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 signi®cantly 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 ®ndings establish the importance of cellular factors in viral RNA-dependent RNA synthesis.

The genome of mouse hepatitis virus (MHV) is a singlestranded, linear, positive-sense, polyadenylated RNA of approximately 31 kb in length (22,24,39). All of the viral proteins are translated from subgenomic mRNAs, except those encoded by the first open reading frame, which are translated from the genome-sized RNA. In MHV-infected cells, six or seven subgenomic mRNAs are transcribed, all of which contain a common leader sequence derived from the 5Ј end of the genome, as well as a common 3Ј end including a 302-to 305-nucleotide (nt)-long untranslated region (3Ј-UTR) (21). When MHV is passaged at a high multiplicity of infection in cell culture, defective-interfering (DI) RNAs which represent deletion mutants of the MHV genome are frequently generated (33, 36). Since DI RNAs can replicate in the presence of a helper virus, they must retain all signals necessary for MHV genome replication.Comparison of various MHV DI RNAs showed that all retain various lengths of both the 5Ј and 3Ј ends of the viral genome (34,35,37,43). Mapping studies of MHV DI RNAs have further revealed that 400 to 859 nt from the 5Ј end and 436 nt from the 3Ј end of the genome are required for DI RNA replication (17, 29). However, only 55 nt from the 3Ј end plus the poly(A) tail are required for synthesis of the negative strand of MHV RNA (30). Thus, it stands to reason that the replication signal from the 5Ј end and the remaining replication signal (nt 55 to 436) from the 3Ј end of the MHV genome are involved in synthesis of the positive-strand RNA. Moreover, since positive-strand RNA synthesis begins from the 5Ј end of the genome, and the 3Ј end will be the last region of the genome reached by the viral polymerase, the replication signal at the 3Ј end likely interacts with signals at the 5Ј end to exert its effect on RNA synthesis. Based on this reasoning, the 5Ј and 3Ј ends of the genome have been proposed to interact during viral RNA replication so that the 3Ј-end sequence can affect the initiation of RNA synthesis (20). Similar observations have also been made for the regulation of MHV subgenomic mRNA transcription (28). Typically, the regulatory sequence for the synthesis of a particular RNA strand resides on the complementary (template) strand; i.e., the signal for synthesizing the positive strand resides on the negative strand, and vice versa. However, the regulatory signals may also reside on the same strand. For example, in brome mosaic virus (BMV) and poliovirus, cis elements that affect synthesis of the positivestrand RNA are mapped to the 5Ј end of the positive-strand RNA (2, 9, 40). Presumably this is due to the possibility that double-stranded replicative-form RNA is used as the template for positive-strand RNA synthesis. Thus, RNA synthesis may be regulated by 5Ј-3Ј-end interaction of both RNA strands.Although 5Ј-3Ј-end interactions of MHV RNA have been suggested from functional studies (28), no apparent sequence complementarity exists between these two regions. Therefore, interaction between the 5Ј and 3Ј ends likely involves p...

A cellular protein, previously described as p55, binds specifically to the plus strand of the mouse hepatitis virus (MHV) leader RNA. We have purified this protein and determined by partial peptide sequencing that it is polypyrimidine tract-binding protein (PTB) (also known as heterogeneous nuclear ribonucleoprotein [hnRNP] I), a nuclear protein which shuttles between the nucleus and cytoplasm. PTB plays a role in the regulation of alternative splicing of pre-mRNAs in normal cells and translation of several viruses. By UV cross-linking and immunoprecipitation studies using cellular extracts and a recombinant PTB, we have established that PTB binds to the MHV plus-strand leader RNA specifically. Deletion analyses of the leader RNA mapped the PTB-binding site to the UCUAA pentanucleotide repeats. Using a defective-interfering RNA reporter system, we have further shown that the PTB-binding site in the leader RNA is critical for MHV RNA synthesis. This and our previous study (H.-P. Li, X. Zhang, R. Duncan, L. Comai, and M. M. C. Lai, Proc. Natl. Acad. Sci. USA 94:9544–9549, 1997) combined thus show that two cellular hnRNPs, PTB and hnRNP A1, bind to the transcription-regulatory sequences of MHV RNA and may participate in its transcription.

The hepatitis B virus posttranscriptional regulatory element (PRE) is an RNA element that increases the expression of unspliced mRNAs, apparently by facilitating their export from the nucleus. We have identified a cellular protein that binds to the PRE as the polypyrimidine tract binding protein (PTB), which shuttles rapidly between the nucleus and the cytoplasm. Mutants of the PRE with mutations in PTB binding sites show markedly decreased activity, while cells that stably overexpress PTB show increased PRE-dependent gene expression. Export of PTB from the nucleus, like PRE function, is blocked by a mutant form of Ran binding protein 1 but not by leptomycin B. Therefore, PTB is important for PRE activity and appears to function as an export factor for PRE-containing mRNAs.Eucaryotic mRNA transcription takes place in the nucleus, but translation occurs in the cytoplasm, necessitating the export of mRNA through nuclear pores. In mammalian cells, this export is strictly controlled, in that only fully spliced and processed mRNA is exported (22,27). Part of the control is at the level of retention of incompletely spliced mRNA, probably by splicing factors binding to splice sites. However, this retention mechanism cannot explain all of the available data. First, some genes give rise to alternatively spliced transcripts, in which some of the mature mRNAs still contain splice sites. Second, for at least some cellular genes (e.g., the ␤-globin gene), removal of all introns leads to a defect in the export of mRNA to the cytoplasm (4). Therefore, the presence of splice sites does not always preclude RNA export, while the absence of splice sites does not always lead to export. These data imply that at least some mRNAs contain cis-acting elements that can effect export independently of splicing.In recent years, the existence of such RNA export elements has been confirmed. The best-studied element is the Rev response element (RRE) of human immunodeficiency virus (HIV) and related lentiviruses (6,14). Like almost all retroviruses, HIV contains only one promoter that gives rise to a transcript that is alternatively spliced, resulting in the export of completely spliced, partially spliced, and unspliced mRNAs into the cytoplasm. HIV codes for a trans-acting protein product that modulates the relative amounts of completely spliced versus incompletely spliced and unspliced messages. This protein, called Rev, binds to the RRE in the nucleus and strongly enhances the export of RRE-containing, unspliced or incompletely spliced transcripts. It has become clear that Rev contains a leucine-rich nuclear export signal (NES) that allows it to form a trimolecular complex with two cellular proteins, Crm1 (exportin) and Ran (8,9,29,34). This complex, together with its RNA cargo, interacts with components of the nuclear pore in order to migrate into the cytoplasm. Rev is then stripped off the mRNA in the cytoplasm and is recycled back into the nucleus by virtue of a nuclear localization signal.Other retroviruses also contain RNA export elemen...