Oxygen-Independent Stabilization of Hypoxia Inducible Factor (HIF)-1 during RSV Infection

Helene A Haeberle,Yashoda M Hosakote,Volkhard A J Kempf,Peter Rosenberger,Roberto P Garofalo,Holger K Eltzschig,Johannes Kuhlicke,Carin Dürrstein,Rory Edward Morty

doi:10.1371/journal.pone.0003352

Abstract

BackgroundHypoxia-inducible factor 1 (HIF)-1α is a transcription factor that functions as master regulator of mammalian oxygen homeostasis. In addition, recent studies identified a role for HIF-1α as transcriptional regulator during inflammation or infection. Based on studies showing that respiratory syncytial virus (RSV) is among the most potent biological stimuli to induce an inflammatory milieu, we hypothesized a role of HIF-1α as transcriptional regulator during infections with RSV.Methodology, Principal FindingsWe gained first insight from immunohistocemical studies of RSV-infected human pulmonary epithelia that were stained for HIF-1α. These studies revealed that RSV-positive cells also stained for HIF-1α, suggesting concomitant HIF-activation during RSV infection. Similarly, Western blot analysis confirmed an approximately 8-fold increase in HIF-1α protein 24 h after RSV infection. In contrast, HIF-1α activation was abolished utilizing UV-treated RSV. Moreover, HIF-α-regulated genes (VEGF, CD73, FN-1, COX-2) were induced with RSV infection of wild-type cells. In contrast, HIF-1α dependent gene induction was abolished in pulmonary epithelia following siRNA mediated repression of HIF-1α. Measurements of the partial pressure of oxygen in the supernatants of RSV infected epithelia or controls revealed no differences in oxygen content, suggesting that HIF-1α activation is not caused by RSV associated hypoxia. Finally, studies of RSV pneumonitis in mice confirmed HIF-α-activation in a murine in vivo model.Conclusions/SignificanceTaking together, these studies suggest hypoxia-independent activation of HIF-1α during infection with RSV in vitro and in vivo.

Highlights

A number of elegant studies, exemplified by those defining induction of the erythropoietin (EPO) gene [1,2], have utilized multidisciplinary approaches to elucidate basic hypoxia-adaptive responses
Previous studies demonstrated that respiratory syncytial virus (RSV) infections are characterized by a prominent inflammation of the pulmonary mucosaboth in natural and experimental infections-and RSV is among the most potent biological stimuli that induce the expression of pro-inflammatory genes [36]
Isotype controls and staining of infected A549 cells with secondary antibody alone were negative. These data reveal that during RSV infection, Hypoxia-inducible factor 1 (HIF)-1a accumulates in the cytosole and the nucleus of infected pulmonary epithelia, suggesting HIF-1a activation during RSV infection in vitro

Summary

Introduction

A number of elegant studies, exemplified by those defining induction of the erythropoietin (EPO) gene [1,2], have utilized multidisciplinary approaches to elucidate basic hypoxia-adaptive responses. Today, convincing evidence confirms a central role of hypoxia-inducible factor (HIF)-1 in mammalian oxygen homeostasis [3,4,5,6]. Such studies demonstrated that HIF-1 is composed of two subunits: constitutively expressed HIF-1b and oxygenregulated HIF-1a. Hydroxylation is inhibited and the VHL protein does not bind to HIF-1, eventually leading to stabilization of the alpha-subunit, heterodymerization, nuclear translocation and transcription of HIF-dependent genes. Hypoxia-inducible factor 1 (HIF)-1a is a transcription factor that functions as master regulator of mammalian oxygen homeostasis. Based on studies showing that respiratory syncytial virus (RSV) is among the most potent biological stimuli to induce an inflammatory milieu, we hypothesized a role of HIF-1a as transcriptional regulator during infections with RSV

Methods

Results

Conclusion