Astrocytoma U373MG cells were provided by Professor Songya Lv from State Key Laboratory of Virology, College of Life Sciences, Wuhan University (Wuhan, China). Human embryonic lung fibroblast MRC-5 cells (Shanghai Institute of Biochemistry and Cell Biology, Shanghai, China), human embryonic kidney HEK293 cells (Shanghai Institute of Cell Biology and Biochemistry) and the astrocytoma U373MG cells were cultured in Dulbecco's modified Eagle's medium (DMEM; GE Healthcare Life Sciences, Logan, UT, USA), which was mixed with 10% fetal bovine serum (GE Healthcare Life Sciences) at 37°C and 5% CO₂. A low-passage clinical isolate Han strain, which was isolated from a urine sample from a congenitally HCMV-infected infant at the Department of Pediatrics and maintained in the Virus Laboratory of The Affiliated Shengjing Hospital, China Medical University (Shenyang, China) was propagated in MRC-5 cells.

The RL13 gene coding sequence was amplified by polymerase chain reaction using HCMV Han strain DNA (Genbank no. KJ426589.1; www.ncbi.nlm.nih.gov) as a template, and the following primers, obtained from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA): Forward 5′-CCGGGCCATGGAGGCCAATAACACGTGCTCC-3′ and reverse 5′-CGCGGATCCGGTACCTTAGGTTTTAGTCCA-3′. The PCR amplification was performed in a volume of 50 µl containing 3.5 µl DNA, 1 µl each primer, 5 µl 10X TaqBuffer (with MgCl₂), 2.5 µl dNTP mix (10 Mm), 1 unit Taq DNA polymerase (1 U/µl; Takara Biotechnology Co., Ltd., Dalian, China) and water up to 50 µl. The reaction was performed in a Bio-Rad MyCycler Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and the reaction conditions were as follows: 95°C for 5 min; 30 cycles of 95°C for 45 sec, 50°C for 45 sec and 72°C for 1 min; followed by a final elongation at 72°C for 10 min. The PCR products were electrophoresed in 1.2% agarose gel containing ethidium bromide (Sigma-Aldrich, St. Louis, MO, USA) for visualization. The PCR products and the plasmids of pGBKT7 vector (Clontech Laboratories, Inc., Mountain View, CA, USA) were purified by wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA) in accordance with the manufacturer's protocol. To construct the pGBKT7-RL13 plasmids, enzyme digestion was performed by BamHI (Takara Biotechnology Co., Ltd.) at 30°C for 2 h and SfiI (Takara Biotechnology Co., Ltd.) at 50°C for 1 h. Following digestion, the products were purified by wizard Genomic DNA Purification Kit (Promega Corporation). Subsequently, the RL13 sequence was inserted into the pGBKT7 vector (Clontech Laboratories, Inc) between the SfiI and BamHI sites downstream of the c-Myc coding sequence by T4 DNA ligase (Takara Biotechnology Co., Ltd.) and T4 DNA ligase buffer (Takara Biotechnology Co., Ltd.) at 16°C overnight, resulting in pGBKT7-RL13 plasmids. The inserted sequence of pGBKT7-RL13 was confirmed by sequencing (Invitrogen; Thermo Fisher Scientific, Inc.). Yeast two-hybrid experiments were performed in accordance with the manufacturer's protocol (Matchmaker GAL4 Two-Hybrid System 3; Clontech Laboratories, Inc.). The human fetus brain cDNA library was provided by Professor Gengfu Xiao from the State Key Laboratory of Virology, College of Life Sciences, Wuhan University, which was cloned into pACT2 (Clontech Laboratories, Inc.), and the pGBKT7-RL13 were co-transformed into the Saccharomyces cerevisiae strain, AH109 (Clontech Laboratories, Inc.) by electroporation (Bio-Rad Laboratories, Inc.) according to the protocol provided by the manufacturer. Positive colonies were screened on synthetic dropout medium (Clontech Laboratories, Inc.) in the absence of tryptophan, leucine, adenine and histidine, and were detected using a chromogenic reaction of x-α-Gal (Clontech Laboratories, Inc.) for α-galactosidase activity (13). The plasmids containing sequences encoding the interaction candidates of RL13, termed pACT2-cDNA, were extracted and rescued via electrotransformation into competent Escherichia coli DH5α (Clontech Laboratories, Inc.). Each pACT2-cDNA plasmid was co-transformed into yeast with either the pGBKT7-RL13 vector or the pGBKT7 empty vector. The transformed cells were selected on QDO plates and underwent assessment for β-galactosidase activity. Human gene sequences from blue yeast colonies were sequenced (Invitrogen; Thermo Fisher Scientific, Inc.), and analyzed using the BLAST database (http://www.ncbi.nlm.gov/blast).

The coding sequence of NUDT14, one of the candidate proteins interacting with RL13 protein, was obtained from a NUDT14-containing cDNA clone (pACT2-NUDT14), and was GST-tagged by cloning into the pGEX-4T-2 vector (Clontech Laboratories, Inc.) between the EcoRI and XhoI sites, yielding pGEX-4T2-NUDT14.

A GST-pull-down experiment was performed, according to the manufacturer's protocol (MagneGST™ Pull-Down System; Promega Corporation). The c-Myc-labeled RL13 (c-Myc-RL13) was expressed from pGBKT7-RL13 in a quick-coupled transcription/translation reaction (TNT) T7 Quick Reaction (14). GST-labeled NUDT14 (GST-NUDT14) was expressed in BL21 (DE3) cells (Tiangen Biotech Co., Ltd., Beijing, China) transfected with pGEX-4T2-NUDT14 and induced with isopropyl β-D-1-thiogalactopyranoside (IPTG; Clontech Laboratories, Inc.). Subsequently, the MagneGST particles were incubated with the reaction products for 30 min at room temperature. As a bait protein, 20 µl of GST-NUDT14 was incubated with 80 µl c-Myc-RL13 for 1.5 h on a rotating platform at room temperature. Following washing with a GST binding/wash buffer (MagneGST™ Pull-Down System) three times, the bound proteins on the MagneGST particles were eluted using elution buffer and solubilized in 2X SDS sample buffer (Beyotime Institute of Biotechnology, Haimen, China). The protein concentration was measured by ultraviolet spectrophotometry as previously described (15). The binding proteins (20 mg) were loaded in each lane of 12% polyacrylamide gels (SDS-PAGE; Sigma-Aldrich) and the proteins were separated by gel electrophoresis at a constant voltage of 100 V. The separated proteins were transferred electrically onto nitrocellulose membranes (Sigma-Aldrich), and then blocked with 5% bovine serum albumin (BSA; Thermo Fisher Scientific, Inc.) for 2 h. Western blot analyses were performed using monoclonal mouse anti-human antibodies against c-Myc (1:1,000; Thermo Fisher Scientific, Inc.; cat. no. AHO0052) or polyclonal goat anti-human antibodies against GST monoclonal antibodies (1:1,000; Thermo Fisher Scientific, Inc.; cat. no. PA5-18394) incubated overnight at 4°C and corresponding goat anti-mouse peroxidase-conjugated secondary antibodies (1:2,000; Beyotime Institute of Biotechnology; cat. no. A0216) and rabbit anti-goat peroxidase-conjugated immunoglobulin G (IgG) secondary antibodies (1:2,000; Absin Bioscience, Inc., Shanghai, China; cat. no. abs20005) incubated for 2 h at room temperature. The membranes were then reacted with the chemiluminescent substrate, which was provided in the Western Chemiluminescent Substrate kit (Pierce; Thermo Fisher Scientific, Inc.). Signals were determined using a Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories, Inc.). In the GST-pull-down assay, parallel experiments were performed in controls containing GST- or myc-tagged protein.

The coding sequence of RL13 in pGBKT7-RL13 was obtained and inserted into the pCMV-myc (Clontech Laboratories, Inc.) between the SfiI and NotI sites, and designated as pCMV-myc-RL13. The coding sequence of NUDT14 in pGEX-4T2-NUDT14 was obtained and cloned into the EcoRI and XhoI sites of pCMV-HA (Clontech Laboratories, Inc.), and designated as pCMV-HA-NUDT14. The constructs were confirmed by gene sequencing (Invitrogen; Thermo Fisher Scientific, Inc.). The plasmids of pCMV-myc-RL13 and pCMV-HA-NUDT14 were transiently co-transfected into HEK293 cells using Lipofectamine 2000, according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.). The cell lysates were harvested with lysis buffer (M-PER™ Mammalian Protein Extraction Reagent; Thermo Fisher Scientific, Inc.) supplemented with the protease inhibitors contained within the Protein G Immunoprecipitation kit (Promega Corporation). The protein concentration was determined using ultraviolet spectrophotometry (15). The co-immunoprecipitation analysis was performed, according to the manufacturer's protocols of the ProFound Mammalian c-myc Tag immunoprecipitation (IP)/Co-IP and hemagglutinin (HA) Tag IP/Co-IP kits (Pierce; Thermo Fisher Scientific, Inc.). The binding proteins (20 mg) were loaded in each lane of 12% polyacrylamide gels (SDS-PAGE) and the proteins were separated by gel electrophoresis. The separated proteins were electrotransferred onto nitrocellulose membranes (Sigma-Aldrich, St. Louis, MO, USA), and then blocked with 5% BSA (Thermo Fisher Scientific, Inc.) for 2 h. The expression levels of myc-RL13 and HA-NUDT14 were detected using Western blotting with mouse anti-human against myc or rabbit anti-human polyclonal antibodies against HA (1:100; Thermo Fisher Scientific, Inc., cat. no. 71-5500) incubated overnight at 4°C and corresponding peroxidase-conjugated goat anti-mouse and goat anti-rabbit (1:2,000; Beyotime Institute of Biotechnology; cat. no. A0208) IgG secondary antibodies incubated at room temperature for 2 h. Imaging was performed using ChemiDoc™ XRS+ (Bio-Rad Laboratories, Inc.) and density analysis was conducted with Image J version 10.2 (National Institutes of Health, Bethesda, MD, USA). Parallel co-immunoprecipitation experiments were performed with specific tagged proteins containing myc or HA.

To establish expression of the RL13 fusion protein with a green fluorescent protein (EGFP) tag, the RL13 coding region was amplified from the HCMV Han strain using the following primers: Forward 5′-CGCTCGAGCCAATAACACGTGCTCC-3′ and reverse 5′-CGCGGATCCTTAGGTTTTAGTCCA-3′. Subsequently, the product was inserted into the pEGFP-C1 (Clontech Laboratories, Inc.) via the XhoI and BamHI sites, yielding pEGFP-C1-RL13. Similarly, the coding sequence of NUDT14 in the NUDT14-containing cDNA clone was fused to the Discosoma sp. red fluorescent protein (DsRed) tag via amplification using PCR with the following primers: Forward 5′-CGGAATTCCGTGTTGGTGAAGCAG-3′ and reverse 5′-CGCGGATCCGGAGCTATGCAAGCC-3′, and cloned into the pDsRed-C1 (Clontech) via the EcoRI and BamHI sites, yielding pDsRed-C1-NUDT14. The constructs were confirmed by gene sequencing (Invitrogen; Thermo Fisher Scientific, Inc.).

The HEK293 cells (5×10⁶) were seeded into a 35 mm confocal microscope dish (Nest Biotechnology Co., Ltd, Jiangsu, China) 24 h prior to transfection. At 75% confluence, the cells were transfected with either 4 µg pEGFP-C1-RL13, 4 µg pDsRed-C1-Nudt14, or a mixture of 2 µg pEGFP-C1-RL13 and 2 µg pDsRed-C1-NUDT14 using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. At 48 h post-transfection, the cells were subjected to DAPI staining (Invitrogen; Thermo Fisher Scientific, Inc.), and the expression levels of EGFP-RL13 and DsRed-NUDT14 were detected using a laser scanning confocal microscope (Nikon Eclipse C1 Plus; Nikon, Tokyo, Japan) with 488 and 543 nm excitation beams, which corresponded to the EGFP-RL13 and DsRed-NUDT14 proteins, respectively.

To generate a U373MG cell line, which stably expressed NUDT14 (U373-S), the pDsRed-C1-NUDT14 vector and empty vector, pDsRed-C1, were transfected into U373 cells using Lipofectamine^® LTX with Plus™ Reagent, according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.), yielding U373-S, and its control cell line, U373-C, respectively. At 48 h post-transfection, neomycin (Thermo Fisher Scientific, Inc.) was at a final concentration of 600 µg/ml following mixing with culture medium. Neomycin-resistant cells were screened with neomycin at ~2 weeks (16,17). Protein extraction and quantification were performed as described previously. Western blot analysis was used to analyze the expression levels of NUDT14 in the selected cell clones, using rabbit anti-human polyclonal antibodies against NUDT14 (1:1,000; Abcam, Cambridge, MA, USA; cat. no. ab139656) and goat anti-rabbit IgG antibody conjugated with horseradish peroxidase. Equal quantities of sample were detected using western blotting, with antibodies against NUDT14. The antibody was characterized and specific bands for the NUDT14 protein were detected. As an internal control, the expression level of cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was analyzed by western blotting using mouse anti-human monoclonal antibody against GAPDH (1:1,000; Abcam; cat. no. ab9482) and goat anti-mouse antibody conjugated with horseradish peroxidase. The primary antibodies were incubated overnight at 4°C and the secondary antibodies were incubated for 2 h at room temperature.

The cells (n=1×10⁵) were cultured and transfected with Silencer^® Select Pre-Designed NUDT14 siRNAs (Ambion; Thermo Fisher Scientific, Inc.) and control siRNA (C-siRNA) (Ambion; Thermo Fisher Scientific, Inc.), respectively, using Lipofectamine RNAiMAX Reagent, according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.). The antisense sequences of the NUDT14-specific siRNAs were: 5′-UUGAAUAAGAGAACGGUCAcg-3′ (S-siRNA-1) and 5′-AAAGAUGACGCCGAGGGUCtt-3′ (S-siRNA-2). In each well, 0.5 µl 10 nM siRNA and 3 µl Lipofectamine (Invitrogen, Carlsbad, CA, USA) were diluted separately in 100 µl Opti-MEM (Invitrogen; Thermo Fisher Scientific, Inc.). Following incubation for 5 min at room temperature, the two solutions were mixed. After 20 min, the mixture was added to the cells. At 10 h post-transfection, the medium containing the siRNA was discarded, and the transfection was repeated once to increase the transfection efficiency and interference efficiency. At 24 h following the secondary transfection, the cells were inoculated with the HCMV Han strain at a multiplicity of infection of one. Total DNAs and proteins were extracted from the infected cells 72 h following infection, and the levels of NUDT14 in the infected cells were determined using Western blot analysis.

In brief, DNA was extracted from the HCMV-infected cells using a TIANamp Genomic DNA kit (Tiangen Biotech Co., Ltd.), according to the manufacturer's protocol. The viral DNAs were amplified and quantified using HCMV UL83-specific primers (forward 5′-GTCAGCGTTCGTGTTTCCCA-3′ and reverse 5′-GGGACACAACACCGTAAAGC-3′) and a SYBR Green PCR Master Mix kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) on an ABI Prism 7300 Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The numbers of viral DNAs were normalized to the numbers of β-actin detected in the same samples (using forward 5′-CGGAACCGCTCATTGCC-3′ and reverse 5′-ACCCACACTGTGCCCATCTA-3′ primers (18). The reaction mixture for qPCR consisted of 4 µl DNA extract, 12.5 µl Power SYBR Green PCR master mix (Applied Biosystems; Thermo Fisher Scientific, Inc.), 0.5 µl of each primer at 10 µM, and an ABI 7300 device (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used. The amplification conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min.

A modified comparative Cq method (2^−ΔΔCq) was used for relative quantification of viral DNAs (19–21). The ΔCq values (Cq_gene−Cq_β-Actin) were calculated, following which the ΔΔCq (ΔCqtreated−ΔCqcontrol) were calculated. The relative numbers of HCMV DNAs were described using the equation 2^−ΔΔCq (22). The experiments were repeated three times.

Statistical significance was analyzed using analysis of variance (ANOVA). P<0.05 was considered to indicate a statistically significant difference. Data are presented as the means ± standard deviation. All statistical analyses were computed using SPSS (Version 13.0; SPSS, Inc., Chicago, IL, USA), and graphs were produced using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA).

The candidate, pACT2-NUDT14, was screened to examine its interaction with RL13. The transformation efficiency was almost 1.1×10⁵ cfu/µg, and 14 yeast colonies produced positive results. Sequencing and BLAST analysis indicated that 10 positive candidates interacted with the RL13 protein (Table I), one of which was 100% identical to that of the human NUDT14 sequence in the National Center for Biotechnology Information (Genbank no. NM177533.3).

To further detect the binding between HCMV RL13 and host NUDT14 in vitro, GST-tagged NUDT14 was used as a bait protein and c-Myc-tagged RL13 was used as the prey protein in the GST-pull down assay. As shown in Fig. 1, a c-Myc-labeled RL13 protein (~34 kDa) was captured, with a GST-tagged NUDT14 protein of ~46 kDa using MagneGST particles, but not with GST alone. These results showed that RL13 had the ability to interact with NUDT14 in vitro.

To further confirm the interaction between RL13 and NUDT14, a co-IP assay was performed. As shown in Fig. 2, the c-Myc-labeled RL13 and HA-labeled NUDT14 proteins were assessed in the recovered products following immunoprecipitation with either anti-c-Myc or anti-HA antibodies (lanes 1 and 2, respectively). The input indicated that the protein levels of c-Myc-labeled RL13 and HA-labeled NUDT14 in the HEK293 cells were detected with anti-c-Myc and anti-HA antibodies, respectively (Fig. 2; lane 3). The positive control showed that the target proteins were correct in position and size (Fig. 2; lane 4). These results confirmed the interaction between the HCMV RL13 and host NUDT14 proteins in human cells.

To determine whether the HCMV RL13 and NUDT14 proteins localized within the same cellular compartment, the HEK293 cells were transfected with pEGFP-RL13 (GFP-RL13), pDsRed-NUDT14 (RFP-NUDT14) or the two plasmids together, in the present study. The resulting merged image represented regions of overlap among the GFP-, RFP- and DAPI-stained images. The GFP-RL13 fusion proteins were localized predominantly in the HEK293 cell membrane and cytoplasm (Fig. 3A). Similarly, The RFP-NUDT14 fusion proteins were predominantly localized in the HEK293 cell membrane and cytoplasm (Fig. 3B). The fluorescent-labeled GPF-RL13 and RFP NUDT14 proteins were spatially co-localized and expressed simultaneously in the HEK293 cell membrane and cytoplasm (Fig. 3C). Furthermore, the results showed that the co-localization of the two proteins had no effects on the distribution of either individually in the uninfected cells.

To investigate the effect of overexpressing NUDT14 on HCMV DNA replication, a stable NUDT14-expressing cell line, U373-S, was constructed. No differences were observed between the stable cell lines and the U373MG cells in growth characteristics or survival. In the stable U373-S cell lines, the protein expression of NUDT14 was five-fold higher than in the U373MG cells and the empty vector-transfected cells (U373-C; Fig. 4A).

To investigate the effect of underexpressing NUDT14 on HCMV DNA replication, the U373-S cells were transfected with NUDT14-specific siRNA molecules (S-siRNA). Compared to the U373-S cells, the underexpression of NUDT14 mediated by specific siRNA showed no effect on cell growth during the investigation. The results of the Western blotting results showed that the protein expression of NUDT14 in the S-siRNA-1- and S-siRNA-2-transfected cells collected 72 h post-HCMV infection were significantly reduced, by 84.14%, compared with the C-siRNA-transfected cells (Fig. 4A).

To determine whether changes in the expression of NUDT14 altered HCMV DNA replication, viral DNA copies were detected using qPCR. At 72 h post-infection, no significant difference in the relative copy number of HCMV, namely UL83/β-actin, in the U373-S cells stably expressing NUDT14, relative to those in the U373-C, U373MG and C-siRNA cells. However, following a decrease in the expression of NUDT14, there was a 20-fold increase in the number of HCMV copies in the infected cells treated with the two NUDT14-specific S-siRNA-1 (ANOVA P=0.036) and S-siRNA-2 (ANOVA P=0.002), compared with those in the cells transfected with C-siRNA and the U373-S cells. The fact that the S-siRNA-1 and S-siRNA-2 molecules exhibited the same phenotype indicated that there was no off-target of the siRNAs in the RNA interference experiments (Fig. 4B). These results suggested that inhibiting the expression of NUDT14 in the HCMV-infected cells increased viral replication.