The experiments with the BMV-coat-antizyme fusion constructs were performed as described previously (Matsufuji et al., 1995) . All other plasmid DNA templates were prepared using QIAGEN Miniprep Kit and then digested with HindIII. Transcripts for SPA in vitro translation were made from PCR templates that had a T7 promoter incorporated into the PCR primers. Linearized DNA (1 mg) was used as a template for in vitro transcription with Ambion MEGAshortscript TM T7 Kit. The DNasetreated RNAs were recovered and resuspended in 40 ml of RNase-free water. One microliter of each speci®ed transcript suspension was used in each in vitro translation reaction [0.5 ml of 1 mM amino acid mix ±Met, 7 ml of reticulocyte lysate (Promega), 0.5 ml of [ 35 S]Met (Amersham)] to a total volume of 10 ml. The reactions were stopped by adding 1 ml of RNase (10 mg/ml). The frameshift ef®ciencies were quanti®ed as described (Ivanov et al., 1998a) . Heterogeneous nuclear ribonucleoprotein A1 regulates RNA synthesis of a cytoplasmic virus Heterogeneous nuclear ribonucleoprotein (hnRNP A1) is involved in pre-mRNA splicing in the nucleus and translational regulation in the cytoplasm. In the present study, we demonstrate that hnRNP A1 also participates in the transcription and replication of a cytoplasmic RNA virus, mouse hepatitis virus (MHV). Overexpression of hnRNP A1 accelerated the kinetics of viral RNA synthesis, whereas the expression in the cytoplasm of a dominant-negative hnRNP A1 mutant that lacks the nuclear transport domain significantly delayed it. The hnRNP A1 mutant caused a global inhibition of viral mRNA transcription and genomic replication, and also a preferential inhibition of the replication of defective-interfering RNAs. Similar to the wild-type hnRNP A1, the hnRNP A1 mutant complexed with an MHV polymerase gene product, the nucleocapsid protein and the viral RNA. However, in contrast to the wild-type hnRNP A1, the mutant protein failed to bind a 250 kDa cellular protein, suggesting that the recruitment of cellular proteins by hnRNP A1 is important for MHV RNA synthesis. Our findings establish the importance of cellular factors in viral RNA-dependent RNA synthesis. Introduction hnRNP A1 is an RNA-binding protein that contains two RNA-binding domains (RBDs) and a glycine-rich domain responsible for protein±protein interaction. It is involved in pre-mRNA splicing and transport of cellular RNAs (reviewed by Dreyfuss et al., 1993) . It is predominantly located in the nucleus, but also shuttles between the nucleus and the cytoplasm (Pin Äol-Roma and Dreyfuss, 1992) . The signal that mediates shuttling has been identi®ed as a 38 amino acid sequence, termed M9, located near the C-terminus of hnRNP A1 between amino acids 268 and 305 (Michael et al., 1995; Siomi and Dreyfuss, 1995; Weighardt et al., 1995) . Yeast two-hybrid screening with M9 as bait resulted in the discovery of a novel transportin-mediated pathway for nuclear import of hnRNP A1 (Pollard et al., 1996; Fridell et al., 1997; Siomi et al., 1997) . The function of the cytoplasmic hnRNP A1 has not been well de®ned. Studies have shown that cytoplasmic and nuclear hnRNP A1 exhibit different RNA-binding pro®les. Cytoplasmic hnRNP A1 is capable of high-af®nity binding to AU-rich elements that modulate mRNA turnover and translation (Hamilton et al., 1993 (Hamilton et al., , 1997 Henics et al., 1994) . It has also been shown to promote ribosome binding to mRNAs by a cap-mediated mechanism, and prevent spurious initiation at aberrant translation start sites (Svitkin et al., 1996) .