A negative elongation factor E inhibits white spot syndrome virus replication by suppressing promoter activity of the viral immediate early genes in red claw crayfish Cherax quadricarinatus
Introduction
White spot syndrome virus (WSSV) is the major viral pathogen of farmed shrimp, which causes huge economic losses to the global shrimp culture industry every year (Escobedo-Bonilla et al., 2008). WSSV is a rod-shaped virus with circular double-stranded DNA that belongs to the virus family Nimaviridae, genus Whispovirus (Yang et al., 2001). After delivery of the WSSV genome into host cell nucleus, the virus expresses immediate early (IE), early, and late genes to start viral replication (Liu et al., 2005, Marks et al., 2005). So far, some WSSV IE genes have been identified, including WSV051, WSV056, WSV069 (also known as IE1), WSV083 and WSV249 et al. (Li et al., 2009, Lin et al., 2011, Liu et al., 2005). The IE1 is one of most important IE genes for WSSV infection, which contains a transactivation domain and a zinc finger DNA-binding domain (Liu et al., 2008). In the studies related to WSSV transcription, it has been proved that WSSV IE genes can hijack host transcription factors to enhance the viral gene transcription. For example, WSSV annexes a shrimp signal transducer and activator of transcription (STAT) to enhance the expression of WSSV IE1 and WSV051 genes (Liu et al., 2007, Yao et al., 2016). The TATA box-binding protein (TBP) from Penaeus monodon interacts with IE1 to enhance the transcription of WSSV (Liu et al., 2011a, Liu et al., 2011b). Meanwhile, shrimp NF-kappaB binds to the IE1 promoter and upregulates its activity (Huang et al., 2010). In addition, activating transcription factor 4 (ATF4), x box binding protein 1 (XBP1), kruppel-like factor (KLF), c-Fos and c-Jun, Yin Yang 1 have also been shown to be transcription factors that activate WSSV transcription (Chang et al., 2012, Huang et al., 2017, Li et al., 2013, Li et al., 2015). So far, most studies have been focusing on the hijacking of host transcription factors by WSSV to enhance the viral transcription, but the molecular mechanism of host cells against transcription of WSSV genes is still largely unclear.
As we all know that the gene transcription of the host or virus follows a similar basic step: assembling the pre-initiation complex (PIC) for transcription, transcription initiation, transcription extension, and transcription end (Shandilya and Roberts, 2012, Zaborowska et al., 2016). Transcriptional elongation is a key link in gene expression of pathogenic viruses (Krauskopf et al., 1991, Ott et al., 2011, Yan et al., 2010). Particularly, the negative elongation factor (NELF) is a transcription factor composed of multi-functional complex containing subunits NELF-A, NELF-B, NELF-C/D, and NELF-E (Narita et al., 2007, Yamaguchi et al., 1999). NELF cooperates with DRB sensitivity-inducing factor (DSIF) to repress RNA polymerase II elongation and to repress the transcription elongation (Yamaguchi et al., 1999, Yamaguchi et al., 2002). As a protein subunit of the NELF complex, NELF-E amino acids consists of an N-terminal leucine zipper (LZ) motif, a central domain rich in Arg-Asp dipeptide repeats (RD motif), and a C-terminal RNA recognition motif (RRM) (Narita et al., 2003). The LZM mediates proteins dimerization and facilitates binding to DNA (Groves and Barford, 1999, Landschulz et al., 1988). While NELF-E RRM domain shows preference for RNA binding over DNA, and it is important for the transcriptional repression activity of NELF complex (Awwad et al., 2017, Yamaguchi et al., 2002). In addition, NELF-E has been shown to modulate the early transcription elongation by interacting with the newly transcribed RNA of RNA polymerase II (Pagano et al., 2014). In the relevant research of viral transcription, it has been reported that NELF represses Human immunodeficiency virus (HIV) transcription by pausing the RNA polymerase II complex (Zhang et al., 2007). And inhibition of NELF expression by RNA interference increased the cell susceptibility to sindbis virus (SINV) and vesicular stomatitis virus (VSV) infection in both Drosophila S2 cells and Kc167 cells accordingly (Xu et al., 2012). However, to our best knowledge, there was no relevant study on the regulation of WSSV gene expression by gene transcription elongation process in host cell so far.
In our present study, a novel NELF-E was identified from red claw crayfish C. quadricarinatus. We analyzed the molecular characteristics of CqNELF-E gene and its expression profiles post WSSV infection. The transcription and replication of WSSV was determined after gene silencing of CqNELF-E. Furthermore, the promoter activity of WSSV IE genes was also determined after overexpression of CqNELF-E by dual luciferase reporter gene assay in SF9 cells. Our data provide the first evidence that CqNELF-E may play an important role in the innate immune defense against the viral infection in a crustacean. This research also provides novel insights on the mechanisms of cellular antiviral response in crustaceans.
Section snippets
Animals, tissue collection
The healthy red claw crayfish C. quadricarinatus averaging 48 ± 5 g in body weight, was purchased from Source Sentai Agricultural Science and Technology Co., Ltd of Zhangzhou, Fujian Province, China. The animals acclimatized in the aerated freshwater tanks and the ambient temperature was 25 °C. Hemocytes were obtained with a sterile syringe and centrifuged for 10 min with 1000 g at 4 °C. The selected tissues, including hemocytes, stomach, gonad, muscle, nerve, intestine, heart, Hpt,
Gene cloning and sequence analysis of CqNELF-E
Previously, we found that an up-regulation of partial CqNELF-E cDNA sequence expression in a transcription library from Hpt cells upon WSSV infection (our unpublished data), indicating that CqNELF-E was involved in immune response against WSSV infection. However, the functional mechanism of CqNELF-E in WSSV infection was yet unknown which needed for further studies. In order to elucidate the role of CqNELF-E in host-WSSV interactions, the full-length cDNA sequence of CqNELF-E was determined by
Conclusion
In summary, a NELF-E gene was identified from a crustacean red claw crayfish C. quadricarinatus, and the transcription and replication of WSSV was significantly increased after gene silencing of CqNELF-E in crayfish Hpt cells. Importantly, the overexpression of CqNELF-E gene significantly inhibited the promoter activity of the WSSV IE genes. Therefore, these data will be helpful for the further study of the molecular mechanism between WSSV and transcription system of host cells, which will
Acknowledgements
This study was supported by the Natural Science Foundation of China (Nos. U1605214, 41676135) and Fundamental Research Funds for the Central Universities (No. 20720180123).
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Cited by (2)
Recent insights into anti-WSSV immunity in crayfish
2021, Developmental and Comparative ImmunologyCitation Excerpt :Interestingly, the replication of WSSV was observably increased in CqNELF-E silenced crayfish Hpt cells. Meanwhile, the overexpression of CqNELF-E gene significantly reduced the promoter activity of the WSSV IE genes WSV051, WSV069 and WSV083 (Gao et al., 2020). Considering that NELF was involved in the process of promoter-proximal pausing, especially binding to the nascent transcript when exited from the RNA polymerase II to repress the transcription elongation (Wu et al., 2003), it can be speculated that CqNELF-E might restrict RNA polymerase II activity to suppress the transcription elongation of WSSV genes during promoter-proximal pausing process, resulting in the weakening of promoter activity of viral IE genes.
- 1
Contribute equally to this work.