Interaction of poly(rC) binding protein 2 with the 5' noncoding region of hepatitis A virus RNA and its effects on translation

Abstract

Utilization of internal ribosome entry segment (IRES) structures in the 5' noncoding region (5'NCR) of picornavirus RNAs for initiation of translation requires a number of host cell factors whose distribution may vary in different cells and whose requirement may vary for different picornaviruses. We have examined the requirement of the cellular protein poly(rC) binding protein 2 (PCBP2) for hepatitis A virus (HAV) RNA translation. PCBP2 has recently been identified as a factor required for translation and replication of poliovirus (PV) RNA. PCBP2 was shown to be present in FRhK-4 cells, which are permissive for growth of HAV, as it is in HeLa cells, which support translation of HAV RNA but which have not been reported to host replication of the virus. Competition RNA mobility shift assays showed that the 5'NCR of HAV RNA competed for binding of PCBP2 with a probe representing stem-loop IV of the PV 5'NCR. The binding site on HAV RNA was mapped to nucleotides 1 to 157, which includes a pyrimidine-rich sequence. HeLa cell extracts that had been depleted of PCBP2 by passage over a PV stem-loop IV RNA affinity column supported only low levels of HAV RNA translation. Translation activity was restored upon addition of recombinant PCBP2 to the depleted extract. Removal of the 5'-terminal 138 nucleotides of the HAV RNA, or removal of the entire IRES, eliminated the dependence of HAV RNA translation on PCBP2.

FIG. 1

Western blot analysis of HeLa and FRhK-4 cell fractions for PCBP2. Thirty micrograms of protein from the A-cut preparations derived from FRhK-4 (lane 2) and HeLa S3 (lane 3) cell RSW were resolved by SDS-PAGE and transferred to a nitrocellulose membrane for immunoblot analysis using antiserum raised against rPCBP2. Immunoreactive proteins recognized by anti-rPCBP2 antiserum are indicated to the right; molecular masses of marker proteins (M; lane 1) are indicated in kilodaltons to the left.

FIG. 2

Mobility and competition assay of RNAs from PV and HAV 5′NCR. ³²P-labeled PV stem-loop IV RNA (nt 220 to 460) was incubated without (lane 1) and with rPCBP2 (lane 2) and separated on a 4% native polyacrylamide gel. Competition for complex formation between ³²P-labeled PV stem-loop IV RNA and rPCBP2 was tested in the presence of 1- to 40-fold molar excesses of transcripts representing the PV 5′NCR (P1-816; lanes 3 to 7) or transcripts representing the HAV 5′NCR (H1-744; lanes 8 to 12). Free labeled PV stem-loop IV (stIV) RNA and the RNA-protein complex are indicated to the left.

FIG. 3

Schematic representation of RNA fragments of the HAV 5′NCR used as competitor RNAs in mobility shift assays with ³²P-labeled PV stem-loop IV RNA and rPCBP2. Open bars represent sequences of the 5′NCR comprising nucleotides indicated below each fragment. The dotted lines indicate the extent of deletions in the 5′NCR. The vertical lines indicate the borders of the predicted stem-loop structures I to V and the pyrimidine-rich tracts at the 5′ (pY1) and 3′ (pY2) ends of the 5′NCR (8, 38) as depicted above the diagram of the RNA fragments. +, (+), or − at the left indicates whether the particular RNA fragment competes, competes weakly, or does not compete with PV stem-loop IV RNA for binding of rPCBP2.

FIG. 4

RNA mobility shift and competition assay with ³²P-labeled PV stem-loop IV RNA (nt 220 to 460), rPCBP2, and various HAV 5′NCR fragments as competitor RNA analyzed on a 4% native polyacrylamide gel. (A) Labeled RNA (PV nt 220 to 460) was incubated with rPCBP2 in the absence (lane 2) or in the presence of unlabeled competitor HAV RNA comprising nt 1 to 744 (lanes 3 and 4) or comprising the 5′ half of the HAV 5′NCR from nt 1 to 354 (lanes 5 and 6) at the molar excess indicated. Lane 1 was loaded with ³²P-labeled PV stem-loop IV (stIV) RNA only. (B) Labeled RNA (PV nt 220 to 460) was incubated with rPCBP2 in the presence of competitor RNA comprising nt 148 to 744 (lanes 3 to 6), nt 292 to 744 (lanes 7 to 10), and nt 1 to 744 with the internal deletion from nt 158 to 292 (lanes 11 to 14). Labeled PV stem-loop IV RNA alone was loaded in lane 1 and as complex with rPCBP2 in lane 2. Free RNA and the RNA-protein complex are indicated to the left.

FIG. 5

Western blot analysis of combined HeLa S10-RSW extract, using antiserum raised against rPCBP2. The extract was depleted of PCBP2 by RNA affinity column chromatography with immobilized PV stem-loop IV RNA (nt 220 to 460). Lane 2, untreated extract; lanes 3 and 4, extract passaged five times over the RNA affinity column; lanes 5 and 6, 1 and 2 M KCl eluates; lanes 7 to 10, control extract passaged over a column without any immobilized RNA (mock-depleted extract) and its high-salt eluates. Molecular masses of marker proteins (M; lane 1) are indicated in kilodaltons to the left; immunoreactive proteins recognized by anti-rPCBP2 antiserum are indicated to the right.

FIG. 6

Translation of truncated HAV and PV RNAs driven by different 5′NCR regions in HeLa S10-RSW extracts. The H1-2024 (lanes 4 to 6) and P1-2954 (lanes 7 to 9) (A), H667-2024 (lanes 3 to 5) and P1-2954 (lanes 6 to 8) (B), and H139-2024 (lanes 3 to 5) and H1-2024 (lanes 6 to 8) (C) transcripts were used to program translation extracts in the presence of [³⁵S]methionine and [³⁵S]cysteine. Translation assays were carried out in mock-depleted extract (mo), PCBP2-depleted extract (de), and PCBP2-depleted extract supplemented with rPCBP2 (de +). PV-encoded polypeptides obtained from PV-infected HeLa cells labeled with [³⁵S]methionine served as protein marker (M; lane 1) and are identified on the left. HeLa S10-RSW extract programmed with 100 ng of PV RNA isolated from purified PV virions was used as internal translation control (panel A, lane 2). Due to the short incubation time used in this assay, the PV polyprotein is mainly uncleaved. HeLa cell extract programmed with no RNA is analyzed in lane 3 of panel A and lanes 2 of panels B and C. The predicted protein sizes of the translation products are indicated to the right.