Growth of mutants by acute infection in the lung and latent infection in the spleen of normal BALB/c mice. The ratios of in vivo to in vitro replication of each mutant are shown. The ratio of mutant that shows same level of replication in vivo and in vitro, relative to the other mutants in a pool, is defined as 1. Individual mutants in a pool are labeled with the numbers 1 to 10. Mutant #6 is the ORF36-null mutant (36T). (B) The same pool of mutant viruses was used to infect IFNAR−/− mice, and the same analysis as (A) was performed. (C) Acute replication of MHV-68 in the lung after infection with WT and 36T individually. (D) Latency establishment of MHV-68 in the spleen after infection with WT and 36T individually. I.C.: infectious center; sp.: splenocytes. 4 mice per group and per time point. Data are represented as mean + standard deviation (SD).

C57BL/6 mice were intranasally infected with 1000 pfu of WT or 36S. (A) The number of latently infected spleen cells was determined using an infectious center assay at 14 and 21 dpi. (B) Analysis of spleen cell numbers at 14 and 21 dpi. (C) Absolute numbers of CD8-positive ORF6_487–495/D^b (left panel) and ORF61_524–531/K^b (right panel) in the lung of infected mice. (D) Absolute numbers of CD8-positive ORF6_487–495/D^b (left panel) and ORF61_524–531/K^b (right panel) in the spleen of infected mice. 5 mice per group and per time point. Data are represented as mean + SD.

The effect of ORF36 expression on the activation of IFN-β reporter which is induced by the co-transfected plasmids expressing IRF-3, IRF-7 or NF-kB (p65), respectively. (B) The effect of ORF36 expression on the activation of IFN-β reporter which is induced by upstream activators of IRF-3 signal transduction pathway, TBK-1, TLR-4 and Sendai virus infection. (C) The effect of ORF36 expression on the activation of interferon stimulatory response element containing reporter. (D) The effect of ORF36 and dominant negative form of IRF-3 (IRF-3dDBD) expression on the activation of IFN-β reporter induced by TBK-1 expression, and the effect of TBK-1 or IRF-3 over-expression in addition to ORF36 or IRF-3dDBD. All the reporter assays were performed in human 293T cells, and the data are represented as mean + SD.

Subcellular localization of IRF-3 upon TBK-1/ORF36 expression in 293T cells. Anti-FLAG and anti-mouse-Alexa405 (Blue) for FLAG-tagged ORF36; anti-HA-FITC (Green) for HA-tagged TBK-1; anti-IRF-3 and anti-rabbit-Cy3 (Red) for endogenous IRF-3. (B) Interaction between ORF36 and the native or activated form of IRF-3 by co-immunoprecipitation assay. 293T cells were transfected with the plasmids expressing the indicated proteins (bottom) and lysed and analyzed at 48 hours post-transfection as described in method. IP: immunoprecipitation using antibody against indicated epitope, IB: immunoblot using antibody against indicated epitope. (C) Interaction between the endogenous IRF-3 activated upon viral infection and the FLAG-tagged ORF36 expressed from MHV-68 during natural infection. 293T cells were uninfected (mock) or infected with a recombinant MHV-68 expressing FLAG-tagged ORF36 at MOI=10 and harvested and analyzed at 24 hours post infection (hpi). (D) Interaction between the endogenous IRF-3 and CBP in the absence/presence of TBK-1/ORF36 in 293T cells. (E) The effect of ORF36 on the recruitment of CBP and RNA Pol II transcription complex to the IFN-β promoter in the transfected 293T cells or infected NIH3T12 cells. (top) Relative amount of the IFN-β promoter precipitated by anti-CBP or anti-Pol II, quantitated by q-PCR. (bottom) The PCR products were run on agarose gels. A set of representative data is shown here. Data are represented as mean + SD. n.s.: not significant; *, P < 0.05.

The effect of homologs of ORF36 in all subfamilies of herpesvirus - alpha (HSV-1 UL13), beta (HCMV UL97) and gamma (EBV BGLF4 and KSHV ORF36) - on the activation of IFN-β reporter induced by TBK-1 expression in human 293T cells. (B) The effect of homologs of ORF36 on the production of IFN-β and the transcription of IFN-β and Mx1 induced by ds polyIC treatment. The murine NIH3T3 cells were transfected as indicated and selected for 3 days with 6 ug/ml of puromycin by the co-transfected pBabe-puro. 3 days after selection, the transfected/selected cells were treated with 10 ug/ml of ds polyIC in liposome-complex. 1 day post treatment, cell supernatant and RNA extracted from the cells were analyzed for endogenous IFN-β production by enzyme-linked immunosorbent assay (ELISA) (top) and transcription level of IFN-β (middle) and Mx1 (bottom) by q-PCR. (C) The effect of ORF36(K107Q), a kinase-null mutant of ORF36, on the activation of IFN-β reporter induced by TBK-1 in human 293T cells. (D) The effect of homologs and a kinase-null mutant of ORF36 on the activation of IFN-β reporter with or without induction by ds polyIC treatment in NIH3T3 cells. Data are represented as mean + SD. *, P < 0.05.

The induction of IFN response by WT, N36S, 36KN and 36R. NIH3T3 cells were infected with WT, N36S, 36KN or 36R virus at MOI=0.05. At 24 hours post infection, cell supernatant and RNA extracted from the infected cells were analyzed for endogenous IFN-β production by ELISA (left) and transcription level of IFN-β (middle) and Mx1 (right) by q-PCR. For ELISA, samples from three independent experiments were combined and tittered. (B) The multi-step growth curve of WT, N36S, 36KN, and 36R. Samples from two independent experiments were combined, tittered, and shown here. (C) Transcript level of ISGs in the lung after WT or 36S MHV-68 infection examined by q-PCR. Viral genome and cellular actin transcript level were also measured as control. Data are represented as mean + SD. (D) Acute replication in the lung after intranasal infection of the indicated viruses in normal C57BL/6 (left) and IFNAR−/− (right) mice. (E) Latency establishment in the spleen after intranasal infection of the indicated viruses in normal C57BL/6 (left) and IFNAR−/− (right) mice. I.C.: infectious center; sp.: splenocytes. 500 pfu/mouse and 4~5 mice per group and per time point. Data are represented as mean + SD. n.s.: not significant; *, P < 0.05.

Bioluminescence imaging of in vivo replication of wildtype M3FL and mutant M3FL-36S in normal mice. Images at different time point are shown to represent the peak of acute infection in the lung (D6 with 5×10⁵ pfu/mouse), the transition of replication from the lung to the spleen (D10), and the establishment of viral replication in the spleen (D14). (B) Bioluminescence imaging of in vivo replication of wildtype M3FL and mutant M3FL-36S in IFNAR−/− mice.