| Background:Viral hepatitis C is a type of viral hepatitis caused by the hepatitis C virus,it has become a global public health problem.According to statistics from the World Health Organization,more than 70 million people(around 1%of the world’s population)worldwide suffering from chronic hepatitis C infection as of 2017,and resulting in approximately 399,000 deaths each year mainly due to cirrhosis and hepatocellular carcinoma.Lower than the Eastern Mediterranean Region(2.3%)and European Region(1.5%),the estimated infection rate of hepatitis C virus in China is nearly equal to global prevalence(1%).However,regarding the population base,the number of hepatitis C virus carriers in China(14 million)is the largest in the world.Although some previous studies on HCV genotypes,conducted on small populations and narrow geographical areas,have been performed in China,little is known about the recombinant variant information and positive selection sites of circulating HCV strains in mainland China.After all sequences are aligned with the reference sequence,a phylogenetic tree is constructed by MEGA 10 software using the maximum likelihood method for phylogenetic analysis.Methods:Based on NCBI nucleotide sequence database(http://www.ncbi.nlm.nih.gov),Virus Pathogen Database(http://www.viprbrc.org)and HCV database(https://hcv.lanl.gov),140 sequences of HCV were included in this study.After aligning all the virus strain sequences with the reference strain H77 strain sequence,phylogenetic analysis was carried out by MEGA 10 software using the maximum likelihood method,and recombination analysis was carried out by RDP5 software and SimPlot software.After the sequences with recombination signals are excluded,and the rest of the sequences are compared with the reference sequences,at last,the Datamonkey network server is used to analyze the selection pressure.Results:By constructing the maximum likelihood tree,it was found that genotype 6 was the most popular hepatitis C genotype in Chinese mainland in recent ten years.Five groups of potential recombination events were identified by RDP5 and Simplot software,including one inter-genotype recombinant virus strain(strain HH075)and four intra-genotype recombinants virus strain(strains WYHCV286,GB28,GZ2983,and HCV156).Using Datamonkey network server to analyze the selection pressure,it was found that most of the codon sites were negative selection,while the number of positive selection sites identified by SLAC,FEL and FUBAR methods were 5,12 and 7,respectively.Five sites identified by SLAC method also appeared in the results of the other two analysis methods.Specifically,two sites in the core gene(codon 72 and 75),codon 395 in the E2 gene and two codon sites in the NS5B gene(codon 2537 and 2540)are being selected.Conclusion:HCV has genetic diversity in Chinese mainland,and the recombination within genotypes and subtypes may be one of the reasons for HCV genetic diversity.The results of selection pressure analysis showed that most codon sites were in negative selection state,indicating that HCV variation was mainly accumulated due to random genetic drift,while codon 2537 and 2540 of NS5B gene were under positive selection,which may have some influences on HCV replication and the resistance of DAAs.Core protein of HCV exerts inhibitory functions in many vitally important immune cells,two positive selection sites identified in this study within the core gene may be partially responsible for the HCV chronic infection.Fully considering the impact of mutations,recombination,and selective pressure in shaping the genetic diversity and evolution of HCV will facilitate the development of broad-spectrum antiviral drugs and effective vaccines,and also helps to establish an appropriate therapeutic DAAs program.Background:Circular RNA(circRNA)was identified in the 1990s as an important type of non-coding RNA,It can be used as a competitive endogenous RNA to adsorb miRNA molecules(miRNA sponge),or it can be combined with RNA binding protein(RBP),both of which can play a role in transcription The role of post-regulation;there is also a part of circRNA that plays a role of transcriptional regulation.However,the above are the functions of some special circular RNAs,and the functions of a large number of circular RNAs are still unknown and require further exploration by researchers.In the previous study,we systematically analyzed the circRNA expression profile of SV40,and discovered a virus-derived circRNA—circ-17kT for the first time,but its formation mechanism and function are still unknown.Methods:After SV40 virus infects Vero cells,the RNase R resistance analysis experiment proved that circ17-kT is a circular RNA molecule.First,we use splice bodies to recognize the splice site of the RNA precursor and catalyze the splicing reaction,as well as the 5’splice site(5’SS)and 3’splice site(3’SS).The classic splice site is the two important features of the joint site where the intron and exon in the RNA precursor can be recognized by the splicing body.The two experiments of splicing body suppression and splicing site mutation are used.The role of the elements related to the RNA splicing process in the formation of the circular RNA is studied.Secondly,we investigate the formation mechanism of circ-17kT by exploring the functions of exons and their flanking introns.Introns play an auxiliary role in the formation of circular RNAs,and some circular RNAs need the help of introns during the formation of circular RNAs.Therefore,after pre-determining the non-repetitive and reverse complementary sequence characteristics of the introns on both sides of the second exon(Exon 2),we designed the second exon(Exon 2)to be truncated.The intron truncation experiment.Exon skipping is an important way to form circular RNA,so we designed an exon skipping experiment from the first exon(Exon 1)to the third exon(Exon 3).Finally,the strategy of plasmid overexpression and antisense oligonucleotide(ASO)interference with circ-17kT expression was used to study the effect of circ-17kT on the large T antigen(LT)gene of its maternal gene.Its relationship with the ability of the virus to multiply.Results:RNase R digestion proved that circ-17kT is a circular RNA produced by SV40 virus infecting Vero cells;when the splicing body is inhibited by Isoginkgetin,the expression of circ-17kT produced by SV40 virus infecting Vero cells has no significant change;The 5’splice site(5’SS)and 3’splice site(3’SS)are mutated to construct plasmid transfected cells,and Vero cells can still be normal after transfection Expression of circ-17kT.Intron truncation experiments found that when the flanking introns of Exon 2 of the T gene forming circ-17kT were truncated,the formation of circ-17kT had no effect,the first exon(Exon 1)to the third The exon skipping experiment of two exons(Exon 3)indicated that circ-17kT may not be produced by lasso precursor processing containing Exon 2.This part of the results requires further experimental verification.Overexpression of circ-17kT can positively promote the expression of SV40 large T antigen gene mRNA,but compared with the control group,the SV40 virus titer does not change significantly;while inhibiting the expression of circ-17kT,the LT expression significantly decreases and the SV40 virus titer Significantly reduced.Conclusion:During the formation of circ-17kT,splicing bodies do not need to provide the splicing function,and the formation of circ-17kT does not require the formation of lasso precursors,nor does it rely on the help of flanking introns.Preliminary experiments show that circ-17kT is not formed by exon skipping.When the splicing site is mutated and the flanking introns of Exon 2 are truncated,it does not affect the formation of circ-17kT,suggesting that there are other cyclization mechanisms driven by RBP or trans factors in the formation of circ-17kT.Preliminary functional analysis showed that circ-17kT can positively promote the expression of its maternal gene LT.The preliminary results suggest that circ-17kT can inhibit the proliferation of SV40 virus when the expression is inhibited. |
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