Figure 1.
Recruitment of the 43S pre-initiation complex for cap-dependent and cap-independent translation initiation.
For cap-dependent translation initiation (Figure 1A), the eIF4F cap-binding complex recognizes and binds to the 5′ cap structure of the mRNA. Following cap binding and ribosome scanning mediated by the 43S pre-initiation complex, initiation occurs at an AUG in a favorable context. After GTP hydrolysis and 60S subunit joining, the ribosome is elongation-competent and protein synthesis begins. In the cap-independent mechanism of translation initiation (Figure 1B), the 43S pre-initiation complex associates with RNA sequences in the IRES either directly or in conjunction with canonical or non-canonical initiation factors, which facilitates initiation at the appropriate AUG start codon. Non-canonical factors are indicated as IRES trans-acting factors, or ITAFs. This figure highlights the major differences in the mechanisms of cap-dependent and cap-independent translation, and is not a comprehensive model for eukaryotic translation initiation. Additional canonical factors (such as eIF4G and eIF4B) as well as non-canonical factors have been shown to bind to the poliovirus IRES and/or stimulate poliovirus translation. (Figure taken from [2], with permission).
Figure 2.
SRp20 re-localization from the nucleus to the cytoplasm of SK-N-SH cells during poliovirus infection.
Cells were transfected with GFP-SRp20 and either mock-infected (A) or infected with poliovirus at an MOI of 25. Cells were fixed at specific times post-infection (1–5 hours, B–F) and imaged. SRp20 localization was determined using confocal microscopy; nuclei were identified by DAPI staining.
Figure 3.
SRp20 is present in cytoplasmic extracts from both mock- or poliovirus-infected cells.
Cytoplasmic extracts from mock- or poliovirus-infected cells (hours 1 through 4 as indicated) generated for sucrose gradient fractionation were also examined for the presence of SRp20. Extracts (100 µg of total protein) were subjected to SDS-PAGE and Western blot analysis, probing with an SRp20 monoclonal antibody. Lanes marked ‘M’ are extracts from mock-infected cells; lanes marked ‘PV’ are extracts from poliovirus-infected cells.
Figure 4.
SRp20 re-localization from the nucleus to the cytoplasm of HeLa cells during poliovirus infection.
Cells were transfected with GFP-SRp20 and either mock-infected (A) or infected with poliovirus at an MOI of 25. Cells were fixed at specific times post-infection (1–5 hours, B–F) and imaged. SRp20 localization was determined using confocal microscopy; nuclei were identified by DAPI staining.
Figure 5.
SRp20 partial co-localization with PCBP2 in the cytoplasm of poliovirus-infected SK-N-SH cells.
Cells were transfected with GFP-SRp20 and mock-infected (A) or infected with poliovirus for 3 hours (B) at an MOI of 25. At 3 hours post-infection, cells were fixed and incubated with a PCBP2 monoclonal antibody, a secondary antibody conjugated to biotin, and streptavidin conjugated to Texas Red. Cells were examined for co-localization of PCBP2 and SRp20 (shown in the merged image in yellow and highlighted by the white arrow) in the cytoplasm of the cells using confocal microscopy; nuclei were identified by DAPI staining.
Figure 6.
SRp20 and PCBP2 co-sedimentation with 40S ribosomal subunits in mock-infected cells.
Extracts were generated from mock-infected HeLa cells, sedimented through 7%–47% sucrose gradients and fractionated. The collected fractions were subjected to Western blot analysis to determine the co-sedimentation of PCBP2 and SRp20 with ribosomal subunits, monosomes, or polysomes. Representative polysome profiles and Western blot analyses are shown.
Figure 7.
SRp20 and PCBP2 co-sedimentation with 40S ribosomal subunits in poliovirus-infected cells (2 hr post-infection).
Extracts were generated from poliovirus-infected HeLa cells after 2 hours of infection and processed as described in the legend for Figure 6. Representative polysome profiles and Western blot analyses are shown.
Figure 8.
SRp20 and PCBP2 co-sedimentation with 40S ribosomal subunits in poliovirus-infected cells (5 hr post-infection).
Extracts were generated from poliovirus-infected HeLa cells after 5 hours of infection and processed as described in the legend for Figure 6. Representative polysome profiles and Western blot analyses are shown. The sedimentation of virus (labeled ‘virus peak’ in the profile) was determined by Western blot analysis of fractions using an anti-VP2 polyclonal antibody that detects the poliovirus VP2 capsid protein (Holzberg, Nguyen, and Semler, unpublished).
Figure 9.
Interaction of PCBP2 and SRp20 on poliovirus stem-loop IV RNA.
Poliovirus stem-loop IV RNA was transcribed and biotinylated with Biotin-CTP. The purified RNA (lane 3 in A and B) or tRNA alone (lane 2 in A and B) was incubated with streptavidin agarose, and the RNA-affinity matrix was subsequently incubated with extracts from poliovirus-infected cells (4 hours post-infection). Bound complexes were examined by Western blot analysis for the presence of PCBP2 and SRp20. Lane 1 in A and B, input extract (20% of experimental). The experiments were repeated exactly as described above, but included an additional experimental control. Extracts were incubated with tRNA alone (lane 2 in C and D), with biotinylated S-L IV RNA (lane 3 in C and D), or biotinylated S-L IV RNA and synthetic poly(rC) RNA (lane 4 in C and D) as a competitor for PCBP2 binding. Lane 1 in C and D, input extract (20% of experimental).
Figure 10.
SRp20ΔRRM re-localization from the nucleus to the cytoplasm of SK-N-SH cells during poliovirus infection.
Cells were transfected with GFP-SRp20ΔRRM and either mock-infected (A) or infected with poliovirus at an MOI of 25. Cells were fixed at specific times post-infection (1–5 hours, B–F) and imaged. SRp20ΔRRM localization was determined using confocal microscopy; nuclei were identified by DAPI staining.
Figure 11.
SRp20ΔRRM partial co-localization with PCBP2 in the cytoplasm of poliovirus-infected SK-N-SH cells.
Cells were transfected with SRp20ΔRRM and mock-infected (A) or infected with poliovirus for 3 hours (B) at an MOI of 25. At 3 hours post-infection, cells were fixed and incubated with a PCBP2 monoclonal antibody, a secondary antibody conjugated to biotin, and streptavidin conjugated to Texas Red. Cells were examined for co-localization of PCBP2 and SRp20ΔRRM (shown in the merged image in yellow and highlighted by white arrows) in the cytoplasm of the cells using confocal microscopy; nuclei were identified by DAPI staining.
Figure 12.
Effect of SRp20ΔRRM expression on poliovirus yield.
Cells were co-transfected with poliovirus cDNA and a plasmid expressing either GFP alone, GFP-SRp20, or GFP-SRp20ΔRRM. Virus was harvested from the cells at 96 hours post-transfection and serially diluted. Dilutions of virus were used to carry out plaque assays (B), and whole cell extracts were also generated to determine the levels of expression of the SRp20 proteins by Western blot using an antibody against SRp20 (A). In (A) lane 1, cells transfected with the plasmid expressing GFP; lane 2, expressing GFP-SRp20; lane 3, expressing GFP-SRp20ΔRRM. Cells expressing the deletion mutant, GFP-SRp20ΔRRM, displayed a two-log decrease in poliovirus yield (C). The observed decrease (∼100 fold) was consistent across three separate experiments, although overall titers for the GFP control between experiments ranged from ∼105 to ∼107 (likely due to the variability of DNA transfection efficiency). Plaque assays to determine poliovirus titers were performed in triplicate.