Fig 2: 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 3: 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.

Fig 4: 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.