We suspected that rMP12-rLuc replication in the presence of ActD may generate dsRNAs and/or 59-triphosphated singlestranded RNAs, which activate PKR, resulting in the phosphorylation of eIF2a. When we analyzed the level of eIF2a phosphorylation in VeroE6 cells infected with rMP12-rLuc in the absence of transcription inhibitor, we found low levels of eIF2a phosphorylation from 8 to 24 h.p.i. and an efficient accumulation of N protein at 8 h.p.i. and onward ( Figure 4A -4C). In contrast, when we treated rMP12-rLuc-infected cells with ActD or aamanitin, either compound induced an efficient accumulation of phosphorylated eIF2a from 8 to 16 h.p.i., with a concomitant poor N protein accumulation ( Figure 4A -4C) and virus replication suppression ( Figure 4D ). The mechanism of reduction in the abundance of the phosphorylated eIF2a at 24 h.p.i. in the presence of ActD or a-amanitin ( Figure 4 , lanes 13 and 19) is unknown. When we treated rMP12-rLuc-infected cells with different concentrations of ActD or a-amanitin, we found that the inhibition of phosphorylated eIF2a accumulation was dependent on the concentrations used ( Figure S2 ). Also a strong correlation was seen between an increase in eIF2a phosphorylation and the decrease in both N protein accumulation and infectious virus production ( Figure S2 ). In contrast, no significant accumulation of phosphorylated eIF2a occurred in MP-12infected VeroE6 cells in the presence of ActD ( Figure 4A -4C). As expected, ActD treatment had little effect on MP-12 replication ( Figure 4D ). These data strongly suggested that the presence of highly phosphorylated eIF2a levels suppressed the translation of viral mRNAs, leading to inefficient virus production, and that the NSs protein somehow suppressed eIF2a phosphorylation, and facilitated efficient viral mRNA translation under the conditions of host transcriptional shutoff.