Abstract
The dicistrovirus Cricket Paralysis virus contains a unique dicistronic RNA genome arrangement, encoding two main open reading frames that are driven by distinct internal ribosome entry sites (IRES). The intergenic region (IGR) IRES adopts an unusual structure that directly recruits the ribosome and drives translation of viral structural proteins in a factor-independent manner. While structural, biochemical, and biophysical approaches have provided mechanistic details into IGR IRES translation, these studies have been limited to in vitro systems and little is known about the behavior of these IRESs during infection. Here, we examined the role of previously characterized IGR IRES mutations on viral yield and translation in CrPV-infected Drosophila S2 cells. Using a recently generated infectious CrPV clone, introduction of a subset of mutations that are known to disrupt IRES activity failed to produce virus, demonstrating the physiological relevance of specific structural elements within the IRES for virus infection. However, a subset of mutations still led to virus production, thus revealing the key IRES-ribosome interactions for IGR IRES translation in infected cells, which highlights the importance of examining IRES activity in its physiological context. This is the first study to examine IGR IRES translation in its native context during virus infection.
Highlights
Canonical eukaryotic translation initiation is a highly orchestrated series of steps involving 40S recruitment to the 5′cap, scanning, 80S assembly and initiation at an AUG codon[1]
The conserved L1.1 loop of the intergenic region (IGR) internal ribosome entry sites (IRES) interacts with the L1 stalk of the 60S ribosomal subunit, which is reminiscent of interactions of the L1 stalk with an E-site deacylated tRNA12,30
To assess the relevance of IGR IRES translation in CrPV infection, we systematically introduced a panel of known mutations that affect specific properties of IGR IRES translation within the CrPV-3 infectious clone (Figs 1 and 2)
Summary
To assess the relevance of IGR IRES translation in CrPV infection, we systematically introduced a panel of known mutations that affect specific properties of IGR IRES translation within the CrPV-3 infectious clone (Figs 1 and 2). Structural studies indicate that SLIV and SLV interact with distinct regions of the 40S subunit, uS7 and eS25, biochemical data suggest that eS25 makes contacts with both loop regions of SLV and SLIV, which may explain how mutations in either stem-loop alone are not sufficient to abolish viral translation in infected cells, yet when combined can prevent viral protein synthesis and virus production (Fig. 4B; see mSLIV/mSLV-L)[14,28] Both SLV loops mutants, SLV-L1 and SLV-L2, yielded different results. As early as 4 hours post-infection there is a noticeable reduction in viral structural protein synthesis when a mutation is present either in the loop (mSLV-L1) or stem (mSLV-S) of SLV with the latter being more detrimental, while no reduction is observed with a SLIV mutation (Fig. 5) These data corroborated viral titres observed after RNA transfection (Fig. 4A) and altogether indicate that IGR IRES-dependent translation is an essential step in promoting virus infection. The use of the CrPV infectious clone provides a powerful biological framework for pinpointing the relevant IGR IRES mechanistic details in a physiological virus system
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