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Human Cytomegalovirus UL141 Protein Interacts with CELF5 and Affects Viral DNA Replication

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Human Cytomegalovirus UL141 Protein Interacts with CELF5 and Affects Viral DNA Replication MOLECULAR MEDICINE REPORTS 17: 4657-4664, 2018 Human cytomegalovirus UL141 protein interacts with CELF5 and affects viral DNA replication FEI ZOU1,2*, ZHI-TAO LU3*, SHUANG WANG1, SI WU1, YING-YING WU1 and ZHENG-RONG SUN1 1Department of BioBank, Affiliated Shengjing Hospital of China Medical University, Shenyang, Liaoning 110004; 2Department of Pediatrics, First Hospital of Jilin University, Changchun, Jilin 130021; 3Department of Pediatrics, Zhangjiagang First People's Hospital, Zhangjiagang, Jiangsu 215600, P.R. China Received June 14, 2017; Accepted January 5, 2018 DOI: 10.3892/mmr.2018.8419 Abstract. Human cytomegalovirus (HCMV) infection is the regulation of HCMV genomic DNA synthesis, which is a key primary viral cause of congenital abnormalities and mental step during HCMV infection leading to neurological disease. retardation in newborns. The HCMV UL141-encoded glyco- protein has been previously revealed to inhibit the cell-surface Introduction expression of cluster of differentiation (CD)155, CD122, tumor necrosis factor-related apoptosis-inducing ligand death Human cytomegalovirus (HCMV) is a ubiquitous herpes virus (TRAIL)-receptor 1 (R1) and TRAIL-receptor 2 (R2), thus with infection rate of ~50-80% in females of reproductive age protecting virally-infected cells by allowing them to escape from different regions of the world, including Australia, Canada, natural killer cell-mediated cytotoxicity. The present study United States, Sweden, Finland, Spain, United Kingdom and investigated the interaction between HCMV UL141 and Ghana (1). Following an HCMV infection, the primary clinical human fetal brain cDNA to elucidate the possible effects of manifestations in healthy people are asymptomatic reces- UL141 on the nervous system. The findings of the current sive or latent infections. When the immune system function study demonstrate that the HCMV UL141 protein directly is reduced, in the event of pregnancy, cancer, HIV infection, interacts with the human protein CUGBP Elav-like family organ transplantation and childbirth, HCMV is activated member 5 (CELF5) via yeast two-hybrid screening, this from its latent infection status and proliferates to a primary interaction was confirmed by glutathione S-transferase or secondary infection. It may also lead to serious illnesses, pull-down and co-immunoprecipitation assays. Additionally, including interstitial pneumonia, encephalitis, hepatitis, retinal the present study demonstrated that the UL141 protein failure and/or nervous systems, gastrointestinal diseases and co-localizes with CELF5 in the cytoplasm of 293 cells using stillbirth. Previous studies have reported that HCMV may fluorescence confocal microscopy. CELF5 overexpression also be associated with the occurrence of atherosclerosis and in a stably‑expressing cell line significantly increased viral malignant tumors (2,3). DNA copy number and titer in HCMV-infected U373MG Currently, the pathogenesis of HCMV infection remains to cells. However, reducing CELF5 expression via specific small be elucidated. Previous studies revealed that the viral genetic interfering RNAs did not affect viral DNA copy number or differences, which are associated with the virulence, the tropism titer in HCMV‑infected cells. The current findings suggest that to tissues, and the ability to escape the immune system, may be the interaction between UL141 and CELF5 may be involved the key factors determining whether HCMV infections lead to in modulating viral DNA synthesis and progeny production. fetal and organ deformities (4,5). Primary clinical isolates carry Therefore, CELF5 may represent a possible mechanism for at least 19 additional genes within the UL/b' genomic region (UL133-151 locus) that have been lost in several commonly-used HCMV strains, which have been passaged extensively in tissue culture (6). The UL141 gene within the UL/b' genomic region is highly conserved between HCMV isolates, encoding a protein containing a potential signal peptide, a hydrophobic transmem- Correspondence to: Mrs. Zheng-Rong Sun, Department of BioBank, Affiliated Shengjing Hospital of China Medical University, brane domain and three potential N-linked glycosylation sites. 36 Sanhao Street, Heping, Shenyang, Liaoning 110004, P.R. China The UL141-encoded protein is a glycoprotein doublet with E‑mail: sunzr@sj‑hospital.org molecular masses of 37-40 kDa that has been localized to the endoplasmic reticulum by immunofluorescence (7). HCMV *Contributed equally UL141 is also requited to inhibit cell surface expression of cluster of differentiation (CD)155, a ligand for the activating Key words: human cytomegalovirus, UL141 protein, CELF5, receptor CD226 (7). UL141 requires assistance from additional human fetus brain, cDNA library HCMV-encoded functions to suppress CD112 expression, also termed nectin cell adhesion molecule 2, by proteasome-medi- ated degradation (8). A previous study revealed that HCMV 4658 ZOU et al: HCMV pUL141 INTERACTS WITH CELF5 UL141 protein may bind tumor necrosis factor-related and expressed protein was termed GST-CELF5. GST-labeled apoptosis-inducing ligand death (TRAIL)-receptor 1 (R1) and CELF5 (GST-CELF5) was expressed in BL21 (DE3) cells TRAIL-receptor 2 (R2) to inhibit apoptosis of infected cells (9). (Tiangen Biotech Co., Ltd., Beijing, China) in logarithm These studies revealed UL141 may effectively participate in growth period transfected with pGEX-4T-2-CELF5 by electro- HCMV immune escape through multiple mechanisms (8,9). poration (Bio-Rad Laboratories, Inc.) according to the protocol When CD155 and TRAIL-R2 accumulate in the endoplasmic provided by the manufacturer and induced with isopropyl reticulum, CD112 is effectively downregulated (8). A previous β‑D‑1‑thiogalactopyranoside (IPTG; Clontech Laboratories, study indicated that HCMV UL141 requisitions US2 to target Inc., Mountainview, CA, USA). For the co-immunoprecipita- CD112 for proteasome degradation and modulates multiple tion (co-IP) assays, the sequences of UL141 and CELF5 were immune-associated pathways (10). It has an important effect on inserted into Myc- and HA-tagged pCMV vector (Clontech cellular processes, such as antigen presentation, natural killer Laboratories, Inc.) for expression in 293T cells. (NK) cell activity, cell migration and coagulation. In addition To generate pEGFP-UL141 and pDsRed-CELF5 constructs, to acting as an immune evasion gene, UL141 is an inhibitor of which were used to express fluorescently-tagged fusion HCMV proliferation in epithelial cell culture (7). A recent study proteins in human cells, the sequences of UL141 and CELF5 suggested that all known mutations in the UL/b' genomic region were amplified using the primers UL141 F, 5'‑CGG AAT TCC in epithelial cells involve loss or mutation of UL141 (11). The TGC CGC CGG GAG TCG CTC CG-3' and R, 5'-CGC GGA TCC interactions between UL141 and human proteins remain to be TCA CCT CTT CAT CTT TCT AA‑3'; CELF5 F, 5'‑CGG AAT fully elucidated. Therefore, the present study performed a yeast TCC CCT CAC ATT GCC CTT CAG-3' and R 5'-CGC GGA TCC two-hybrid screen to identify binary interactions that may yield ACC TTC TCG CTG CTG CTG-3', then inserted into pEGFP important information regarding the process of viral entry and and pDsRed vectors (Clontech Laboratories, Inc.), respec- replication. tively. The resultant constructs were confirmed by restriction In the present study, the human protein CUGBP Elav-like digestion profiles and sequencing (Shanghai Invitrogen family member 5 (CELF5) was identified as a directly‑interacting Biotechnology Co., Ltd., Shanghai, China). partner of HCMV-pUL141. Additionally, the viral DNA level was increased in HCMV-infected U373 cells overexpressing CELF5. Viruses, antibodies and cells. In this study, 10 patients (6 male, However, reduced expression of CELF5 had no significant effect 4 female) aged <5 months in the Affiliated Shengjing Hospital on HCMV DNA synthesis. These findings suggest that CELF5 of China Medical University who met the CMV infection has a key role in modulating viral DNA synthesis. The interactions diagnostic criteria (13) were enrolled between December 2006 identified between HCMV UL141 and the CELF5 human protein and March 2007. The urine of the patient was collected and may elucidate the molecular mechanisms underlying HCMV treated with an antibiotic cocktail containing 500 U/ml peni- diseases. cillin, 500 µg/ml streptomycin and 500 µg/ml kanamycin. Following centrifugation at 300 x g for 15 min, the supernatant Materials and methods was applied onto a human embryonic lung fibroblasts (HELFs) monolayer, and incubated at 37˚C overnight to allow virus Oligonucleotides and plasmid constructs. Oligonucleotide adsorption. Then the inoculum was removed and the cells primers used for the current study were purchased from washed with Hank's buffer. The inoculated HELFs were main- Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, tained at 37˚C in Modified Eagle's medium (Gibco; Thermo USA) and the recombinant plasmids used are presented Fisher Scientific, Inc.) supplemented with 2% fetal bovine in Table I. To generate pGBKT7-UL141 for the yeast two-hybrid serum (FBS), penicillin (100 U/ml)/streptomycin (100 µg/ml) screen, the coding sequence of HCMV UL141 was amplified and 2 mM l‑glutamine (Gibco; Thermo Fisher Scientific, Inc.). by polymerase chain reaction (PCR) (12) using HCMV H The culture medium was refreshed every 5 days and the cells strain DNA (Genbank no. GQ981646) as a template with the passaged when necessary. The cultures were observed
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