Study On Human Cytomegalovirus Reactivation From Monocyte And The Structure Of UL16-UL17 Transcripts

Background : Human cytomegalovirus(HCMV)belongs to betaherpesvirinae of herpesviridae family.The population infection rate around the world is 60%?90%.Once infected people,the virus set up a latent infection state all over the life.The current research reveals that the HCMV mainly latent in Hematopoietic stem cells and monocyte system.The latency and reactivation process is associated with part of HCMV genes,such as LUNA and UL138.The reactivation is also related with monocyte differentiation.The molecular mechanism of HCMV latency and reactivation is not well understood up to now.It could reactivation when hypoimmunity and lead to severe clinical symptoms.The infection in pregnancy could lead to cytomegalic inclusion disease.A small number of newborns present with microcephaly and low intelligence.The severe infection leads to abortion or dead fetus.Perinatal period infection could lead to newborn jaundice or hearing loss.Reactivation of organ transplant patient could lead to transplant failure even death.The reveal to the core part of reactivation will make big progress to the prevention and treatment of reactivation related disease.The major aspects of reactivation,including cell differentiation,receptors,cell to cell interaction,will be studied in latency model and natural infection cells.Then,the mechanism of the factors to play function will be study.HCMV is a double strand DNA virus,which consists of 230-235 kilo bases.It is predicted to contain 751 open reading frames(ORFs).In previous studies,three transcripts were detected from the UL16-17 region by northern blot analysis for Merlin strain.Transcriptions of UL16 and UL17 were also studied by 5' rapid amplification of c DNA ends(5'RACE)and deep sequencing for AD169 and Towne strains,respectively.However,details of 3' end of UL16 and UL17 transcripts have never been confirmed by3'RACE.The expressing phage of the UL16-17 region needs further research by northern blot,too.The uncertainty of start site,stop position and accurate phase of transcription will hinder protein function study in the field of exogenous expression and gene edit.c DNA library screening,northern blot and RACE technology was used in this study to reveal the exactly transcription information of UL16 and UL17 for future functional research.Methods:1.The difference of infection rate and mechanism between monocyte and fibroblast to HCMV was studied.THP-1 cell was infected with HCMV Han strain and passaged or sustained to form latency infected monocyte model.Real time quantitative PCR were used to detect the infection rate.Flow cytometric cell sorting and magnetic beads cell sorting were used to separate CD34+ hematopoietic progenitor cells and CD14+monocytes from mobilized peripheral blood.PCR and q PCR were used to examine HCMV DNA.q PCR was used to detected the mRNA expression level of HCMV receptors and endocytosis associated proteins CAV1 in THP-1 and HFF.The possibility that the receptor difference affect the infection rate will be analysed.Lentivirus technology was used to construct PDGFRA or CAV1 over expression cell lines.q PCR and IFA were used to detect the expression and location of PDGFRA and CAV1 in the stable transfection cell line.HCMV infection rate was detected with q PCR.Improving detection stability by improve infection rate will be evaluated.2.The difference of HCMV transcription activity between monocyte and fibroblast was researched.PCR was used to detect the gene expression trend with time of HCMV in latency model.Mononuclear cells from patients with active infection were isolated with density gradient centrifugation.10 X single cell sequencing was used to detect HCMV transcripts in the natural infected monocyte.The difference between subsets of mononuclear cells wil be analysed.Mass spectrometry analysis technology was used to analyze the protein profile of different cells lines,which supply different environment to HCMV.The proteome associated with HCMV activity will be analysed with bioinformatics.3.The requirement and factors of reactivation from latency was researched.THP-1was differentiated into macrophage with TPA.PCR and q PCR were used to study the impact of differentiation to the infection rate and HCMV activity.The virus titration method was used to detect the progeny virions titer in the medium before and after TPA treatmentment.The impact of differentiation to the HCMV activity was analysed.Latency infected THP-1 was co-cultured with HFF.PCR,q PCR and virus titration methods were used to analyse the impact to HCMV activity of cell to cell contact.4.The structure of UL16,UL17 transcripts was studied.c DNA library screening with UL16 and UL17 special primers was used to screen the UL16 and UL17 transcripts from HCMV late phase c DNA library.Northern blot were used to identify the transcription phase of UL16 and UL17.RACE was used to identify the transcription start and stop sites of the UL16-17 region.Bioinformatics technology was used to analyse the cis acting elements,such as promoter and transcription stop signal.Results:1.The infection rate of monocyte to HCMV is partially restricted by the low expression level of receptors.The infection rate of Han to THP-1 is smaller than 1% even with 10 MOI.HCMV DNA is not detected in the CD34+ hematopoietic progenitor cells and CD14+ monocytes with one round PCR.HCMV DNA can be detected occasionally from CD14+ monocytes with nested PCR.The HCMV receptors,PDGFRA,NRP2 and EGFR,were high transcribed in HFF and low expressed in THP-1.Overexpression of CAV1 alone did not increase the infection rate of Han to THP-1.Overexpression of PDGFR alone could slightly increase the infection rate of Han to THP-1.2.The transcription activity of HCMV was restricted in monocyte.There is a decreasing trend of HCMV gene expression in THP-1,both with sustained culture and passaged culture.Lytic gene and latent gene have no obvious different expression trend in THP-1.HCMV transcripts were not found with 10 X single cell sequencing from11593 mononuclear cells(including 1244 monocytes).In the supportive cell lines,ARPE-19,U373,and HELF,the high expressed genes are enrichment at cytoskeleton assembling and cell differentiation compared to THP-1 cell.Transcription factors,Rel,NFATc2,MEF2 D,IRF5 and Elf1 are high expressed in THP1.3.Differentiation and contaction to fibroblast were beneficial to HCMV reactivation from THP-1.TPA treatment could improve HCMV infection rate 4 times in THP-1.HCMV DNA replication improved 8 times after TPA treatment at 7 days post infection.Coculture with HFF could lead to HCMV activation in THP-1.TPA treatmen could improve the speed of activation.4.3 clusters of transcripts with the same 3' end were found to be expressed from the UL16-17 region in both Han and AD169 strains.The lengths of the core transcripts among the 3 clusters were 1,254 nt,718nt and 468 nt,respectively.The corresponding 5'ends are at nt23119,nt23655,nt23905 in the HCMV Han genome.The consistent 3' end is located at nt24372 in the Han genome.The 1,254 nt and 468 nt transcripts are transcribed in early and late phases,and the 718 nt transcript is transcribed only in the late phase.Conclusion: The infection rate of HCMV to hematopoietic progenitor cells,monocytes and THP-1 is low.The low infection rate may be due to the low expression of HCMV receptors.There was no obviously expression style difference between UL138 and UL123 during infection of HCMV to THP-1.Co culture could improve HCMV activity in THP-1.TPA treatment could significantly decorate the internal environment of THP-1and make it friendly to HCMV.There are mainly 3 clusters of transcripts in UL16-UL17 region.There was no significant difference in transcription between Han strain and AD169 strain in UL16-UL17 region.