( Figure 3B ); these cells express the MHV nucleocapsid (N) protein in the cytoplasm ( Figure 3A ). By comparison, in the uninfected cells, which did not have N protein staining, hnRNP A1 was predominantly localized to the nucleus (arrow in Figure 3B ). In DBT-A1DC cells, very few cells were stained positive for the MHV N protein at 7 h p.i. ( Figure 3C ). Signi®cantly, the viral N protein was detected only in the cells that were stained weakly or not at all for Flag-hnRNP A1 ( Figure 3D ), suggesting that the expression of a high level of hnRNP A1DC interfered with viral replication. The effects of wt and mutant hnRNP A1 on MHV protein production We further investigated the effects of the wt and mutant hnRNP A1 on the production of MHV structural and nonstructural proteins. Cytoplasmic protein was extracted from infected cell lines at different time points after infection for immunoblot analysis to detect an open reading frame (ORF) 1a product, p22 (Lu et al., 1998) and the N protein. p22 expression in DBT-VEC cells was clearly detected at 6 h p.i. and peaked at~16 h p.i. ( Figure 4A ). In DBT-A1 cells, p22 appeared at 5 h p.i. and peaked at~8 h p.i. In DBT-A1DC cells, no p22 protein was detected until 16 h p.i. Similar patterns of differences were observed for the N protein in these three cell lines. Actin levels in different cell lines remained relatively constant throughout the infection, except that, in DBT-A1 cells, actin was not detected at 16 and 24 h p.i. due to the loss of the dead cells ( Figure 4A ). These results clearly demonstrated that overexpression of the wt hnRNP A1 accelerated viral protein production, whereas expression of the mutant hnRNP A1 delayed it. We also performed immuno¯uorescent staining of the N protein at 7 h p.i. to further con®rm the western blot results. As represented by images shown in Figure 4B , there were more DBT-A1 cells stained positive for the N protein than DBT-VEC cells. Very few cells were found to express the N protein in DBT-A1DC cells. The p22 and N proteins appeared as doublets in some of the lanes of Figure 4A , but the results varied from experiment to experiment. The N protein is known to be phosphorylated (Stohlman and Lai, 1979) . Whether p22 is post-translationally modi®ed is not known. Figure 5A ). DBT-A1 cells showed a signi®cantly higher level of [ 3 H]uridine incorporation, which peaked at~8 h p.i. DBT-A1DC cells did not show any detectable level of incorporation of the radioactivity. These results suggest that hnRNP A1 regulates MHV RNA synthesis. We further assessed the production of genomic and subgenomic MHV RNAs in these cell lines by northern blot analysis. The genomic and the six subgenomic RNA species were detected at 8 h p.i. in both DBT-VEC and DBT-A1 cells; there were signi®cantly higher steady-state levels of all of the RNA species in DBT-A1 cells ( Figure 5B ). In contrast, no viral RNA was detected in DBT-A1DC cells at that time point. At 16 h p.i., MHV RNA levels in DBT-VEC and DBT-A1 cells decreased generally because of the loss of the dead cells, while the smaller subgenomic RNAs became detectable in DBT-A1DC cells. By 24 h p.i., most viral RNA species became detectable in DBT-A1DC cells ( Figure 5B , lane 10), while most of the DBT-A1 cells were dead (lane 9). These results con®rmed that the synthesis of all of the viral RNA species is accelerated by overexpression of the wt hnRNP A1 and delayed by a dominant-negative mutant of hnRNP A1.