Analysis The Transcriptional Differences In T98G Cells During Human Cytomegalovirus From Latency To Reactivation

Objective:Human cytomegalovirus(HCMV)is universally infected in the population,and it is a herpesvirus with both latent and lytic life cycles.HCMV are more susceptible to reactivation than healthy people in neonates,organ transplants and HIV-immunocompromised patients,with leading to severe complications and even death.HCMV only infects humans because of it`s strict species specificity and there is no good animal model to learn about it.The study of latency to activation often uses cell models.In this study,the latency and reactivation model of HCMV in glioma cell T98G was established,and the infected cells with latent and activated states at different time points were collected for high-throughput sequencing of transcriptome.Analyze transcriptional regulation related genes of host cell with significant differences under latent and activated infection states.To explore the role of gene transcriptional regulation mechanism in the reactivation of HCMV infection.To provide an experimental basis for further exploring the mechanism of HCMV latency to reactivation.Methods:1.1.Establish a latent model of HCMV-infected T98G cells:Establish a latent model of HCMV-infected T98G cells:Serum starved(78 h)T98G cells were plated in 150 mm cell culture dishes(2×10~6 cells/dish).After the cells adhered to the wells within 2 hours,the T98G cells were infected with HCMV at multiplicity infection of 10.Removed the inoculum and washed the cells once with PBS 16 hours post infection then replaced with new medium.The cells were cultured for 7 days and passaged.During the passage,cell samples of each generation were collected to detect viral genome copy number and RNA transcription levels of related genes.The cell supernatants were collected for viral titer determination.The first generation of cells was defined as P0,followed by P1,P2,...PX.Infected cells that still maintained viral genomes but no detectable infectious virus particles in the supernatant were defined as latently infected cells.2.To establish a latency to reactivation model of HCMV in T98G cell:latent cells were added with c AMP/IBMX(using a final concentration of100uM)to change the medium every 4 days,and the supernatants were collected for virus titer determination.Collect infected cells at 4,8,and 12 days.3.The Illumina Hiseq4000 sequencing platform was used to perform transcriptome high-throughput sequencing of T98G cells induced at different times.Using HISAT and Bowtie2software to compare the reference genome and the reference gene.The sequencing results were analyzed using BGI Dr.Tom system.4.Using siRNA interference technology to interfere with CEBPD expression and verify the effect of CEBPD on HCMV reactivation.Results:1.The latency to reactivation model of HCMV in T98G was successfully established.There was almost no expression of green fluorescent protein(GFP)in T98G infected cells of P7 generation.The RT-PCR method cannot detect the transcription of HCMV UL54,UL99 and other lytic infectious genes,and there were no infectious virus particles in the supernatants of infected cells.The above results indicate that the T98G cell model of HCMV latent infection was successfully established.Latently infected T98G cells were added with cAMP/IBMX for 4 days.We found that some cells expressed GFP and detected the transcription of UL54,UL82 and other genes by RT-PCR and observed infectious virus particles in the supernatant of infected cells.These indicated that latently infected HCMV in T98G cells was successfully activated.2.High-throughput sequencing analysis of cellular transcriptome.Compared with latently infected cells,8367 cell differential genes were obtained after adding the inducer,and 485 transcription factors were enriched.The mRNA expression of the transcription factor CEBPD after induced for 24 hours compared to latency cells increased significantly.3.High-throughput sequencing analysis of viral transcriptome.The virus genes were compared by using the Towne strain(FJ616285.1)of HCMV as a reference.A total of 145 genes were matched with Towne strain.Most of the up-regulated genes were immediate early genes and early genes.4.Compared with the DMSO control group,the expression of p-AKT protein in PI3K-AKT pathway was up-regulated after adding cAMP/IBMX.5.Using siRNA interference technology to successfully down-regulate CEBPD protein expression,the phenomenon of HCMV from latency to reactivation in T98G cells was suppressed.Conclusion:1.cAMP/IBMX added to cytomegalovirus latently infected T98G cells after 24 hours,resulting in changes in the intracellular molecules which is a key factor for the latency to reactivation of human cytomegalovirus.2.c AMP combined with IBMX for 24 hours induced latent infection to reactivation of cytomegalovirus inT98G cells,which made differences in the transcription factor CEBPD could promote the process of viral reactivation.3.The changes in the PI3K-AKT pathway that may play a role in viral reactivation as a result of jointly using cAMP and IBMX for 24 hours in cytomegalovirus latently infected T98G cells.