The DNA Virus White Spot Syndrome Virus Uses an Internal Ribosome Entry Site for Translation of the Highly Expressed Nonstructural Protein ICP35

Abstract

Although shrimp white spot syndrome virus (WSSV) is a large double-stranded DNA virus (ϳ300 kbp), it expresses many polycistronic mRNAs that are likely to use internal ribosome entry site (IRES) elements for translation. A polycistronic mRNA encodes the gene of the highly expressed nonstructural protein ICP35, and here we use a dual-luciferase assay to demonstrate that this protein is translated cap independently by an IRES element located in the 5= untranslated region of icp35. A deletion analysis of this region showed that IRES activity was due to stem-loops VII and VIII. A promoterless assay, a reverse transcription-PCR together with quantitative real-time PCR analysis, and a stable stem-loop insertion upstream of the Renilla luciferase open reading frame were used, respectively, to rule out the possibility that cryptic promoter activity, abnormal splicing, or read-through was contributing to the IRES activity. In addition, a Northern blot analysis was used to confirm that only a single bicistronic mRNA was expressed. The importance of ICP35 to viral replication was demonstrated in a double-stranded RNA (dsRNA) interference knockdown experiment in which the mortality of the icp35 dsRNA group was significantly reduced. Tunicamycin was used to show that the ␣ subunit of eukaryotic initiation factor 2 is required for icp35 IRES activity. We also found that the intercalating drug quinacrine significantly inhibited icp35 IRES activity in vitro and reduced the mortality rate and viral copy number in WSSV-challenged shrimp. Lastly, in Sf9 insect cells, we found that knockdown of the gene for the Spodoptera frugiperda 40S ribosomal protein RPS10 decreased icp35 IRES-regulated firefly luciferase activity but had no effect on cap-dependent translation.

Transcriptome Analysis of the Spodoptera frugiperda AscovirusIn VivoProvides Insights into How Its Apoptosis Inhibitors and Caspase Promote Increased Synthesis of Viral Vesicles and Virion Progeny

“…Later, HSV was found to use the noncanonical internal ribosome entry site (IRES) strategy in translation (42). Although the recruitment of the ribosome by IRES secondary structures is known for many RNA viruses, it has only rarely been reported for DNA viruses, including herpes simplex virus 1, Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV), and white spot syndrome virus (WSSV) (42)(43)(44)(45). Therefore, given that ascoviruses destroy the nucleus prior to viral vesicle formation, and that eukaryotic cells favor the switch to an IRES mechanism during apoptosis (46,47), the ability of ascoviruses to use the IRES mechanism may be advantageous to ensure that replication and translation proceed efficiently.…”

Cell of Origin and Immunologic Events in the Pathogenesis of Breast Implant–Associated Anaplastic Large-Cell Lymphoma

“…The discovery of ribosomal protein S10 in BIA-ALCL samples suggests that viral etiology is another possibility because ribosomal protein S10 contributes to formation of the internal ribosome entry site by which viral transcripts gain entry to the ribosome. 17,18 Innate Immune Response Cellular and cytokine studies suggest that an inflammatory milieu may be a necessary component of the pathobiology of BIA-ALCL. 19e21 In response to bacteria or other yet undefined antigens, acute inflammation is initiated by innate immune cells (eg, mast cells, neutrophils, and macrophages).…”

Molecular Mechanisms of White Spot Syndrome Virus Infection and Perspectives on Treatments

“…Furthermore, only around 30% of the ORFs in the WSSV genome contain a polyadenylation signal. ORFs without polyadenylation signals are usually part of a cluster of ORFs with small intergenic regions and identical transcriptional orientation that can produce polycistronic mRNAs (e.g., the vp60b / wssv478 / wssv479 / vp28 cluster) [ 59 , 60 , 61 , 62 ]. Translation from these polycistronic mRNAs is likely to be facilitated by internal ribosome entry sites (IRES) [ 62 , 63 ].…”

“…ORFs without polyadenylation signals are usually part of a cluster of ORFs with small intergenic regions and identical transcriptional orientation that can produce polycistronic mRNAs (e.g., the vp60b / wssv478 / wssv479 / vp28 cluster) [ 59 , 60 , 61 , 62 ]. Translation from these polycistronic mRNAs is likely to be facilitated by internal ribosome entry sites (IRES) [ 62 , 63 ]. Several IRES have been identified thus far including in the 5′ UTR of wssv480 ( vp28 ) [ 64 ], in the coding regions of wssv396 (vp31) and wssv395 ( vp39b ) [ 63 ], and in the 5′ UTR of icp35 [ 62 ].…”

“…Translation from these polycistronic mRNAs is likely to be facilitated by internal ribosome entry sites (IRES) [ 62 , 63 ]. Several IRES have been identified thus far including in the 5′ UTR of wssv480 ( vp28 ) [ 64 ], in the coding regions of wssv396 (vp31) and wssv395 ( vp39b ) [ 63 ], and in the 5′ UTR of icp35 [ 62 ]. Translation initiation through IRES enables viral protein production even under unfavorable conditions such as during host response to viral infection, therefore making the virus more robust to host interference [ 63 , 65 ].…”