Rivera C, Lloyd RE.. Modulation of enteroviral proteinase cleavage of poly(A)-binding protein (PABP) by PABP-associated factors. Virology 375: 59-72

Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Virology (Impact Factor: 3.32). 06/2008; 375(1):59-72. DOI: 10.1016/j.virol.2008.02.002
Source: PubMed


Poliovirus (PV) causes a drastic inhibition of cellular cap-dependant protein synthesis due to the cleavage of translation factors eukaryotic initiation factor 4G (eIF4G) and poly(A) binding protein (PABP). Only about half of cellular PABP is cleaved by viral 2A and 3C proteinases during infection. We have investigated PABP cleavage determinants that regulate this partial cleavage. PABP cleavage kinetics analyses indicate that PABP exists in multiple conformations, some of which are resistant to 3C(pro) or 2A(pro) cleavage and can be modulated by reducing potential. Cleavage reactions containing a panel of PABP-binding proteins revealed that eukaryotic release factor 3 (eRF3) and PABP-interacting protein 2 (Paip2) modulate and interfere with the cleavage susceptibility of PABP, whereas all other PABP-binding proteins tested do not. We show that PABP on cellular polysomes is cleaved only by 3C(pro) and that Paip2 does not sediment with polysomes. Also, viral polysomes contained only full-length PABP, however, cellular or viral ribosomes were equally susceptible to 3C(pro) cleavage in vitro. Finally, we determined that precursor 3CD and mature 3C(pro) have equivalent cleavage activity on purified PABP, but only 3C(pro) cleavage activity was stimulated by PABP-binding viral RNA. The results further elucidate complex mechanisms where multiple inherent PABP conformations and protein and RNA interactions both serve to differentially regulate PABP cleavage by 3CD, 3C(pro) and 2A(pro).

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    • "/ml heparin-Na by Dounce homogenization, and post nuclear lysates were collected by sedimentation at 5000 × g. The cell lysates were then subjected to 10–50% continuous sucrose gradient centrifugation at 43,000 rpm in a SW55 rotor at 4 °C for 60 min and the eluted fractions were continuously monitored by UV spectroscopy with an ISCO gradient fractionator as described (Rivera and Lloyd, 2008). For analysis of distribution of ubiquitin mRNAs in polysomes, pooled polysome sucrose gradient fractions were combined with 50 μg carrier yeast tRNA then RNA precipitated with three volumes ethanol overnight. "
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    • "CTD, separated by HIV-1 PR, could reduce the availability of translation factors such as eIF4B, Paip-1 or eRF3. Alternatively, lack of CTD could inhibit the oligomerization of PABP on poly(A) tail [3], [20]. HIV-1 PR also cleaves PABP within RRM3, rendering a product containing RRM1-2 [16]. "
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