Heterogeneous nuclear ribonucleoprotein a1 binds to the 3'-untranslated region and mediates potential 5'-3'-end cross talks of mouse hepatitis virus RNA

Abstract

The 3'-untranslated region (3'-UTR) of mouse hepatitis virus (MHV) RNA regulates the replication of and transcription from the viral RNA. Several host cell proteins have previously been shown to interact with this regulatory region. By immunoprecipitation of UV-cross-linked cellular proteins and in vitro binding of the recombinant protein, we have identified the major RNA-binding protein species as heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). A strong hnRNP A1-binding site was located 90 to 170 nucleotides from the 3' end of MHV RNA, and a weak binding site was mapped at nucleotides 260 to 350 from the 3' end. These binding sites are complementary to the sites on the negative-strand RNA that bind another cellular protein, polypyrimidine tract-binding protein (PTB). Mutations that affect PTB binding to the negative strand of the 3'-UTR also inhibited hnRNP A1 binding on the positive strand, indicating a possible relationship between these two proteins. Defective-interfering RNAs containing a mutated hnRNP A1-binding site have reduced RNA transcription and replication activities. Furthermore, hnRNP A1 and PTB, both of which also bind to the complementary strands at the 5' end of MHV RNA, together mediate the formation of an RNP complex involving the 5'- and 3'-end fragments of MHV RNA in vitro. These studies suggest that hnRNP A1-PTB interactions provide a molecular mechanism for potential 5'-3' cross talks in MHV RNA, which may be important for RNA replication and transcription.

FIG. 1

Identification of p35/38 UV cross-linked to the MHV 3′-UTR. (A) Immunoprecipitation of UV-cross-linked cell extract. DBT whole-cell extract cross-linked to ³²P-labeled 3′-UTR RNA was immunoprecipitated by various antibodies in lanes 2 to 5; lane 1 is the input of UV-cross-linked DBT whole-cell extract without immunoprecipitation (IP). The arrow indicates the position of p35/38. The immunoprecipitates were resolved on a SDS–10% polyacrylamide gel. (B) UV cross-linking of recombinant GST (lane 1) and GST-A1 (lane 2) to ³²P-labeled MHV 3′-UTR. The arrow indicates the position of GST-A1. Positions of molecular weight standards (in kilodaltons) are shown on the left of each gel.

FIG. 2

Competition of RNA-A1 binding in a UV cross-linking assay. GST-hnRNP A1 was incubated with different amounts (fold excess over the labeled RNA) of various unlabeled RNAs and then UV cross-linked to ³²P-labeled MHV 3′-UTR. Lanes 1 to 4, 3′-UTR; lanes 5 to 8, negative-strand leader; lanes 9 to 12, positive-strand leader; lanes 13 to 16, unrelated RNA transcribed from pBluescript. The arrow indicates the position of GST-A1.

FIG. 3

Mapping of hnRNP A1-binding sites on the MHV 3′-UTR. The 3′-UTR (350 nt) was separated into four fragments as indicated above the gel. ³²P-labeled in vitro transcript of each fragment was subjected to UV cross-linking with GST-A1. Numbering of the nucleotides is from the 3′ end of MHV RNA for convenience. The position of the 68-kDa size marker is shown on the left.

FIG. 4

Identification of nucleotides from positions 170 to 90 that are important for hnRNP A1 binding. (A) Computer-predicted secondary structure of the 3′-most 299 nt of MHV viral RNA, generated by the Mulfold2 program (55). (B) Expanded structure of nt 52 to 150. Stretch C nucleotides are marked. (C) UV cross-linking assay using wild-type (lane 1), subsC (lane 2), and ΔC (lane 3) mutants with GST-A1.

FIG. 5

RNP complex formation between 5′ and 3′ ends of both strands. (A) RNP complex formation between the leader and 3′-UTR RNA (positive strand). Various proteins or protein combinations were incubated with ³²P-labeled MHV 3′-UTR and biotin-labeled leader sequence. RNP complexes were precipitated by streptavidin-agarose beads. RNA was extracted from the beads and resolved on a 6% denaturing polyacrylamide gel. (B) RNP complex formation between negative-strand leader [Leader(−)] and c3′-UTR.

FIG. 6

RNP complex formation using mutant MHV 3′-UTR. ΔC (lane 3) and subsC (lane 4) mutants and wild-type (lane 2) 3′-UTR RNA were used in an RNP complex formation assay as described for Fig. 5. The arrow indicates the position of ³²P-labeled 3′-UTR. Relative efficiencies of RNP complex formation assay are shown at the bottom.

FIG. 7

Northern blot analysis of replication of DI RNAs harboring mutations at the hnRNP A1-binding sites. (A) Northern blot analysis of the replication of wild-type (wt; lane 1), ΔC (lane 2), and subsC (lane 3) 25CAT DI RNAs detected by a probe complementary to the 3′-UTR of MHV RNA. The arrow indicates the position of 25CAT DI RNA; the seven mRNA species from the helper virus are indicated at the left. (B) Northern blot analysis using a probe complementary to the 5′-end of MHV RNA just downstream of the leader sequence. Arrows indicate the positions of genomic RNA and 25CAT DI RNA.