TRAF6 Plays a Proviral Role in Tick-Borne Flavivirus Infection through Interaction with the NS3 Protease.

Tick-borne flaviviruses (TBFVs) can cause life-threatening encephalitis and hemorrhagic fever. To identify virus-host interactions that may be exploited as therapeutic targets, we analyzed the TBFV polyprotein in silico for antiviral protein-binding motifs. We obtained two putative tumor necrosis factor receptor-associated factor 6 (TRAF6)-binding motifs (TBMs) within the protease domain of the viral nonstructural 3 (NS3) protein. Here, we show that TBFV NS3 interacted with TRAF6 during infection and that TRAF6 supports TBFV replication. The proviral role of TRAF6 was not seen with mosquito-borne flaviviruses, consistent with the lack of conserved TBMs. Mutation of the second TBM within NS3 disrupted TRAF6 binding, coincident with reduced abundance of mature, autocatalytically derived form of the NS3 protease and significant virus attenuation in vitro. Our studies reveal insights into how flaviviruses exploit innate immunity for the purpose of viral replication and identify a potential target for therapeutic design.

Roles of Toll-Like Receptor 2 (TLR2), TLR4, and MyD88 during Pulmonary Coxiella burnetii Infection.

Coxiella burnetii, the causative agent of Q fever, is an obligate intracellular, primarily pulmonary, bacterial pathogen. Although much is known about adaptive immune responses against this bacterium, our understanding of innate immune responses against C. burnetii is not well defined, particularly within the target tissue for infection, the lung. Previous studies examined the roles of the innate immune system receptors Toll-like receptor 2 (TLR2) and TLR4 in peripheral infection models and described minimal phenotypes in specific gene deletion animals compared to those of their wild-type controls (S. Meghari et al., Ann N Y Acad Sci 1063:161-166, 2005,http://dx.doi.org/10.1196/annals.1355.025; A. Honstettre et al., J Immunol 172:3695-3703, 2004,http://dx.doi.org/10.4049/jimmunol.172.6.3695) . Here, we assessed the roles for TLR2, TLR4, and MyD88 in pulmonary C. burnetii infection and compared responses to those that occurred in TLR2- and TLR4-deficient animals following peripheral infection. As observed previously, neither TLR2 nor TLR4 was needed for limiting bacterial growth after peripheral infection. In contrast, TLR2 and, to a lesser extent, TLR4 limited growth (or dissemination) of the bacterium in the lung and spleen after pulmonary infection. TLR2, TLR4, and MyD88 were not required for the general inflammatory response in the lungs after pulmonary infection. However, MyD88 signaling was important for infection-induced morbidity. Finally, TLR2 expression on hematopoietic cells was most important for limiting bacterial growth in the lung. These results expand on our knowledge of the roles for TLR2 and TLR4 in C. burnetii infection and suggest various roles for these receptors that are dictated by the site of infection.

Tick-borne flaviviruses antagonize both IRF-1 and type I IFN signaling to inhibit dendritic cell function.

Tick-borne encephalitis virus (TBEV), a member of the Flaviviridae family, is a leading cause of viral encephalitis in Europe and Asia. Dendritic cells (DCs), as early cellular targets of infection, provide an opportunity for flaviviruses to inhibit innate and adaptive immune responses. Flaviviruses modulate DC function, but the mechanisms underpinning this are not defined. We examined the maturation phenotype and function of murine bone marrow-derived DCs infected with Langat virus (LGTV), a naturally attenuated member of the TBEV serogroup. LGTV infection failed to induce DC maturation or a cytokine response. Treatment with LPS or LPS/IFN-γ, strong inducers of inflammatory cytokines, resulted in enhanced TNF-α and IL-6 production, but suppressed IL-12 production in infected DCs compared with uninfected "bystander" cells or mock-infected controls. LGTV-mediated antagonism of type I IFN (IFN-I) signaling contributed to inhibition of IL-12p40 mRNA expression at late time points after stimulation. However, early suppression was still observed in DCs lacking the IFN-I receptor (Ifnar(-/-)), suggesting that additional mechanisms of antagonism exist. The early IFN-independent inhibition of IL-12p40 was nearly abolished in DCs deficient in IFN regulatory factor-1 (IRF-1), a key transcription factor required for IL-12 production. LGTV infection did not affect Irf-1 mRNA expression, but rather diminished IRF-1 protein levels and nuclear localization. The effect on IRF-1 was also observed in DCs infected with the highly virulent Sofjin strain of TBEV. Thus, antagonism of IRF-1 is a novel mechanism that synergizes with the noted ability of flaviviruses to suppress IFN-α/ß receptor-dependent signaling, resulting in the orchestrated evasion of host innate immunity.

Toll-like receptor 7 suppresses virus replication in neurons but does not affect viral pathogenesis in a mouse model of Langat virus infection.

Toll-like receptor 7 (TLR7) recognizes guanidine-rich viral ssRNA and is an important mediator of peripheral immune responses to several ssRNA viruses. However, the role that TLR7 plays in regulating the innate immune response to ssRNA virus infections in specific organs such as the central nervous system (CNS) is not as clear. This study examined the influence of TLR7 on the neurovirulence of Langat virus (LGTV), a ssRNA tick-borne flavivirus. TLR7 deficiency did not substantially alter the onset or incidence of LGTV-induced clinical disease; however, it did significantly affect virus levels in the CNS with a log(10) increase in virus titres in brain tissue from TLR7-deficient mice. This difference in virus load was also observed following intracranial inoculation, indicating a direct effect of TLR7 deficiency on regulating virus replication in the brain. LGTV-induced type I interferon responses in the CNS were not dependent on TLR7, being higher in TLR7-deficient mice compared with wild-type controls. In contrast, induction of pro-inflammatory cytokines including tumour necrosis factor, CCL3, CCL4 and CXCL13 were dependent on TLR7. Thus, although TLR7 is not essential in controlling LGTV pathogenesis, it is important in controlling virus infection in neurons in the CNS, possibly by regulating neuroinflammatory responses.

Identification and characterization of the host protein DNAJC14 as a broadly active flavivirus replication modulator.

Viruses in the Flavivirus genus of the Flaviviridae family are arthropod-transmitted and contribute to staggering numbers of human infections and significant deaths annually across the globe. To identify cellular factors with antiviral activity against flaviviruses, we screened a cDNA library using an iterative approach. We identified a mammalian Hsp40 chaperone protein (DNAJC14) that when overexpressed was able to mediate protection from yellow fever virus (YFV)-induced cell death. Further studies revealed that DNAJC14 inhibits YFV at the step of viral RNA replication. Since replication of bovine viral diarrhea virus (BVDV), a member of the related Pestivirus genus, is also known to be modulated by DNAJC14, we tested the effect of this host factor on diverse Flaviviridae family members. Flaviviruses, including the pathogenic Asibi strain of YFV, Kunjin, and tick-borne Langat virus, as well as a Hepacivirus, hepatitis C virus (HCV), all were inhibited by overexpression of DNAJC14. Mutagenesis showed that both the J-domain and the C-terminal domain, which mediates self-interaction, are required for anti-YFV activity. We found that DNAJC14 does not block YFV nor HCV NS2-3 cleavage, and using non-inhibitory mutants demonstrate that DNAJC14 is recruited to YFV replication complexes. Immunofluorescence analysis demonstrated that endogenous DNAJC14 rearranges during infection and is found in replication complexes identified by dsRNA staining. Interestingly, silencing of endogenous DNAJC14 results in impaired YFV replication suggesting a requirement for DNAJC14 in YFV replication complex assembly. Finally, the antiviral activity of overexpressed DNAJC14 occurs in a time- and dose-dependent manner. DNAJC14 overexpression may disrupt the proper stoichiometry resulting in inhibition, which can be overcome upon restoration of the optimal ratios due to the accumulation of viral nonstructural proteins. Our findings, together with previously published work, suggest that the members of the Flaviviridae family have evolved in unique and important ways to interact with this host Hsp40 chaperone molecule.