequently, LAINeffs∆-1 was often replaced by wild type LAI. Unexpectedly, LAINeffs∆-1 and LAINeffs∆-13 Nefs were specifically defective for CD4 downregulation activity. Viruses with these mutant nefs were used to infect BLT mice. LAINeffs∆-1 and LAINeffs∆-13 exhibited three-fold reduced viral replication (compared to LAI) and a 50% reduction of systemic CD4+ T cells (>90% for LAI) demonstrating the importance of CD4 downregulation. These results also demonstrate that functions other than CD4 downregulation enhanced viral replication and pathogenesis of LAINeffs∆-1 and LAINeffs∆-13 compared to LAINeffs. To gain insight into the nature of these activities, we constructed the double mutant P72A/P75A. Multiple Nef activities can be negated by mutating the SH3 domain binding site (P72Q73V74P75L76R77) to P72A/P75A and this mutation does not affect CD4 downregulation. Virus with nef mutated to P72A/P75A closely resembled the wild-type virus in vivo as viral replication and pathogenesis was not significantly altered. Unlike LAINeffs described above, the P72A/P75A mutation had a very weak tendency to revert to wild type sequence. CONCLUSIONS: The in vivo phenotype of Nef is significantly dependent on CD4 downregulation but minimally on the numerous Nef activities that require an intact SH3 domain binding motif. These results suggest that CD4 downregulation plus one or more unknown Nef activities contribute to enhanced viral replication and pathogenesis and are suitable targets for anti-HIV therapy. Enforced evolution studies in BLT mice will greatly facilitate identification of these critical activities.

Overlapping effector interfaces define the multiple functions of the HIV-1 Nef polyproline helix.

BACKGROUND: HIV-1 Nef is a multifunctional protein required for full pathogenicity of the virus. As Nef has no known enzymatic activity, it necessarily functions through protein-protein interaction interfaces. A critical Nef protein interaction interface is centered on its polyproline segment (P69VRPQVPLRP78) which contains the helical SH3 domain binding protein motif, PXXPXR. We hypothesized that any Nef-SH3 domain interactions would be lost upon mutation of the prolines or arginine of PXXPXR. Further, mutation of the non-motif "X" residues, (Q73, V74, and L75) would give altered patterns of inhibition for different Nef/SH3 domain protein interactions. RESULTS: We found that mutations of either of the prolines or the arginine of PXXPXR are defective for Nef-Hck binding, Nef/activated PAK2 complex formation and enhancement of virion infectivity (EVI). Mutation of the non-motif "X" residues (Q, V and L) gave similar patterns of inhibition for Nef/activated PAK2 complex formation and EVI which were distinct from the pattern for Hck binding. These results implicate an SH3 domain containing protein other than Hck for Nef/activated PAK2 complex formation and EVI. We have also mutated Nef residues at the N-and C-terminal ends of the polyproline segment to explore interactions outside of PXXPXR. We discovered a new locus GFP/F (G67, F68, P69 and F90) that is required for Nef/activated PAK2 complex formation and EVI.MHC Class I (MHCI) downregulation was only partially inhibited by mutating the PXXPXR motif residues, but was fully inhibited by mutating the C-terminal P78. Further, we observed that MHCI downregulation strictly requires G67 and F68. Our mutational analysis confirms the recently reported structure of the complex between Nef, AP-1 µ1 and the cytoplasmic tail of MHCI, but does not support involvement of an SH3 domain protein in MHCI downregulation. CONCLUSION: Nef has evolved to be dependent on interactions with multiple SH3 domain proteins. To the N- and C- terminal sides of the polyproline helix are multifunctional protein interaction sites. The polyproline segment is also adapted to downregulate MHCI with a non-canonical binding surface. Our results demonstrate that Nef polyproline helix is highly adapted to directly interact with multiple host cell proteins.

Nef functions in BLT mice to enhance HIV-1 replication and deplete CD4+CD8+ thymocytes.

BACKGROUND: The outcome of untreated HIV-1 infection is progression to AIDS and death in nearly all cases. Some important exceptions are the small number of patients infected with HIV-1 deleted for the accessory gene, nef. With these infections, disease progression is entirely suppressed or greatly delayed. Whether Nef is critical for high levels of replication or is directly cytotoxic remains controversial. The major problem in determining the role of Nef in HIV/AIDS has been the lack of tractable in vivo models where Nef's complex pathogenic phenotype can be recapitulated. RESULTS: Intravenous inoculation (3000 to 600,000 TCIU) of BLT humanized mice with HIV-1LAI reproducibly establishes a systemic infection. HIV-1LAI (LAI) replicates to high levels (peak viral load in blood 8,200,000 ± 1,800,000 copies of viral RNA/ml, range 3,600,000 to 20,400,000; n = 9) and exhaustively depletes CD4+ T cells in blood and tissues. CD4+CD8+ thymocytes were also efficiently depleted but CD4+CD8- thymocytes were partially resistant to cell killing by LAI. Infection with a nef-deleted LAI (LAINefdd) gave lower peak viral loads (1,220,000 ± 330,000, range 27,000 to 4,240,000; n = 17). For fourteen of seventeen LAINefdd-infected mice, there was little to no loss of either CD4+ T cells or thymocytes. Both LAI- and LAINefdd-infected mice had about 8% of total peripheral blood CD8+ T cells that were CD38+HLA-DR+ compared <1% for uninfected mice. Three exceptional LAINefdd-infected mice that lost CD4+ T cells received 600,000 TCIU. All three exhibited peak viral loads over 3,000,000 copies of LAINefdd RNA/ml. Over an extended time course, substantial systemic CD4+ T cell loss was observed for the three mice, but there was no loss of CD4+CD8+ or CD4+CD8- thymocytes. CONCLUSION: We conclude Nef is necessary for elevated viral replication and as a result indirectly contributes to CD4+ T cell killing. Further, Nef was not necessary for the activation of peripheral blood CD8+ T cells following infection. However, CD4+CD8+ thymocyte killing was dependent on Nef even in cases of elevated LAINefdd replication and T cell loss. This depletion of thymic T cell precursors may be a significant factor in the elevated pathogenicity of CXCR4 trophic HIV-1.

Analysis of CFTR folding and degradation in transiently transfected cells.

Misfolding and premature degradation of F508del-CFTR is the major cause of cystic fibrosis. Components of the ubiquitin-proteasome system function on the surface of the endoplasmic reticulum to select misfolded proteins for degradation. The folding status of F508del-CFTR is monitored by at least two ER quality control checkpoints. The ER-associated Derlin-1/RMA1 E3 complex appears to recognize folding defects in CFTR that involve misassembly of NBD1 into a complex with the R-domain. In contrast, the cytosolic Hsp70/CHIP E3 complex appears to sense folding defects that occur after synthesis of NBD2. Herein we describe methods that allow for the study of how modulation of these ER quality control factors by siRNA impacts CFTR folding and degradation. The experimental system described employs transiently transfected HEK293 cells and is utilized to monitor the biogenesis of CFTR by both Western blot and pulse chase studies. Methods to detect complexes formed between CFTR folding intermediates and ER quality control factors will also be described.