Recombinant UL128 protein was obtained as reported previously.7 The UL128 gene sequence position 1243-2003 was selected from the Genbank (Accession no: GU574790.1). UL128 gene product was cloned using the pIRES-AcGFP vector (Qiagen, Valencia, CA) and was employed for the bacterial expression of pIRES-AcGFP with a 6-Histag fused to its N-terminus. Chinese hamster ovary cells were used for production of recombinant UL128 protein and the target protein was obtained through His·Bind Kit (Novagen, Germany). Protein concentration was quantified using the Bradford assay (Biorad) according to the manufacturer's instruction.

Since recombinant UL128 protein was labeled by 6-Histag, western blot analysis was carried out using 6×His Rabbit Polyclonal Antibody (Epitomics, Cat.: S2917) to confirm the presence of UL128 protein. The purified recombinant UL128-histag from SDS PAGE was transferred to PVDF (polyvinylidene difluoride) membrane. The membrane was blocked in 5% defatted milk dissolved in PBST (PBS containing 0.5% Tween-20) with gentle shaking at room temperature for 2h. The membrane was incubated in a 1:1000 dilution of rabbit anti-his-polyclonal antibody in blocking buffer with gentle shaking at room temperature for 1h, then changed to 4°C overnight. After washing with PBST three times for 10min each, the membrane was incubated in a 1:2000 dilution of sheet anti-rabbit IgG horseradish peroxidase (HRP) conjugated as secondary antibody (Epitomics, Cat.: 3053-1) in blocking buffer, with gentle shaking at room temperature for 1h after the membrane was washed with PBST for three times.

Silver stain was applied to detect trace amounts of proteins in gels. (1) Fixed: after electrophoresis (see above of SDS-PAGE), the gel was put in 100mL fixative liquid and shook for 20min at room temperature, placing overnight to further reduce background; (2) Washing: remove the from the fixed liquid, add 100mL of 30% ethanol and shake for 10min at room temperature. Discard ethanol, add 200mL double distilled water and shake for 10min at room temperature; (3) Sensitization: remove water, then add 100mL silver staining sensitizing solution (1×), shake for 2min at room temperature at a shaking speed of 60–70rpm (revolutions per minute); (4) Silver staining: add 100mL silver solution (1×), shake for 10min at a shaking speed of 60–70rpm, and washed by double distilled water for 1min; (5) Color: discard water, add 100mL silver staining solution, shake for 3–10min until the ideal protein band appears; (6) Termination: remove from silver staining solution, add 100mL silver staining terminated liquid (1×), shake for 10min at room temperature, and washed by double distilled water for 2min.

PBMC from whole blood of healthy volunteers were isolated by standard density centrifugation (Ficoll Separation Solution: GIBCO, Germany). The mononuclear cells were collected and washed twice in phosphate-buffered saline (PBS, pH 7.2). By adjusting to 1×106cells/mL, the cells were re-suspended in MEM medium with 10% heated-activated fetal calf serum. Informed consents were provided before sample collection.

As immune cells activation is countered by calcium fluxion, the intracellular calcium concentration in response to UL128 was monitored using fluo-3 AM (Calbiochem; La Jolla, CA USA) in PBMCs. The PBMC calcium level was determined by flow cytometry (Millipore, easyCyte8). Cells were pre-treated with or without 10μM fluo-3 AM (intracellular calcium chelator) for 1h, then washed with PBS and diluted to 5×106cells/mL in Hank's balanced salt solution containing Ca2+ and Mg2+ and 1% FBS. Different concentrations of U.L128 were added to 100nM and incubated at 37°C for 30min, and then analyzed by flow cytometry. Relative intra-cellular calcium levels were measured as the ratio of emissions at 490nm to the emissions at 400nm according to the manufacturer's instructions.

The cross-linking assay was used to prove the ability of UL128 protein to bind the membrane receptor of PBMC and compare with the positive control MIP-1α. PBMC (1×107cells/mL) were suspended in MEM medium pretreated with recombinant UL128 (10ng/mL) or MIP-1α(10ng/mL) for 24h then made into slides. Slides were washed four times with D-hanks (100nmol/L) containing 0.5% BSA and fixed with 4% paraformaldehyde for 20min. Slides were incubated with either anti-UL128 antibody (1:200, supplied by HangZhou NeuroPeptide Biological Science and Technology Incorporation) or rabbit anti-MIP-1αantibody (Rockland, 109-401-315) or normal rabbit serum at room temperature for 45min. Slides were washed four times with D-hanks and incubated with biotin-conjugated goat anti-rabbit IgG (1:200) at room temperature for 30min. The results were observed under microscope by counting the positive cells. The experiment was repeated three times for data analysis.

qPCR analyses were performed to quantify the CC receptor gene expression. Purified recombinant UL128 protein was co-cultured with PBMC for 24h before CC receptor detection. PBS added in PBMC was used as negative control and MIP-1α co-cultured PBMC was tested as positive control. Conserved regions at appropriate distances of CC receptors were used for designing the primers employed in qPCR reactions (Table 1). This was achieved using Primer Express 3.0 software. Total RNA was extracted using the RNeasy kit (Qiagen). RNA concentration and purity was estimated using a NanoVue Spectrophotometer and assessed for quality using agarose gel electrophoresis. Total RNA (2mg) was reverse transcribed to cDNA using the Fermentas First Strand Synthesis kit (Invitrogen). Amplifications were performed in an ABI Prism 7500 sequence detection system (Applied Biosystems). Fifteen μL reactions were prepared in clear 96 well fast plates. Amplification mixture concluded with the Maxima SYBR Green qPCR Master Mix (2×) (Thermo Scientific, USA), and a 0.2μM end concentration of each of the forward and reverse primers, 267nM Rox, and 25ng template cDNA. Amplification was carried out with a preliminary 2min UDG pretreatment at 50°C followed by a 10min initial denaturation at 95°C, followed by 40 cycles of denaturation for 15s at 95°C and annealing/extension for 60s at 60°C. Each experiment contains no template and no reverse transcriptase controls.

Results from real-time PCR and FCM were derived from at least three independent experiments. Statistical significance between siRNA and control groups was evaluated with one-way ANOVA followed by the Dunnett post-test using the Prism3 GraphPad software. Significance level was calculated using an assigned confidence interval of 95%. p<0.05 was considered significant.

The rUL128 production system was successfully constructed by using eukaryotic expression vector (pIRES-AcGFP-UL128) with 6× His-Tag fused to its N-terminal, then transferred into Chinese Hamster Ovary (CHO) cell line. Purified rUL128 was about 26kilodalton (KD) and was condensed into 0.1mg/mL (Table 2). Silver stain experiment confirmed high purification of the destined protein (Fig. 1). As shown in the figure, the red arrow refers to purified recombinant UL128 protein, which was consistent with western blot analysis.

Fig. 1.

Silver stain of recombinant UL128 protein. M: Marker, L1: Supernatant of CHO-Vector, L2: Purification lipid of CHO-Vector supernatant, L3: Blank control, L4: Supernatant of CHO-UL128; L5: Purification lipid of CHO-UL128 supernatant, rUL128: Western Blot of recombinant UL128 (using anti-Histag).

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Chemotactic agonists induce a rapid and transient rise of Ca2+ in target cells. Our experiment showed that UL128 protein increased Ca2+ in PBMC at chemotactic concentrations which was similar to the function of MIP-1α (Fig. 2).

Fig. 2.

Sample1: The negative control group (PBMC+Fluo-3/AM). Fluorescent indicator did not detect significant Ca2+ when PBMC without treatment factor. Sample2: The positive control group (PBMC+Fluo-3/AM+MIP-1α). Intracellular Ca2+ concentration increased instantly when adding MIP-1α in the cell-culture medium. Sample3: The experimental group (PBMC+Fluo-3/AM+UL128). The level of intracellular Ca2+ concentration was markedly increased after treatment of UL128.

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Immunohistochemistry results of cross-linking assay showed UL128 protein binding the cytomembrane receptors of PBMC. Human CC chemokine (MIP-1α) was used as positive control. The mean percentage of positive cells that UL128 and MIP-1αbinding with PBMC were 13% and 12.6%, respectively (Fig. 3). There was no significant difference between them (p>0.05).

Fig. 3.

UL128 binding the cytomembrane receptor of PBMC. BLANK: purification elute of CHO-Vector supernatant (negative control), MIP-1α: 10ng/mL (positive control), UL128: 10ng/mL. White arrows in the picture indicate the positive cells.

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In our experiment, UL128 protein could enhance production of CC chemokine receptors (CCR) according to PCR detection. CCR1, CCR3, and CCR4 were up-regulated when PBMC was co-cultured with UL128 at low concentration (10ng/mL). Transcription of CCR2 and CCR5 were improved with the increase of UL128 concentration, but they could hardly be detected with UL128 at low concentration. Based on these results, we infer that HCMV encoded UL128 protein may interact with different kinds of CCRs of PBMC and active at low concentration (Fig. 4).

Fig. 4.

Rt-PCR detection of CC chemokine receptors in PBMC.

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