PMC

MAVS/IPS-1 and RNase L are essential for HSV-induced cytokine expression. (a-d) RIG-I −/−, MDA5 −/−, MAVS/IPS-1 −/−, and RNase L −/− MEFs, and their respective heterozygote or wild type cells were seeded at 7×10⁵ cells per 6 well in culture plate and treated with HSV-2, 3 MOI. Four hours post infection RNA was harvest with High Pure RNA Isolation Kit (Roche) and levels of IFN-β were measured by Q-PCR and normalized to β-actin. Data is shown as mean of triplicates +/− SEM.

RIG-I and TLR9 activate distinct signaling pathways and co-ordinately stimulate activation of IRF-3. (a-d) RAW-pcDNA3 and RAW-dnRIG-I cells (a-b) or RAW264.7 cells incubated in the presence of 3 μM ODM2088 (c-d) were infected with HSV-2 (MOI 3), and total cell lysates (Bio-Plex cell lysis kit, Bio-Rad) were harvested after the indicated amounts of time. Phosphorylation of P-IκBα, (a, c), and p38 (b, d) was measured by Luminex Technology, using a kit from Bio-Rad. (e) RAW264.7 cells were infected with HSV-2 at MOI 3, and total cell lysates were made at the indicated time points p.i. Phospho-IRF-3 and GAPDH were detected by Western Blotting. (f) RAW-pcDNA3 and RAW-dnRIG-I cells were incubated in the presence of 3 μM ODN2088 and infected with HSV-2 (MOI 3) for 3 h. Total cell lysates were made, and proteins bound to the IFN-β promoter PRD I-III region were precipitated. Phospho-IRF-3 was detected by Western Blotting. Similar results were obtained in at least 3 independent experiments.

RIG-I and TLR9 cooperatively induce expression of cytokines during HSV-2 infection. (a-c) Parental RAW264.7 cells or stably transfected with the empty vector pcDNA3 or a dominant-negative RIG-I construct were seeded at 8×10⁴ cells per well in 96 well culture plates and stimulated with EMCV, 5 MOI; VSV, 1,6 MOI or PolyIC (Invivogen), 25 μg/ml. Four and 16 hours post infection, RNA and cell culture supernatants, respectively, were harvested for measurement of IFN-β (Q-PCR) and RANTES (ELISA, using matched antibody pairs from R&D Systems). (d–e) RAW-pcDNA3 and RAW-dnRIG-I cells were treated with HSV-2, 3 MOI. Supernatants were harvested at indicated time points, and RANTES and IFN-α/β were measured by ELISA and bioassay, respectively. (f) RAW264.7 cells were treated with LPS (100 ng/ml) or ODN1826 (1 μM) in the presence or absence of the TLR9 antagonist ODN2088 (3μM). Supernatants were harvested at 20 h p.i. and IL-6 and IFN-α/β were measured by ELISA and bioassay. (g) RAW264.7 cells incubated in the presence or absence of ODN2088 as indicated were infected with HSV-2 at MOI 2 for the indicated amounts of time. Supernatants were harvested and levels of IFN-α/β were determined by bioassay. (g) MyD88+/− and MyD88−/−xTRIF−/− MEFs were seeded at 7×10⁵ cells per 6 well in culture plate and treated with HSV-2, 3 MOI. Four hours post infection RNA was harvest and levels of IFN-β were measured by Q-PCR and normalized to β-actin.. All data are shown as mean of triplicates +/− SEM.