Chromatin State Of Partial Viral Genome And Its Relation With Replication After Infection Of Neuro-2a Cells With Pseudorabies Virus

PRV(Pseudorabies virus),also known as Aujeszky's disease virus(ADV)or suid herpesvirus type 1,can cause pseudorabies(PR).PRV has a broad host range,infecting most mammals and some avian species.However,higher primates including humans are not susceptible.Pigs are the natural host of PRV,and infected herds are the natural reservoir of PRV.Since 2012,an unprecedented large-scale outbreak of PR of pigs in China has caused great economic losses in the swine industry.Therefore,Pseudorabies is a continuing focus on controlling pseudorabies in the pig industry.It is vital to study the PRV infection mechanism.The fundamental unit of chromatin is the nucleosome which is composed of145-147 bp of DNA sequence wrapped around a octamer of core histone(an H3-H4 histone protein tetramer associating with two H2A-H2 B dimer).There is 30-40 bp DNA to connect between adjacent nucleosomes.Core histones are highly conserved among organisms in evolution,H3 and H4 particularly.In 1991,Paul Laybourne and Kadonaga found that core histones(H2A,H2 B,H3,H4)wrapped the cloned DNA to form the nucleosomes which made a mild inhibitory effect on genome activity(approximately four fold).Upon entry of the viral DNA into the cell nucleus,host functions will assemble chromatin on the naked viral DNA to silence incoming genes,as is observed for transfected DNA.The assembly into chromatin poses a physical barrier to DNA access.It happens to herpes simplex virus type 1(HSV-1),human herpes simplex virus type 2(HSV-2),varicella-zoster virus(VZV),human cytomegalovirus(HCMV)and human herpes virus type 4(EBV).To date,during the process of PRV infection,the chromatin hasn't been detected,which provides a new prospect for the study of PRV infection mechanism.Therefore we employed chromatin immunoprecipitation(Ch IP)to determine the chromatin state of the partial PRV genome during lytic infection.Chromatin immunoprecipitation(CHIP)is a method to study the chromatin environment in vivo [1].It is used to capture and accurately determine the target geneinteracted with histone H3 in vivo,which is valuable for the relationship between histone H3 and DNA replication and transcription.Establishing the CHIP method will lay a foundation for the relationship between PRV replication and the viral genome chromatin structure.In the view of the above background,this study revealed three parts.The detailed contents of this project are as follows:1.Development of a SYBR Green-based real-time quantitative PCR assay for detecting Pseudorabies virus gE GeneThis study describes the development of SYBR Green based real-time polymerase chain reaction(real-time PCR)for detection and quantitation of PRV gE gene.The primers were designed and custom-synthesized based on nucleotide sequence of gE gene of PRV.A standard curve was plotted using 10-fold serial dilution of standard plasmid DNA and Ct value.The standard curve was found to be linear.The real-time PCR results were expressed as the number of DNA copies of PRV per mg of plasmid and showed range of 103 to 107 copies of viral DNA per mg of plasmid.The analytical sensitivity of the SYBR Green based real-time PCR was shown to be equivalent to 100 copies.Comparison between the results of three different assays revealed that real-time PCR is more sensitive than conventional PCR and allow the detection of low titers of PRV.A SYBR Green-based real-time quantitative PCR assay for detecting PRV gE gene was developed for the basis on the early and rapid detection and quantitatively evaluating the infect degree of PRV.2.Establishment of the CHIP-qPCR methods for detecting the mouse housekeeping genes binding with histone H3We developed a CHIP-qPCR methods for detecting the mouse housekeeping genes binding with histone H3,which offers a method and reference gene for further research on PRV chromatin.Neuro-2a cells were fixed with formaldehyde,sonicated to shear the DNA into 100-500 bp fragments and then immunoprecipitated with the specific anti-H3 monoclonal antibody.Antibody-enriched DNA was then amplified,labeled,and mixed with equal amounts of differentially labeled input DNA and amplified by qPCR.The results revealed that DNA fragments of 100-500 bp were obtained using 6 seconds on and 9.9 seconds off each time at the power of 150 W.The whole sonicated time is 20 minute.The correlation coefficient of recombinant mouse GAPDH-p MD18-T plasmid was 0.99,and it had a fine linear relationship between threshold cycle and template concentration during 103-107 copies/?L.The results ofqPCR show thatCt =|Ct(? GAPDH)-Ct(Ig G)|=11.14,in accordance with CHIP-qPCR conditionsCt =|Ct(? GAPDH)-Ct(Ig G)|?3.In our study,the CHIP-qPCR methods were successfully established to detecte the immunoprecipitated mouse GAPDH gene,which lay the foundation for further research on PRV chromatin after infecting Neuro-2a cells.3.Study of the relationship between part of PRV DNA sequences binding with host histone H3 with viral replicationFirstly,according to the established PRV gE qPCR method,the dynamic replication of the viral genome was determined after 0.1MOI PRV infected Neuro-2a cells during 12 h.Then cells that PRV infected Neuro-2a cells was collected and used to CHIP.It was found that part of the PRV genome was assmbled to chromatin structure,and this structure caused suppression to the replication of the viral genome.In our study,the CHIP-qPCR of PRV chromatin structure after infecting Neuro-2a cells was established successfully.This will provides a new prospect for the study of PRV infection mechanism,and lay a foundation for the research of PRV epigenerics.