The polypyrimidine tract binding protein (PTB) requirement for internal initiation of translation of cardiovirus RNAs is conditional rather than absolute
Picornavirus RNAs are translated by an unusual mechanism of internal ribosome entry that requires a substantial segment of the viral 5′-untranslated region, generally known as the internal ribosome entry segment (IRES), and in some circumstances may require cellular trans-acting proteins, particularly polypyrimidine tract binding protein (PTB). It is shown here that for encephalomyocarditis virus (EMCV), the PTB dependence of IRES function in vitro is determined partly by the nature of the reporter cistron, and more especially by the size of an A-rich bulge in the IRES. With a wild-type EMCV IRES (which has a bulge of 6 As), translation is effectively independent of PTB provided the IRES is driving the synthesis of EMCV viral polyprotein. With an enlarged (7A) bulge and heterologous reporters, translation is highly dependent on PTB. Intermediate levels of PTB dependence are seen with a 7A bulge IRES driving viral polyprotein synthesis or a wild-type (6A) bulge IRES linked to a heterologous reporter. None of these parameters influenced the binding of PTB to the high-affinity site in the IRES. These results argue that PTB is not an essential and universal internal initiation factor, but, rather, that when it is required, its binding to the IRES helps to maintain the appropriate higher-order structure and to reverse distortions caused, for example, by an enlarged A-rich bulge.(Received December 4 1997)
(Revised January 20 1998)
(Accepted March 19 1998)
Key Words: cardiovirus; IRES; picornavirus; RNA affinity chromatography; RNA-binding proteins.
c1 Reprint requests to: Richard J. Jackson, Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB21GA, United Kingdom; e-mail: firstname.lastname@example.org.