Identificaion Of UL15 And UL41 Genes From Duck Enteritis Virus

Duck enteritis virus (DEV), also known as Anatid herpesvirus-1 (AnHV-1), is an important pathogen of birds from order Anseriformes, including ducks, geese and swans, causing the acute contagious disease duck viral enteritis (DVE), which results in substantial mortality and reduction of egg production in domestic as well as in wild waterfowl. The DEV genomic DNA sequence was not determined until most recently, most of which were determined merely on the basis of the sequence analysis with their counterparts from other herpesviruses. Exploring these protein encoding genes may facilitate our better understanding on the biology feature of the virus. Therefore, two DEV genes and/or their encoding proteins that are homologous to UL15 and UL41 of herpes simplex virus-1 (HSV-1) were then identified in this study.DEV UL15 consists of two exons with a 3.5 kilobases (kb) inron and transcribes into two transcripts: the full-length UL15 and an N-terminally truncated UL15.5. The 2.9 kb UL15 transcript encodes a protein of 739 amino acids with an approximate molecular mass of 82 kilo Daltons (kDa), whereas the UL15.5 transcript is 1.3 kb in length, containing a putative 888 base pairs (bp) ORF that encodes a 32-kDa polypeptides. Coding sequences of UL15 is highly conserved within the Herpesviridae, containing Walker A (261VPRRHGKT267) and Walker B (354LLFVDE359) motifs homologous to the catalytic subunit of the bacteriophage terminase as revealed by sequence analysis. Phylogenetic tree constructed with the amino acid sequences of 23 herpesvirus UL15 homologues suggests a close relationship of DEV to the Mardivirus genus within the Alphaherpesvirinae. It was also demonstrated that UL15 gene belonged to the late kinetic class as its expression was sensitive to cycloheximide (CHX) and phosphonoacetic acid (PAA). Further, the UL15 and UL15.5 proteins can be detected in the infected cell lysate but not in the sucrose density gradient-purified virion when reacting with the antiserum against UL15. Within the CEF cells, the UL15 and/or UL15.5 localize(s) in the cytoplasm at 6 h post infection (hpi) and mainly in the nucleus at 12 hpi and at 24 hpi, while accumulate(s) in the cytoplasm in the absence of any other viral protein.DEV UL41 encodes a virion host shutoff (VHS) homolog that destabilizes both host and viral mRNAs as demonstrated in its counterparts. Amino acid sequences searching by PSI-BLAST program suggested that DEV UL41 shares homology with proteins from nuclease family, in addition, the modeled 3 dimension structure of DEV UL41 matched well with those of T4 RNase H and Taq DNA polymerase with respect to the potential active regions. DEV is preferably classified as a member within Mardivirus genus according to the phylogenetic tree constructed with respect to UL41 amino acid sequences.To further characterize the encoding protein, UL41 ORF was cloned into the pMAL-c4x vector, expression of full-length soluble MBP-UL41 fusion protein and generation of anti-UL41 antiserum were achieved thereafter. The expressed MBP-UL41 fusion protein exhibited ribonuclease activity in absence of other cellular or viral proteins as revealed by in vitro VHS assay. Transiently expressed UL41 in CEF cells resulted in reduced Luc activity by cotransfection of UL41 and Luciferase gene expression plasmids, confirming the RNase activity of intracellular expressed UL41. UL41 is expressed as an early gene during DEV infection concerning that its expression is sensitive to CHX and insensitive to PAA.The UL41 protein can be detected in DEV-infected cell lysates as well as in sucrose density gradient-purified virion as indicated by Western blot assay. In DEV-infected cells, UL41 protein accumulated in small punctuate granules dispersed in the cytoplasm and prinuclear region at 6 hpi and 12 hpi, and localizes mainly in the cytoplasm and occasionally scattered in nucleus in transfected cells.Finally, individually mutated conserved residues of UL41 protein exhibited reduced or lose of VHS activity, D253 and D299 are the potential metal binding sites based on the mutagenesis assay and homology prediction.Taken together, the identification and characterization of the putative UL15 and UL41 genes and/or their encoding proteins in this study may provide data to aid the classification of DEV within the Herpesviridae family and provides clues for better understanding the possible role of the two proteins in DEV infection.