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140?Inhibition of AKT Kinase Activity Decreases Replication of Human Respiratory Syncytial Virus.

ABSTRACT: BackgroundHuman Respiratory Syncytial Virus (RSV) is a leading cause of pediatric pulmonary disease and severe RSV infection predisposes to wheezing later in life. RSV infection has also been shown to be an environmental trigger for asthma. We are investigating whether targeting host factors important for RSV infection is a viable antiviral strategy. Lowering viral burden through these therapies will result in decreased severity of infection and may also prevent the occurrence of pathologic sequelae.MethodsInhibition of AKT by chemical inhibitors, siRNA, or dominant-negative mutants, was tested for activity against RSV replication in cultured cells. We examined the effect of viral protein expression on Akt activation and downstream signal transduction by Western blot and promoter assay. In addition, we examined the effect of Akt on specific viral processes (entry, macromolecular synthesis, and assembly) and proteins both in vitro and in RSV-infected cells, using kinase assays, Western blotting, and qRT-PCR.ResultsWe found that AKT inhibition decreases RSV protein expression and viral titers. Expression of RSV NS2 protein activates AKT, leading to NFkB-dependent transcription, and inhibition of AKT blocks this effect. Activated AKT also phosphorylates RSV P protein at a specific site. Interestingly, AKT inhibitors that target the pleckstrin homology (PH) domain of AKT showed decreased efficacy against RSV compared to those that target AKT kinase activity.ConclusionsInhibition of AKT can effectively decrease RSV replication in culture, likely by decreasing P phosphorylation and thus viral protein transcription and expression. Activation of AKT during RSV infection likely involves the NS2 protein and does not depend on the PH domain of AKT. AKT inhibitors have been found to be safe and efficacious in clinical trials for a number of different cancers; thus, AKT inhibition may be a potential therapeutic treatment for severe RSV infection.

SUBMITTER: Tran K

PROVIDER: S-EPMC3513011 | biostudies-other | 2012 Feb

REPOSITORIES: biostudies-other

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Project description:Respiratory syncytial virus (RSV) is the main cause of acute lower respiratory tract infection (ALRI) in children worldwide. Virus-host interactions affect the progression and prognosis of the infection. Autophagy plays important roles in virus-host interactions. Respiratory epithelial cells serve as the front line of host defense during RSV infection, However, it is still unclear how they interact with RSV. In this study, we found that RSV induced autophagy that favored RSV replication and exacerbated lung pathology in vivo Mechanistically, RSV induced complete autophagy flux through reactive oxygen species (ROS) generation and activation of the AMP-activated protein kinase/mammalian target of rapamycin (AMPK-MTOR) signaling pathway in HEp-2 cells. Furthermore, we evaluated the functions of autophagy in RSV replication and found that RSV replication was increased in HEp-2 cells treated with rapamycin but decreased remarkably in cells treated with 3-methylademine (3-MA) or wortmannin. Knockdown key molecules in the autophagy pathway with short hairpinp RNA (shRNA) against autophagy-related gene 5 (ATG5), autophagy-related gene 7 (ATG7), or BECN1/Beclin 1 or treatment with ROS scavenger N-acetyl-l-cysteine (NAC) and AMPK inhibitor (compound C) suppressed RSV replication. 3-MA or shATG5/BECN1 significantly decreased cell viability and increased cell apoptosis at 48 hours postinfection (hpi). Blocking apoptosis with Z-VAD-FMK partially restored virus replication at 48 hpi. Those results provide strong evidence that autophagy may function as a proviral mechanism in a cell-intrinsic manner during RSV infection.IMPORTANCE An understanding of the mechanisms that respiratory syncytial virus utilizes to interact with respiratory epithelial cells is critical to the development of novel antiviral strategies. In this study, we found that RSV induces autophagy through a ROS-AMPK signaling axis, which in turn promotes viral infection. Autophagy favors RSV replication through blocking cell apoptosis at 48 hpi. Mechanistically, RSV induces mitophagy, which maintains mitochondrial homeostasis and therefore decreases cytochrome c release and apoptosis induction. This study provides a novel insight into this virus-host interaction, which may help to exploit new antiviral treatments targeting autophagy processes.

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140?Inhibition of AKT Kinase Activity Decreases Replication of Human Respiratory Syncytial Virus.

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