Characterization of stable cell lines expressing the wt and a C-terminus-truncated hnRNP A1 To explore a potential role for hnRNP A1 in MHV RNA synthesis, we established murine DBT cell lines stably expressing the Flag-tagged wt hnRNP A1 (DBT-A1) or a mutant hnRNP A1, which has a 75 amino acid deletion from the C-terminus (DBT-A1DC) ( Figure 1A ). This mutant lacks part of the glycine-rich domain and the M9 sequence responsible for shuttling hnRNP A1 between the nucleus and the cytoplasm. Immunoblot of the whole-cell lysates with an anti-Flag antibody detected a 34 kDa protein in DBT-A1 cells and a 27 kDa protein in three independent clones of DBT-A1DC cells ( Figure 1B ), whereas no protein was cross-reactive to the anti-Flag antibody in the control cell line stably transfected with the pcDNA3.1 vector (DBT-VEC). The amounts of the Flagtagged wt and truncated hnRNP A1 were comparable in these cell lines. A chicken polyclonal antibody against hnRNP A1 detected two endogenous hnRNP A1 isoforms or hnRNP A1-related proteins in the whole-cell lysates of all of the cell lines. The bottom band (34 kDa) overlaps the Flag-tagged wt hnRNP A1 in DBT-A1 cells. There was only a slight increase in the overall amount of hnRNP A1 in DBT-A1 cells as compared with DBT-VEC cells, indicating that the exogenous hnRNP A1 constituted a small fraction of the total hnRNP A1 in the cells. In DBT-A1DC cells, an additional band of smaller size (27 kDa) corresponding to the mutant hnRNP A1 was detected. The overall expression levels of the exogenous hnRNP A1 and hnRNP A1DC were~3-fold lower than that of the endogenous hnRNP A1 in whole-cell lysates ( Figure 1B ). Similar to the endogenous hnRNP A1 protein (Pin Äol-Roma and Dreyfuss, 1992) , the Flag-tagged wt hnRNP A1 was localized almost exclusively in the nucleus ( Figure 1C ). The mutant hnRNP A1, however, was localized predominantly in the cytoplasm ( Figure 1C) , consistent with the previous ®nding that the M9 nuclear localization signal is necessary to localize hnRNP A1 to the nucleus Weighardt et al., 1995) . Thus, hnRNP A1DC was much more abundant than the endogenous hnRNP A1 in the cytoplasm. The expression levels of the wt or mutant hnRNP A1 varied among individual cells based on immuno¯uorescent staining ( Figure 1C ). The growth rate ( Figure 1D ) and cell morphology (data not shown) were similar among the different cell lines.