Molecular analysis of the factorless internal ribosome entry site in Cricket Paralysis virus infection

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.

Translational Control in Virus-Infected Cells

“…Activation of the unfolded protein response and PERK by hepatitis C virus (HCV) and vesicular stomatitis virus (VSV), however, may also favor virus replication by accelerating type I IFN receptor degradation to attenuate IFN responses (Liu et al 2009). Finally, several RNA viruses including cricket paralysis virus (CrPV) and Sindbis virus (SINV) contain cis-elements that obviate the requirement for eIF2 altogether (Wilson et al 2000;Spahn et al 2004;Kerr et al 2016;Sanz et al 2017). Although in vitro studies suggested a mechanism in which eIF2A or eIF2D supply Met-tRNA i Met , recent work using knockout cell lines shows that these factors are dispensable (Sanz et al 2017;Gonzales-Almela et al 2018).…”

IRES-mediated ribosome repositioning directs translation of a +1 overlapping ORF that enhances viral infection

“…The pellet was resuspended in PBS and sterilized through a 0.2 μM filter. Viral titres and yield were determined as previously described (20). All viruses were sequence verified via RT-PCR with primers directed against the CrPV IGR IRES.…”

“…From here, in an elongation factor 2 dependent manner, the IGR IRES pseudo-translocates to the ribosomal P site, followed by aminoacyl-tRNA delivery to the A site and a second round of eEF2-dependent pseudo-translocation of the IGR IRES to the E site of the ribosome (17–19). Altogether, the IGR IRES acts as a complete RNA machine that supersedes initiation factors and commandeers the ribosome, a strategy that is essential for viral protein synthesis in dicistrovirus-infected cells (20).…”

UGA stop codon readthrough to translate intergenic region of Plautia stali intestine virus does not require RNA structures forming internal ribosomal entry site

“…The development of a cultivation system together with an infectious cDNA clone, which is currently unavailable in PSIV but has been reported in another dicistrovirus (Kerr et al 2016), will advance the virological understanding of PSIV readthrough. The roles of PSIV readthrough should be addressed via a multidisciplinary approach using virological, biochemical, bioinformatic, and any other methods available.…”