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. 2010 May;84(9):4229-42.
doi: 10.1128/JVI.02198-09. Epub 2010 Feb 17.

Mechanistic consequences of hnRNP C binding to both RNA termini of poliovirus negative-strand RNA intermediates

Kenneth J Ertel  1 , Jo Ellen Brunner, Bert L Semler
Affiliations

Mechanistic consequences of hnRNP C binding to both RNA termini of poliovirus negative-strand RNA intermediates

Kenneth J Ertel et al. J Virol. 2010 May.

Abstract

The poliovirus 3' noncoding region (3' NCR) is necessary for efficient virus replication. A poliovirus mutant, PVDelta3'NCR, with a deletion of the entire 3' NCR, yielded a virus that was capable of synthesizing viral RNA, albeit with a replication defect caused by deficient positive-strand RNA synthesis compared to wild-type virus. We detected multiple ribonucleoprotein (RNP) complexes in extracts from poliovirus-infected HeLa cells formed with a probe corresponding to the 5' end of poliovirus negative-strand RNA (the complement of the genomic 3' NCR), and the levels of these RNP complexes increased during the course of viral infection. Previous studies have identified RNP complexes formed with the 3' end of poliovirus negative-strand RNA, including one that contains a 36-kDa protein later identified as heterogeneous nuclear ribonucleoprotein C (hnRNP C). We report here that the 5' end of poliovirus negative-strand RNA is capable of interacting with endogenous hnRNP C, as well as with poliovirus nonstructural proteins. Further, we demonstrate that the addition of recombinant purified hnRNP C proteins can stimulate virus RNA synthesis in vitro and that depletion of hnRNP C proteins in cultured cells results in decreased virus yields and a correspondingly diminished accumulation of positive-strand RNAs. We propose that the association of hnRNP C with poliovirus negative-strand termini acts to stabilize or otherwise promote efficient positive-strand RNA synthesis.

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Figures

FIG. 1.
FIG. 1.
The 5′ end of poliovirus negative-strand RNA and hnRNP C proteins. (A) Mfold-predicted structure of the 5′ end of poliovirus negative-strand RNA, the complement of the genomic-strand 3′ NCR. Shown are the X(−) stem-loop (formed by base pairing that includes a stretch of uridine residues) and the Y(−) stem-loop. The 5′ and 3′ ends of the RNA structure are indicated (59). (B) Scheme depicting the domains of wild-type hnRNP C1 and C2 proteins. NLS, nuclear localization signal; auxiliary domain, region implicated in protein-protein interactions. Indicated is the additional 13-amino-acid (aa) region of C2 proteins. The location of the CID mutation engineered to create hnRNP C1-L187Q is also indicated.
FIG. 2.
FIG. 2.
Binding of proteins from poliovirus-infected cells to the 5′ end of virus negative-strand RNA. (A) Mfold-predicted structure of the 5′ end of poliovirus negative-strand RNA with a 5′ hammerhead ribozyme. The “2-G start” (boxed) resulting from RNA transcription from the T7 promoter is indicated, followed by the nucleotide sequence forming the hammerhead ribozyme structure. The arrow indicates the site of autocatalytic cleavage of the ribozyme and the poly(U) sequence of poliovirus negative-strand RNA, followed by a portion of the 5′ end of the negative-strand RNA corresponding to the genomic 3′ NCR (underlined) (59). (B) UV cross-linking assays with poliovirus 5′(−) RNA probe and cytoplasmic extracts from mock-infected HeLa (lane 3) and mock-infected NGP (lane 4) cells. Lanes 5 to 7 show complexes from UV cross-linking assays carried out in the presence of 50 μg of cytoplasmic extracts generated from poliovirus-infected HeLa cells harvested at 2, 4, and 6 h postinfection, respectively. Samples were boiled in the presence of Laemmli sample buffer and resolved on a 12.5% polyacrylamide-SDS-containing gel. Analysis was carried out using a phosphorimager (Bio-Rad). Lane M, marker proteins ([35S]methionine-labeled in vitro translation of poliovirus virion RNA). FP, free probe cross-linked in the absence of any extract and treated with RNase as described in Materials and Methods. (C) UV cross-linking assay using extracts from poliovirus-infected cells in the presence of unlabeled competitor RNAs. These studies were carried out using cytoplasmic extracts generated from poliovirus-infected HeLa cells at 6 h postinfection. Competitor RNAs corresponding to nonradiolabeled 5′(−) RNA probe (lanes 4 to 6), nonradiolabeled 3′(−) RNA probe (lanes 7 and 8), tRNA (lanes 9 and 10), or nonradiolabeled transcript of 20 uridine residues (lanes 11 and 12) were added to reaction mixtures at the indicated molar excess compared to the amount of [32P]UTP-radiolabeled 5′(−) RNA probe. Analysis was carried out using a phosphorimager. Lane M, marker proteins ([35S]methionine-labeled in vitro translation of poliovirus virion RNA). FP, free probe in the absence of any extract.
FIG. 3.
FIG. 3.
The cellular protein hnRNP C binds the 5′ end of poliovirus negative-strand RNA. (A) Increased concentrations of hnRNP C proteins in HeLa S10 cytoplasmic extracts generated from poliovirus-infected cells. Cytoplasmic extracts of HeLa cells were prepared from either mock-infected cells (three separate extracts; lanes 1 to 3) or poliovirus-infected cells (lane 4) at 5 h postinfection. Equivalent amounts of total protein (10 μg) from each extract were resolved on a 12.5% polyacrylamide-SDS-containing gel. Western blot analysis was carried out using mouse monoclonal anti-C1/C2 as the primary antibody (Abcam) and an alkaline phosphatase-conjugated anti-mouse IgG (Zymed) as a secondary antibody. Molecular masses are indicated on the left. As a loading control, the blots were also probed with mouse monoclonal anti-β-actin antibody (Abcam). (B) Immunoprecipitation of UV cross-linked complexes confirms binding of hnRNP C to the 5′(−) RNA probe. UV cross-linking assays were carried out as described in Materials and Methods with the [32P]UTP-radiolabeled 5′(−) RNA probe and cytoplasmic extracts generated from mock-infected (Mock inf) (lanes 3 to 5) or poliovirus-infected (PV inf) (lanes 6 to 8) HeLa cells at 5 h postinfection. [32P]UTP-radiolabeled UV cross-linked samples from mock- or poliovirus-infected HeLa cells were immunoprecipitated by the inclusion of normal IgG (lanes 4 and 7) or hnRNP C1/C2 antibody (lanes 5 and 8) in reaction mixtures following RNase digestion. The immunoprecipitated complexes were resolved on a 12.5% polyacrylamide-SDS-containing gel. Analysis was carried out using a phosphorimager. Lane M, marker proteins ([35S]methionine-labeled in vitro translation of poliovirus virion RNA). FP, free probe in the absence of any extract. (C) Electrophoretic mobility shift assays using extracts generated from poliovirus-infected cells in the presence or absence of antibody to analyze the contents of RNP complexes. Cytoplasmic extracts (1 μg) from poliovirus-infected HeLa cells were incubated with radiolabeled poliovirus 5′(−) RNA probe. The reaction mixtures in lanes 3 and 4 contained 0.1 μg of either IgG or C1/C2 monoclonal antibody, respectively. The unbound probe is indicated at the bottom left, and the appearance of a supershifted complex in lane 4 is indicated by an arrow at the top right.
FIG. 4.
FIG. 4.
Binding of hnRNP C to the 5′ end of poliovirus negative-strand RNA under nondenaturing conditions. (A) Binding of recombinant hnRNP C to poliovirus negative-strand RNA. UV cross-linking assays were carried out using 50 μg of extracts generated from poliovirus-infected HeLa cells at 2 h (lane 3), 4 h (lane 4), and 6 h (lane 5) postinfection. Lanes 6 and 7 show UV cross-linked complexes with bacterially expressed His-tagged hnRNP C recombinant proteins containing a carboxyl-terminal hexahistidine tag. Lane 6, 0.5 μg hnRNP C1-His; lane 7, 0.5 μg of hnRNP C2-His. Image analysis was carried out using a phosphorimager. Lane M, marker proteins ([35S]methionine-labeled in vitro translation of poliovirus virion RNA). FP, free probe in the absence of any extract, as described in the legend to Fig. 2. (B) RNA mobility shift assays were carried out as described in Materials and Methods. Recombinant purified hnRNP C1 (lanes 2 to 4) or hnRNP C2 (lanes 5 to 8) was incubated with radiolabeled 5′(−) RNA probe. The competition reaction mixtures (lanes 4 and 8) contained nonradiolabeled 5′(−) RNA in molar excess. Following incubation, complexes were resolved on a 4% native polyacrylamide gel. The unbound probe is indicated on the left, as are the putative monomeric (L) and multimeric (H) complexes.
FIG. 5.
FIG. 5.
Differential binding capabilities of wild-type and mutated hnRNP C proteins. Electrophoretic mobility shift assays were carried out as described in Materials and Methods. Recombinant purified hnRNP C1-His (lanes 1 to 4), hnRNP C1-K50Q (lanes 5 to 8), or hnRNP C1-L187Q (lanes 9 to 12) was incubated at the indicated concentrations with radiolabeled poliovirus 5′(−) RNA probe, and the resulting complexes were resolved on a 4% native polyacrylamide gel. Lane 13 contained free probe in the absence of any protein, with the unbound probe indicated at the lower right. L, lower-molecular-mass RNP complex; H, higher-molecular-mass RNP complex.
FIG. 6.
FIG. 6.
Interaction of infected-cell viral proteins 3C/3CD with the 5′ end of poliovirus negative-strand RNA. Electrophoretic mobility shift assays were performed as described in Materials and Methods. Recombinant purified 3CD protein containing a C147A mutation (a generous gift of R. Perera) was incubated with poliovirus 5′(−) RNA probe at the indicated concentrations. Supershift assays were carried out as described in Materials and Methods using a monoclonal antibody to hnRNP C1/C2 or a polyclonal antibody capable of recognizing virus proteins containing poliovirus 3C sequences (41). Cytoplasmic extracts from poliovirus-infected cells (1 μg) were incubated with the radiolabeled poliovirus 5′(−) RNA probe. The reaction mixtures in lanes 5, 6, and 7 contained 0.1 μg of normal mouse IgG (Zymed), mouse monoclonal hnRNP C1/C2 antibody (Abcam), or polyclonal antibody raised against recombinant poliovirus protein 3C, respectively. The presence of the supershifted complexes in lanes 6 and 7 is indicated by an arrow at the top right. An asterisk on the right indicates the disappearance of RNP complexes in lane 7.
FIG. 7.
FIG. 7.
Stimulation of poliovirus RNA synthesis by the addition of wild-type (WT) hnRNP C1 proteins. Recombinant wild-type hnRNP C1 (lanes 3 to 5) or hnRNP C1-L187Q (lanes 6 to 8) protein or protein buffer alone (lane 1) was added to in vitro replication/translation reaction mixtures containing HeLa S10 cytoplasmic extracts from uninfected cells. Each reaction mixture was split into two fractions as described previously (15). (A) Replication of poliovirus virion RNA. Replication fractions containing purified poliovirus vRNA, HeLa S10 cytoplasmic extracts, and recombinant hnRNP C proteins were incubated at 30°C to allow translation of nonstructural proteins. Following addition of [32P]CTP to the reaction mixtures, RNA synthesis was allowed to proceed for 2 h. The replication reaction mixtures were then purified using an RNaqueous spin column and resolved on a 1.1% Tris-borate-EDTA-agarose gel containing ethidium bromide. As a negative control, 2 mM guanidine hydrochloride (GuaHCl) was added to the reaction mixture shown in lane 2 to inhibit negative-strand RNA synthesis. 18S and 28S rRNAs were used to confirm equal loading of samples. Comparative density values of the signals for RNA synthesis are depicted as fold increases compared to lane 1. (B) Translation fractions of the samples depicted in panel A were incubated with [35S]methionine and, following incubation at 30°C for 4 h, the reaction mixtures were boiled in Laemmli sample buffer and resolved on a 12.5% polyacrylamide-SDS-containing gel.
FIG. 8.
FIG. 8.
Poliovirus growth kinetics and viral-RNA accumulation in hnRNP C-depleted HeLa cells. Knockdown of hnRNP C via retrovirus infection and selection of infected cells is described in Materials and Methods. Plaque assays were carried out as described previously (13) and in Materials and Methods. (A) Whole-cell lysates of mock-treated (lane 1), HCScram-treated (lane 2), and HC12KD-treated (lane 3) cells were generated as described in Materials and Methods. Ten micrograms of each cell lysate was resolved on a 12.5% polyacrylamide-SDS-containing gel. Western blot analysis was carried out with a mouse monoclonal anti-hnRNP C1/C2 antibody (Abcam) and an alkaline phosphatase-conjugated anti-mouse secondary antibody (Promega). As a loading control, mouse monoclonal antibodies recognizing β-actin were utilized (Abcam). (B) One-step growth analysis was carried out in HeLa, HeLaScram, or HeLaHC12KD monolayers in 60-mm plates at 37°C. The cells were infected at an MOI of 25 with wild-type or PVΔ3′NCR virus. Infected HeLa, HeLaScram, and HeLaHC12KD cells were harvested beginning at 0 h postinfection and every 2 h thereafter. The virus particles were released by freeze-thaw, and the total virus yield was determined from the plaque assay titer divided by the total initial cell count. (C) Quantitative real-time PCR of poliovirus positive-strand RNA accumulation was carried out using total-RNA harvests of monolayers from wild-type HeLa cells or retrovirus-infected HeLaHC12KD cells infected with either wild-type poliovirus or PVΔ3′NCR. The cells were infected at an MOI of 25, and total RNA was harvested as described in Materials and Methods. (D) Quantitative real-time PCR of poliovirus negative-strand RNA accumulation was carried out as described for panel C, except that DNA oligonucleotides annealing to poliovirus negative-strand RNA were used. (E) Viral-RNA synthesis is depicted as the ratio of detected positive- to negative-strand RNAs, with the mean ratio depicted above the bars for each sample. As an internal control for RNA content in individual samples, quantitative real-time PCR of cellular β-actin RNAs was carried out for each sample in positive-strand vRNA accumulation (C) and negative-strand vRNA accumulation (D) to normalize poliovirus RNA detection results. Note that for the quantitative real-time PCR assays, shRNA-mediated knockdown was compared to untreated HeLa cells because there were no significant differences in poliovirus growth curves when poliovirus infections of untreated HeLa cells were compared to HeLaScram-treated cells. The error bars indicate standard deviations.
FIG. 9.
FIG. 9.
Model of the initiation of positive-strand RNA synthesis. Negative-strand template RNA, usually found as part of the double-stranded RF, is depicted in green as single stranded for simplicity. Nascent positive-strand RNA (B) is depicted by a black dashed arrow. (A) Prior to positive-strand RNA synthesis, the 5′ and 3′ termini associate with known (hnRNP C) and unknown (host) cellular proteins, in addition to viral proteins 3C/3CD and 2C. Binding of hnRNP C, possibly in conjunction with other viral/cellular proteins, allows interaction of 3C/3CD with the negative-strand RNA termini. (B) These interactions may allow contact of the 5′ and 3′ ends of the RF (indicated by the double-headed arrow), possibly facilitating initiation of positive-strand RNA synthesis by the RNA-dependent RNA polymerase (3Dpol). The activity of hnRNP C in the virus RNA replication cycle may be a rate-limiting step during the initial stages of poliovirus infection, where the hnRNP C cytoplasmic concentration is low. At later times postinfection, hnRNP C distribution shifts to the cytoplasm of the infected cell. VPg, the genome-linked protein of poliovirus; pUpU, the first two nucleotides of poliovirus genomic RNA.
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