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.