Although the molecular mechanisms by which host cells defend themselves against

Although the molecular mechanisms by which host cells defend themselves against viral infection have been studied in great depth and countermeasures viruses employ to suppress such defensive responses have been widely documented relatively little attention has been devoted toward elucidating how such interactions between virus and host are resolved over multiple rounds of infection. (ISG) involved in the interference of viral protein translation and a marker of antiviral defense activation. The presence of the fluorescent protein reporters had minimal effects on the normal behavior of the cells or viruses. Moreover expression of the virus and cell reporters correlated with the kinetics of viral replication and activation of an anti-viral response respectively. This two-color system enabled us to track and quantify in live cells how viral replication and activation of host defensive responses play out over multiple rounds of infection. Initial study of propagating infections demonstrated that antiviral activation over multiple rounds was critical for slowing and ultimately halting the spread of infection. INTRODUCTION Shortly after a virus enters a cell and initiates replication pathogen recognition receptors (PRRs) of the host cell detect the presence of viral nucleic Rabbit Polyclonal to RPS2. acids and proteins and trigger signaling cascades that activate antiviral defenses. A A 922500 competition ensues pitting the ability of the virus to replicate and suppress the host-cell response against the ability of its host cell to launch defenses to inhibit the viral replication and induce signaling to warn neighboring cells. Decades of research have elucidated many diverse molecular mechanisms of the virus-host arms race within an infected cell; however less attention has been paid to how the release of virus and signals from an infected cell affects the subsequent spread of infection. PRRs such as the toll-like receptors and RNA helicases are responsible for the initial detection of protein or A 922500 nucleic A 922500 acid species produced by the virus. For infection by RNA viruses it is thought to be primarily RNA helicases such as RIG-I or MDA5 that detect aberrant RNA intermediates such as double-stranded RNA (dsRNA) A 922500 or uncapped unencapsidated forms of single-stranded RNA (ssRNA) (reviewed in Gerlier and Lyles 2011 Kawai and Akira 2006 Randall and Goodbourn 2008 These sensors then set off signaling cascades that lead to the production of type-I interferons (IFNs) and other secreted cytokines. These signaling molecules can then feedback through the type I interferon receptor (IFNAR) on the infected cell or stimulate further gene expression in neighboring cells. Such interferon stimulated genes (ISGs) can have additional signaling roles A 922500 or direct the degradation of viral RNA prevent translation of viral proteins or carry out other antiviral activities. To effectively replicate in the face of potentially diverse antiviral responses viruses have evolved a wide range of strategies to either suppress or evade these defenses (Andrejeva et al. 2004 García-Sastre and Biron 2006 Unterstab et al. 2005 In the species used in this work vesicular stomatitis virus (VSV) its matrix(M) protein translocates to nuclear pores and prevents the export of the host mRNAs through a Rae1 dependent interaction with the nucleoporin Nup98. This interaction largely suppresses the activation of host defensive response genes (Petersen et al. 2000 Rajani et al. 2012 Stojdl et al. 2003 Here we employ a VSV mutant that carries a methionine-to-arginine point mutation in its M protein (M51R) that largely abolishes this function and is a useful tool for probing the ability of the host to respond in the absence of viral suppression (Ahmed et al. 2003 Rajani et al. 2012 The transcriptional activity of type I IFNs and ISGs have been probed by promoter-reporter constructs originally with luciferase or CAT reporters (Bluyssen et al. 1994 Wang et al. 2000 but more recently with fluorescent protein readouts (Martínez-Sobrido et al. 2006 Nguyen et al. 2009 Such fluorescent reporters have allowed for the discrimination of individual cell activity and demonstrated a spatially heterogeneous A 922500 response of individual cells to viral stimuli. An IFN-β-GFP reporter cell line was used to show that in response to parainfluenza infection only a small minority of cells are able to activate their IFN-β gene but that this minority is sufficient to produce enough secreted cytokine to activate ISGs in the majority of neighboring cells.