Heterogeneous nuclear ribonucleoprotein A1 binds to the transcription-regulatory region of mouse hepatitis virus RNA - PubMed (original) (raw)

Heterogeneous nuclear ribonucleoprotein A1 binds to the transcription-regulatory region of mouse hepatitis virus RNA

H P Li et al. Proc Natl Acad Sci U S A. 1997.

Abstract

A cellular protein, previously described as p35/38, binds to the complementary (-)-strand of the leader RNA and intergenic (IG) sequence of mouse hepatitis virus (MHV) RNA. The extent of the binding of this protein to IG sites correlates with the efficiency of the subgenomic mRNA transcription from that IG site, suggesting that it is a requisite transcription factor. We have purified this protein and determined by partial peptide sequencing that it is heterogeneous nuclear ribonucleoprotein (hnRNP) A1, an abundant, primarily nuclear protein. hnRNP A1 shuttles between the nucleus and cytoplasm and plays a role in the regulation of alternative RNA splicing. The MHV(-)-strand leader and IG sequences conform to the consensus binding motifs of hnRNP A1. Recombinant hnRNP A1 bound to these two RNA regions in vitro in a sequence-specific manner. During MHV infection, hnRNP A1 relocalizes from the nucleus to the cytoplasm, where viral replication occurs. These data suggest that hnRNP A1 is a cellular factor that regulates the RNA-dependent RNA transcription of the virus.

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Figures

Figure 1

Two-dimensional PAGE analysis of the partially purified HeLa cytoplasmic extracts. (A) Silver-stained, 2-D nonequilibrium pH gradient electrophoresis gel of the partially purified p35/38 (spot nos. 1–4) that have been crosslinked to the 32P-labeled (−)-strand leader. Molecular weight markers in kDa are indicated, and eEF-1α is identified. (B) Autoradiogram of the same silver-stained 2-D gel. The arrow indicates the radiolabeled spot that matches spot no. 1 in the silver-stained gel. (C) UV-crosslinking of gel-purified spots 1–4 (lanes 1–4) and eEF-1α (lane 5) (0.5 μg each) with 32P-labeled (−)-strand leader RNA. The products were resolved by SDS/PAGE on a 10% polyacrylamide gel and autoradiographed.

Figure 2

Immunoprecipitation of UV-crosslinked proteins. Cytoplasmic extracts from HeLa (lanes 1–4), DBT (lanes 5–8), and A59-infected DBT cells (lanes 9–12) were UV-crosslinked with 32P-labeled (−)-strand leader RNA. Different antibodies were used for immunoprecipitation, and the precipitated proteins were analyzed by SDS/PAGE, a, mAb for hnRNP A1, 4B10; b, polyclonal antibody for TFIIB; and c, polyclonal antibody for Sam68. The asterisk indicates the potential protein specific for MHV-infected cells.

Figure 3

UV-crosslinking of recombinant hnRNP A1 with various RNAs. Three different RNAs: MHV (−)-strand leader, (+)-strand leader, and adenovirus early pre-mRNA were UV-crosslinked with 60 μg of (a) HeLa nuclear extract, (b) E. coli expressed (human) hnRNP A1 lysates, and (c) GST-fused (murine) hnRNP A1 lysates.

Figure 4

The specificity of binding between the recombinant hnRNP A1 and IG sequences. The sequences of the (−)-strand leader and wild-type and mutant IG are shown. (A) The relative transcription efficiencies from the various IG sites were derived from Zhang and Lai (12). (B) 32P-labeled RNA probes were UV-crosslinked with 60 μg of (a) HeLa nuclear extract, (b) E. coli expressed (human) hnRNP A1 lysates, and (c) E. coli (vector) lysates. (C) Similar to (B), except that a GST or GST-hnRNP A1 (murine) fusion protein (GSTmA1) was used. Two different concentrations (2 and 10 μg) of each protein were used for UV crosslinking.

Figure 5

The relocalization of hnRNP A1 in MHV-infected cells. Immunofluorescent staining of MHV-infected cells (×200 magnification) at various time points postinfection. hnRNP A1 or Sam68 antibodies were used.

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Heterogeneous nuclear ribonucleoprotein A1 binds to the transcription-regulatory region of mouse hepatitis virus RNA - PubMed (original) (raw)