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

Varicella zoster virus glycoprotein C increases chemokine-mediated leukocyte migration

Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV spreads within the host by hijacking leukocytes, including T cells, in the tonsils and other regional lymph nodes, and modifying their activity. In spite of its importance in pathogenesis, the mechanism of dissemination remains poorly understood. Here we addressed the influence of VZV on leukocyte migration and found that the purified recombinant soluble ectodomain of VZV glycoprotein C (rSgC) binds chemokines with high affinity. Functional experiments show that VZV rSgC potentiates chemokine activity, enhancing the migration of monocyte and T cell lines and, most importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation of chemokine activity occurs through the C-terminal part of gC ectodomain, containing predicted immunoglobulin-like domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the chemokine receptor. Finally, we show that VZV viral particles enhance chemokine-dependent T cell migration and that gC is partially required for this activity. We propose that VZV gC activity facilitates the recruitment and subsequent infection of leukocytes and thereby enhances VZV systemic dissemination in humans

CD4 T Cell Immunity Is Critical for the Control of Simian Varicella Virus Infection in a Nonhuman Primate Model of VZV Infection

Primary infection with varicella zoster virus (VZV) results in varicella (more commonly known as chickenpox) after which VZV establishes latency in sensory ganglia. VZV can reactivate to cause herpes zoster (shingles), a debilitating disease that affects one million individuals in the US alone annually. Current vaccines against varicella (Varivax) and herpes zoster (Zostavax) are not 100% efficacious. Specifically, studies have shown that 1 dose of varivax can lead to breakthrough varicella, albeit rarely, in children and a 2-dose regimen is now recommended. Similarly, although Zostavax results in a 50% reduction in HZ cases, a significant number of recipients remain at risk. To design more efficacious vaccines, we need a better understanding of the immune response to VZV. Clinical observations suggest that T cell immunity plays a more critical role in the protection against VZV primary infection and reactivation. However, no studies to date have directly tested this hypothesis due to the scarcity of animal models that recapitulate the immune response to VZV. We have recently shown that SVV infection of rhesus macaques models the hallmarks of primary VZV infection in children. In this study, we used this model to experimentally determine the role of CD4, CD8 and B cell responses in the resolution of primary SVV infection in unvaccinated animals. Data presented in this manuscript show that while CD20 depletion leads to a significant delay and decrease in the antibody response to SVV, loss of B cells does not alter the severity of varicella or the kinetics/magnitude of the T cell response. Loss of CD8 T cells resulted in slightly higher viral loads and prolonged viremia. In contrast, CD4 depletion led to higher viral loads, prolonged viremia and disseminated varicella. CD4 depleted animals also had delayed and reduced antibody and CD8 T cell responses. These results are similar to clinical observations that children with agammaglobulinemia have uncomplicated varicella whereas children with T cell deficiencies are at increased risk of progressive varicella with significant complications. Moreover, our studies indicate that CD4 T cell responses to SVV play a more critical role than antibody or CD8 T cell responses in the control of primary SVV infection and suggest that one potential mechanism for enhancing the efficacy of VZV vaccines is by eliciting robust CD4 T cell responses

Current gene therapy using viral vectors for chronic pain

The complexity of chronic pain and the challenges of pharmacotherapy highlight the importance of development of new approaches to pain management. Gene therapy approaches may be complementary to pharmacotherapy for several advantages. Gene therapy strategies may target specific chronic pain mechanisms in a tissue-specific manner. The present collection of articles features distinct gene therapy approaches targeting specific mechanisms identified as important in the specific pain conditions. Dr. Fairbanks group describes commonly used gene therapeutics (herpes simplex viral vector (HSV) and adeno-associated viral vector (AAV)), and addresses biodistribution and potential neurotoxicity in pre-clinical models of vector delivery. Dr. Tao group addresses that downregulation of a voltage-gated potassium channel (Kv1.2) contributes to the maintenance of neuropathic pain. Alleviation of chronic pain through restoring Kv1.2 expression in sensory neurons is presented in this review. Drs Goins and Kinchington group describes a strategy to use the replication defective HSV vector to deliver two different gene products (enkephalin and TNF soluble receptor) for the treatment of post-herpetic neuralgia. Dr. Hao group addresses the observation that the pro-inflammatory cytokines are an important shared mechanism underlying both neuropathic pain and the development of opioid analgesic tolerance and withdrawal. The use of gene therapy strategies to enhance expression of the anti-pro-inflammatory cytokines is summarized. Development of multiple gene therapy strategies may have the benefit of targeting specific pathologies associated with distinct chronic pain conditions (by Guest Editors, Drs. C. Fairbanks and S. Hao)

Cyclin-Dependent Kinase 1/Cyclin B1 Phosphorylates Varicella-Zoster Virus IE62 and Is Incorporated into Virions▿

Varicella-zoster virus (VZV), an alphaherpesvirus restricted to humans, infects differentiated cells in vivo, including T lymphocytes, keratinocytes, and neurons, and spreads rapidly in confluent cultured dermal fibroblasts (HFFs). In VZV-infected HFFs, atypical expression of cyclins D3 and B1 occurs along with the induction of cyclin-dependent kinase (CDK) activity. A specific CDK1 inhibitor blocked VZV spread, indicating an important function for this cellular kinase in VZV replication. CDK activity assays of infected cells revealed a large viral phosphoprotein that was identified as being the major immediate-early transactivator, IE62. Since IE62 colocalized with CDK1/cyclin B1 by confocal microscopy, we investigated whether this cellular kinase complex interacts with IE62. Using recombinant fragments of IE62 spanning the entire amino acid sequence, we found that purified CDK1/cyclin B1 phosphorylated IE62 at residues T10, S245, and T680 in vitro. Immunoprecipitation of cyclin B1 from VZV-infected HFFs indicated that IE62 was included in the complex within infected cells. The full-length IE62 protein, obtained by immunoprecipitation from infected cells, was also phosphorylated by purified CDK1/cyclin B1. Based on IE62/CDK1/cyclin B1 colocalization near viral assembly regions, we hypothesized that these cellular proteins could be incorporated into VZV virions with IE62. Purified virions were analyzed by immunoblotting for the presence of CDK1 and cyclin B1, and active CDK1 and cyclin B1 were present in the VZV tegument with IE62 and were sensitive to detergent treatment. Thus, IE62 is a substrate for CDK1/cyclin B1, and virions could deliver the active cellular kinase to nondividing cells that normally do not express it

Roscovitine, a Cyclin-Dependent Kinase Inhibitor, Prevents Replication of Varicella-Zoster Virus

Understanding the interactions between varicella-zoster virus (VZV) and host cells can be addressed by using small molecule inhibitors of cellular enzymes. Roscovitine (Rosco) is a purine derivative that inhibits cyclin-dependent kinase 1 (cdk1), cdk2, cdk5, cdk7, and cdk9, which are key regulators of the cell cycle and transcription. Herpesviruses are known to interact with cell cycle proteins; thus, the antiviral effects of Rosco on VZV growth were evaluated. In a plaque reduction assay, 25 μM Rosco prevented VZV replication, and the antiviral effect was reversible for at least up to 24 h posttreatment. Rosco also reduced expression of the major transactivator, IE62, over 48 h. Confocal microscopy studies indicated that Rosco caused the immediate-early proteins ORF4 and IE62 to abnormally localize in infected cells and prevented cell-cell spread of VZV over 48 h. Rosco was found to inhibit VZV DNA synthesis as measured by real-time PCR, and this technique was used to estimate the 50% effective concentration (EC(50)) of 14 μM. This value was close to the EC(50) estimate of 12 μM determined from plaque reduction assays. At 25 μM, Rosco was not cytotoxic over 48 h in a neutral red uptake assay, and proliferation was slowed as the cells accumulated in a G(2)-like state. These results demonstrate the importance of cdk's in VZV replication and suggest that cdk inhibitors could serve as useful VZV antivirals