TRIM5alpha Restricts Flavivirus Replication by Targeting the Viral Protease for Proteasomal Degradation

Tripartite motif-containing protein 5alpha (TRIM5alpha) is a cellular antiviral restriction factor that prevents early events in retrovirus replication. The activity of TRIM5alpha is thought to be limited to retroviruses as a result of highly specific interactions with capsid lattices. In contrast to this current understanding, we show that both human and rhesus macaque TRIM5alpha suppress replication of specific flaviviruses. Multiple viruses in the tick-borne encephalitis complex are sensitive to TRIM5alpha-dependent restriction, but mosquito-borne flaviviruses, including yellow fever, dengue, and Zika viruses, are resistant. TRIM5alpha suppresses replication by binding to the viral protease NS2B/3 to promote its K48-linked ubiquitination and proteasomal degradation. Importantly, TRIM5alpha contributes to the antiviral function of IFN-I against sensitive flaviviruses in human cells. Thus, TRIM5alpha possesses remarkable plasticity in the recognition of diverse virus families, with the potential to influence human susceptibility to emerging flaviviruses of global concern

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

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

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

DNAJC14 inhibits YFV in a temporal and dose-dependent manner.

<p>(A) SW13 cells were mock transduced (Mock) or transduced with Trip-RFP-hNZAP (NZAP), Trip-RFP-hDNAJC14-FL (FL) or Trip-RFP-hDNAJC14-NT1 (NT1) and infected 2 d later with YFV-Venus (moi = 5). Cells were fixed at 1.5 d post infection and analyzed by flow cytometry. The Venus (y-axis) and RFP (x-axis) fluorescence intensities of the cells are shown; gates to indicate expression of the transduced protein or productive infection were set on Mock transduced, uninfected cells (not shown). (B) SW13 cells were transduced with Trip-RFP-hDNAJC14-NT1 and infected 2 d later with YFV-Venus (moi = 5) and analyzed as in (A) at the indicated days after infection. (C) SW13 cells were left untransduced (closed circles) or were transduced with Trip-RFP-hDNAJC14-NT1 (RFP-NT1, open circles) and infected 2 d later with YFV-Venus (moi = 5). At the indicated times, the medium was removed and YFV present in the medium was quantified by plaque assay. Each data point represents the mean titer obtained from duplicate wells; error bars indicating the range are obscured by the symbols. The dotted line indicates the sensitivity of the plaque assay.</p

DNAJC14 is recruited to YFV replication complexes.

<p>(A) SW13 cells were left untransduced (top row) or were transduced (lower 2 rows) with the noninhibitory V1-hDNAJC14 mutants FL-H471Q or CT1 as indicated. Two d later the cells were mock treated (left panels) or were challenged with YFV (moi = 5, right 3 panels). After an additional 2 d, the cells were fixed and immunostained with rabbit anti-YFV NS3 polyclonal antibodies (NS3), and mouse anti-calnexin antibody (calnexin) or mouse anti-myc monoclonal antibody (myc) as indicated. AF488-conjugated anti-mouse IgG and AF594-conjugated anti-rabbit IgG antibodies were used as secondary antibodies. The cells were analyzed by confocal microscopy and representative images are shown. Calnexin or DNAJC14 mutants are shown in green, YFV NS3 is shown in red, and the merged images are shown on the right. (B) SW13 cells were left untransduced or were transduced with the V1-hDNAJC14-CT1 mutant (CT1-myc) as indicated and were infected 2 d later with YFV (moi = 1). After 2 d of infection, myc-tagged DNAJC14-CT1 was immunoprecipitated using anti-myc antibody. Western blots were performed using antibodies against NS3, calnexin and the myc epitope tag as indicated. (C) SW13 cells were left uninfected or were infected with YFV (moi = 1) as indicated. The cells were fixed 1 d later and analyzed by confocal microscopy for endogenous DNAJC14 (red) and double stranded RNA (dsRNA, green). The merged image is shown on the right. Arrows indicate several areas of colocalized DNAJC14 and dsRNA.</p