and AI104887 to S.W.) and The American Association of Immunologists Careers in Immunology Fellowship Program (AAI EIN 52-2317193 to C.B.L.). drives the persistence of otherwise acute viruses. Introduction Persistent viral genomes are observed after a number of acute viral infections in humans, including respiratory syncytial virus (RSV), measles, and Ebola1C3. A number of host factors, such as impaired or altered cytokine production and progressive loss of immunological functions, support the maintenance of persistent infections4. However, the processes and cellular mechanisms determining the onset of viral persistence after acute viral infections remain unknown. The innate immune response is the first active host barrier to virus replication and is essential to control the infection and activate adaptive responses that result in virus clearance. The antiviral innate response is initiated upon recognition of viral molecular patterns by cellular sensor molecules. Activation of these sensor pathways leads to the expression of genes with pro-inflammatory, antiviral, and pro-apoptotic activities that control virus growth and spread. During infections with important human pathogens including RSV, parainfluenza virus, and measles virus, the antiviral response is triggered by replication defective copy-back viral genomes (DVGs) that accumulate during viral replication5C8. DVGs potently stimulate intracellular RIG-I-like receptors (RLRs) that signal through the mitochondrial antiviral-signaling (MAVS) protein to stimulate the expression of genes that control virus replication and spread, and direct clearance of infected cells9, 10. Paradoxically, some species of DVGs can promote the establishment of persistent RSV, parainfluenza virus, measles virus, and other viruses during infections in tissue culture11C14 and are proposed to be responsible for establishing persistent Ebola virus infections in humans1. This pro-persistence activity of DVGs has been related to the continuous competition for the viral polymerase between full-length genomes and Cefuroxime sodium DVGs, resulting in alternating cycles of replication of full-length and defective genomes15C17. However, this mechanism cannot explain the survival of virus-infected cells in the presence of strong pro-apoptotic and Cefuroxime sodium antiviral molecules, including type I IFNs and TNF, that are induced in response to sensing of DVGs10. In order to better understand the hostCvirus interactions driving the establishment of persistent infections of otherwise acute viruses, we developed a technology that allowed us to investigate at a single cell level the mechanisms behind the different activities of DVGs in infected cells. Using fluorescent in situ hybridization targeting ribonucleic acid molecules (RNA FISH) to Cefuroxime sodium distinguish DVGs from standard viral genomes during infection, we reveal that during infection with the murine parainfluenza virus Sendai (SeV) or RSV DVGs accumulate only in a subpopulation of infected cells, and that these cells survive the infection longer than cells enriched in full-length virus Survival of DVG-high cells is dependent on MAVS signaling, and we identify TNF produced in response to MAVS signaling as pivotal in determining cell fate during SeV infection. We show that while cells harboring full-length viral genomes die from virus-induced TNF-mediated apoptosis, cells enriched in DVGs regulate the Rabbit polyclonal to ZNF33A expression and activity of a TNFR2/TRAF1 pro-survival Cefuroxime sodium program that protects them from TNF-induced apoptosis. Overall, this study reveals a mechanism by which distinct viral genomic products determine cell fate upon infection by taking advantage of the dual functions of TNF to perpetuate both virus and host. Results DVGs dominate in a subpopulation of infected cells To better understand the impact of DVGs during infection, we established a RNA FISH assay that allowed us to differentiate SeV full-length genomes (FL-gSeV) from SeV DVGs at a single cell level. As copy-back DVGs are generated from the 5? end of the viral genome and thus have a high sequence homology with the FL-gSeV18, 19, we designed a two-color probing strategy to distinguish DVGs from FL genomes within infected cells (Fig.?1a). To detect replicating virus, a set of probes labeled with Quasar-570 (pseudo-colored red) was prepared against the 5? end of the positive sense viral RNA and a different set of probes labeled with Quasar-670 (pseudo-colored green) was prepared against the 3? end of the positive sense SeV genome, which covers the viral genomic sequence shared with DVGs. As a result of this design, DVGs are only bound by Quasar-670-labeled probes (denoted DVG), while FL-gSeV are bound by a combination of Quasar-570 and Quasar-670-labeled probes (denoted FL-gSeV and appearing as orange in the images) (Fig.?1a). To test the specificity.