Background
Duck plague (or duck viral enteritis) caused by the duck enteritis virus (DEV) is an acute, febrile, septic disease of waterfowl (ducks, geese, and swans of all ages and species) that leads to sizeable economic losses worldwide in the avian industry due to the associated high mortality rate and decreased duck egg production [
1‐
5]. DEV belongs to the Alphaherpesvirinae subfamily, with a genome consisting of linear, double-stranded DNA comprising a unique long (UL) region, a unique short (US) region, a unique short internal repeat (IRS) region, and a unique short terminal repeat (TRS) region. The genomic arrangement pattern is UL-IRS-US-TRS [
6].
The UL41 gene is highly conserved in alphaherpesviruses [
7,
8], and UL41-encoded proteins have been described in other Alphaherpesvirinae subfamily members, including bovine herpesvirus 1 (BHV-1) [
9,
10], monkey B virus (macacine herpesvirus 1; BV) [
11], equine herpesvirus-1 (EHV-1) [
12], pseudorabies virus (PRV) [
7], and varicella-zoster virus (VZV) [
13,
14]. The HSV-1 virion host shutoff (VHS) protein, a late (γ2) tegument protein, is encoded by the UL41 gene and is packaged into the virion; essential for viral infection [
15‐
19], VHS is released into the host cell cytoplasm, selectively degrading cellular mRNAs via its RNase activity and contributing to the shutoff of host cell protein synthesis early in infection [
20,
21]. In addition, VHS-RNase activity is tightly modulated by several HSV proteins. Early in the infection, VHS-RNase first binds tegument proteins VP13/14 and ICP27, encoded by UL47 and UL54 genes, respectively, and selective degradation of mRNAs by the VHS-RNase is regulated by the VP13/14. Then, late in the infection, VHS-RNase activity is neutralized by VP16 and VP22, encoded by UL48 and UL49 genes, respectively [
21].VHS functions in BHV-1 [
9], BV [
10] and EHV-1 [
12] may serve the same purpose in the mechanisms of these viruses. The PRV VHS protein also exhibits RNase activity, but it is not identical to that of HSV-1 VHS [
7,
22]. VZV open reading frame 17 (ORF17) is a late nonstructural protein with a delayed cellular RNA shutoff and a homologue of the VHS protein but has no major function in VZV-mediated delayed host shutoff [
13,
14].
The characteristics of some DEV genes have been reported [
23‐
35]; however, information regarding the DEV UL41 gene is limited. Bioinformatic analyses have revealed that the full-length sequence of the DEV UL41 ORF is 1494 bp and encodes a 497 amino acid protein with a molecular mass of 56 kDa and shares 31% homology with the HSV-1 VHS protein. In this study, we characterized the DEV UL41-encoded protein (pUL41) using western blot, real-time quantitative reverse-transcription PCR (RT-qPCR), indirect immunofluorescence assays (IFAs), a pharmaceutical test and mass spectrometry. Our results will provide guidance for further study of DEV UL41.
Methods
Viruses, cells and vectors
The DEV CHV strain (Gene bank: JQ647509.1) was isolated and preserved in our laboratory [
22].
Duck embryo fibroblasts (DEF) were propagated in minimal essential medium (MEM; Gibco, Meridian Road Rockford, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, Meridian Road Rockford, USA) at 37 °C with 5% CO2. For viral infections, maintenance medium supplemented with 2% FBS was added. Commonly used reagents were prepared in our laboratory.
The recombinant β-actin plasmid [
32] and rabbit anti-UL47 protein polyclonal antibody were prepared in our laboratory.
Construction of the recombinant expression vector
All primers were designed by Primer Premier 5 software (Table
1). The wild-type DEV UL41 (GenBank: AFC61841.1) coding region was constructed in the pET-32a(+) vector (Novagen, Podenzano, Italy) using specific primers (P
1 and P
2) to create pET-32a(+)-UL41, and in the eukaryotic plasmid pEGFP-C2 using specific primers (P
3 and P
4) to create pEGFP-C2-UL41. The wild type DEV UL47 (GenBank:
AFC61835.1) coding region was sub-cloned into pcDNA3.1(+) using specific primers (P
5 and P
6), creating pcDNA3.1(+)-UL47. Next, we constructed the eukaryotic plasmids pcDNA3.1(+)-UL47
Δ40–50 and pcDNA3.1(+)-UL47
Δ768–777, which carry deletions from 40 to 50 and 768 to 777 amino acids (aa) of the DEV UL47-encoded protein (pUL47), respectively.
Table 1
Sequences and primer pair characteristics
P1 | G/AATTCATGGGGCTGTATGGTTGTATAAGC | DEV UL41 | 1526 |
P2 | C/TCGAGTCTTACAACAGTTAATCTTAGTCCCAAT |
P3 | G/AATTCGCCACCATGGGGCTGTATGGTTGTATAAGC | DEV UL41 | 1532 |
P4 | G/GTACCTCTTACAACAGTTAATCTTAGTCCCAAT |
P5 | CGCG/GATCCGCCACCATGGATAAATCACGAAGACAGCG | DEV UL47 | 2391 |
P6 | CCGC/TCGAGTTAATGTAACTCTCTCCGCCCAG |
P7 | TGATTTACACCGCTACCCTA | DEV UL41(RT-PCR) | 107 |
P8 | TCTCACTTCTTTCAGCCATT |
P9 | CCGGGCATCGCTGACA | Duck β-actin(RT-PCR) | 177 |
P10 | GGATTCATCATACTCCTGCTTGCT |
P11 | GAACAACCGCCGAACAC | DEV UL54 | 127 |
P12 | TCAAACATCCGCCTCAA |
P13 | GCCACCAACCCTACCAAG | DEV UL13 | 131 |
P14 | GTCGTCAGCCCATCACCA |
P15 | AGACGGTTCCGAAAGTACAG | DEV Us2 | 111 |
P16 | TCGGCAGCACCAATAATCC |
Prokaryotic expression of UL41-his fusion proteins in E. coli
pET-32a(+)-UL41 was transformed into
E. coli BL21 (DE3)-competent cells. Bacterial cultures were kept at 37 °C in Luria-Bertani (LB) medium containing 50μg/ml ampicillin. Protein expression was induced by adding IPTG (St Louis, MO, USA). Different induction concentration effects (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mM), induction times (2, 4, 6, 8 and 10 h) and induction temperatures (25, 30 and 37 °C) were examined to optimize conditions to obtain the highest level of UL41 protein expression [
33]. After IPTG induction, bacteria were collected at different time points by centrifugation at 4 °C and disrupted by ultrasonication. The vector control culture was analyzed in parallel. All expression levels were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 12% resolving gels and 5% stacking gels.
Purification of the fusion protein and preparation of the polyclonal antibody
The UL41 protein was purified by gel and electric elution, and the purified protein was used to generate a polyclonal antibody in mice. Approximately 1.1 mg of purified protein mixed with an equal volume of QuickAntibody-Mouse3W adjuvant (Biodragon, Beijing, China) was used to immunize eleven mice by intramuscular injection. Then, the mice were exsanguinated by eyeball removal 1 week after the last immunization, and antisera were harvested by centrifugation (9600×g, 20 min, 4 °C).
Western blotting
As previously described [
29], DEV-infected DEF cells in 6-well dishes were collected at 7, 24, 36, 48, 60, 72 h post-infection (hpi), removing the supernatant. Proteins that were resolved by 12% SDS-PAGE were further transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked for 2 h in 5% skim milk, then incubated with primary antibody and probed with HRP-conjugated secondary antibody (Bio-rad Lab, CA, USA) for 1 h at 37 °C. All antibodies were diluted in 1% skim milk. After several washes with PBST to remove unbound antibodies, the signal was detected using Western BLoT Chemiluminescence HRP Substrate (Takara, Dalian, China) according to the manufacturer’s instructions.
Indirect immunofluorescence assay (IFA)
As previously described [
35], DEV-infected DEF cells in 6-well dishes were collected on coverslips at 0, 7, 12, 24, 36, 48, 60, 72 hpi, fixed with 4% paraformaldehyde in PBS for 30 min, permeabilized with 0.25% Triton X-100 in PBS for 30 min at 4 °C and blocked for 1 h with 5% BSA PBS at 37 °C. Additionally, cells were incubated for 1 h with primary antibodies, then secondary antibodies (Thermo Fisher Scientific, Meridian Road Rockford, USA). All antibodies were diluted in 1% BSA PBS. Finally, the cell nuclei were visualized with DAPI (Roche, Mannheim, Germany)for 15 min at room temperature. Coverslips were sealed on glass slides with glycerin buffer, and the cells were examined using a confocal microscope (Nikon A1, Japan).
RT-qPCR
Total RNA from the DEV-infected DEF cells at different time points (0, 2, 6, 12, 24, 36, 42, 45, 48, and 54 hpi) was extracted using TRIzol reagent l (Invitrogen, CA, USA) according to the manufacturer’s recommendations. Extracted RNA integrity and purity were evaluated by 1% agarose gel electrophoresis and by optical density measurements (OD260/OD280 ratio) using a NanoDrop spectrophotometer, respectively. Subsequently, the RNA was reverse transcribed into cDNA using the PrimeScript®RT reagent kit with the gDNAeraser (Takara, Beijing, China). The UL41 (P
7 and P
8) and β-actin (P
9 and P
10) [
35] genes were detected using the previously described primers (Table
1). The RT-qPCR conditions were set as follows: initial denaturation at 95 °C for 1 min, followed by 45 cycles of denaturation at 95 °C for 5 s, annealing at 59 °C for 20 s, and extension at 72 °C for 25 s. All reactions were performed in triplicate and in at least two independent experiments. The cycle number at threshold (Ct value) was determined to analyze UL41 and β-actin gene transcription, and the results were calculated using the 2
-ΔΔCT method [
25].
Pharmaceutical identification virion gene type
DEV-infected DEF cells cultured in 6-well dishes were incubated with 300 μg/ml acyclovir (Glaxo SmithKline) or 50 μg/ml cycloheximide (Meilunbio, Dalian, China) for 2 h. Total RNA was extracted at 24 hpi with TRIzol and subsequently reverse transcribed into cDNA. PCR was conducted using primers for UL41 (P
1 and P
2) and β-actin (P
9 and P
10) and confirmed by 1% agarose gel. The immediate-early (IE) gene UL54 (P
11 and P
12) [
35], early (E) gene UL13 (P
13 and P
14) [
36], and late (L) gene Us2 (P
15 and P
16) [
37] were detected as experimental controls.
Virion purification
DEV-infected DEF cells were collected, and cellular debris was removed by low-speed centrifugation (2000×g, 30 min, 4 °C). Extracellular DEV virions were harvested by ultracentrifugation (40,000×g, 2 h, 4 °C) in a Beckman Ti70 rotor through a 30% (wt/vol) sucrose cushion. Viral bands were collected by isopycnic gradient ultracentrifugation in a continuous 30 to 60% (wt/vol) potassium tartrate gradient in TBS (40,000×g, 2 h, 4 °C) in a Beckman SW60 rotor, diluted ten-fold in TBS and then pelleted by ultracentrifugation (20,000×g, 60 min, 4 °C). The pellet was finally resuspended in TBS and stored at − 80 °C.
Mass spectrometry
Purified virion samples were analyzed by 12% SDS-PAGE. The gel was stained with Coomassie brilliant blue (Sigma) and sent to Sangon Biotech Company (Shanghai, China) for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis [
38]. All data were also searched against the NCBI
Bos taurus database. Only sequences identified with a Mascot score greater than 30 were considered. Proteins identified by at least one unique peptide were accepted. And the exponentially modified protein abundance index (emPAI) was used to estimate protein relative abundance for the complete virion extracts [
39‐
41].
Discussion
Until now, information regarding the DEV UL41 gene has been limited. Jinxiong Liu and colleagues [
43] constructed a recombinant virus, rDEV-ul41HA, to serve as a bivalent live-attenuated vaccine against both DEV and H5N1 infections in ducks, using the UL41 gene of the DEV genome as the insertion site. UL41 in other herpesvirus genomes was identified as a region nonessential for viral replication [
44]; however, the results indicated that UL41 was not an ideal site for foreign gene insertion. We further explored the function of the DEV pUL41 as the first step in studying the properties and characteristics of the DEV UL41 protein, and the salient features of the results presented here were as follows.
The herpesvirus cycle is characterized by two distinct phases: latency and the productive or lytic cycle. During the productive cycle, herpesviruses exhibit strictly regulated temporal cascades of gene expression that are divided into three stages: IE, E, and L. IE genes are expressed first and encode nuclear regulatory proteins that act at the transcriptional and post-transcriptional levels to stimulate viral E and L gene expression. Second, E genes participate in viral DNA replicative machinery. Viral DNA replication then augments expression of L genes that encode most of the structural components of the viral particles. L genes are subdivided into two categories as leaky late (γ1) or strict late (γ2) based on their apparent DNA replication requirements. The expression of γ1 genes is delayed compared to that of early genes, whereas γ2 gene strictly requires the onset or completion of lytic DNA amplification [
45,
46]. The HSV-1 VHS protein is a γ2 tegument protein that contributes to the degradation of cellular messenger RNAs (mRNAs) and an overall decrease in host protein synthesis [
20,
21]. Additionally, VZV ORF17 is also expressed as an L protein that shuts off cellular RNA during the viral replication cycle [
14]. The UL41 gene of the infectious laryngotracheitis virus (ILTV) is classified as an E/L gene because it has both E and L properties [
47].
To identify the DEV UL41 gene type, we studied its transcription and expression kinetics at different time points post-infection using RT-qPCR and western blot analysis, respectively. Based on previous studies [
32,
37,
48,
49], we suggested that accumulation of the DEV pUL41 may occur during the late stage of infection. IE genes are expressed in the presence of CHX, a protein synthesis inhibitor, while transcription of E and L genes is suppressed in the presence of CHX [
50]. To investigate this further, we treated the infected cells with a DNA polymerase inhibitor, ACV, and a protein synthesis inhibitor, CHX, and observed that the UL41 gene was inhibited in both the ACV and CHX groups. Thus, the DEV UL41 gene was a γ2 gene and strictly dependent on the onset or completion of lytic DNA amplification. Mass spectrometry is an efficient means to identify protein composition of complex samples and has been performed in the analysis of various herpesviruses [
39,
40,
51‐
53]. Based on the emPAI,DEV pUL41 is a low-abundance virion component, which means that DEV pUL41 may share identical characteristics with the HSV-1 VHS protein, which is essential for viral infection.
Intracellular viral protein localization is associated with viral protein function, but intracellular DEV pUL41 localization is undocumented. We analyzed DEV pUL41 intracellular localization by IFA using the mouse anti-UL41 protein polyclonal antibody. The results showed that DEV UL41 protein-specific fluorescence was distributed throughout the cells and that the signal was mainly present in the cytoplasm. According to previous studies [
19,
45], the HSV-1 VHS protein is delivered to the cytoplasm of newly infected cells to degrade cellular mRNAs. This degradation process contributes to shutting off host protein synthesis, redirecting the cellular machinery from host to viral gene expression, and disrupting pre-existing polyribosomes. These processes comprise the major early mechanism through which the virus blocks the host’s response to infection. Studies by other investigators [
21] have shown that the wild-type HSV-1 VHS protein localizes initially to the nucleus and then is translocated to the cytoplasm. The protein, which can shuttle between the nucleus and the cytoplasm, contains a putative nuclear export signal (NES), but no obvious NLS has been identified. Another late tegument HSV-1 protein, pUL47 (or VP13/14), which binds RNA and shuttles between the cytoplasm and the nucleus [
54], can regulate subcellular localization of some viral and cellular proteins that interact with it, may be involved in translocating VHS to the nucleus [
55‐
57]. VHS-RNase and VP13/14 must assemble with newly synthesized and processed mRNA in the nucleus prior to VHS-RNase/VP13/14 translocation to the cytoplasm, where VHS-RNase then degrades the mRNA. Early in the infection, VHS function is regulated by VP13/14, which attenuates degradation of all kinetic classes of viral mRNAs and stable host mRNAs in the infected cells with no apparent effect on the stability of AU-rich mRNAs [
42]. Sequence prediction reveals that DEV UL41 contains NES motifs but no NLS motifs. Based on these reports, we also found that DEV pUL41 could not localize autonomously to the nucleus and that DEV pUL47 translocated pUL41 into the nucleus in the absence of other viral proteins; these processes were dependent on predicted pUL47 NLS signals.
From these data, we concluded that DEV UL41 is a γ2 gene that encodes a structural protein similar to HSV-1 VHS. DEV pUL41 may be essential for viral infection, functioning in the cytoplasm of infected cells and interacting with pUL47 to affect viral and host genes, but additional studies are required to confirm this.