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

Highlights

• CqNELF-E is involved in antiviral immunity.

• CqNELF-E may inhibit the replication of WSSV.

• CqNELF-E functions in suppressing the promoter activity of WSSV IE genes.

• CqNELF-E function may provide new strategy against white spot disease.

Abstract

Invertebrates rely solely on the innate immune system to protect against virus infection, while the viral infection must rely on the transcriptional system of the host cell to achieve the expression of viral genes, which is naturally regulated by the host's transcriptional system. However, the mechanism of the host against viral transcription in host cells is still poorly understood in crustaceans. Previously, we found that the partial transcript sequence of a negative elongation factor E (named as CqNELF-E) was up-regulated in a differentially expressed transcriptome library of the haematopietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus upon white spot syndrome virus (WSSV) infection, suggesting a possible role of CqNELF-E in WSSV-host interaction. In the present study, we revealed the function of CqNELF-E. The full-length cDNA sequence of CqNELF-E was identified with 1726 bp from red claw crayfish, which contained an open reading frame of 816 bp, encoding 271 amino acids. Amino acid sequencing analysis revealed that the CqNELF-E had a conserved RNA recognition motif (RRM) and a leucine zipper motif (LZM). Tissue distribution analysis showed that CqNELF-E was widely expressed in various tissues with the highest expression in muscle, relatively abundant in Hpt and the lowest presence in heart. Interestingly, the gene expression of CqNELF-E was significantly up-regulated at both 6 and 12 hpi after WSSV infection in Hpt cell cultures in red claw crayfish. In addition, the expression of both the viral immediately early gene (IE) 1 (IE1) and a late gene envelope protein VP28 were significantly increased after gene silencing of CqNELF-E in Hpt cells, indicating the potential suppression role of CqNELF-E against the viral infection. Further study revealed that the CqNELF-E had an inhibitory effect on the promoter activity of WSSV IE genes WSV051, WSV069 (IE1) and WSV083 by a dual luciferase reporter gene assay. Taken together, these results suggest that CqNELF-E plays an antiviral role, probably via inhibition on the viral transcription activity in WSSV infection in a crustacean.

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.