HIV-1 Nef associates with p22-phox, a component of the NADPH oxidase protein complex

Abstract

Altered neutrophil function may contribute to the development of AIDS during the course of HIV infection. It has been described that Nef, a regulatory protein from HIV, can modulate superoxide production in other cells, therefore altered superoxide production in neutrophils from HIV infected patients, could be secondary to a direct effect of Nef on components of the NADPH oxidase complex. In this work, we describe that Nef, was capable of increasing superoxide production in human neutrophils. Furthermore, a specific association between Nef and p22-phox, a membrane component of the NADPH oxidase complex, was found. We propose that this association may reflect a capability of Nef to modulate by direct association, the enzymatic complex responsible for one of the most efficient innate defense mechanisms in phagocytes, contributing to the pathogenesis of the disease.

Introduction

The NADPH oxidase complex is composed of two flavocytochrome subunits located in the plasma membrane, gp91-phox and p22-phox constituting the catalytic core of the enzyme, and by three cytosolic subunits, named p47-phox, p67-phox and p40-phox. Upon stimulation, the cytosolic factors are activated and translocated to the plasma membrane and bind the flavocytochrome, thereby completing the active form of the enzyme [1]. Whereas the role of p40-phox remains undefined, there is evidence that points to a role for p47-phox as an adaptor protein between flavocytochrome b558 and p67-phox [2]. On the other hand, p67-phox represents an essential activating cofactor, which includes a domain that regulates the reduction of FAD by NADPH [3], [4]. During activation, inhibitory intramolecular SH3 domain interactions in p47-phox are relieved via phosphorylation events [5], allowing the protein to make direct contact with p22-phox, thereby bringing p67-phox to the membrane where it makes direct contact with gp91-phox [6]. Nevertheless, translocation of cytosolic phox proteins to the flavocytochrome b558 is not enough to activate the oxidase. The presence of the small cytosolic GTPase Rac (1 or 2) is absolutely required for electron transfer reactions [7], and it has been proposed that Rac regulates electron transfer reactions of the assembled oxidase [8] via direct interactions with p67-phox and gp91-phox.

During HIV infection an impaired function of neutrophils has been demonstrated, which compromise the ability to kill bacterial and fungal pathogens predisposing patients to certain opportunistic infections [9]. These abnormalities include decreased number of neutrophils [10], accelerated neutrophil death [11], secondary to an altered oxidative stress [12], impairment of chemotaxis and phagocytosis [13]. These dysfunctional activities have been demonstrated in neutrophils from both symptomatic and asymptomatic HIV infected patients [14], [15]; hence an increased basal superoxide production has been described in HIV infected patients at early stages of the disease [16].

Altered ROS production by HIV-1 infection may contribute to the development of AIDS pathogenesis by inducing an inflammatory status which may lead to recruitment and activation of immune cells [17] for optimal virus spread. Since neutrophils have not been proved to be reservoirs for HIV, they may become targets of the virus by direct contact between viral proteins and elements of the neutrophil cell membrane [18].

Nef is a 27–34 kDa myristoylated protein produced exclusively by HIV-1/2 and simian immunodeficiency virus, playing a pivotal role in AIDS pathogenesis. It is produced shortly after virus infection, and it is expressed throughout all stages of the disease [19]. While Nef is known to enhance virus production and infectivity, it has been recently recognized that Nef also exerts pathogenic effects independently of viral replication [20], [21]. Despite its small size this protein presents a relatively large surface area for protein interaction. Binding of Nef to a multitude of host proteins including plasma membrane receptors, sorting receptors and signaling proteins has been documented [22], [23].

Because it has been shown that Nef can regulate superoxide production in macrophages and microglias [24], [25], we hypothesized that the altered superoxide production observed in neutrophils from HIV infected patients, could be secondary to an interaction between Nef and components of the NADPH oxidase complex. In this work, we show that Nef, was capable of increasing superoxide production in human neutrophils. Furthermore, Nef associated with the p22-phox component of the NADPH oxidase complex, from neutrophil cell lysates and to purified p22-phox. We propose that the dysfunctional activity observed in neutrophils from HIV infected patients may be explained in part by an interaction between viral proteins (such as Nef) and elements of the NADPH oxidase enzymatic complex.