HIV-1 Nef associated PAK and PI3-kinases stimulate Akt-independent Bad-phosphorylation to induce anti-apoptotic signals.

A highly conserved signaling property of Nef proteins encoded by human or simian immunodeficiency virus is the binding and activation of a PAK kinase whose function is unclear. Here we show that Nef-mediated p21-activated kinase (PAK) activation involves phosphatidylinositol 3-kinase, which acts upstream of PAK and is bound and activated by Nef similar to the manner of Polyoma virus middle T antigen. The Nef-associated phosphatidylinositol-3-PAK complex phosphorylated the pro-apoptotic Bad protein without involving the protein kinase B-Akt kinase, which is generally believed to inactivate Bad by serine phosphorylation. Consequently, Nef, but not a Nef mutant incapable of activating PAK, blocked apoptosis in T cells induced by serum starvation or HIV replication. Nef anti-apoptotic effects are likely a crucial mechanism for viral replication in the host and thus in AIDS pathogenesis.

Induction of Fas ligand expression by HIV involves the interaction of Nef with the T cell receptor zeta chain.

During HIV/SIV infection, there is widespread programmed cell death in infected and, perhaps more importantly, uninfected cells. Much of this apoptosis is mediated by Fas-Fas ligand (FasL) interactions. Previously we demonstrated in macaques that induction of FasL expression and apoptotic cell death of both CD4(+) and CD8(+) T cells by SIV is dependent on a functional nef gene. However, the molecular mechanism whereby HIV-1 induces the expression of FasL remained poorly understood. Here we report a direct association of HIV-1 Nef with the zeta chain of the T cell receptor (TCR) complex and the requirement of both proteins for HIV-mediated upregulation of FasL. Expression of FasL through Nef depended upon the integrity of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR zeta chain. Conformation for the importance of zeta for Nef-mediated signaling in T cells came from an independent finding. A single ITAM motif of zeta but not CD3epsilon was both required and sufficient to promote activation and binding of the Nef-associated kinase (NAK/p62). Our data imply that Nef can form a signaling complex with the TCR, which bypasses the requirement of antigen to initiate T cell activation and subsequently upregulation of FasL expression. Thus, our study may provide critical insights into the molecular mechanism whereby the HIV-1 accessory protein Nef contributes to the pathogenesis of HIV.

A natural variability in the proline-rich motif of Nef modulates HIV-1 replication in primary T cells.

In the infected host, the Nef protein of HIV/SIV is required for high viral loads and thus disease progression. Recent evidence indicates that Nef enhances replication in the T cell compartment after the virus is transmitted from dendritic cells (DC). The underlying mechanism, however, is not clear. Here, we report that a natural variability in the proline-rich motif (R71T) profoundly modulated Nef-stimulated viral replication in primary T cells of immature dendritic cell/T cell cocultures. Whereas both Nef variants (R/T-Nef) downregulated CD4, only the isoform supporting viral replication (R-Nef) efficiently interacted with signaling molecules of the T cell receptor (TCR) environment and stimulated cellular activation. Structural analysis suggested that the R to T conversion induces conformational changes, altering the flexibility of the loop containing the PxxP motif and hence its ability to bind cellular partners. Our report suggests that functionally and conformationally distinct Nef isoforms modulate HIV replication on the interaction level with the TCR-signaling environment once the virus enters the T cell compartment.

A mechanistic, model-based approach to safety assessment in clinical development.

Assessing the safety of pharmacotherapies is a primary goal of clinical trials in drug development. The low frequency of relevant side effects, however, often poses a significant challenge for risk assessment. Methodologies allowing robust extrapolation of safety statistics based on preclinical data and information from clinical trials with limited numbers of patients are hence needed to further improve safety and efficacy in the drug development process. Here, we present a generic systems pharmacology approach integrating prior physiological and pharmacological knowledge, preclinical data, and clinical trial results, which allows predicting adverse event rates related to drug exposure. Possible fields of application involve high-risk populations, novel drug candidates, and different dosing scenarios. As an example, the approach is applied to simvastatin and pravastatin and the prediction of myopathy rates in a population with a genotype leading to a significantly increased myopathy risk.CPT: Pharmacometrics & Systems Pharmacology (2012) 1, e13; doi:10.1038/psp.2012.14; advance online publication 7 November 2012.

The N-terminus of Nef from HIV-1/SIV associates with a protein complex containing Lck and a serine kinase.

The Nef protein of human and primate lentiviruses is a key factor in HIV/SIV pathogenesis. Here we report that Nef associates with two different kinases, forming a multiprotein complex at the far N-terminus of the viral protein. One of the kinases was identified as Lck, whereas the second protein was found to be a serine kinase that phosphorylated Nef and Lck in vitro and could be discriminated from the serine kinase identified previously. The Nef-associated kinase complex (NAKC) was demonstrated in COS cells, in HIV-infected cells, and in vitro using recombinant Lck and Nef proteins. Deletion of a short amphipathic alpha-helix in the N-terminus, which was found to be conserved in all Nef proteins, inhibited association of the NAKC and significantly reduced virion infectivity.