Human Cytomegalovirus Influences Host CircRNA Transcriptions During Productive Infection

Objective: Human cytomegalovirus(HCMV)is a beta-herpesvirus and has a double-stranded genome of approximately 230-kb in size.HCMV can infect many human cells,including fibroblasts,endothelial cells,neuronal cells,monocytes/macrophages,and dendritic cells.HCMV infection is ubiquitous among people.HCMV is a widespread virus that can cause life-long latent infection in large populations.It's estimated that the global seroprevalence of HCMV Ig G antibodies is 83%in the general population and 86% in women of childbearing,respectively.HCMV often causes asymptomatic infection in healthy individuals.However,HCMV is a life-threaten pathogen in congenitally infected newborns and immunocompromised populations.Congenitally infected babies manifest many kinds of clinical symptoms,such as congenital deafness,impairment in the central nervous system at birth or later in life.Although much research was focused on HCMV infection,the precise pathogenesis of HCMV infection still lacks till now.Circular RNAs(circ RNAs)are non-coding RNAs with a circular structure whose 5'and 3' terminus are covalently linked together.Circ RNAs were firstly discovered in viroid-infected plant cells in 1976.Nevertheless,circ RNAs were regarded as a byproduct of the RNA splicing process concerned with their low abundance and unclear functions for a long time.Gradually,more and more evidence revealed that circ RNAs exist extensively and play crucial roles in many biological processes in organisms,including serving as mi RNA or protein sponges and protein scaffolding.In recent years,studies were focused on the relationship between circ RNAs and herpes virus infections.It was reported that Kaposi's sarcoma herpesvirus(KSHV)infection significantly changed host circ RNA transcriptions in primary human umbilical vein endothelial cells(HUVECs)and MC116(a KSHV-infected B cell line).Those influenced host circ RNAs could repress viral gene expressions during infection.Circ BARTs produced by Epstein-Barr virus(EBV)were highly expressed in infected tissue and cell lines with potential functions in viral oncogenesis.Nonetheless,little is known about the relationship between host circ RNAs and HCMV productive infection.In our study,host circ RNA profiles of mock-infected and HCMV-infected human embryonic lung fibroblasts(HELF)cells were achieved by using RNA deep sequencing and bioinformatics analysis.Host circ RNAs significantly influenced by HCMV productive infection were compared and analyzed.Transcriptions and sequences of noteworthy circ RNAs(circ SP100,circ MAP3K1,circ PLEKHM1,and circ TRIO)were validated using RT-PCR and Sanger sequencing.Characteristics of circ SP100 were determined using northern blot and RT-quantitative PCR.circ SP100 binding proteins were investigated using RNA antisense purification and mass spectrometry,and the potential function of these proteins was predicted by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genome(KEGG).Methods:1.Circ RNA profiles of the mock-infected and HCMV-infected(72hpi)HELFs were achieved by using RNA deep sequencing and predicted by CIRI.The predicted circ RNAs were aligned with circ Base and classified by using ANNOVAR database and UCSC.2.Differential circ RNA transcriptions were calculated according to spliced reads per billion mappings(SRPBM)with edge R.Compared to circ RNAs in mock-infected cells,circ RNAs with >2(or <-2)fold changes and q-value < 0.05 were determined to be significantly differential circ RNAs.Cluster analysis of the parental genes of differential circ RNAs for Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genome(KEGG)were performed to assess the potential biological function of differential circ RNAs.3.Selected circ RNAs(circ SP100,circ MAP3K1,circ PLEKHM1,and circ TRIO)were validated by RT-PCR using RNase R treated total RNA extracted from HCMV-infected(72hpi)HELF cells.Linear SP100 and GAPDH were set as RNase R non-resistant control.Then,the sequence of the selected circ RNAs was achieved by Sanger sequencing.4.Characteristics of circ SP100 were determined using northern blot and RT-quantitative PCR(RT-q PCR).Potential functions of circ SP100 were investigated using RNA antisense purification and mass spectrometry and predicted by GO and KEGG.Results:1.A total number of 27409 and 35515 distinct host circ RNAs were identified in mock-infected and HCMV-infected HELF cells,respectively.The backspliced read of more than 96% of the recognized host circ RNAs was less than 100.More than 82% of host circ RNAs were derived from exons;the others were obtained from introns or intergenic regions.Host circ RNAs were transcribed extensively throughout the genomes and showed no difference in the distribution between mock-infected and HCMV-infected cells.Excluding intergenic circ RNAs,there were 40274 circ RNAs produced from 8138 host genes due to alternative splicing.By comparison,approximately 37% of circ RNAs identified in this study were annotated in circ Base,which the rest of was novel circ RNAs.2.It's demonstrated that 19724 host circ RNAs were found in both mock-infected and HCMV-infected cells,15791 host circ RNAs newly appeared after infection,and7,685 circ RNAs were found only in mock-infected cells.According to the threshold value of fold-change >2(or<-2)and q-value < 0.05,283 host circ RNAs were identified to be significantly changed by HCMV infection.Among them,transcriptions of 184 host circ RNAs were up-regulated considerably after infection,whereas transcriptions of 99 circ RNAs were down-regulated.GO term analysis and KEGG enrichment suggested that circ RNAs significantly changed by HCMV infection might play potential roles in regulating viral entry,apoptosis,and cell proliferation.3.RT-PCR indicated products of linear SP100 and GAPDH markedly decreased after RNase R treatment.No decrease was observed for circ RNA products after RNase R digestion.The above PCR products were cloned,and the entire sequence information of circ SP100,circ MAP3K1,circ PLEKHM1,and circ TRIO was achieved by Sanger sequencing.The information of these circ RNAs was consistent with our previous RNA-seq results and data in Circ Interactome.It's confirmed that the validated circ RNAs were exonic circ RNAs and consist of multiple exons except for circ PLEKHM14.Northern blots verified that cric SP100 was a sense transcript as predicted and increased after HCMV infection.Subcellular fractionation followed by RT-PCR suggested that circ SP100 predominately localized in the cytoplasm.The kinetics of SP100 m RNA and circ SP100 showed that circ SP100 levels were increased continuously post-infection,while SP100 m RNA levels were increased before 24 hpi and then dropped gradually.5.RNA antisense purification accomplished with mass spectrometry identified 291 proteins and 42 proteins in UV-crosslinked cells and the control cells,respectively.Excluding the 34 proteins in UV-crosslinked cells and control cells,257 proteins were identified as circ SP100-binding proteins,among which 10 were HCMV encoding proteins.About 59% of the circ SP100-binding proteins are located in the cytoplasm,whereas 37% of which were distributed in the nucleus.6.KEGG enrichment of the circ SP100-binding proteins suggested that these proteins were mainly involved in the spliceosome,protein processing in ER,ribosome,and phagosome pathways.It indicates that the multifunctional characteristic of circ SP100 during infection.Conclusion: HCMV productive infection influence the host circ RNA transcriptions.283 host circ RNAs were identified to be significantly changed by HCMV infection;the full-length sequence of circ SP100,circ MAP3K1,circ PLEKHM1,and circ TRIO are identified.Circ SP00 is a sense transcript and is mainly located in the cytoplasm.A total number of 257 circ SP100-binding proteins were identified.