Fig 1.
Subcellular fractionation of HRV16-infected HeLa cells for protein distribution analysis.
(A) Mass spectrometry analysis of protein distribution. Mock- or HRV16-infected HeLa cells were separated into cytoplasmic and nuclear fractions, digested with trypsin, isotopically labeled, pooled, and subjected to nanoLC-MS/MS. (B) Fractionation of HeLa cells following mock, 4, or 8 hours post-HRV16 infection (hpi) was confirmed by Western blot analysis. Vinculin (VCL) was used as a cytoplasmic (C) marker protein and lamin A/C (LMNA) as a marker of the nucleus (N). As confirmation of a productive infection, fractions were assayed for the expression of HRV16 RNA-dependent RNA polymerase 3D and its precursor 3CD. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control.
Table 1.
Most highly enriched terms in the “GO complete” Panther database applicable to the 276-protein set of S2 Table, with respect to a reference database comprising the merged experimental HeLa nuclear proteomes (nuc1 and nuc2, S1 Table).
Fold enrichment: Proportion of accessions with the given GO term in the 276-protein set / proportion in the 6700-member merged experimental nuclear reference proteome. Raw P-value: Raw statistical probability that the given fold-enrichment could have occurred by chance. FDR (false discovery rate): Proportion of decoy search results above raw P-value in paired target/decoy search. Terms are ranked by FDR: For “GO biological process” and GO molecular function”, all GO terms with an FDR > 1E-6 are shown. For “GO cellular component”, the threshold was 1E-10 since this was the approximate value for the GO term “nuclear part”, the compartment of origin.
Table 2.
Proteins whose abundance consistently increased in the cytoplasm while decreasing in the nucleus at 8 hr post-infection of HeLa cells with HRV16.
This table is an abstraction of those proteins in S2 Table detected in all four datasets (‘Nuc1’, ‘Nuc2’, ‘Cyto1’, ‘Cyto2’). Delimited values ‘x/y/z’ refer to an 8hr:mock abundance ratio of x based on z tryptic peptide species, y of which tracked the direction (< 1 or > 1) of x. For additional details see S2 Table legend.
Fig 2.
HRV16 induces a coordinated redistribution of proteins into the cytoplasm of infected cells.
Scatter plots show 8hpi:mock abundance ratios for all tryptic peptides detected in replicate in (A) nuclear and (B) cytoplasmic fractions. On the theoretical diagonal line of reproducibility (lower-left to upper-right), the apparent overrepresentation of tryptic peptides in the lower-left quadrant with respect to the upper-right for nuclear tryptic peptides is consistent with a net efflux of proteins from the nucleus, and the corresponding apparent overrepresentation of the upper-right quadrant for cytoplasmic tryptic peptides is consistent with a net influx of proteins into the cytoplasm. (C) Tryptic peptides scoring < 0.9, reproducibly, in nuclear 8hpi:mock abundance ratio were recorded (red) and the subset shared with both cytoplasmic replicates was then highlighted (bold red) on the cytoplasmic scatter plot (D), indicating a clear coordination between nuclear depletion and cytoplasmic enrichment.
Fig 3.
Heterogeneous nuclear ribonucleoprotein M (hnRNP M) redistributes to the cytoplasm of HRV16-infected HeLa cells.
(A) Tryptic peptides in Fig 2 that correspond to hnRNP M are highlighted red, clearly reflecting the nucleocytoplasmic redistribution of this protein 8 hours post infection (hpi) by HRV16. (B) HeLa cells were mock- or HRV16-infected (MOI 10) then fixed 4 or 8 hpi. Cells were permeabilized then probed, via indirect immunofluorescence, for HRV16 2C (red), a marker of HRV16 RNA replication sites, and cellular protein hnRNP M (green). DNA was counterstained with DAPI to indicate location of nuclei (blue). Cells were then imaged using confocal microscopy. (C) HeLa cells were mock- or HRV16-infected (MOI 10), fractionated at the indicated times, and fractions were analyzed by Western blot. Cleaved hnRNP M was observed 8 hpi in both the cytoplasmic and nuclear fractions (cp*). HRV16 3D and its precursor 3CD were used as markers of infection. VCL and LMNA were used as markers of the cytoplasmic (C) and nuclear fractions (N), respectively, and GAPDH was used as a general loading control.
Fig 4.
Serine and arginine rich splicing factor 2 (SRSF2) remains within the nucleus of HRV16-infected HeLa cells.
HeLa cells were mock- or HRV16-infected (MOI 10) then fixed on coverslips at the indicated times post-infection. HRV16 2C (red) and SRSF2 (green) were labeled by indirect immunofluorescence. DNA was counterstained with DAPI to indicate the location of nuclei (blue). Cells were imaged using confocal microscopy. Hpi: hours post infection.
Fig 5.
Splicing factor proline and glutamine rich (SFPQ) migrates from the nucleus to the cytoplasm following HRV16 infection.
(A) Tryptic peptides in Fig 2 that correspond to SFPQ are highlighted red as described in the legend for Fig 3. (B) HeLa cells were mock- or HRV16-infected (MOI 10) and fixed then imaged as described in the legend for Fig 4. (C) Western blot analysis of cytoplasmic (C) and nuclear (N) fractions of mock- or HRV16-infected HeLa cells. A C-terminal cleavage product (cp*) was detected in the cytoplasm at 8 hours post infection (hpi). A second cleavage fragment of SFPQ was detected in the nucleus at 8 hpi (cp*). HRV16 3D/3CD,VCL, LMNA, and GAPDH used as in Fig 4. (D) Schematic of SFPQ domains (adapted from [86]) with putative 3CD/3C cleavage sites. The proposed 3CD/3C cleavage site resulting in the fragment observed 4 hpi is indicated in red. The N-terminal portion of SFPQ includes the glycine, proline, and glutamine-rich (GPQ-rich) and DNA-binding domain (DBD). The C-terminal portion contains two RNA-recognition motifs (RRM1 and RRM2), a NonA/paraspeckle (NOPS) domain, a coiled-coiled domain, glycine-rich (G-rich) region, and nuclear localization signal (NLS).
Fig 6.
SFPQ is differentially cleaved during HRV16 or poliovirus infection of HeLa cells; cleavage is independent of caspase activity and is carried out by recombinant 3CD in vitro.
(A) HeLa cells were mock- or HRV16-infected (MOI 10), cell lysates were generated at the indicated times, and lysates were subjected to Western blot analysis. SFPQ cleavage products are indicated (cp*). HRV16 2C and its precursor 2BC were used as indicators of infection and GAPDH was used as a loading control. (B) HeLa cells were mock- or poliovirus-infected (MOI 10) followed by the generation of cell lysates at the indicated times, and which were then subjected to Western blot analysis. The single SFPQ cleavage product is indicated as in (A). Poliovirus 3A and its precursor 3AB served as markers of infection and GAPDH was used as above. Poliovirus and HRV16 infections were both carried out at 34°C. (C) HeLa cells were infected with HRV16 in the presence or absence of 50 μM zVAD-FMK after which lysates were generated at the indicated times and subjected to Western blot analysis. SFPQ cleavage was observed with or without zVAD-FMK. HRV16 3D/3CD and GAPDH were used as above. Cleavage product of poly(ADP-ribose) polymerase 1 (PARP) is indicated (cp*). Data are representative of at least two independent experiments. (D) HeLa cell nuclear extract was incubated with bovine serum albumin (BSA) or different forms of recombinant HRV16 3CD and subjected to Western blot analysis. Cleavage of SFPQ (cp*) was observed in the presence of wild type (WT) 3CD and a form of 3CD containing a mutation in the 3C/3D autoproteolysis site (uncleavable, μ10). Catalytically inactive 3CD (C146A) did not cause cleavage of SFPQ. Hpi: hours post infection; ns: non-specific.
Fig 7.
SFPQ knockdown correlates with reduced HRV16 replication.
(A) Transfection of non-targeting siRNA (siNT) or SFPQ-targeting siRNA (siSFPQ) did not result in statistically significant differences in cell viability 96 h post-transfection, as assessed by trypan blue exclusion prior to infection (P > 0.05). Mean percent viable values are displayed with error bars representing one standard deviation. (B) HeLa cells transfected with siRNA for 96 h were infected with HRV16 (MOI 0.1) and cells and cell culture fluids were harvested at the indicated times. Virus titer was determined by plaque assay. Data represent the means of three biological replicate experiments with error bars indicating standard error of the means (SEM) (* P < 0.005, ** P < 0.0005). (C) Single cycle growth analysis of HRV16 from siRNA-transfected and HRV16-infected HeLa cells (MOI 10). As in panel B, data represent the means of three biological replicate experiments with error bars displaying SEM (* P <0.005, ** P <0.0005). (D) siRNA-transfected and HRV16-infected HeLa cell lysates corresponding to the time-points in panel B (MOI 0.1) were generated and subjected to Western blot analysis. Knockdown of SFPQ was confirmed and levels of 2C and precursor (2BC) represented viral protein production. GAPDH was used as a loading control. (E) Western blot analysis of lysates from siRNA-transfected and HRV16-infected HeLa cells corresponding to time points in panel C (MOI 10). Western blots in panels D and E are representative results from three biological replicate experiments. (F) RNA isolated from siRNA-transfected and HRV16-infected HeLa cells at time-points corresponding to panel B (MOI 0.1) was subjected to RT-PCR. Primers for PCR were specific for HRV16 RNA, SFPQ mRNA, or actin (ACTB) mRNA. PCR products were separated by agarose gel electrophoresis. (G) RT-PCR analysis of RNA isolated from siRNA-transfected and HRV16-infected HeLa cells at time-points corresponding to panel C (MOI 10). Data in panels F and G are representative results from biological duplicate experiments. hpi: hours post infection.
Fig 8.
An SFPQ cleavage product associates with in vitro transcribed HRV16 RNA.
(A) HeLa cells were mock- or HRV16 infected, fractionated at 2 h intervals, and the subcellular distribution of proteins was analyzed by Western blot. The cleavage product (cp*) of polypyrimidine tract binding protein 1 (PTBP1) is indicated and HRV16 3D and its precursor 3CD served as markers of infection. VCL and LMNA served as markers for the cytoplasm (C), nucleus (N); GAPDH was used as a general loading control. (B) Biotinylated, in vitro transcribed control or HRV16 RNA were assayed for binding to cellular proteins present in HeLa cell lysates following mock infection or 8 hours post-infection (hpi) with HRV16 by Western blot analysis. A representative experiment is shown. (C) Quantification of four separate RNA affinity experiments was carried out using Quantity One software. Means are shown and error bars represent standard deviations (* P < 0.05).
Fig 9.
Proposed model of HRV16-SFPQ interactions during the infectious cycle.
Following translation of viral proteins, HRV16 3CD/3C enters the nucleus where it targets SFPQ for cleavage at Q257 within the N-terminus. Cleavage releases SFPQ from interactions with nuclear-resident anchors such as DNA and allows the C-terminal fragment to migrate to the cytoplasm through degraded nuclear pore complexes. Once in the cytoplasm, the SFPQ fragment interacts with HRV16 RNA, directly or through other protein partners, and may promote viral replication.