1/journal.pone.0123087.g002 UV-inactivated viruses, even at higher MOIs, indicating that viral replication is essential for cells to activate an PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19770275 antiviral response. Inhibition of cellular IFN response enhances HAstV replication In order to determine whether blocking of intracellular IFN defense mechanism benefits HAstV replication, the effect of treatment of CaCo-2 cells with BX795 inhibitor was examined. BX795 is an aminopyrimidine compound which inhibits the catalytic activity of TBK1/IKK by blocking its phosphorylation. CaCo2 wells were pre-treated with 10 M BX795 for 3 hours at 37C before infecting them with a MOI of 10. After infection, DMSO, 1M or 5 M of BX795 was added in the culture media, and HAstV production was measured by Fig 3. Level of antiviral activity released into the supernatant during HAstV infection. Antiviral activity in the supernatant of poly:IC-transfected cells, mock-infected cells and HAstV-infected cells at a MOI of 1 and 10 was measured by a virus infectivity reduction bioassay using EMCV, which is sensitive to IFN. HeLa cells were treated for 24 hours with serial 2-fold dilutions of the indicated supernatant, and were infected with EMCV. Results expressed as level of antiviral activity relative values to the reference control are shown. Data are pooled from three independent experiments and error bars represent the SEM. doi:10.1371/journal.pone.0123087.g003 8 / 18 HAstV Delays Interferon Induction Fig 4. HAstV replication is essential for induction of a cellular antiviral response. Antiviral activity in the supernatant of cultures infected with HAstV at a MOI of 10, and harvested at 48 hpi, was measured using a virus infectivity reduction bioassay. Results are expressed as level of antiviral activity relative values to the reference control. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19768759 Data represent mean values of 2 independent experiments and error bars represent the SEM.– UV: Non-inactivated virus; +UV: Inactivated virus. doi:10.1371/journal.pone.0123087.g004 quantification of viral RNA by qRT-PCR, assessment of YM-155 web infectious viruses released in the supernatant, and measurement of HAstV capsid protein in total cell lysates by ELISA. Lack of cellular toxicity was estimated by observation of cells under the microscope and by detection of unchanged levels of GAPDH mRNA by qRT-PCR in all BX795-treated cells, and absence of cellular IFN responses were confirmed by the lack of expression of IFN- mRNA by qRT-PCR. Results show that treatment of cells with 5 M of BX795 resulted in a significant 2-fold increase in total HAstV RNA produced as well as in the amount of infectious progeny released in the supernatant compared to untreated cells. However, quantification of total capsid protein by ELISA indicated a 4-fold increase compared to untreated cells, suggesting that although a higher amount of viral capsid proteins were produced, only half of these proteins were able to be fully converted into infectious virions. 9 / 18 HAstV Delays Interferon Induction Exogenous IFN inhibits viral replication In order to understand whether HAstV replication is sensitive to IFN, CaCo-2 cells were pre-treated with 1,000 U/ml of type I IFN for 24 hours before 
infection at three different MOIs. EMCV and and RV SA11 were used as controls for an IFN-sensitive virus and an IFN-resistant virus, respectively. The number of HAstV-infected cells observed by IF at 24 hpi was slightly reduced after IFN treatment. Virus progeny released into the supernatant was measured for HAstV by Fig
