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Host protein Snapin interacts with human cytomegalovirus pUL130 and affects viral DNA replication GUILI WANG1,2 , GAOWEI REN1 , XIN CUI1 , ZHITAO LU1 , YANPIN MA1 , YING QI1 , YUJING HUANG1 , ZHONGYANG LIU1 , ZHENGRONG SUN1,* and QIANG RUAN1,* 1 Virus Laboratory, The Affiliated Shengjing Hospital, China Medical University, Shenyang 110004, China 2 Department of Pediatrics, The Third Affiliated Hospital of Liaoning Medical College, Jinzhou 121001 Liaoning, China *Corresponding author (Email, ZS – [email protected]; QR – [email protected]) The interplay between the host and Human cytomegalovirus (HCMV) plays a pivotal role in the outcome of an infection. HCMV growth in endothelial and epithelial cells requires expression of viral proteins UL128, UL130, and UL131 proteins (UL128-131), of which UL130 is the largest gene and the only one that is not interrupted by introns. Mutation of the C terminus of the UL130 protein causes reduced tropism of endothelial cells (EC). However, very few host factors have been identified that interact with the UL130 protein. In this study, HCMV UL130 protein was shown to directly interact with the human protein Snapin in human embryonic kidney HEK293 cells by Yeast two-hybrid screening, in vitro glutathione S-transferase (GST) pull-down, and co-immunoprecipitation. Additionally, heterologous expression of protein UL130 revealed co-localization with Snapin in the cell membrane and cytoplasm of HEK293 cells using fluorescence confocal microscopy. Furthermore, decreasing the level of Snapin via specific small interfering RNAs decreased the number of viral DNA copies and titer in HCMV-infected U373-S cells. Taken together, these results suggest that Snapin, the pUL130 interacting protein, has a role in modulating HCMV DNA synthesis. [Wang G, Ren G, Cui X, Lu Z, Ma Y, Qi Y, Huang Y, Liu Z, Sun Z and Ruan Q 2016 Host protein Snapin interacts with human cytomegalovirus pUL130 and affects viral DNA replication. J. Biosci. 41 173–182] DOI 10.1007/s12038-016-9604-2 1. Introduction Human cytomegalovirus (HCMV) is ubiquitous throughout populations worldwide and represents a significant public health challenge in both developed and developing countries (Mocarski et al. 2007). Severe forms of the disease are most commonly observed when immune systems have been compromised by infection (e.g. HIV/AIDS) or immunosuppressive therapy (e.g. in transplant recipients). HCMV is also the leading viral cause of congenital abnormalities and mental retardation in newborns. Keywords. HCMV can infect a wide range of cell types in vivo (Sinzger et al. 1996; Kahl et al. 2000; Riegler et al. 2000; Bissinger et al. 2002) and establish lytic, persistent, and latent infections in many different cells (Wills et al. 2005; Britt 2008). HCMV utilizes two different pathways for host cell entry. Infection of fibroblasts occurs by direct fusion of the virion envelope with the plasma membrane, and this requires glycoproteins gB and gH/gL. In contrast, in epithelial and endothelial cells (EC), membrane fusion takes place in vesicles following internalization by endocytosis or micropinocytosis and requires an additional complex DNA replication; HCMV; Snapin; UL130 Abbreviations used: Co-IP, Co-immunoprecipitation; HCMV, Human cytomegalovirus; ORF, open reading frame; PCR, polymerase chain reaction; siRNAs, small interfering RNAs http://www.ias.ac.in/jbiosci Published online: 7 April 2016 J. Biosci. 41(2), June 2016, 173–182 * Indian Academy of Sciences 173 174 Guili Wang et al. composed of gH, gL, and UL128, UL130, and UL131 (UL128-131) (Ryckman et al. 2006; Sinzger 2008; Haspot et al. 2012; Wussow et al. 2014). Genes encoding UL128131 are transcribed with the 3′ coterminal at late kinetics (Akter et al. 2003; Hahn et al. 2004), although their mRNA initiation points may be different (Sun et al. 2010). Their proteins are produced when HCMV replication is at the stage of virion assembly and release (Akter et al. 2003; Hahn et al. 2004). The pUL128-131 is required for efficient infection and cell-to-cell spread in several cell types, including monocytes, epithelial, endothelial, and myeloid cells (Revello and Gerna 2010; Straschewski et al. 2011; Schuessler et al. 2012; Nogalski et al. 2013), either by binding to cell surface receptors or promoting nuclear translocation of virions (Sinzger et al. 2000; Ryckman et al. 2008a; Sinzger 2008). However, pUL128-131 restricts either cell-to-cell transmission or virus production in fibroblast cultures (Stanton et al. 2010). Further investigation into the interactions between virus proteins and host cell proteins will yield important information regarding the process of viral entry and replication, which is critical for developing new strategies and novel compounds for the treatment and prevention of HCMV infection. Of the genes in the pUL128-131, the UL130 is centrally located, is the largest gene, and is the only one that is not interrupted by introns (Akter et al. 2003; Hahn et al. 2004). pUL130 is a luminal glycoprotein that is inefficiently secreted from infected cells but is incorporated into the virion envelope in a Golgi-matured form (Patrone et al. 2005). The C terminus of pUL130 serves an important function for infection of endothelial cells by HCMV, and mutation of the C-terminal of pUL130 causes a reduction of EC tropism (Schuessler et al. 2010). However, few host factors that interact with pUL130 have been identified to date. In this study, the human protein Snapin was identified as directly interacting with HCMV pUL130. In addition, the viral DNA level was found to increase in HCMV-infected U373 cells over-expressing Snapin and decrease in cells treated with Snapin specific siRNA. These results suggest that Snapin, the pUL130 interacting protein, has a role in modulating HCMV DNA synthesis. 2. Material and methods 2.1 Cells and viruses Human embryonic lung fibroblast MRC-5 cells, astrocytoma U373MG cells, and human embryonic kidney HEK293 cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum at 37°C in 5% CO2. HCMV clinical strain Han was at ages of <5 months and isolated from urine samples of neonates J. Biosci. 41(2), June 2016 admitted to the Pediatrics Department, Affiliated Shengjing Hospital of China Medical University. The virus was inoculated into MRC-5 cells maintained in minimal essential medium (MEM) supplemented with 2% fetal calf serum (HyClone, Logan UT, USA) and penicillin-streptomycin in an incubator at 37°C, 5% CO2. 2.2 Yeast two-hybrid screening The coding sequence of HCMV UL130 was amplified by polymerase chain reaction (PCR) using HCMV Han strain DNA (Genbank No: GQ981646) as a template with primers 5′-CCGGAATTCTTGTCGACCCTGCGGCTTCTGC TTCGTCAC-3′ and 5′-CGCGGATCCGGTACCTCAAA CGATGAGATTGGGATG -3′. After digestion, the UL130 sequence was cloned into the pGBKT7 vector (Clontech, Mountain View, CA, USA) between the EcoRI and BamHI sites downstream of the c-Myc coding sequence, resulting in pGBKT7-UL130. The inserted sequence of the pGBKT7UL130 was confirmed by sequencing (Invitrogen Biotechnology Co., Ltd., Shanghai, China). Yeast two-hybrid experiments were performed according to the manufacturer's instructions (Matchmaker GAL4 TwoHybrid System 3, Clontech, USA). Briefly, Saccharomyces cerevisiae strain AH109 was co-transformed with pGBKT7UL130 and a human fetus brain cDNA library, which was cloned into pACT2 (pACT2-cDNA; Clontech Laboratories, Inc.). Positive colonies were selected on synthetic dropout (SD) medium lacking tryptophan, leucine, adenine, and histidine (SD-minus Trp/Leu/Ade/His (Quadruple dropouts, QDO)) and tested for β-galactosidase activity by a colonylift filter assay as described previously (To et al. 2011). Plasmids containing sequences encoding the interaction partners of UL130 (designated as pACT2-cDNA) were extracted from the positive colonies and rescued by electrotransformation into competent Escherichia coli DH5α. Each pACT2-cDNA plasmid rescued from E. coli was cotransformed into yeast together with the pGBKT7-UL130 or the pGBKT7 empty vector. The transformed cells were plated on QDO plates and subjected to a β-galactosidase activity test. Human gene sequences in pACT2-cDNA from blue yeast colonies were identified using the sequencing primer 5′-AATACCACTACAATGGAT-3′, and analysed by the BLAST network server at the National Center for Biotechnology Information (http://www.ncbi.nlm.gov/blast). 2.3 Glutathione S-transferase (GST) pull-down assay The coding sequence of one of the HCMV UL130 candidate interacting proteins, Snapin, was obtained from a selected cDNA clone (pACT2-Snapin) and GST-tagged by cloning 175 Snapin interacts with HCMV pUL130 into the pGEX-4T-2 vector between the EcoRI and SamI sites, yielding pGEX-4T2-Snapin. A GST-pull-down experiment was performed according to the manufacturer's instructions (MagneGST™ Pull-Down System, Promega, Madison, WI, USA). Briefly, c-Myclabelled UL130 protein (c-Myc-UL130) was expressed from the pGBKT7-UL130 in a TNT (Quick coupled transcription/ translation reaction) T7 Quick Reaction (McMahon and Anders 2002). GST-labelled Snapin protein (GST-Snapin) was expressed in BL21 (DE3) cells transfected with the pGEX-4T2-Snapin and induced with isopropyl β-D-1-thiogalactopyranoside (IPTG). As a bait protein, 20 μL of GSTSnapin was incubated with 80 μL of c-Myc-UL130 at room temperature for 1.5 h on a rotating platform. Then, the reaction products were incubated with MagneGST particles for 30 min. After washing three times with buffer, the bound proteins on the MagneGST particles were eluted with elution buffer and solubilized in 2× SDS sample buffer. The recovered proteins were subjected to electrophoresis on a 12% SDS-polyacrylamide gel, and the separated proteins were transferred electrically onto nitrocellulose membranes (Sigma–Aldrich, St. Louis, MO, USA). Western blot analyses were performed using mouse anti-c-Myc or goat antiGST monoclonal antibodies (Pierce, Rockford, IL, USA) and corresponding peroxidase-conjugated secondary antibodies. The membranes were then incubated with chemiluminescent substrate provided in the Western Chemiluminescent Substrate kit (Thermo Pierce, Rockford, IL, USA). Signals were revealed using a Molecular Imager ChemiDoc XRS System (Bio-Rad, Inc., Hercules, CA, USA). 2.4 Co-immunoprecipitation (co-IP) analysis The coding sequence of UL130 in the pGBKT7-UL130 was obtained and cloned into the pCMV-HA vector between SfiI and NotI sites, designated as pCMV-HA-UL130. The coding sequence of Snapin in the pGEX-4T2-Snapin was also obtained and cloned into SfiI and XhoI sites of pCMVmyc, designated as pCMV-myc-Snapin. All constructs were confirmed by DNA sequencing (Invitrogen Biotechnology Co., Ltd, Shanghai, China). HEK293 cells were cotransfected with pCMV-myc-Snapin and pCMV-HAUL130 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The cell lysates were harvested at 48 h post transfection. Co-immunoprecipitation experiments were performed using the ProFound Mammalian HA Tag IP/Co-IP and c-Myc Tag IP/Co-IP kits following the manufacturer’s protocols (Pierce). The Myc-Snapin and HA-UL130 were detected by Western blot using mouse anti-Myc or rabbit anti-HA antibodies (Clontech, USA), respectively, and corresponding peroxidase-conjugated secondary antibodies. 2.5 Cellular localization assay To express UL130 fusion protein labelled with an enhanced green fluorescent protein (EGFP) tag, the UL130 coding region was amplified from the HCMV Han strain using primers 5′-CGGAATTCCCTGCGGCTTCTGCTTCGT-3′ and 5′-CGCGGATCCTCAAACGATGAGATTGGG-3′. Then, the product was cloned into the pEGFP-C1 vector (Clontech, USA) via the EcoRI and BamHI sites, yielding pEGFP-C1-UL130. Similarly, the coding sequence of Snapin in the Snapin-containing cDNA clone was amplified by PCR with primers 5′-CGGAATTCCATGGCGG G GG C T G GT T C C - 3′ and 5 ′ - C G CG GA T C C T T AT TTGCCTGGGGAGCC-3′. The products were cloned into the pDsRed-C1 vector (Clontech, USA) via the EcoRI and BamHI sites, yielding pDsRed-C1-Snapin. All constructs were confirmed by DNA sequencing (Invitrogen Biotechnology Co., Ltd., Shanghai, China). HEK293 cells (5×106) were seeded in 6-cm dishes 24 h before transfection. At 75% confluence, the cells were transfected with 4 μg pEGFP-C1-UL130, 4 μg pDsRed-C1Snapin, or a mixture of 2 μg of pEGFP-C1-UL130 and 2 μg of pDsRed-C1-Snapin using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. At 48 h post-transfection, the cells were subjected to DAPI staining (Invitrogen, Carlsbad, CA, USA) and expression of the fluorescently labelled EGFP-UL130 and DsRed-Snapin was analysed using a TCS SP2 Leica laser scanning confocal microscope (Nikon Eclipse C1 Plus, Tokyo, Japan) at 488 nm or 543 nm, respectively. 2.6 Stable expression of Snapin and transfection of Snapin-specific small interfering RNA (siRNA) into cells To generate a U373MG cell line that stably expressed Snapin (U373-S) and a control cell line, pDsRed-C1Snapin and the empty vector pDsRed-C1 were transfected into U373 cells, respectively. At 48 h post-transfection, neomycin was added to the culture medium at a final concentration of 600 μg/mL. Neomycin-resistant cells were subsequently selected in the presence of neomycin for 2 weeks. Total protein was extracted from the cells and equal amounts of the protein were subjected independently to Western blot analysis. The expression level of Snapin in the selected cell clones was determined by Western blot using a mouse anti-Snapin antibody (Abcam, Hong Kong, China) and goat anti-mouse IgG antibody conjugated with horseradish peroxidase (Vector Laboratories, Burlingame, CA, USA) as following. As an internal control, the expression level of cellular glyceraldehyde -3-phosphate dehydrogenase (GAPDH) in the cells was detected using a mouse antiGAPDH antibody and goat anti-mouse antibody conjugated with horseradish peroxidase (Vector Laboratories). J. Biosci. 41(2), June 2016 176 Guili Wang et al. U373-S cells (1×105) were seeded in 12-well plates and transfected with Silencer® Select Pre-Designed Snapin siRNAs (Ambion, Grand Island, NY, USA) or control siRNA (C-siRNA) molecules (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The antisense sequences of the Snapin specific siRNAs were: 5′-UUAGCCGUCUCAGU CGUUCct-3′ (S-siRNA-1) and 5′-UACGUGAGAGUC GAGCUGCtg-3′ (S-siRNA-2). For each well, 0.5 μl of 10 nM siRNA and 3 μL of Lipofectamine (Invitrogen, Carlsbad, CA, USA) were diluted separately in 100 μL of Opti-MEM (Invitrogen, Carlsbad, CA, USA). After 5 min of incubation at room temperature, both solutions were mixed together. Twenty min later, the mixture was added to the cells. At 10 h post-transfection, siRNA-containing medium was removed. The cells were washed and incubated with complete DMEM supplemented with 10% fetal bovine serum. At 10 h after the first round of transfection, the transfection was repeated. The transfected cells, together with U373MG (U373), U373-C (U373-C), and U373-S (U373-S) cells that were cultured in parallel, were infected with the HCMV Han strain at a multiplicity of infection (MOI) of 1 at 24 h after the second round of transfection. Total protein was extracted from the infected cells at 72 h post infection. The level of Snapin in the infected cells was determined by Western blot as described above. The experiments were repeated three times. 2.7 Viral growth and quantitative PCR analysis of viral infection To assay viral growth, we infected cells (n=1×105) with HCMV at an MOI of 1. We collected the cells and medium at 5 days postinfection and prepared viral stocks. We determined the titers of the viral stocks by infecting 1×105 human foreskin fibroblasts and counting the number of plaques 10– 14 days after infection. The values obtained were averages from triplicate experiments. Quantitative PCR (qPCR) analysis of viral DNA was conducted as follows. Briefly, DNA was isolated from the HCMV-infected cells using the TIANamp Genomic DNA Kit (Tiangen Biotech Co. Ltd., Beijing, China) according to the manufacturer’s instructions. Viral DNA was amplified and quantified using HCMV UL83-specific primers (5′-GTCAGCGTTCGTGTTTCCCA -3′ and 5′-GGGACACAACACCGTAA AGC-3′) and the SYBR Green PCR Master Mix kit (Applied Biosystems, USA) on an ABI Prism 7300 Sequence Detection System (Applied Biosystems, USA). The number of viral DNA copies was normalized to that of β-actin detected in the same sample using primers 5′-CGGAACCGCTCATTGCC-3′ and 5′-ACCCACACTGTG CCCATCTA-3′. The amplification conditions were 95°C for 10 min and 40 cycles of 95°C for 15 s and 60°C for 1 min. J. Biosci. 41(2), June 2016 The relative quantity of viral DNA copies was calculated using a modified comparative CT method (2−ΔΔCT), in which CT was defined as the cycle number at which fluorescence reached a set threshold value. The difference in the CT value between the target gene and the corresponding internal control β-actin gene, ΔCT (CTgene – CTβ-Actin), was calculated. Then, the change in ΔCT of the treated group relative to the control group, ΔΔCT (ΔCTtreated – ΔCTcontrol), was computed. The relative number of HCMV DNA copies was described using the equation 2−ΔΔCT. The experiments were repeated three times. 2.8 Statistics Data are shown as mean±SD. Statistical significance was determined by analysis of variance (ANOVA). A p-value <0.05 was considered to be statistically significant. 3. 3.1 Results Snapin was selected as a candidate binding protein of HCMV UL130 by a yeast two-hybrid screen To identify cellular factors that potentially interact with HCMV pUL130, a yeast two-hybrid screen was carried out by transforming S. cerevisiae AH109 with the pGBKT7UL130 and a cDNA library derived from human fetal brain. Two of the identified constructs, Homo sapiens SNAPassociated protein (pACT2-Snapin), which contain the full length coding sequence of Snapin, were consistently found to interact with pUL130. The nucleotide sequence of pACT2-Snapin was 99% identical to that of the human Snapin sequence in NCBI (Genbank No: NM_012437.5) (table 1). 3.2 Interaction between HCMV pUL130 and host Snapin was identified by GST pull-down assay To detect the binding ability between HCMV pUL130 and host Snapin in vitro, GST-tagged Snapin was used as a bait protein and c-Myc-tagged pUL130 was used as the prey protein in GST pull-down experiments. The recovered products were detected with a mouse anti-c-Myc monoclonal antibody and a goat anti-GST monoclonal antibody, respectively. As shown in figure 1, an approximate 50 kDa c-Myc labelled UL130 protein was captured by an approximate 41 kDa GST-tagged Snapin protein stabilized on the MagneGST particles. These results reveal that UL130 protein has the ability to interact with Snapin protein in vitro. 177 Snapin interacts with HCMV pUL130 Table 1. Homologous genes interacted with HCMV UL130 protein were analysed by comparing the gene sequences with human genome from Genebank Homologous genes Identical clone Homology (%) Homo sapiens DnaJ (Hsp40) homolog Homo sapiens forkhead box G1(FOXG1) Homo sapiens glycoprotein, synaptic2 Homo sapiens SNAP-associated protein Homo sapiens chromosome 9 open reading Homo sapiens chromosome 12 open reading Homo sapiens similar to putative DNA dependent ATPase and helicase 2 1 1 2 1 1 1 1 96 100 98 99 100 99 99 96 Note: The sequencing results of positive clones, which were screened by Yeast two-hybrid experiment between HCMV UL130 and a human fetus brain cDNA library, were analysed by the BLAST network server at the National Center for Biotechnology Information (http:// www.ncbi.nlm.gov/blast). Seven homologous genes were obtained by comparing with human genome of Genbank. 3.3 Interaction between HCMV pUL130 and host Snapin protein was identified by co-IP To further determine whether the interaction between pUL130 and Snapin occurs in vitro, a co-IP assay was performed using proteins expressed from pCMV-MycSnapin and pCMV-HA-UL130 in co-transfected HEK293 cells. As shown in figure 2, both the c-Myc-tagged Snapin and HA-tagged UL130 proteins were detected in the recovered products immunoprecipitated with either anti-c-Myc or anti-HA antibodies. These results confirm the specific Figure 1. Interaction between host Snapin and HCMV pUL130 protein was analysed by GST pull-down. GST-tagged Snapin was used as the bait protein and c-Myc-tagged UL130 was used as the prey protein. The recovered products were detected with either mouse anti-c-Myc monoclonal antibody or goat anti-GST monoclonal antibody. The ‘GST pull-down’ shows the c-Myc-tagged pUL130 and only GST control (lane 1) or GST-snapin (lane 2) captured by the MagneGST particles; The ‘input’ shows the c-Myclabelled protein that was expressed from pGBKT7-UL130 in vitro (lane 3) or the protein lysates of BL21(DE)3 transfected with GSTSnapin expressing plasmid pGEX-4T2-Snapin (lane 4). interaction between the HCMV pUL130 and host Snapin protein in human cells. 3.4 Co-localization of pUL130 and Snapin proteins in human cells was identified by fluorescence confocal microscopy To determine whether HCMV pUL130 and Snapin proteins localize within the same cellular compartment, HEK293 cells were transfected with pEGFP-UL130, pDsRedSnapin, or both plasmids. The fusion proteins of GFPUL130 (figure 3A) and DsRed-Snapin (figure 3B) were mainly localized in the membrane and cytoplasm of Figure 2. Interaction between c-Myc-tagged Snapin and HAtagged pUL130 was identified by co-immunoprecipitation. HEK293 cells were co-transfected with plasmids pCMV-c-mycSnapin and pCMV-HA-UL130. Cell lysates were prepared at 48 h post transfection. The input protein samples (lane 1) and samples immunoprecipitated with either anti-c-myc (lane 2) or anti-HA antibodies (lane 3) were separated on SDS-containing polyacrylamide gels and analysed with anti-c-myc and anti-HA antibodies, respectively. Negative control was analysed simultaneously (lane 4). Both the c-Myc-tagged Snapin and HA-tagged UL130 protein were detected in the recovered products. J. Biosci. 41(2), June 2016 178 Guili Wang et al. Figure 3. Localization of pUL130 and Snapin expressed in HEK293 cells. HEK293 cells were transiently transfected with pEGFP-UL130 (A), pDsRed-Snapin (B), or both (C). At 48 h post transfection, nuclear DNA was stained with DAPI (blue image). Localization of the expressed proteins was observed by fluorescent confocal microscopy. ‘Merge’ represents the overlapping image of fluorescence from expressed proteins and nuclear staining in the same field of view. HEK293 cells and were expressed simultaneously (figure 3C). 3.5 Effect of Snapin protein on HCMV DNA synthesis and lytic infection To study the effect of Snapin on HCMV DNA replication, a stable Snapin-expressing cell line, U373-S, was constructed. U373-S cells were then transfected with Snapin-specific siRNA. U373-S cells and Snapin-specific siRNA-transfected U373-S cells showed no obvious change in the cell growth during compared to U373MG cells (data not shown), suggesting that changes in Snapin expression did not result in J. Biosci. 41(2), June 2016 significant cytotoxicity. Western blot results showed that the expression level of Snapin in U373-S cells was five-fold higher than in the parental U373MG cells and cells transfected with the empty vector (U373-C). The levels of the over-expressed and endogenous Snapin proteins in the SsiRNA-1 and S-siRNA-2 transfected cells collected at 72 h post-HCMV infection were significantly reduced (75.59% and 92.45%, respectively) compared with cells transfected with control siRNA (figure 4A). To determine whether changes in Snapin expression alter HCMV DNA synthesis and lytic infection, the number of viral DNA copies and titer was detected. At 72 h postinfection, an approximate 5-fold increase in the number of viral DNA copies (UL83/β-Actin) was observed in the 179 Snapin interacts with HCMV pUL130 Figure 4. Effects of Snapin on HCMV DNA synthesis were detected by real-time PCR. U373-S cells were transfected with Snapinspecific siRNAs (S-siRNA-2 or S-siRNA-1) or control siRNA (C-siRNA). The cells, together with U373MG (U373), U373-C (U373-C), and U373-S (U373-S) cells were infected with HCMV Han strain at MOI of 1. (A) At 72 h post-infection, the level of Snapin protein was detected by Western blot analysis using a mouse anti-Snapin antibody (Abcam). The expression of cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. (B) The values of the relative levels of HCMV DNA and titers (mean±SD from triplicate experiments). The relative level of HCMV DNA and titers are shown compared to the number of viral DNA copies in the parental U373MG cells (U373). Snapin-expressing U373-S cells compared with U373-C cells (p=0.018) and the parental U373MG cells (p=0.033). Moreover, the number of viral DNA copies was significantly reduced in cells treated with anti-Snapin S-siRNA-1 molecules (84%, p=0.020) and anti-Snapin S-siRNA-2 molecules (89%, p=0.028) compared with U373-C and parental U373MG cells (figure 4B). At the same time, viral progeny production in these cells was determined. Virus stocks were prepared from the infected cultures (cells and culture medium together), and their titers were determined as similar with viral DNA copies. The two Snapin specific siRNA molecules (S-siRNA-1 and S-siRNA-2) yielded similar effects on Snapin expression and HCMV DNA replication and their titer, indicating that these results were not due to off-targets effects of the siRNAs. These findings suggest that the increase of Snapin expression in HCMV-infected cells benefits HCMV DNA synthesis and vice verse. 4. Discussion Similar to other viral infections, HCMV is known to perturb a number of host cell functions. The majority of these perturbations presumably optimize host cell functions and J. Biosci. 41(2), June 2016 180 Guili Wang et al. conditions to support viral persistence and productive viral replication (Kalejta and Shenk 2002; Landolfo et al. 2003). Despite the clear importance of UL128-131 in virion cell tropism, the mechanisms that regulate their relative abundance during HCMV infection have for the most part remained elusive. Our results have revealed that host protein Snapin interacts with pUL130, as determined by two yeasthybrid screening, GST pull-down assay, and co-immunoprecipitation. Co-localization of these two proteins in both the cytosol and the plasma membrane was confirmed by confocal microscopic analysis. These observations suggest that the host factor Snapin may play an important role at the time of HCMV entry into cells. A few host factors have been identified that interact with HCMV proteins and are involved in all stages of the virus life cycle (Scholz et al. 2003). Host factors may have important roles in viral entry, replication, budding, release, pathogenesis, and restricting cross-species transmission. Snapin is small protein of 15 kDa and only 136 amino acids. Snapin is a ubiquitous cellular protein, and it is located in both the cytosol and the plasma membrane in neuronal and non-neuronal cells (Ilardi et al. 1999; Vites et al. 2004; Ruder et al. 2005). In neuronal cells, Snapin is a component of the SNARE (soluble Nethylmaleimide-sensitive fusion protein attachment protein receptor) complex that is required for synaptic vesicle docking and fusion (Buxton et al. 2003). Despite this fact, Snapin interacts with a number of molecules to regulate their function (Wang et al. 2009). In other types of cells, Snapin interacts with components of the biogenesis of lysosome-related organelles complex 1 (BLOC-1), which is a ubiquitously expressed multi-subunit protein complex required for normal biogenesis of specialized organelles of the endosomal-lysosomal system, such as melanosomes and platelet-dense granules (Starcevic and Dell'Angelica 2004; Ruder et al. 2005). HCMV transcriptionally regulates expression of some cellular soluble Nethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) genes whose products are involved in membrane fusion events (Hertel and Mocarski 2004). Given that SNARE proteins drive many membrane fusion events within eukaryotic cells, it is likely that in infected cells, HCMV exploits/manipulates these cellular proteins to produce new virions. The export of gH/gL complexes from the endoplasmic reticulum (ER) to the Golgi apparatus and cell surface has previously been shown to dramatically increase when UL128, UL130, and UL131 were coexpressed with gH/gL (Ryckman et al. 2008b). Snapin also plays a role in cell vesicle transport and fusion and has been implicated in the regulation of exocytosis and endocytosis (Hertel and Mocarski 2004). Taken together, this suggests that the interaction between Snapin and HCMV UL130 may impact the transport and fusion of J. Biosci. 41(2), June 2016 HCMV from the endoplasmic reticulum to the Golgi or from the Golgi to the cell membrane. In our study, the number of HCMV DNA copies and synthesis in an HCMV-infected Snapin-expressing U373 cell line was significantly higher than that in normal U373MG cells. In addition, decreased Snapin expression in the Snapin-expressing U373 cell line via siRNA resulted in a significant decrease in the number of viral DNA copies in HCMV-infected Snapin-expressing U373 cells. These results suggest that the increase of Snapin expression in HCMV-infected cells benefits viral DNA replication synthesis and vice verse. In contrast, Snapin over-expression in U373 cells has previously been shown to down-modulate HCMV viral DNA synthesis and lytic infection by interaction with viral primase UL70 (Shen et al. 2011). This opposing observation maybe because of the differences of using strains. The HCMV Towne strain was used in the study (Shen et al. 2011), however, the HCMV clinical strain was used in our study. According to our previous published article (Sun et al. 2009), the coding region of UL130 gene in clinical strains is 645 bp in length, whereas that in the Towne strain is 690 bp. UL130 gene in Towne strain has a frameshift near the 3′ end of the gene since double T insertion. It was observed that a cysteine codon, which was conserved in clinical strains, was substituted by serine codon at the 3′ end of UL130 in Towne strain. In addition, UL130 protein in Towne strain contains an additional casein kinase II phosphorylation site and an N-myristoylation site, which were absent in clinical strains and Merlin strain. The UL130 putative protein of clinical strains contains a putative chemokine domain from amino acids 46 to 120, although it lacks two of the four cysteines that are strictly conserved in a chemokine of this type. U373MG is an epithelia-like cell line derived from neuronal tissues. It is highly permissive to HCMV replication and has been widely used in HCMV infection studies (Mocarski et al. 2007). In our study, Snapin was identified as interacting with pUL130 and modulating HCMV DNA replication in U373MG cells. Because of the necessary roles of HCMV pUL128-131 in epithelial cell infection, it could be speculated that Snapin may be involved in the HCMV infection by interacting with pUL130, except for the known mechanism (Patrone et al. 2005; Schuessler et al. 2010). It is interesting to understand whether the influence of pUL130, together with the other proteins of pUL128-131, on HCMV infection or the regulation of Snapin on HCMV DNA synthesis is mediated by their interaction. In conclusion, the present study demonstrated the interaction between HCMV pUL130 and Snapin and confirmed the modulating role of Snapin on HCMV DNA replication. 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PLoS Pathog. 10 e1004524 MS received 01 August 2015; accepted 03 March 2016 Corresponding editor: SHAHID JAMEEL J. Biosci. 41(2), June 2016