The Varicella-Zoster Virus ORF47 Kinase Interferes with Host Innate Immune Response by Inhibiting the Activation of IRF3

The innate immune response constitutes the first line of host defence that limits viral spread and plays an important role in the activation of adaptive immune response. Viral components are recognized by specific host pathogen recognition receptors triggering the activation of IRF3. IRF3, along with NF-κB, is a key regulator of IFN-β expression. Until now, the role of IRF3 in the activation of the innate immune response during Varicella-Zoster Virus (VZV) infection has been poorly studied. In this work, we demonstrated for the first time that VZV rapidly induces an atypical phosphorylation of IRF3 that is inhibitory since it prevents subsequent IRF3 homodimerization and induction of target genes. Using a mutant virus unable to express the viral kinase ORF47p, we demonstrated that (i) IRF3 slower-migrating form disappears; (ii) IRF3 is phosphorylated on serine 396 again and recovers the ability to form homodimers; (iii) amounts of IRF3 target genes such as IFN-β and ISG15 mRNA are greater than in cells infected with the wild-type virus; and (iv) IRF3 physically interacts with ORF47p. These data led us to hypothesize that the viral kinase ORF47p is involved in the atypical phosphorylation of IRF3 during VZV infection, which prevents its homodimerization and subsequent induction of target genes such as IFN-β and ISG15

The Nitric Oxide Pathway Provides Innate Antiviral Protection in Conjunction with the Type I Interferon Pathway in Fibroblasts

The innate host response to virus infection is largely dominated by the production of type I interferon and interferon stimulated genes. In particular, fibroblasts respond robustly to viral infection and to recognition of viral signatures such as dsRNA with the rapid production of type I interferon; subsequently, fibroblasts are a key cell type in antiviral protection. We recently found, however, that primary fibroblasts deficient for the production of interferon, interferon stimulated genes, and other cytokines and chemokines mount a robust antiviral response against both DNA and RNA viruses following stimulation with dsRNA. Nitric oxide is a chemical compound with pleiotropic functions; its production by phagocytes in response to interferon-γ is associated with antimicrobial activity. Here we show that in response to dsRNA, nitric oxide is rapidly produced in primary fibroblasts. In the presence of an intact interferon system, nitric oxide plays a minor but significant role in antiviral protection. However, in the absence of an interferon system, nitric oxide is critical for the protection against DNA viruses. In primary fibroblasts, NF-κB and interferon regulatory factor 1 participate in the induction of inducible nitric oxide synthase expression, which subsequently produces nitric oxide. As large DNA viruses encode multiple and diverse immune modulators to disable the interferon system, it appears that the nitric oxide pathway serves as a secondary strategy to protect the host against viral infection in key cell types, such as fibroblasts, that largely rely on the type I interferon system for antiviral protection

Genetic and Molecular In Vivo Analysis of Herpes Simplex Virus Assembly in Murine Visual System Neurons

Herpes simplex virus (HSV) infects both epithelial cells and neuronal cells of the human host. Although HSV assembly has been studied extensively for cultured epithelial and neuronal cells, cultured neurons are biochemically, physiologically, and anatomically significantly different than mature neurons in vivo. Therefore, it is imperative that viral maturation and assembly be studied in vivo. To study viral assembly in vivo, we inoculated wild-type and replication-defective viruses into the posterior chamber of mouse eyes and followed infection in retinal ganglion cell bodies and axons. We used PCR techniques to detect viral DNA and RNA and electron microscopy immunohistochemistry and Western blotting to detect viral proteins in specific portions of the optic tract. This approach has shown that viral DNA replication is necessary for viral DNA movement into axons. Movement of viral DNA along ganglion cell axons occurs within capsid-like structures at the speed of fast axonal transport. These studies show that the combined use of intravitreal injections of replication-defective viruses and molecular probes allows the genetic analysis of essential viral replication and maturation processes in neurons in vivo. The studies also provide novel direct evidence for the axonal transport of viral DNA and support for the subassembly hypothesis of viral maturation in situ

ICP0 and the U(S)3 protein kinase of herpes simplex virus 1 independently block histone deacetylation to enable gene expression

SK-N-SH cells exposed to low ratios of ICP0-null (ΔICP0) mutants of herpes simplex virus per cell express the viral α proteins, but the progression to β and γ gene expression does not ensue. In these restrictive cells, post-α gene expression can be induced after exposure of the infected cells to sodium butyrate, an indication that VP16 brought into cells by the virus and the α gene products made after infection cannot block the silencing of viral post-α genes by histone deacetylases (HDACs). This observation is consistent with evidence reported earlier that ICP0 dissociates HDAC1/2 from the CoREST/REST complex. In permissive U2OS cells, replication is independent of the ratio of ΔICP0 mutant per cell. To determine whether other viral genes are involved in blocking HDACs, we used a surrogate system consisting of baculoviruses carrying viral or cellular genes driven by CMV immediate–early promoter. Expression of these genes requires blocking of histone deacetylation. We report that (i) cotransduced U(S)3 or U(S)3.5 protein kinase substitutes for sodium butyrate in enabling the expression of a reporter gene in restrictive cells and enhancing it in permissive cells; (ii) HDAC1 is phosphorylated concomitant with the expression of reporter genes; and (iii) the amounts and appearance of HDAC1 are altered in transduced cells expressing U(S)3 protein kinase in the absence of other viral proteins. We conclude that the U(S)3 protein kinase blocks histone deacetylation by a mechanism distinct from that of ICP0 and that debilitated histone deacetylation contributes to the permissiveness of U2OS cells for ΔICP0 mutants