As expected, no signal for the variants was detected in HeLa cells

r idea, only a low percentage of infected cells show translocation of IRF3 to the nucleus, suggesting that IFN induction does not take place in each infected cell. Although it cannot be completely ruled out that IFN- synthesis may occur independent of IRF3 activation, our working hypothesis is that IFN-b mRNA would not be produced within every infected cell. In addition, nuclear translocation of IRF3 occurs at late times post-infection, and antiviral activity in the supernatant of infected cells is not detected until 48 h after infection, confirming that in any case, IFN production takes place at the late stages of the viral replication cycle and requires infectious viruses. Finally, when we examined the ability of HAstV to inhibit the IFN response induced by polyI:C transfection, we observed that HAstV infection is not able to block the ability of the cell to respond to dsRNA as it has been observed for other well-studied ssRNA viruses. HAstV behavior would be more similar to what has been described for some other viruses such as rhinovirus or mouse hepatitis virus. Taken together, our results may be explained by the observation that HAstV capsid acts as an inhibitor of complement activation, and that intracellular uptake of complement 13 / 18 HAstV Delays Interferon Induction Fig 8. Effect of nsP1a/4 genotype on the level of IFN- response. Normalized levels of IFN-/GAPDH response at 24 hpi were plotted against the average number of HAstV genome copies per infected cell after infecting differentiated CaCo-2 cells with different nsP1a/4 mutants at the same MOI. Data summarize results from three independent experiments. doi:10.1371/journal.pone.0123087.g008 factors bound to viral particles may trigger innate immune responses within cells. Since FBS was added to the post-infection medium in all experiments, it is plausible that the activation of innate responses was delayed due to PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19768747 the action of the HAstV capsid limiting deposition of complement factors on the viral surface. In addition to this, other mechanisms may also contribute to minimize IFN production within HAstV-infected cells. Although active antagonists of the type I IFN response have been characterized for other well-studied gastroenteritis viruses such as rotavirus, as AZD-6244 site reviewed in, viral countermeasures to limit cellular antiviral responses may also be achieved by several mechanisms such as tightly controlling virus transcription and replication to minimize the production of dsRNA, encapsidating all forms of viral RNAs produced within the cell, protecting the 5′ end of their RNAs with a cap structure or a VPg protein from recognition by RIG-I, or by replicating within intracellular membrane vesicles to “hide” any viral RNA from the cytoplasmic cellular innate recognition machinery. Recent work in our laboratory has experimentally confirmed that HAstV genome is covalently linked to a VPg protein on its 5′ end, and large membrane rearrangements can be observed in HAstV-infected cells, with virus aggregates surrounded by double-membrane vacuoles. Indeed, HAstV assembly seems to start at cellular membranes, at the same site where nonstructural proteins replicate the genome. Cell fractionation studies have shown that both PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19768759 positive and negative HAstV RNAs and also nonstructural proteins localize in vesicles where capsid protein gets anchored after synthesis. Despite these strategies to minimize innate antiviral responses, IFN- would be expressed at the late stages of the viral repli