A Novel Cre Recombinase Imaging System for Tracking Lymphotropic Virus Infection In Vivo

BACKGROUND:Detection, isolation, and identification of individual virus infected cells during long term infection are critical to advance our understanding of mechanisms of pathogenesis for latent/persistent viruses. However, current approaches to study these viruses in vivo have been hampered by low sensitivity and effects of cell-type on expression of viral encoded reporter genes. We have designed a novel Cre recombinase (Cre)-based murine system to overcome these problems, and thereby enable tracking and isolation of individual in vivo infected cells. METHODOLOGY/PRINCIPAL FINDINGS:Murine gammaherpesvirus 68 (MHV-68) was used as a prototypic persistent model virus. A Cre expressing recombinant virus was constructed and characterised. The virus is attenuated both in lytic virus replication, producing ten-fold lower lung virus titres than wild type virus, and in the establishment of latency. However, despite this limitation, when the sEGFP7 mouse line containing a Cre-activated enhanced green fluorescent protein (EGFP) was infected with the Cre expressing virus, sites of latent and persistent virus infection could be identified within B cells and macrophages of the lymphoid system on the basis of EGFP expression. Importantly, the use of the sEGFP7 mouse line which expresses high levels of EGFP allowed individual virus positive cells to be purified by FACSorting. Virus gene expression could be detected in these cells. Low numbers of EGFP positive cells could also be detected in the bone marrow. CONCLUSIONS/SIGNIFICANCE:The use of this novel Cre-based virus/mouse system allowed identification of individual latently infected cells in vivo and may be useful for the study and long-term monitoring of other latent/persistent virus infections

Involvement of TLR2 in Recognition of Acute Gammaherpesvirus-68 Infection

Toll-like receptors (TLRs) play a crucial role in the activation of innate immunity in response to many viruses. We previously reported the implication of TLR2 in the recognition of Epstein-Barr virus (EBV) by human monocytes. Because murine gammaherpesvirus-68 (MHV-68) is a useful model to study human gammaherpesvirus pathogenesis in vivo, we evaluated the importance of mouse TLR2 in the recognition of MHV-68.In studies using transfected HEK293 cells, MHV-68 lead to the activation of NF-κB reporter through TLR2. In addition, production of interleukin-6 (IL-6) and interferon-α (IFN-α) upon MHV-68 stimulation was reduced in murine embryonic fibroblasts (MEFs) derived from TLR2-/- and MyD88-/- mice as compared to their wild type (WT) counterpart. In transgenic mice expressing a luciferase reporter gene under the control of the mTLR2 promoter, MHV-68 challenge activated TLR2 transcription. Increased expression levels of TLR2 on blood granulocytes (CD115(-)Gr1(+)) and inflammatory monocytes (CD115(+)Gr1(+)), which mobilized to the lungs upon infection with MHV-68, was also confirmed by flow cytometry. Finally, TLR2 or MyD88 deficiency was associated with decreased IL-6 and type 1 IFN production as well as increased viral burden during short-term challenges with MHV-68.TLR2 contributes to the production of inflammatory cytokines and type 1 IFN as well as to the control of viral burden during infection with MHV-68. Taken together, our results suggest that the TLR2 pathway has a relevant role in the recognition of this virus and in the subsequent activation of the innate immune response

Gammaherpesvirus Latency Accentuates EAE Pathogenesis: Relevance to Epstein-Barr Virus and Multiple Sclerosis

Epstein-Barr virus (EBV) has been identified as a putative environmental trigger of multiple sclerosis (MS), yet EBV's role in MS remains elusive. We utilized murine gamma herpesvirus 68 (γHV-68), the murine homolog to EBV, to examine how infection by a virus like EBV could enhance CNS autoimmunity. Mice latently infected with γHV-68 developed more severe EAE including heightened paralysis and mortality. Similar to MS, γHV-68EAE mice developed lesions composed of CD4 and CD8 T cells, macrophages and loss of myelin in the brain and spinal cord. Further, T cells from the CNS of γHV-68 EAE mice were primarily Th1, producing heightened levels of IFN-γ and T-bet accompanied by IL-17 suppression, whereas a Th17 response was observed in uninfected EAE mice. Clearly, γHV-68 latency polarizes the adaptive immune response, directs a heightened CNS pathology following EAE induction reminiscent of human MS and portrays a novel mechanism by which EBV likely influences MS and other autoimmune diseases

Viral Bcl-2-Mediated Evasion of Autophagy Aids Chronic Infection of γHerpesvirus 68

γ-herpesviruses (γHVs) have developed an interaction with their hosts wherein they establish a life-long persistent infection and are associated with the onset of various malignancies. One critical virulence factor involved in the persistency of murine γ-herpesvirus 68 (γHV68) is the viral homolog of the Bcl-2 protein (vBcl-2), which has been implicated to counteract both host apoptotic responses and autophagy pathway. However, the relative significance of the two activities of vBcl-2 in viral persistent infection has yet to be elucidated. Here, by characterizing a series of loss-of-function mutants of vBcl-2, we have distinguished the vBcl-2-mediated antagonism of autophagy from the vBcl-2-mediated inhibition of apoptosis in vitro and in vivo. A mutant γHV68 virus lacking the anti-autophagic activity of vBcl-2 demonstrates an impaired ability to maintain chronic infections in mice, whereas a mutant virus lacking the anti-apoptotic activity of vBcl-2 establishes chronic infections as efficiently as the wild-type virus but displays a compromised ability for ex vivo reactivation. Thus, the vBcl-2-mediated antagonism of host autophagy constitutes a novel mechanism by which γHVs confer persistent infections, further underscoring the importance of autophagy as a critical host determinant in the in vivo latency of γ-herpesviruses

An In Vitro System for Studying Murid Herpesvirus-4 Latency and Reactivation

The narrow species tropisms of Epstein-Barr Virus (EBV) and the Kaposi's Sarcoma -associated Herpesvirus (KSHV) have made Murid Herpesvirus-4 (MuHV-4) an important tool for understanding how gammaherpesviruses colonize their hosts. However, while MuHV-4 pathogenesis studies can assign a quantitative importance to individual genes, the complexity of in vivo infection can make the underlying mechanisms hard to discern. Furthermore, the lack of good in vitro MuHV-4 latency/reactivation systems with which to dissect mechanisms at the cellular level has made some parallels with EBV and KSHV hard to draw. Here we achieved control of the MuHV-4 lytic/latent switch in vitro by modifying the 5′ untranslated region of its major lytic transactivator gene, ORF50. We terminated normal ORF50 transcripts by inserting a polyadenylation signal and transcribed ORF50 instead from a down-stream, doxycycline-inducible promoter. In this way we could establish fibroblast clones that maintained latent MuHV-4 episomes without detectable lytic replication. Productive virus reactivation was then induced with doxycycline. We used this system to show that the MuHV-4 K3 gene plays a significant role in protecting reactivating cells against CD8+ T cell recognition

Interferon Regulatory Factor-1 (IRF-1) Shapes Both Innate and CD8+ T Cell Immune Responses against West Nile Virus Infection

Interferon regulatory factor (IRF)-1 is an immunomodulatory transcription factor that functions downstream of pathogen recognition receptor signaling and has been implicated as a regulator of type I interferon (IFN)-αβ expression and the immune response to virus infections. However, this role for IRF-1 remains controversial because altered type I IFN responses have not been systemically observed in IRF-1-/- mice. To evaluate the relationship of IRF-1 and immune regulation, we assessed West Nile virus (WNV) infectivity and the host response in IRF-1-/- cells and mice. IRF-1-/- mice were highly vulnerable to WNV infection with enhanced viral replication in peripheral tissues and rapid dissemination into the central nervous system. Ex vivo analysis revealed a cell-type specific antiviral role as IRF-1-/- macrophages supported enhanced WNV replication but infection was unaltered in IRF-1-/- fibroblasts. IRF-1 also had an independent and paradoxical effect on CD8+ T cell expansion. Although markedly fewer CD8+ T cells were observed in naïve animals as described previously, remarkably, IRF-1-/- mice rapidly expanded their pool of WNV-specific cytolytic CD8+ T cells. Adoptive transfer and in vitro proliferation experiments established both cell-intrinsic and cell-extrinsic effects of IRF-1 on the expansion of CD8+ T cells. Thus, IRF-1 restricts WNV infection by modulating the expression of innate antiviral effector molecules while shaping the antigen-specific CD8+ T cell response

γ-Herpesvirus-Induced Protection Against Bacterial Infection Is Transient

Herpesviruses are widely disseminated in the population and establish lifelong latency, which is associated with a variety of pathological consequences. A recent report showed that mice latently infected with either murine γ-herpesvirus-68 (γHV68) or murine cytomegalovirus (mCMV), mouse pathogens genetically similar to the human herpesviruses, Epstein-Barr virus, Kaposi's sarcoma–associated herpesvirus, and cytomegalovirus, had enhanced resistance to subsequent bacterial infection, suggesting protective as well as deleterious effects of latency. Here we confirm that latent γHV68 infection confers protection against subsequent infection with Listeria monocytogenes. However, the effect is transient, lasting only a few months