Human Herpes Virus 7-invasive Preliminary Study Of The Molecular Mechanism Of Cd4 ~ + T Cells

Human herpesvirus 7 (HHV-7) was first isolated by Frenkel fromactivated CD4~+ peripheral blood T cells of a healthy individual in 1990. Itis currently known to belong to theβ-herpesvirus subfamily. HHV-7 has ahigh infection rate and can be mainly transmitted through saliva. HHV-7appears to be persistent throughout life after initial infection in earlychildhood. It has been suggested to be a cause of exanthema sbuitum andpityriasis rosea.HHV-7 mainly infects CD4~+ T cells and CD4 molecule can serves asa critical receptor for both HHV-7 and HIV. The antagonistic effectbetween HHV-7 and HIV could be exploited to devise therapeuticapproaches to AIDS. However, the precise mechanism by which HHV-7enters into CD4~+ T cells is still unknown. This study aimed to indentifythe essential viral proteins for HHV-7 entry into CD4~+ T cells and studythe potential interactions between these proteins and CD4 molecule.Since the basic entry machinery is conserved among theherpesviruses, based on the study of other herpesvirus, we hypothesizedthat glycoprotein B (gB), gH, gL, gO were sufficient and necessary forHHV-7 entry. In this dissertation, we cloned HHV-7 gB, gH and gO genes.Using yeast two-hybrid system, we studied the potential interactionamong gB, gH, gO and CD4. Finally, we characterized the essential viralproteins for HHV-7 entry. The main contents and results are as follows:1. Molecular cloning and sequence analysis of HHV-7 gB,gH and gO genes Human CBMCs were infected with HHV-7 (Nanjing local strainYY5). After CPE were observed, DNA was extracted. gB, gH and gOgenes were amplified by PCR and cloned into pUCM-T vectors toconstruct pUCM-T-gB, pUCM-T-gH and pUCM-T-gO. Then, therecombinant plasmids were transformed into E. coli Top10. RecombinantTop10 clones were identified by means of blue/white plaque screeningand PCR test. The inserted segments in the recombinant plasmids weresequenced. Nucleotide homology analysis indicated that the sequencesshowed about 99％identity to the sequences of both RK and JI strain inGenbank.2. Studying the potential interactions among HHV-7 gB, gH,gO and CD4 with yeast two-hybrid system.The ectodomain DNA fragments of gB, gH and gO genes wereanalyzed by bioinformatics and amplified by PCR. The amplified fulllength genes and the ectodomain DNA fragments of gB, gH and gO werecoloned into prey vectors. The recombinant prey plasmids and thepositive control plasmid pACT2-gp120 were transformed intoSaccharomyces cerevisiae Y190 containing the bait plasmid pAS2-1-CD4,respectively. The positive colonies were identified by means ofnutrient-deficient media screening, PCR test and Western blot. The resultsofβ-galactosidase assay showed that CD4 could combine with gp120,but not interact with HHV-7 gB, gH or gO.3. Characterization of HHV-7 glycoprotein-induced cell-cellfusionHHV-7 infection is dependent on the functions of structural glycoproteins at multiple stages of the viral life cycle. These proteinsmediate the initial attachment and fusion events that occur between theviral envelope and a host cell membrane, as well as virion-independentcell-cell spread of the infection. To characterize the HHV-7 glycoproteinsthat can mediate cell fusion, a cell-based fusion assay was used. 293Tcells expressing the HHV-7 glycoproteins of interest along with aluciferase reporter gene under the control of the T7 promoter werecocultivated with SupT1 cells transfected with T7 RNA polymerase.These results showed that HHV-7 glycoproteins gB, gH, gL and gO couldmediate the fusion of 293T cells with SupT1 cells, and the fusion couldbe inhibited by anti-CD4 mAbs.In summary, these results above showed that thecoexpression of gB, gH, gL and gO was sufficient and necessaryfor HHV-7 enrtry. And the ligand for CD4 might be a proteincomplex. This study provides the groundwork for an in-depthanalysis of the glycoprotein functions during HHV-7 entry.Understanding the molecular mechanisms about HHV-7 entrywill leads to the development of a new method for AIDSprevention and treatment.