Molecular characterization of the duck enteritis virus US10 protein

Abstract

BackgroundThere is little information regarding the duck enteritis virus (DEV) US10 gene and its molecular characterization.MethodsDuck enteritis virus US10 was amplified and cloned into the recombinant vector pET32a(+). The recombinant US10 protein was expressed in Escherichia coli BL21 cells and used to immunize rabbits for the preparation of polyclonal antibodies. The harvested rabbit antiserum against DEV US10 was detected and analyzed by agar immunodiffusion. Using this antibody, western blotting and indirect immunofluorescence analysis were used to analyze the expression level and subcellular localization of US10 in infected cells at different time points. Quantitative reverse-transcription PCR (qRT-PCR) and pharmacological inhibition tests were used to ascertain the kinetic class of the US10 gene. A mass spectrometry-based strategy was used to identify US10 in purified DEV virions and quantify its abundance.ResultsThe recombinant pET32a(+)/US10 protein was expressed as inclusion bodies, purified by gradient urea washing, and used to prepare specific antibodies. The results of qRT-PCR, western blotting, and pharmacological inhibition tests revealed that US10 is mainly transcribed in the late stage of viral replication. However, the presence of the DNA polymerase inhibitor ganciclovir and the protein synthesis inhibitor cycloheximide blocked transcription. Therefore, US10 is a γ2 (true late) gene. Indirect immunofluorescence analysis showed that US10 proteins were initially diffusely distributed throughout the cytoplasm, but with the passage of time, they gradually relocated to a perinuclear region. The US10 protein was detected in purified DEV virions by mass spectrometry, but was not detected by western blotting, indicating that DEV US10 is a minor virion protein.ConclusionsThe DEV US10 gene is a γ2 gene and the US10 protein is localized in the perinuclear region. DEV US10 is a virion component.

Duck enteritis virus UL21 is a late gene encoding a protein that interacts with pUL16

“…In recent years, numerous methods have been developed to analyse the structural proteins of herpesviruses [35,36]; among these methods, mass spectrometry has emerged as the most widely used. Indeed, this method is very sensitive to low-abundance proteins, such as HSV-1 UL6 (12 copies of which exist in mature virus particles), and small virus proteins, such as HSV-1 US9 and UL11 (which are 90 and 96 aa long, respectively) [19,35]. According to emPAI, DEV pUL21 is a low-abundance virion component.…”

“…The band containing the virions was collected, diluted tenfold in TBS, and pelleted by ultracentrifugation (20, 000×g, 30 min, 4°C). The pellets were resuspended in TBS and stored at − 80°C [19].…”

“…Studies investigating its subcellular location have shown that pUL21 is distributed in both the cytoplasm and nucleus but mainly in the former [7,18]. Although the characteristics of many DEV genes have been reported [19,20], the molecular properties and functions of the DEV UL21 protein have not been described to date.…”

Duck enteritis virus UL21 is a late gene and encodes a protein that interacts with pUL16

“…(which are 90 and 96 aa long, respectively) (20,34). According to emPAI, DEV pUL21 is a lowabundance virion component.…”

“…Studies on its subcellular location have shown that pUL21 is distributed in both the cytoplasm and nucleus, but mainly in the former (7,19). Although the characteristics of many DEV genes have been reported (20)(21), the molecular properties and functions of the DEV UL21 protein have not yet been described.…”