5'UTR Of Human Bocavirus Capsid Transcripts Regulates Its MRNA Biogenesis And Alternative Translation

Human Bocavirus has been identified as a second human pathogen in Parvoviridae family which is thought to be associated with respiratory diseases in infants less than 2 years of age.It was firstly identified from a nasopharyngeal aspirate from an infected child in year 2005.There are four differing HBoV variants,HBoV1 to-4,of which,HBoV1 is the only one respiratory virus and is used in this study.HBoV1 is a single strand DNA virus with genome DNA about 5.5kb in length,and possess heterogeneous terminal hairpin at both ends which participates viral genome replication process.HBoV1 uses promoter P5 to transcribe a pre-mRNA,followed by alternatively splicing and alternatively polyadenylation,respectively,to generate mature mRNA transcripts which encode viral nonstructural and structural proteins.The HBoV1 genome containing three main ORFs consist of nonstructural protein(NS1-4),NP1 and capsid protein(VP1,2,3)genes,respectively.Three variously spliced mRNA transcripts(R6,R7,and R8)encode HBoV1 capsid proteins.During HBoV1 infection,VP1/2/3 are expressed at a ratio of ~1:1:10.Since VP genes are overlapped,the obvious difference of expression efficiency of these three proteins theoretically suggests a precise regulation in the VP1/2/3 alternative translation process.Here we reported an example of capsid protein alternative translation as a mean of HBoV1 used to proliferation within a limited genome size.In this study,we firstly proved that the alternative translations of VPs are independent events depends on ribosomal leaky scanning,since the translation efficiency of these proteins could be regulated by Kozak sequence.We excluded the potential that the alternative translation of VP1,VP2 and VP3 is IRES dependented by identifying no IRES motif in neither the HBoV1 5'UTRs nor VP1 u.We focus on the essential cis-elements in HBoV1 R6-5'UTR which regulate VPs expression.This cis-element only exists in HBoV1 R6 mRNA transcripts,and its function is regulating VP mRNA transcription abundance.By sequence analysis,three upstream ATGs(uATGs)are distributed in R6-5'UTR(termed uATG1,uATG2 and uATG3).We verified the essential regulation function of uATG2&3 in VPs alternative translation and HBoV1 RNA biogenesis,which therefore influenced the viron production.Another 5'-cap proximal uATG,uATG1,is irrelevant in these regulating processes.Thus,the 5'UTR is important both in alternative translation of VPs and viral RNA biogenesis.HBoV1 is a small non-enveloped icosahedral virus that is important pathogens in humans.The viral capsid proteins determinate several essential physiological status of virus infection,such as genome package,assembly and antigenicity.However,the capsid proteins of HBoV1 are all encoded by polycistronic mRNAs.The regulation mechanism within the translation process is important for HBoV1 expressing all the capsid proteins to full fill its life cycle.In this study,we propose a uATG-dependent leaky scanning model of HBoV1 regulating the alternative translation of capsid proteins,indicating the relevance to virus proliferation.