Human Mannose-binding Lectin Inhibits HCMV Infection In Human Embryonic Pulmonary Fibroblast

Objective:Human cytomegalovirus (HCMV) is the leading viral cause of congenital disorders and has high sero-prevalence rates among human. Primary HCMV infection occurs early in life and followed by the establishment of a life long latency in the infected host. A primary infection or reactivation of endogenous virus in immunosuppressive individuals is associated with substantial morbidity and mortality. To date, a limited number of drugs have been used for treatment of HCMV and disease, including ganciclovir, cidofovir, foscamet, fomiversen and maribavir. These drugs all share the similar antiviral mechanism. Although combating HCMV by inhibiting virus replication is effective and has been established for many years, problems with toxicity and emergence of drug resistance underscore the need to develop new and improved antiviral drugs with novel molecular targets. The blocking of virus entry into cells before their replication is a promising way to research new antiviral agents.Mannose-binding lectin (MBL), a soluble C-type lectin, constitutes an important part of the innate immune defence. MBL binds to repetitive mannose and N-acetylglucosamine residues on microorganisms, activating the complement system. MBL can inhibit the infection of HIV, influenza A virus and SARS-CoV. MBL deficiency may lead to susceptibility to HCMV and secondary infection. However, the antiviral activities of human MBL against HCMV are not well studied.This study expressed human MBL gene in mammary cells and obtained recombinant MBL (rMBL). Then the anti-HCMV activities of both rMBL and isolated native MBL (hMBL) from human serum were tested and the possible mechanisms were explored.Methods:1. Establishment of MBL-CDNA TA cloneThe target sequence of MBL (747bp) was produced by oligo synthesis, and the gene was cloned to pCR2.1-TOPO vector and then transformed to competent cell DH5a. The transformed cells were proliferated and the plasmids were extracted and sequenced to confirm the MBL-CDNA clone was established.2. Construction of recombinant expression vector PIRES2-AcGFP-MBLThe above plasmids from MBL-CDNA clone were amplified and the restriction sites for XhoI and EcoRI were added by PCR. The PCR products and the expression vector PIRES2-AcGFP were both cleaved by endonucleases XhoI and EcoRI and followed by the sequential insertions of the MBL genes into the expression vector PIRES2-AcGFP. After transformation to E. coli DH5a, the recombinants were selected, amplified and sequenced. The interested sequences were then compared with the MBL gene sequences to confirm the correct insertion.3. Stable transfection of CHO cells by recombinant expression vector PIRES2-AcGFP-MBLThe recombinant expression vector PIRES2-AcGFP-MBL and the empty vector were transfected to CHO cells by using lipofectamine 2000 kit. After a sensitive toxicity test, G418 with certain dilution were put into cell culture medium and cell morphological changes and green fluorescence were observed every day until G418-resistant cell clones were selected. These clones were stable transfected by recombinant expression vector.4. RT-PCR analyse MBL expression in stable transfedted CHO cellsTotal cell RNAs were extracted and were reverse transcripted to CDNA. The MBL expression level in PIRES2-AcGFP-MBL transfected CHO cells were compared t to that in empty vector transfected CHO cells.5. Purificatioin of MBL protein by affinity chromatographyThe rMBL were purified utilizing the calcium-dependent carbohydrate binding by MBL. The culture supernatant of stable transformants was concentrated by centrifugal ultrafiltration and pretreated before pass a chromatography column packed with mannose-Sepharose. The bound MBL were eluted and concentrated and then identified by SDS-PAGE electrophoresis and Commassie Blue Staining.6. Quantitation of MBL by ELISAThe ELISA procedure was performed according to the Hbt human MBL ELISA kit and the absorbance was measured and the MBL level of tested sample was calculated according to standard curve.7. HCMV neutralization testHCMV was treated with each diluted MBL solution or both with mannan, and inoculated onto the MRC-5 cell monolayers for 2 hours. The cells were then washed and the HCMV-DNAs in cells were quantified by Q-PCR and the HCMV-PP65 antigens were tested by flow cytometry (FCM). Cells infected by PKH-26 stained HCMV were fixed and anti-vimentin was used to obtain immunofluorescence. The cells and HCMV were observed by confocal microscope.8. HCMV growth inhibition testAfter HCMV inoculation onto monolayers of MRC-5 cell for 2 hours, the cells were washed and incubated with several dilutions of MBL or with mannan at the same time. Then the cells were incubated for 3 days, every 24 hours, the supernatant of cell culture was tested for HCMV-DNA by Q-PCR. At 72 hour, cytopathic effect (CPE) were observed by the inverted microscope and cells were collected for HCMV-DNA quantitation and HCMV-PP65 test.Results:1. The sequence of extracted plasmid from pCR2.1-TOPO vector transformed cell DH5αclone were consistent with the MBL sequence and confirmed the establishment of MBL-CDNA TA clone.2. The MBL sequence from MBL-CDNA clone was inserted into expression vector. The resulting recombinant vector was transformed to DH5αcell to proliferation and then extracted and digested with endonuclease and two correct sequences were obtained with one is 5.3kb (vector) and one is 774bp (target gene).3. The recombinant expression vector and empty vector was transinfected into CHO cells.400μg/ml G418 was used to select resistant cells according to sensitive toxicity test. After 3 weeks, two cell lines transfected with recombinant expression vector and empty vector separately were obtained. The former was named as CHO-MBL.4. The total RNAs extracted from the above 2 cell lines were reverse transcripted and MBL gene were amplified by Q-PCR. The MBL expression level in CHO-MBL was 103 times higher than the empty vector transfected cell.5. CHO-MBL culture supernatant was concentrated from 2000ml to 200ml and 1.5ml rMBL obtained after affinity chromatography and enrich. The MBL concentration of the above 1.5ml sample was 800μg/ml according to ELISA. So the MBL concentration in culture supernatant was 600μg/L. SDS-PAGE electrophoresis and followed Commassie Blue Staining showed rMBL was a 32-KD protein under reducing condition and multimer>170KD under unreducing condition.6. HCMV neutralization test revealed 10μg/ml rMBL/hMBL significantly decreased the HCMV invasion in MRC-5 cell. However, the invasion of HCMV incubated with both 10μg/ml rMBL/hMBL and 20mg/ml mannan had no difference with untreated HCMV. 1μg/ml and 5μg/ml rMBL/hMBL have no such function. The infective rate of 10u.g/ml rMBL incubated HCMV was higher than 10μg/ml hMBL incubated HCMV. Confocal pictures showed after 2 hours of HCMV invasion, the virus were mostly located in cytoplasma and less HCMV invasion after MBL treatment were confirmed.7. HCMV growth inhibition test showed at 24th hour and 48th hour after HCMV invasion, there was no difference in the HCMV-DNA concentrations between MBL incubated cells supernatant and control cells supernatant. At 72nd hour, the HCMV-DNA concentrations in supernatant of 10μg/ml rMBL/hMBL incubated cells were lower than control cells. After 3 days of infection, both HCMV-DNA and HCMV-PP65 in 10μg/ml rMBL/hMBL incubated cells were lower than control cells.Conclusin:1. The CHO cell line express rMBL and the purified rMBL with high bioactivity were obtained.2. The concentration of rMBL in cell supernatant was 600μg/L and the majority of the purified rMBL was estimated to contain multimers larger than 170 KD.3.10μg/ml MBL can inhibit invasion of HCMV and prevents viral spreading to contiguous cells after 72 hours.4. MBL inhibit HCMV infection by binding the glucoprotein of HCMV and blocking its attachement and invasion to cytomembrane.