Functional And Molecular Characterization Of Bovine Herpes Virus-1 (BoHV-1) PUL21

Bovine herpesvirus 1(BoHV-1),an enveloped double-stranded-DNA virus that belongs to the subfamily Alphaherpesvirinae of the Herpesviridae family,causes bovine respiratory disease,vulvovaginitis,abortion,conjunctivitis,balanoposthitis,and severe neonatal disease in cattle.BoHV-1 infects cattle of all ages and breeds worldwide,and it causes severe economic fall to the cattle industry.The BoHV-1 virion comprise of a multilayered architecture that is common in herpesviruses: a capsid enclosing the doublestranded DNA genome,an envelope,and the tegument.Tegument is a cluster of proteins that occupy the space between the envelope and nucleocapsids.All alpha herpesviruses possess more than 15 tegument proteins.These proteins functions in the early stage of viral particle production by regulating the viral immediate-early genes expression and in the late stage of infection via their involvement in viral morphogenesis,assembly,and egress.These proteins play critical roles in each step of envelopment,de-envelopment,and re-envelopment process.The BoHV-1 tegument proteins have been poorly characterized.Only a few tegument proteins of BoHV-1 have been studied in detail;thus,the functions and interactions of the majority of tegument proteins remained uncharacterized.Sequence analysis showed UL21 is a multifunctional conserved tegument protein among Alphaherpesvirinae subfamily.BoHV-1 pUL21 is a 575-amino acid tegument protein.Functions of pUL21 have been well characterized only in herpes simplex virus(HSV)and pseudorabies virus(PRV).However,function of UL21 of BoHV-1 remains uncharacterized.The present study was conducted to determine the functions of UL21 in BoHV-1.Following main findings were obtained.1.Mutant construction In this study,we generated a recombinant virus bearing a UL21 deletion(vBoHV-1-?UL21)and its revertant virus(?UL21R)in which the UL21 gene was restored using a bacterial artificial chromosome system(BAC)and transgenic techniques.Single step growth kinetics showed that the replication of vBoHV-1-?UL21 was 1,000-fold lower than those of the wild-type virus in the extracellular and intracellular environments at 48 hpi.,respectively.In contrast,the titers of vBoHV-1,?UL21R and BoHV-1 UL21-HA viruses were similar.Moreover,the plaque size of the UL21 mutant viruses also severely decreases than the other viruses.The plaque size of UL21 mutant was almost the 85% to the size of wild type and revertant viruses.While the plaque sizes of the BoHV-1,vBoHV-1-?UL21R and vBoHV-1-UL21-HA viruses did not differ significantly.These data predicts that the HA tag did not interfere with UL21 function and furthermore,UL21 is critical for cytoplasmic exit of the virions.2.Effect of UL21 on virus egress To characterize the UL21 protein,we linked HA tag UL21 gene using two-step redmediated recombination an immunofluorescence analysis showed that UL21 localized predominantly to the cytoplasm,and it only exhibited punctate nuclear staining.To investigate,the function of UL21 in nuclear egress pathway,we linked HA tag with the small capsid protein(UL35)in UL21 deletion and wild type viruses.The immunofluorescence assay of capsid protein demonstrates that pUL21 mutation did not impair nucleocapsids egress.To corroborate our findings we performed TEM analysis of UL21 deletion and wild type viruses.As showed from the immunofluorescence results,TEM analysis also evidences no significant difference in the total number of nucleocapsids in the nuclei infected with UL21 mutant and wild type viruses.These results support our previous observations that the UL21 mutation did not affect nuclear egress of the nucleocapsids.An ultra-structural analysis confirmed that the UL21 deletion caused an accumulation of partially enveloped or non-enveloped virions in the cytoplasm,as well as depletion in the number of mature virions.This data showed that UL21 deletion virus exhibited a defect in secondary envelopment as evidenced by large numbers of capsid clusters that accumulated in the cytoplasm,which resulted in the failure of the capsids to become fully enveloped.Furthermore,TEM quantitation of the virion distribution within cells,showed no significant difference in the total number of nucleocapsid virus particles in the nuclei,while the number of intracellular enveloped viral particles in the cytoplasm of the v BoHV-1?UL21-infected cells was reduced by ~ 80% and the number of extracellular mature virions was reduced by ~ 90% compared with the wild-type infected cells or v BoHV-1-?UL21R viruses.3.The identification of UL21 interactive protein The UL21 interactive proteins were identified using Mass spectrometry analysis.After immunoprecipitation of HA tag protein the band was subjected for mass spectrometry analysis.The results revealed that the tagged protein was UL21 and that HA-tagged UL21 pulled down UL16,suggesting that these two proteins form a complex,and further o-immunofluorescence assay showed that UL21 interacted with UL16.Taken together,these data provide evidence that mutation of pUL21 resulted in severe viral growth defects but it did not affect the nuclear egress.Furthermore,UL21 plays critical roles in BoHV-1 secondary envelopment and cell-to-cell spreading in tissue culture and UL16 as a reactive protein of UL21 is likely to be involved in these activities.