Study On Human Cytomegalovirus On Transcription And Translation Mechanism Of Human Ribosome Large Subunit Protein 4 Gene

Objective:Human cytomegalovirus（HCMV）,a double-stranded DNA virus,belongs to the subfamilyβ-herpesviruses and has a genome of approximately 230-235 kb,which is the largest genome of any known herpesvirus^[1].HCMV is commonly infected in the population and can be latent for life,with a 95%prevalence of previous infections in adults in China.HCMV is a latent infection and is usually asymptomatic in immunocompetent populations,but in immunocompromised patients（e.g.,AIDS patients,organ transplant recipients,etc.）latent HCMV infection can be reactivated into active infection,and in severe cases,lethal infection can occur^[2].HCMV is also the most important pathogen of fetal congenital infection leading to birth defects^[3],and can cause jaundice HCMV can cause multi-organ and multi-systemic pathologies such as jaundice,biliary atresia,congenital megacolon,microcephaly,mental retardation,and deafness^[4,5].Therefore,HCMV infection is a great danger and brings a great burden to families and society.HCMV is one of the most complex viruses in terms of structure,replication process and host interaction,and the pathogenic mechanism of its infection is complex and far from clear.The study of the interaction with host cells during HCMV infection,especially the regulation of the biological functions of host cells by the virus,is of great importance to reveal the pathogenic mechanism of the virus.The damage of HCMV infection to the organism involves cell cycle,apoptosis,metabolism and other aspects,while protein is the most basic and important molecule in all cellular life processes,and its synthesis mechanism determines the fate of viral infection and host cells,and is closely related to the success or failure of viral infection and pathological changes of host cells^[6].HCMV is significantly different from those viruses whose acute infection shuts down the host protein translation process and inhibits host cell protein synthesis;On the one hand,HCMV completely relies on the translation mechanism of the host cell to synthesize viral proteins and competes with host m RNA for the limited ribosomes to translate viral m RNA into viral proteins^[7,8];On the other hand,host cells rapidly activate multiple defense mechanisms after HCMV infection,while the virus inhibits host cell defenses by interfering with multiple host cell signaling pathways to ensure the continued synthesis of host and viral proteins required for viral replication^[9,10].Thus,viral regulation of host cell protein synthesis mechanisms is key to viral replication,in which ribosomes,the site of protein synthesis,play a crucial role.The relative amount of ribosomal proteins（RPs）that make up the ribosome varies in different tissues or different growth environments of cells in the human body.Ribosomes with different ratios of RPs composition regulate the translation of specific m RNAs,resulting in differences in protein synthesis in different cellular environments to adapt to various cellular stress environments such as cell growth and differentiation as well as nutrition or infection,a phenomenon known as ribosomal heterogeneity^[11].To ensure efficient translation of viral m RNAs,HCMV selectively regulates the expression of RPs to favor the coding capacity of viral genes and promote viral replication.In a previous study,we found that protein expression differences between HCMV-infected and uninfected human embryonic lung fibroblasts at 72h were significantly enriched in ribosomal structure-and function-related proteins and their signaling pathways.RPs expression levels were generally upregulated 1-3-fold after infection,with RPL4expression levels upregulated more than 5-fold.In contrast,in the high-throughput sequencing analysis of RNA from HCMV-infected and uninfected cells at 72h,no difference in m RNA transcript levels between infected and uninfected cells was found to be enriched in ribosomal structure and function-related genes and their signaling pathways.m RNA transcript levels of RPL4 were not significantly up-regulated after HCMV infection,as in the case of proteins.These results suggest that the upregulation of ribosomal protein expression in host cells by HCMV infection does not result from the upregulation of their m RNA transcript levels,and that other unknown mechanisms may exist.The core promoter of a gene,located upstream of the m RNA transcription start site,contains the most important regulatory elements that interact with transcription factors and can strictly regulate the transcription initiation process,playing a central role in the transcriptional regulatory mechanism.Most human genes are regulated by their respective core promoters and exhibit different TSS transcripts in different tissue cell types,growth and development processes,and pathophysiological states.and transcripts of the same gene can have different lengths and structures of the5’UTR.The 5’UTR of m RNA is the most important region for the regulation of translation efficiency,where the cis-acting elements are critical for the effect of protein translation efficiency.We used Cap Analysis of Gene Expression and Deep Sequencing technique to detect the effect of HCMV infection on TSS of HELF cell genes.The TSS of RPGs with up-regulated protein levels but unaltered m RNA levels before and after infection identified in the previous study were analyzed and validated,and their m RNA and protein expression levels before and after infection were further verified.The regulation of protein expression levels by different 5’UTRs of RPL4 gene was examined in transiently and stably transfected cells,respectively,and the effect of mammalian rapamycin target protein complex 1 on the regulation of different 5’UTRs and the effect of HCMV infection on the regulation was investigated.To capture and identify the cellular or viral proteins that may be involved in the different 5’UTRs of RPL4 gene to regulate m RNA translation,and to investigate the mechanism by which HCMV infection alters the TSS of RPL4 gene to cause different 5’UTRs of RPL4 m RNA and thus affect the translation efficiency.Methods:1.Transcription start site information of m RNA was obtained by Deep CAGE-seq of HCMV Han-BAC infected 12h,72h and uninfected HELF cells in two independent replicates.Genes with major TSS shifts were selected,and biological function and pathway enrichment analysis were performed for these genes.Combined with the results of high-throughput sequencing of m RNA and protein profiling,we searched for the commonality of major transcription start site shift sequences in genes playing the same biological function and the biological significance caused by such shifts.2.For RPGs with significant shifts in the first exon TSS by Deep CAGE-seq under HCMV infection,c DNA 5’end rapid amplification technique,RNA Array and Parallel Reaction Monitoring techniques were used to verify the TSS,m RNA and protein expression of the genes.3.Transient transfection of expression vectors inserted with different 5’UTRs before and after RPL4 infection was performed,and the expression levels of downstream fusion proteins were detected by Western blot;The RNA-pulldown technique was used to screen the host and/or viral proteins involved in the translational regulation of different RPL4 5’UTR sequences.We established HELF stable transient cell lines with different 5’UTR stably expressed fluorescent reporter genes and used Western blot to detect the protein expression levels of downstream fluorescent protein genes;m TORC1 activation and inhibition assays were used to study the regulatory role of m TORC1 on the 5’UTR after infection and the effect of HCMV infection on this regulatory role.Results:1.Deep CAGE-seq results showed that the number of total reads in host cells decreased gradually after HCMV infection compared with that in uninfected host cells,and decreased by about 40%after 72h of infection,which was consistent with the trend of changes in m RNA expression levels of host genes in the previous study.HCMV infection caused TSS shift in about 25%of host cells.2.CAGE-seq results of HCMV-infected and uninfected HELF cells showed that genes with shifted TSS before and after infection were significantly enriched in ribosomal structure and function-related genes and their signaling pathways.The protein profiles and m RNA high-throughput sequencing results showed that the protein expression levels of the above enriched genes and signaling pathways were significantly up-regulated after infection,while the m RNA levels did not change significantly,indicating that the genes TSS shifts occurring after HCMV infection are probably an important factor in the elevated protein expression levels of RPs.3.The major TSS,m RNA levels and protein levels of some ribosomal protein genes（RPL4,RPL22,RPL38,RPS11 and RPS23）were verified by 5’RACE experiments,RNA Array,PRM assays and Western blot techniques,and the results showed that:a.The shift of the dominant TSS of the above RPGs before and after infection was consistent with the CAGE-seq sequencing results.b.No significant differences in the m RNA levels of RPGs was observed after infection;c.The protein levels of RPs were significantly up-regulated after infection.4.RPL4,which was validated by the above experiments,was selected as the study subject.The expression vectors with different 5’UTRs before and after insertion of RPL4 infection were transiently transfected and stably transfected,and the up-regulation of downstream protein expression by 5’UTR after RPL4 infection was verified by Western blot;RNA-pulldown combined with protein profiling was used to capture the cellular proteins bound to the 5’UTR before and after RPL4 infection.The results showed that the 5’UTR bound33 proteins enriched in the positive regulatory pathway of translation after RPL4 infection;In cells stably transfected with a fluorescent reporter gene expression vector inserted into the 5’UTR after RPL4 infection,addition of the m TORC1 inhibitor Torin1 down-regulated the expression of the fluorescent reporter motif;addition of the activator 3BDO up-regulated the expression level of the fluorescent reporter motif,and this regulation was made more pronounced by HCMV infection.Conclusion:1.HCMV infection decreases the transcription initiation activity and m RNA expression levels of host cell genes.It also caused shift of major TSS of a large number of genes in host cells.2.HCMV infection of HELF cells caused upregulation of protein expression levels of genes related to ribosome structure and function,but no significant changes in m RNA levels.Their dominant TSSs were significantly shifted before and after infection.3.HCMV infection caused displacement of the major TSS of the RPL4 gene in host cells,and the addition of the TOP sequence at the 5’end of the m RNA 5’UTR upregulated the protein translation level of the RPL4 gene.4.The binding of more translation initiation-related proteins to the m RNA 5’UTR of the HELF RPL4 gene after infection compared to uninfected may be an important mechanism for the upregulation of protein expression levels of RPL4 by infection.