Fang Li

Identification of three immediate-early genes of white spot syndrome virus.

Viral immediate-early (IE) genes generally encode regulatory proteins that are critical for viral replication. Their transcription, which is independent of de novo viral protein synthesis, is driven directly by host transcription factors. In this study, we examined promoter activities of 12 predicted regulatory genes of white spot syndrome virus (WSSV) belonging to the zinc finger protein family by EGFP-reporter assays in High Five cells. The results showed that the promoters of three genes (wsv056, wsv403 and wsv465) could drive reporter gene expression, and RT-PCR analysis revealed that their expression in WSSV-infected primary crayfish hemocytes was insensitive to the protein synthesis inhibitor cycloheximide (CHX). Therefore, they are IE genes of WSSV.

Analysis of white spot syndrome virus envelope protein complexome by two-dimensional blue native/SDS PAGE combined with mass spectrometry

White spot syndrome virus (WSSV) is a large enveloped virus, but the organization of its envelope proteins remains largely unknown. In the present study, we used blue native polyacrylamide gel electrophoresis (BN-PAGE) and SDS-PAGE in combination with mass spectrometry to analyze the envelope protein complexome of WSSV. Our results show that the viral envelope consists of multi-protein complexes (MPCs). Within them, the envelope protein VP19 exists as a homotrimer, while another major envelope protein, VP28, mainly exists as a homotetramer. The most notable feature is that the majority of MPCs include VP26 and VP24, suggesting that these two proteins might serve as hub proteins to recruit low-abundance proteins to MPCs and play crucial roles in the process of protein complex formation. Furthermore, we found significant evidence for interactions between several low-abundance proteins, such as VP52B/VP38/VP33 and VP12/VP150. The result of this study may promote the further research on WSSV envelope assembly.

Isolation and preliminary characterization of a new pathogenic iridovirus from redclaw crayfish Cherax quadricarinatus.

We report the preliminary characterization of a new iridovirus detected in diseased Cherax quadricarinatus collected from a farm in Fujian, China. Transmission electron microscopy identified numerous icosahedral particles (~150 nm in diameter) in the cytoplasm and budding from the plasma membrane of hematopoietic tissue cells. SDS-PAGE of virions semi-purified from the hemolymph of moribund C. quadricarinatus identified 24 proteins including a 50 kDa major capsid protein (MCP). By summing the sizes of DNA restriction endonuclease fragments, the viral genome was estimated to be ~150 kb in length. A 34 amino acid sequence deduced from a 103 bp MCP gene region amplified by PCR using degenerate primers targeted to MCP gene regions conserved among iridoviruses and chloriridoviruses was most similar (55% identity) to Sergestid iridovirus. Based on virion morphology, protein composition, DNA genome length, and MCP sequence relatedness, the virus identified has tentatively been named Cherax quadricarinatus iridovirus (CQIV). In addition, experimental infection of healthy C. quadricarinatus, Procambarus clarkii, and Litopenaeus vannamei with CQIV caused the same disease and high mortality, suggesting that CQIV poses a potential threat to cultured and wild crayfish and shrimp.

Identification of the interaction domains of white spot syndrome virus envelope proteins VP28 and VP24.

VP28 and VP24 are two major envelope proteins of white spot syndrome virus (WSSV). The direct interaction between VP28 and VP24 has been described in previous studies. In this study, we confirmed this interaction and mapped the interaction domains of VP28 and VP24 by constructing a series of deletion mutants. By co-immunoprecipitation, two VP28-binding domains of VP24 were located at amino acid residues 46-61 and 148-160, while VP24-binding domain of VP28 was located at amino acid residues 31-45. These binding domains were further corroborated by peptide blocking assay, in which synthetic peptides spanning the binding domains were able to inhibit VP28-VP24 interaction, whereas same-size control peptides from non-binging regions did not.

Characterization of white spot syndrome virus immediate-early gene promoters

Twenty-one immediate-early (IE) genes of white spot syndrome virus (WSSV) have been identified so far. However, the transcriptional regulation of WSSV IE genes remains largely unknown. In this report, the 5 flanking regions of 18 WSSV IE genes were cloned and eight functional promoter regions were identified. WSSV IE gene promoters normally contained a TATA box approximately 30 bp upstream of the transcriptional initiation site. Also, the cyclic AMP response element (CRE; TGACGTCA) was frequently found within the WSSV IE promoter regions. Mutations of the CREs of WSSV IE promoters P403 and P465 reduced their activity significantly, suggesting that these elements have a role in WSSV IE gene transcription. Our findings provide a more global view of WSSV IE gene promoters and will facilitate the in-depth investigation of viral gene transcriptional regulation.

Effects of high salinity, high temperature and pH on capsid structure of white spot syndrome virus

The structural stability of white spot syndrome virus (WSSV) capsids at high salinity, high temperature and various pH values was studied. To obtain the viral capsids, the nucleocapsids were treated with high salinity. The results showed that high salinity treatment can cause the dissociation of VP15 and most of VP95 from the nucleocapsid, but there were no noticeable alterations in morphology and ultrastructure of the nucleocapsid and capsid. The capsids retained morphological integrity at temperatures <45°C but became aberrant at >60°C. In addition, the capsids were relatively resistant to strong acid conditions and were tolerant to a broad pH range of 1 to 10. However, morphological change occurred at pH 10.5. The capsids broke up into small pieces at pH 11 and completely degraded in 0.1 and 1.0 M NaOH. These results indicated that the WSSV capsid is acid-stable and alkali-labile.

Characterization of white spot syndrome virus VP52B and its interaction with VP26

White spot syndrome virus (WSSV) is one of the major pathogens of cultured shrimp. Identification of envelope protein interactions has become a central issue for the understanding of WSSV assembly. In this paper, WSSV envelope protein VP52B was fused with GST-tag and expressed in Escherichia coli BL-21(DE3). Immunogold-electron microscopy revealed that VP52B was located on the outside surface of WSSV virions. Far-Western blotting analysis suggested that VP52B might directly interact with a major viral envelope protein VP26, and their interaction was confirmed by GST pull-down assay. Further investigation showed that the VP52B binding domain was located between residues 135–170 of VP26. These findings will enhance our understanding of the molecular mechanisms of WSSV morphogenesis.

Low-abundance envelope protein VP12 of white spot syndrome virus interacts with envelope protein VP150 and capsid protein VP51

VP12 and VP150 are two minor envelope proteins of white spot syndrome virus (WSSV). In our previous studies, VP12 was found to co-migrate with 53-kDa form of VP150 on two-dimensional Blue Native/SDS-PAGE, suggesting that there is an interaction between them. In this study, we confirmed the interaction by co-immunoprecipitation assay and demonstrated that the binding region with VP12 is located between residues 207 and 803 of VP150. Further studies found that VP12 can be attached to WSSV capsids by interacting with capsid protein VP51. These findings suggest that VP12 may function as a linker protein participating in the linkage between VP12/VP150 complex and viral nucleocapsid.

Characterization of the promoter of white spot syndrome virus immediate-early gene wsv249

White spot syndrome virus immediate early (IE) gene wsv249 encodes an E3 ubiquitin ligase that can interact with a shrimp ubiquitin-conjugating enzyme to mediate ubiquitination. In this study, to understand the transcriptional regulation of wsv249, a serial of 5-truncated mutations were made on its promoter and the activities of mutated promoters was analyzed. Four 25u202fbp regions potentially containing either positive or negative regulatory elements were identified. Notably, the deletion of -275/-250, which abolished a cAMP-response element (CRE), greatly reduced the promoter activity by 84.2%. CRE serves as the binding site for proteins belong to the cAMP responsive element-binding proteins (CREBs) family and the activator protein 1 (AP-1) family. Electrophoretic mobility shift assay (EMSA) showed that Lvc-Jun could directly bind to the CRE element in the promoter region of wsv249. In addition, the regulation of shrimp homolog of c-Jun and CREB on wsv249 promoter was further investigated. We found that Lvc-Jun greatly upregulated the activity of wsv249 promoter by ∼12.4 fold, and the CRE at -212/-205 but not the one at -256/-249 was essential for the regulation. In contrast, LvCREB-3 could not activate wsv249 promoter activity. These findings extend our knowledge of the transcriptional regulation of WSSV IE genes.