Discussion Of The Relationship Between Human Herpesvirus 7 Surface Glycoprotein Gh, Gl Of Cells And The Cd4

Human herpesvirus 7(HHV-7) was first isolated from the peripheral blood of a healthy individual in 1990 and has shown to be a CD4~+ T-cell-tropic herpesvirus. HHV-7 has a high infection rate and can be mainly transmitted through saliva. HHV-7 appears to be persistent throughout life after initial infection in early childhood. It has been suggested to be a cause of exanthema sbuitum, pityriasis rosea and CMV syndrome after renal transplantation.HHV-7 and HHV-6 are included in the β -herpesvirus subfamily. They were found to contain sequences homologous with the average relatedness amounting to 60% in the nucleotide sequences and 53.4% in the corresponding amino acid sequence. They have similarity in host range, infection mode and clinical symptom. During the course of studying HHV-6 glycoprotein, gH (binding to gL) was found to be a ligand of CD46, the receptor of HHV-6, and play an important role in virus invading host cell. In other herpesvirus, it was also found gH (binding to gL) to act in concert to induce membrane fusion of virus to host cells and of infected cells to adjacent cells. As we known, CD4 is an important receptor of HIV and HHV-7. HIV gp120 can combine with CD4 and induce virus entry. The glyprotein of HHV-7 that combine with CD4 is still unknown. Finding this protein will give an aid in the prevention and cure of HIV infection. Whether HHV-7 gH can bind to gL and show important role in viral invade and binding to CD4 need to been study.In this dissertation, we cloned HHV-7 gH, gL genes and the gene encoding CD4. Using yeast two-hybrid and co-immunoprecipitation, we studied the interaction among gH, gL and CD4. Finally, we analysed the influence of gH, gL to CPE, induced by HHV-7. The main results are as following:1. Cultivation and identification of HHV-7HHV-7 (strain Glasgow) was cultured with human CBMC andSupT1. After 10-15 days, CPE were observed. The infected cell DNA has been amplified by PCR and the amplified 439bp fragments were sequenced. The nucleotide homology analysis indicated that the sequence show 99.9%-100% identity to HHV-7 (strain JI and strain RK).2. Molecular cloning and sequence analysis of HHV-7 glycoprotein gH, gL and CD4 gene(1)SupT1 was infected by HHV-7 (strain Glasgow). After CPE were observed, extract DNA and amplified gH and gL gene by PCR. The amplified fragments were cloned into pBS-T vector to construct pBS-T-gH and pBS-T-gL, and transformed into E. coli DH5 α . Recombinant DH5 a were identified by means of blue/white plaque screening and PCR test. The cloned DNA in the recombinant plasmid (pBS-T-gH and pBS-T-gL) was sequenced. Nucleotide homology analysis indicated that the sequence was identical to HHV-7 (strain RK) gH and gL genes.(2)Extracted SupTl RNA and amplified CD4 cDNA by reverse transcription-PCR. The amplified fragments were cloned into pAS2-l vector and transformed into E. coli DH5 α. Recombinant DH5 a were identified by means of ampicillin screening, PCR and endoenzyme digestion test. The cloned DNA in the recombinant plasmid pAS2-1-CD4 was sequenced. Nucleotide homology analysis indicated that the sequence show 99.8% identity to the sequence in documents and in Genbank.3. Studying the interaction of HHV-7 gH, gL and CD4 with yeast two-hybrid.In yeast two-hybrid, two protein genes respectively insert into specified vectors to construct bait plasmid and prey plasmid. Interactions between proteins can be judged according to the expression of the reporter gene.Studying interactions between CD4 and HHV-7 gH, gL: Bait plasmid pAS2-1-CD4 was constructed and transformed intoSaccharomyces cerevisiae Y190. Recombinant Y190 were identified by means of nutrient-deficient media screening and PCR test. Then test pAS2-1-CD4 for their autonomous activation. At the same time, construct prey plasmids, pACT2-gH, pACT2-gL and positive control prey plasmid pACT2-gp120, respectively. The prey plasmids were transformed into Y190/pAS2-1-CD4 and identified by means of nutrient-deficient media screening and PCR test. Results of β-galactosidase assay showed the CD4 can combine with gp120, but have no interactions with HHV-7 gH,gL.Studying interactions between HHV-7 gH and gL: Construct bait plasmid, pAS2-l-gL, and prey plasmid, pACT2-gH respectively. Transformed them into Y190. Results of β -galactosidase assay showed HHV-7 gH and gL can interac on each other.4. Eukaryotic expression of HHV-7 glycoprotein gH, gL and preliminary study of their functionsAmplified gH and gL DNA by PCR from pBS-T-gH and pBS-T-gL vector. The amplified fragments were cloned into yeast expression vector pPICZaA to construct pPICZaA-gH and pPICZaA-gL. After identify, the vectors were linearized with endoenzyme SacI and transformed into Pichia pastoris GS115 with electroporation. Recombinant GS115 were identified by means of zeocin screening and PCR test. During fermentation, methanol was used to induce the expression of protein. The recombinant proteins in culture supernant were identified by SDS-PAGE, and the recombinant gH was further identified by western blot. The results showed recombinant GS115/pPICZαA-gH 和 GS115/pPICZaA-gL were found to express gH and gL.The culture supernant, containing recombinant proteins, were supplemented into the cultures of SupT1, which were infected with HHV-7. Observed the results. No obverse effects to CPE of SupTl were found after adding gH or gL culture supernant. After adding gH and gL culture supernant together, the CPE of SupT1 was reduced.5. Studying the interaction of HHV-7 gH, gL and CD4 with co-immunoprecipitation.Adding the Protein A/G Plus-Agarose, which chelating recombinant gH, into GS115/pPICZaA-gL culture supernant. Incubate and then centrifuge to get precipitation. The proteins in precipitation were separated and identified by SDS-PAGE. The result of SDS-PAGE suggested that gH and gL can interact with each other during co-immunoprecipitating.Adding the Protein A/G Plus-Agarose, which chelating recombinant sCD4, into GS115/pPICZaA-gH culture supernant, GS115/pPICZaA-gL or their mixture. Incubate and then centrifuge to get precipitation. The proteins in precipitation were separated and identified by SDS-PAGE. The results of SDS-PAGE suggested that CD4 cann't interact with gH, gL or their mixture.In summary, the results above suggested that HHV-7 gH, gL can form complex, this complex can reduce CPE of HHV-7 infected host cell. Although CD4 can combine with gpl20, there were no direct interactions between CD4 and gH, gL or their mixture, so the viral ligand of CD4 still need to be investigate. Such results provide experimental and theoretical basis for the further researching in mechanism of glycoprotein functions during HHV-7 infection.