| Bovine herpesvirus 1(BoHV-1) is an important pathogen that causes pneumonia, conjunctivitis, genital disorders, and abortions in cattle. Additionally, BoHV-1 is an important factor in shipping fever. As a result, BoHV-1 causes significant economic losses to the cattle industry. BoHV-1 is a member of the Alphaherpesvirinae subfamily. The mature BoHV-1 virion consists of defined structures that are found in all herpesviruses: the nucleocapsid, tegument, and envelope. The nucleocapsid contains a linear, double-stranded DNA genome encircled by a proteinaceous layer of tegument and an outermost envelope. The viral genome consists of double-stranded DNA that encodes approximately 70 proteins, of which 33 are known to be structural and up to 15 are non-structural. The tegument comprises approximately 20 viral-encoded proteins. These proteins function to establish suitable conditions for efficient viral growth, assembly, and egress. Virion assembly and egress proceed from the nucleus to the cytoplasm. These processes are explained by an envelopment, de-envelopment, and re-envelopment model. Tegument proteins play important roles at each point of virion assembly and egress, in particular during primary envelopment, secondary envelopment, and viral particle trafficking.The tegument proteins of BoHV-1 have been poorly characterized. Only a few tegument proteins of Bo HV-1 have been studied in detail; thus, the functions and interactions of the majority of tegument proteins remain largely unknown.Sequence analysis showed that the BoHV-1 protein pUL51 is a protein that is conserved in all herpesvirus family members. BoHV-1 pUL51 is a 243-amino acid tegument protein whose function is unknown. HSV-1 UL51 belongs to viral genes of the γ2 class, while BoHV-1 pUL51 belongs to the γ1 class. The association of pUL51 with viral assembly and egress in different viruses of the herpesvirus family demonstrates that pUL51 interacts with different viral proteins to perform its functions. Nevertheless, the phenotype of a BoHV-1 pUL51 mutant has not been characterized in cell culture or in vivo.In this study, we constructed and characterized a mutant BoHV-1 virus that does not express pUL51. For this, we have constructed BoHV-1 bacterial artificial chromosome(BAC) and we used BAC recombinant and transgenic techniques to delete a major part of the UL51 open reading frame along with construction of different recombinant viruses. Growth kinetic analysis showed that there was almost 2 fold reduction of the UL51 titer in single step and multi step growth kinetic as compared to other viruses. Moreover the plaque size of the UL51 mutant viruses also severly reduced than the other viruses. The plaque size of UL51 mutant was almost the 10% to the size of wild type and revertant viruses. Interestingly there was almost 1 fold reduction in extracellular titer of UL51 mutant virus than the intracellular titer of UL51 mutant virus. While no reduction in extracellular titer observed in other viruses. This predicts the role of UL51 in cytoplasmic exit of the virions. To characterize the UL51 protein, we attached HA tag UL51 gene using BAC recombinant techniques. Mass spectrometry analysis after immunoprecipitation of HA tag protein showed that the tagged protein was UL51 protein. Immunofluorescence analysis confirm that UL51 protein localize in the cytoplasm of the cell and it co localize with cis-Golgi marker protein GM130 during early(12 hrs post infection) and late stages(18 hrs post infection) of infection. That predicts the role of UL51 in secondary envelopment of the viruses.To explore the role of UL51 in egress pathway, we attached HA tag with minor capsid protein(UL35) in UL51 mutant and wild type viruses. Immunofluorescence analysis of capsid protein showed that there was a high density of concentrated, punctate fluorescence in cells infected with the wild-type virus compared with those infected with UL51 mutant. Meanwhile, in the UL51 mutant virus pUL35 protein localized in the cytoplasm and at the edges of the cells compared with the wild-type pUL35 protein that distributed throughout cytoplasm. The data showed that the UL51 mutation may impair the nuclear egress of capsids. To confirm our findings we performed TEM analysis of UL51 mutant and wild type viruses. As predicted from the immunofluorescence experiments, in TEM analysis a huge number of nucleocapsids were present on the inner side of the nuclear membrane of UL51 mutantinfected cells.This analysis supports our previous finding that the UL51 mutation may decrease the nuclear egress of the nucleocapsids. Furthermore, in UL51 mutant-infected cells, only a few mature virions were seen in the cytoplasm, while most of the cytoplasmic capsids remained non-enveloped or partially enveloped. These analyses confirmed the role of UL51 protein in nuclear egress, secondary envelopment and cytoplasmic exit of virions. Moreover, in vivo analysis showed that the pUL51 mutant exhibited reduced virulence in rabbits, with no clinical signs, no nasal shedding of the virus, and no detectable serum neutralizing antibodies. Therefore, we conclude that the Bo HV-1 pUL51 is indispensable for efficient viral growth in vitro and is essential for virulence in vivo. |
