Cell-specific proteins regulate viral RNA translation and virus-induced disease

Abstract

Translation initiation of the picornavirus genome is regulated by an internal ribosome entry site (IRES). The IRES of a neurovirulent picornavirus, the GDVII strain of Theiler's murine encephalomyelitis virus, requires polypyrimidine tract-binding protein (PTB) for its function. Although neural cells are deficient in PTB, they express a neural-specific homologue of PTB (nPTB). We now show that nPTB and PTB bind similarly to multiple sites in the GDVII IRES, rendering it competent for efficient translation initiation. Mutation of a PTB or nPTB site results in a more prominent decrease in nPTB than PTB binding, a decrease in activity of nPTB compared with PTB in promoting translation initiation, and attenuation of the neurovirulence of the virus without a marked effect on virus growth in non-neural cells. The addition of a second-site mutation in the mutant IRES generates a new PTB (nPTB) binding site, and restores nPTB binding, translation initiation and neurovirulence. We conclude that the tissue-specific expression and differential RNA-binding properties of PTB and nPTB are important determinants of cell-specific translational control and viral neurovirulence.

Fig. 1. Toeprint analysis of 48S complexes assembled on wt GDVII RNA in the presence of increasing amounts of PTB and nPTB. Reactions were assembled with translation components as indicated in the table above the gel; molar ratios of PTB- and nPTB-dimers to mRNA are listed. Reference lanes T, A, C and G depict the negative-strand sequence and the position of the initiator AUG is indicated to the left. Positions of cDNA products terminated due to 48S complex formation are labeled as 48S to the right. Other RT stops that changed in intensity in the presence of PTB and nPTB are labeled by an asterisk on the right.

Fig. 2. A model of the structure of the GDVII IRES showing the location of PTB- and nPTB-binding sites. Stem–loops are named according to Duke et al. (1992). A pseudoknot is designated as pk. Thin lines with arrows connect different structural domains. Some regions of the IRES that are not detailed are shown with a bold line connecting the number of the downstream and upstream nucleotides of the region. The initiator AUG is boxed. Nucleotides susceptible to DMS and CMCT are marked by circles and squares, respectively, while cleavages produced by RNase ONE and RNase V1 are marked by arrowheads and arrows, respectively. Nucleotides and bonds with no change in susceptibility to chemicals and RNases following PTB or nPTB incubation are marked by black symbols. Nucleotides and bonds protected by PTB or nPTB are marked by red symbols. The names of the PTB- and nPTB-binding sites are shown near the protected RNA segments (see text). Nucleotides and bonds in which susceptibility to RNases and chemicals was enhanced following PTB or nPTB binding are marked by green symbols.

Fig. 3. Chemical and enzymatic footprints of wt GDVII IRES alone and in complexes formed with PTB or nPTB (as noted in the table above the gel). RNA probes were extended from a primer complementary to nt 740–758 (A) or nt 1107–1121 (B). Reference lanes T, A, C and G depict the negative-strand sequence derived using the relevant primer with GDVII plasmid DNA. Positions of PTB- and nPTB-binding sites are indicated on the right.

Fig. 4. Chemical and enzymatic footprints of the mutant GD-105 (A and C) and GD-106 (B and D) IRES. RNA was incubated with protein(s) and chemical(s) in reactions as listed in the tables above the gels. RNA probes were extended from a primer complementary to nt 740–758 (A and B) or nt 1107–1121 (C and D). Positions of PTB- and nPTB-binding sites in wt GDVII IRES are indicated on the right. Note that compared with wt GDVII (Figure 3A) and GD-105 (A), there is now protection with PTB and nPTB at a new site, 5ss, in GD-106 (B). In GD-105, there is decreased protection with PTB and nPTB at sites 4ss, 4ds and 5ds, as well as decreased protection with nPTB at site 7ss, while in GD-106 protection of the above sites with PTB and nPTB had been restored. Reference lanes T, A, C and G depict the negative-strand sequence derived using the relevant primer with GD-105 plasmid DNA.

Fig. 5. Chemical and enzymatic footprints of the mutant GD-121 (A and B) and GD-122 (C) IRES. RNA probes were extended from a primer complementary to nt 740–758 (B and C) or nt 1107–1121 (A). For more details, see the legends to Figures 3 and 4. Note that compared with wt GDVII (Figure 3) and GD-122 (C), there is decreased protection with nPTB at sites 2ds, 3ss, 3ds and 4ds in GD-121 (B). In GD-122, PTB and nPTB protect a new site, 5ss, and there is now restoration of protection with nPTB that had been perturbed in GD-121. Reference lanes T, A, C and G depict the negative-strand sequence derived using the relevant primer with GD-121 plasmid DNA.

Fig. 6. Toeprint analysis of 48S complexes assembled on wt GDVII and mutant RNAs in the presence of PTB, nPTB and Nova-1. Reactions were assembled with translation components as indicated in the tables above the gels. For more details, see the legend to Figure 1.