Expression And Function Of The Protein Expressed By Human Cytomegalovirus UL49 Open Reading Frame

Background and objectivesUL49 is a herpes virus conserved Open Reading Frame (ORF).Up to now, besides that Walter Dunn and his team found UL49 was a essential gene for HCMV growth when they recovered the bacterial artificial chomosome containing human cytomegalovirus genome deleted UL49 ORF, the study of UL49 gene function limited only to the biophysical information analysis. For studying the biophysical function of UL49 gene during HCMV infection, it is necessary to unclose firstly the question as below:weather is the mRNA expression and how are the cDNA sequences? Can detect the UL49 ORF encoding protein? When do the protein express and where do the protein locate in virion or host cells? After answering the above question, we will observe pUL49 location in host cells infected HCMV and the biophysical function of pUL49 during HCMV infection and production, and also observed the target proteins interacting with the bait protein pUL49.MethodsHCMV AD 169 (HCMVA) was inoculated to well growth primary human embryonic lung fibroblasts, the HELFs were collected while the cytopathic effect appearing, and HCMVA RNA was extracted from the collected cells.5'RACE and 3'RACE of cDNA of HCMV UL49 were amplified by SMART rapid amplification of cDNA ends technology. The full lengh cDNA of HCMV UL49 was amplified by RT-PCR, and cloned the cDNA into pMD18-T simple vector and sequenced subsequently. The secondary structure and transmembrane domain were predicted by SOPMA and TMHMM respectively. Hydrophilicity, accessibility, polarity, flexibility, surface probability and antigenicity index predicted by methods of Kyte&Doolittle, Emini, Zimmerman and average flexibility. EMBOSS and Bopped Wu'method were combined and the possible B cell epitopes of pUL49 protein were predicted. The B cell epitome of pUL49 protein was synthesized and purified using chemical method then linked it with KLH and condensed with EDC to form immunogen KRFDARADLAVY-KLH. When New Zealand Rabbits were immuned by the immunogen. After collected serum from the immuned rabbits, the serum titer was detected using ELISA. pGEX-4T-3-UL49A from HCMV UL49 (aa3-246) was contracted and pUL49A-GST was induced using IPTG, pCDNA3.1-UL49A-myc from HCMV UL49 was contracted and the pUL49A-myc protein was obtained using instantaneous transfection method. Cell-free supernatants were collected from the medium infected HCMV seven days later. The pUL49 was finally detected from above three samples using Western blotting.Human foreskin fibroblasts (HFFs), after infected RV-Towne 3,6,12,24,48,72,96 and 120 hours, were collected and cracking, samples were confirmed by Western blotting using the monoclonal antibody anti-IE (CMVpp72/86,CH160:sc-69748, Santa Cruz Biotechnology, Inc.), anti-E (CMV gB, CH28, sc-69742, Santa Cruz Biotechnology, Inc.), anti-LA (CMVpp28, CH19, sc-69749, Santa Cruz Biotechnology, Inc.) and anti-HCMV pUL49. HCMV virion were collected from the medium HFFs appearing near 100% cytopathic effect, and purified using 58,750xg at 4℃.The purified virion were pretreated with triton X-100 or trypsin, then treated with 100000 g at 4℃. The pUL49 from above samples were finally detected using WB.HCMV BACs were extracted and purified by using a Nucleobond AX kit. The fragment of UL82 was amplified by PCR and cloned into the vector pcDNA3.1 (+). The vector pcDNA3.1 (+)-UL82 or pcDNA3.1 (+)-UL82 and pcDNA3.1 (+)-UL49 together with BACs were co transformed into HFFs respectively. Confluent cytopathic effect was observed from the infected cells by microscope. HFFs, after infected RV-Towne 3,6,12,24,48,72,96 and 120 hours, were collected and confirmed by immunohistochemistry using the monoclonal antibody anti-E, anti-LA, hochest33258 and anti-HCMV pUL49. The cells images were observed by fluorescence microscope. The purified RV were treated with anti-pUL49 at 37℃for 30 minutes, then infected HFFs with 1 PFU/cell. The cell-free supernatants after infected 1-7 days were collected to observe the growth curve. As above, the cells after infected 1-6 hours were collected and extracted total DNA from these cells, then amplified RV-Towne special gene IE1 and analyzed the product titer. The fragments of UL49 was amplified by PCR, and then cloned into the bait expression vector pGBKT7. The bait vector pGBKT7-UL49, being verified by sequencing, was transformed into AH 109 yeast cells. Then Western Blotting analyzed the bait protein pUL49. Toxicity and self-activation of the bait protein were detected by cultured in different SD cultures. Human embryonic kidney cells CDNA library together with pGBKT7-UL49 were co transformed into yeast cells AH109. The positive clones were selected in different medium deleted nutrient, and detected by filter assay. The positive vectors were transformed to DH5 a and extracted plasmid.Designed PCR primer of cDNA library vector, and the positive gene sequences were amplified, the PCR products also digested by HaeⅢ. The positive sequences were identified by DNA sequence analyzer. With Genebank BLAST and other analysis software, the positive genes were recovered by hybrid and filter assay. Amplified the genes CYB5D2, C11ORF17, COL3A1 and UCHL3, the four fragments were cloned into the vectors pGEX4T-3, pCDNA3.1 (+)-UL49 was constructed meantime. The fusion proteins of CYB5D2, C11ORF17, COL3A1 and UCHL3 with GST were induced by IPTG respectively, pUL49 was expressed also. According to reference, pUL49-3Xflag together with the GST fusion CYB5D2, C11ORF17, COL3A1 and UCHL3 proteins were done GST-pull down test respectively. pCDNA3.1(+)-COL3A1 and pCDNA3.1(+)-C11ORF17 were constructed, and they were transformed into HFFs respectively,24 hours later the cells were infected RV with 1.OPFU/cell. The cell-free supernatants after infected 1-7 days were collected to observe the RV growth curve. The cell-free supernatants after infected 1-7 days were collected to observe the RV growth curve.ResultsWe infected HCMVA successfully to well growth HELFs. and HCMV typical genes IE1 and LA were identified using PCR. Using Smart assay, we obtained and cloned the 3'and 5'termination of UL49 gene. Sequencing results suggested its 3'- untranslated region and 5'-untranslated regions were 315bp and 90bp respectively. The full-length cDNA of UL49 gene,2118bp, was amplified using RT-PCR.The pUL49 protein N-terminal amino acid residues locating at 228-243 regions were predicted by all prediction methods, and this regions containedβsheet and coils structure, which regions or their surrounding regions were the predominant epitomes. The B cell epitomes of pUL49 protein were synthesized and purified, linked with KLH and condensed successfully with EDC. Immunogen KRFDARADLAVY-KLH was formed finally. After immuned, immuned rabbit serum was collected and then detected the titer being 1:8000. pGEX-4T-3-UL49A (3-246) was contracted and the pUL49A-GST was induced, pCDNA3.1-UL49 was contracted and pUL49-myc protein was obtained, the pUL49 from above three samples were detected.pUL49 protein began to express after HFF infected RV-Towne 12 hours and the maxim titer appeared after infected RV-Towne 96 hours. pUL49 expression kinetics was related with the type HCMV gene gB.pUL49 WB image was related with the type HCMV gene pp28 after the samples treated with TX-100 and trypsin.We succeed in constructed the pcDNA3.1(+)-UL82 and pcDNA3.1(+)-UL49, the HFFs co transformed with pcDNA3.1(+)-UL82 or together with pcDNA3.1(+)-UL49 and Towne-BACs were observed green fluorescence 15 days later. pcDNA3.1(+)-UL82 together only with pcDNA3.1(+)-UL49 and delTowne-BACs were observed green fluorescence 20 days later. pUL49 appeared red fluorescence in infected cells 12 hours later and located in cytoplasm, and pUL49 appeared from spreading uniformly before infected 24 hours to condense somewhere region. pUL49 was also collocated with pp28 at infected 72-96 hours. The growth curve of RV treated with anti-HCMV pUL49 reduced 10 times than untreated with anti-HCMV pUL49. The DNA of RV treated with anti-HCMV pUL49 was not different to untreated with anti-HCMV pUL49 during virus invasive period.UL49 was amplified and cloned into pGBKT7 successfully. The vector pGBKT7-UL49 was transformed into AH 109 as well and those cells exhibited neither toxicity nor self-activation. Western Blotting detected the expression of the bait protein pUL49. Human embryonic kidney cells CDNA library together with pGBKT7-UL49 were co transformed into yeast cells AH 109.30 positive clones were selected in different medium deleted nutrient and filter assay.11 none repeated positive vectors were identified by PCR and HaeⅢdigestion.4 positive genes were recovered hybrid and filter assay, gene C11ORF17 and COL3A1 appeared stronger ability to interact with the bait protein pUL49. The vectors pGEX4T-3 with above four fragments were constructed, pCDNA3.1 (+)-UL49 was constructed meantime. The fusion GST proteins, pUL49 protein were expressed. pC11ORF17 and pCOL3A1 proteins appeared positive pull down test result. pCDNA3.1(+)-COL3A1 and pCDNA3.1(+)-C11ORF17 were constructed. The HFFs expressed pCDNA3.1(+)-C11ORF17 appeared lower virus growth than normal HFFs, but the pCDNA3.1(+)-COL3A1 appeared no affection virus growth to normal HFFs.ConclusionWe observed the mRNA expression of HCMV UL49. The cDNA sequence were 2118bp in size, and its 5'-untranslated region were 90bp, while its 3'-untranslated region were 315bp. HCMV UL49 open reading frame were 1710bp and capable of encoding 570 amino acids.Prediction of the secondary structure and B cell epitopes of pUL49 protein laid the foundation for studying the characteristics of the protein and developing the epitope-based the monoclonal antibody against pUL49 protein. ImmunogenKRFDARADLAVY-KLH was synthesized to the B cell epitopes of pUL49 protein. We obtained high titer and strong specific rabbit serum anti-HCMV pUL49. pUL49, HCMV UL49 ORF encoding protein, expressed in the host cells infected HCMV, and its expression kinetic was early period. That pUL49 endured TX-100 and trypsin was near to pp28, these two proteins maybe located one region in virion. We succeed in recovering the recombinant HCMV (RV) from the recovered BACs containing HCMV genome. pUL49 was the essential gene for Towne growth. In HFFs expressed pUL49. delTowne-BACs only recovered partly. pUL49 was located at cytoplasm in infected cells and was recruited to somewhere region during infected later period. pUL49 appeared collocated in endoplasmic reticulum with pp28 at 72-96 hours.Anti-pUL49 affected the growth of RV, but did not affect virion entry to its host cells.The bait expression vector of UL49 was constructed successfully, which laid the foundation for screening target proteins interacting with the bait protein pUL49 using the yeast two-hybrid technique. With hybrid test, we obtained 11 none repeated positive vectors. From 4 positive genes, gene C11ORF17 and COL3A1 appeared stronger ability to interact with the bait protein pUL49. Which result was reinsured by GST pull down test. The interaction between pUL49 with pC11ORF17 maybe affects growth and proliferation of HCMV in HFFs, but the interaction between pUL49 with pCOL3A1 maybe not affects growth and proliferation of HCMV in HFFs.