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.

Functional characterization of the human immunodeficiency virus type 1 Nef acidic domain.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef downregulates major histocompatibility complex class I (MHC-I) from the cell surface. It has been proposed that the direct interaction of the acidic cluster (AC) of Nef, (62)EEEE(65), with the furin binding region (fbr) of PACS-1 is crucial for this Nef function. Contrary to this proposal, evidence is presented here that the four glutamates in Nef do not functionally engage the PACS-1 fbr. (i) The binding of Nef to the PACS-1 fbr in vitro is much weaker than the binding of the canonical furin AC to the PACS-1 fbr. (ii) The mutation of two of the four glutamates in Nef's AC to alanines does not alter Nef's ability to downregulate MHC-I, and triply mutated Nefs exhibit 50% activity. (iii) The introduction of lysine into the AC has little effect on Nef function. (iv) The mutation of all four glutamates to alanine does debilitate Nef MHC-I downregulation, but this quadruple mutation also impairs the ability of Nef to regulate p21-activated protein kinase and enhance viral particle infectivity. (v) The replacement of the Nef AC with the bona fide AC from furin results in the loss of the expected regulatory properties of the furin AC. (vi) The insertion of the conformation-disrupting amino acid proline into the Nef AC does not disrupt MHC-I downregulation. Our results are consistent with an alternative model in which (62)EEEE(65) plays a stabilizing role in the formation of a ternary complex between Nef, the MHC-I cytoplasmic domain, and AP-1.

Intra- and intersubtype alternative Pak2-activating structural motifs of human immunodeficiency virus type 1 Nef.

The design of antiviral strategies against human immunodeficiency virus type 1 (HIV-1) has been largely derived from studies of subtype B viruses, although they constitute only 12% of infections worldwide. At 50% of all HIV-1 infections worldwide, subtype C viruses are the most predominant. Here, we present evidence that subtype C Nefs display functional Pak2-activating motifs that differ from those found in subtype B and E Nefs. The identification of multiple Pak2-activating structural motifs that singly affect one Nef activity revealed a functional plasticity that has implications for future drug and vaccine design aimed at HIV-1 Nef and its effects on the deregulation of the immune system.

Reconstitution and molecular analysis of an active human immunodeficiency virus type 1 Nef/p21-activated kinase 2 complex.

Human immunodeficiency virus type 1 (HIV-1) Nef activation of p21-activated kinase 2 (PAK-2) was recapitulated in a cell-free system consisting of in vitro-transcribed RNA, rabbit reticulocyte lysate, and microsomal membranes on the basis of the following observations: (i) Nef associated with a kinase endogenous to the rabbit reticulocyte lysate that was identified as PAK-2, (ii) Nef-associated kinase activity was detected with Nefs from HIV-1(SF2), HIV-1(YU2), and SIV(mac239), (iii) kinase activation was not detected with a myristoylation-defective Nef (HIV-1(SF2)NefG2A) or with a Nef defective in PAK-2 activation but fully competent in other Nef functions (HIV-1(SF2)NefF195I), and (iv) Nef-associated kinase activation required activated endogenous p21 GTPases (Rac1 or Cdc42). The cell-free system was used to analyze the mechanism of Nef activation of PAK-2. First, studies suggest that the p21 GTPases may act transiently to enhance Nef activation of PAK-2 in vitro. Second, addition of wortmannin to the cell-free system demonstrated that Nef activation of PAK-2 does not require PI 3-kinase activity. Third, ultracentrifugation analysis revealed that whereas the majority of Nef and PAK-2 partitioned to the supernatant, Nef-associated PAK-2 activity partitioned to the membrane-containing pellet as a low-abundance complex. Lastly, Nef activation of PAK-2 in vitro requires addition of microsomal membranes either during or after translation of the Nef RNA. These results are consistent with a model in which activation of PAK-2 by Nef occurs by recruiting PAK-2 to membranes. As demonstrated herein, the cell-free system is a new and important tool in the investigation of the mechanism of PAK-2 activation by Nef.