TANK-binding kinase-1 plays an important role during in vitro and in vivo type I IFN responses to DNA virus infections

Abstract

TANK-binding kinase-1 (TBK1) and the inducible IkappaB kinase (IKK-i) have recently been shown to activate type I IFN responses elicited by intracellular detection of RNA or DNA from infecting viruses. Detection of viral RNA is mediated by retinoic acid inducible gene-I or melanoma differentiation-associated gene-5 pathways in which TBK1 and IKK-i have been demonstrated to play redundant roles in IFN activation. In this study, we have examined whether such redundancy occurs in the type I IFN response to DNA viral challenges by examining induction of IFNs and IFN-mediated signaling and gene programs in TBK1(-/-) macrophages. In contrast to the normal IFN responses in TBK1(-/-) macrophages infected with an RNA virus, IFN responses were severely abrogated during DNA virus infections in TBK1(-/-) macrophages. Because both TBK1 and IKK-i are expressed in macrophages, our studies suggest that TBK1 and IKK-i differ functionally in DNA virus-mediated IFN responses; however, they are redundant in RNA virus-mediated IFN responses. Confirmatively, reconstitution of TBK1(-/-)IKK-i(-/-) fibroblasts revealed that TBK1 rescued IFN responses to transfected B-DNA to a much stronger degree than IKK-i. Finally, we demonstrate the requirement for the TBK1-IFN regulatory factor-3 pathway in host defense against a DNA virus infection in vivo.

Figure 1. TBK1 is required for IFN responses to DNA virus infections

TBK1^+/+TNFR1^−/− and TBK1^−/−TNFR1^−/− BMMs were infected with the indicated virus at a multiplicity of infection (MOI) of 1−3. At the indicated times total RNA was extracted and expression of IFNβ, IFNα5, IP-10, and MX1 was analyzed by Q-PCR (A, D, and G) or IFNα and IFNβ levels in supernatants were quantitated by ELISA 24 hours after infection (B, E, and H). Asterisks (*) indicate IFN levels were undetectable by ELISA. 24 hours after infection or treatment with media alone CD86 expression was measured by flow cytometry and mean fluorescence intensity of the infected sample is indicated (C, F, and I). Results shown are representative of at least two independent experiments.

Figure 2. TBK1 is required for IFN responses to transfected B-DNA

TBK1^+/+TNFR1^−/− and TBK1^−/−TNFR1^−/− BMMs were transfected with 1 ug/ml B-DNA (poly dA-dT:dT-dA) or stimulated with 10ng/ml LPS. At the indicated times total RNA was extracted and expression of IFNβ, IFNα5, IP-10, and MX1 was analyzed by Q-PCR (A and B) or IFNα and IFNβ levels in supernatants were quantitated by ELISA 24 hours after stimulation (C and D). Asterisks (*) indicate IFN levels were undetectable by ELISA. 24 hours after stimulation or treatment with media alone CD86 expression was measured by flow cytometry and mean fluorescence intensity of the stimulated sample is indicated (E and F). Results shown are representative of at least two independent experiments.

Figure 3. B-DNA and DNA virus activation of type I IFN mediated signal transduction require TBK1

TBK1^+/+TNFR1^−/− and TBK1^−/−TNFR1^−/− BMMs were transfected with 1 ug/ml B-DNA (poly dA-dT:dT-dA) or infected with the indicated virus at a multiplicity of infection (MOI) of 1−3. At the indicated time points, cells were harvested, fractionated and nuclear fractions were probed for phospho-STAT1 and USF2 as a loading control for nuclear proteins (A, B, and C) or total cell extracts were probed for phospho-STAT1 and total STAT1(D). E, WT or TBK1^−/−IKK-i^−/− MEFs reconstituted with a pBABE vector or with HA-tagged TBK1 or IKK-i were transfected with 1 ug/ml B-DNA for the indicated time points. Total cell extracts were probed for phospho-STAT1 and HA. A non-specific (n.s.) HA band is shown as a loading control. Results shown are representative of at least two independent experiments.

Figure 4. Macrophages use MyD88-independent IRF3-dependent pathways to recognize DNA and RNA virus infections

WT and MyD88^−/− or IRF3^−/− BMMs were infected with MHV-68 or Sendai virus at a multiplicity of infection (MOI) of 1−3. At the indicated time points total RNA was extracted and analyzed by Q-PCR for expression of IFNβ, IFNα5, IP-10, and ISG-15 (A, B, E, and F) or total cell extracts were probed for phospho-STAT1 and total STAT1 (C, D, G,and H).

Figure 5. IRF3 is required for in vivo resistance to DNA viruses

WT and IRF3−/− mice were infected intranasally with 5,000 pfu of M3FL. On days 5 (A) and 7 (B) following infection, mice were injected with D-luciferin and imaged. The maximum photon flux value (Log₁₀ photons/sec/cm²/sr) was determined for each mouse.

Figure 6. TBK1 is required for in vivo resistance to DNA viruses

TBK1^+/+TNFR1^−/− and TBK1^−/−TNFR1^−/− mice were infected intranasally with 5,000 pfu of M3FL. On days 5 (A) and 7 (B) following infection, mice were injected with D-luciferin and imaged. The maximum photon flux value (Log₁₀ photons/sec/cm²/sr) was determined for each mouse. Viral load in lung tissue was determined on day 7 by standard plaque assays (C) and luciferase values (D).