The Formation And Modification Of Chromatin-like Structure Of Human Parvovirus B19 Regulate Viral Genomic Replication And RNA Processing

B19 virus(B19V)is a member of the genus of Erythroparvovirus in the family of Parvoviridae.B19 V contains a linear single-stranded DNA genome of approximately 5.6kb.B19 V and human bocavirus(HBoV)are the only two members of the Parvoviridae family to infect and cause illness in humans.B19 V infection causes a number of human diseases,such as erythema infectiosum,transient aplastic crisis,chronic anemia and hydrops fetalis when pregnant women are infected.B19V viral genome contains proximal polyadenylation site [(pA)p] and distal polyadenylation site [(pA)d].One of the limiting steps of B19 V infection is the viral genome replication,which resulted in more RNA transcripts reading through(pA)p and polyadenylated at(p A)d.The efficient splicing of the B19 V pre-mRNA within the second intron interfered with polyadenylation at(pA)p.It has been reported that the splicing within the second intron was also inhibited by the viral genome replication.However,the mechanism how B19 V genome replication affects its RNA processing is still unclear.Minute virus of mice(MVM)and adeno-associated virus(AAV)are members of the parvovirus and have been reported to form chromatin-like structure within hours after infection of cells,suggesting that the organization of viral DNA in nucleosomes is an essential step in the virus cycle.However,the function of chromatin-like structure in parvovirus is still unclear.In the present study,we sought to study whether B19 V genome DNA forms chromatin-like structure and whether the modification and state of viral chromatin affect viral genome replication and RNA processing.To investigate whether B19 V genome forms chromatin-like structure during replication.The nuclei were isolated from transfected HEK293 T cells at indicated time(12h,24 h,36h and 48h)and incubated with micrococcal nuclease.After blotting and hybridization with B19 V genome probe,the chromatin-like ladder of B19 V DNA was detected at 12 h post-transfection which was prior to the replication.The inhibitor of DNA methyl-transferase,5-Aza-2'-deoxycytidine(DAC),was used to treat HEK293 T cells that were co-transfected with B19 V genome DNA and pHelper plasmid.Hirt DNA was isolated after 48 h and digested with Dpn I.After treated with methylation detection kit and PCR followed by sequencing,we found that DAC treatment modulated the status of DNA methylation.To test whether the chromatin state or modification affect the viral genome replication and RNA processing,HEK293 T cells co-transfected with B19 V infectious clone and pHelper plasmid were treated with 20 ?M of DAC.At 48 h post-transfection,Hirt DNA,nuclei and total RNA were harvested for Southern blot and RNase protection assay(RPA)analysis.The results showed that DAC inhibited not only the chromatin-like structure formation and the replication of B19 V genome,but also B19 V RNA alternative processing.To study the mechanism of B19 V replication affecting polyadenylation at(pA)p sites,the 5'and 3' parts of B19 V genome was inserted into IRES2-eGFP and pBluescript KS II vectors,respectively.RNase protection assay was performed to detect RNA splicing at D1 and D2 sites.The results showed that B19 V genome replication regulates polyadenylation at(pA)p by affecting splicing of the second intron.Our results demonstrated that the formation and modification of B19 V chromatin-like structure play an important role in the regulation of DNA genome replication and RNA processing,suggesting a new layer to regulate the B19 V gene expression.Our study improves new insights to virus control,prevention and treatment.