Identification and characterization of bovine herpesvirus 1 minor glycoproteins L, H and M

Bovine herpesvirus-1 (BHV-1) encodes a number of glycoproteins, which are present in the virion envelope and play a pivotal role in herpesvirus biology. Glycoproteins gL, gH, and gM are minor glycoproteins of BHV-1, which are well conserved among herpesviruses. The objective of this investigation was to isolate and identify the gene encoding glycoprotein gL and to identify and characterize the glycoprotein gL. In addition other minor glycoproteins, gH and gM, were identified and characterized. Sequencing of 3113 nucleotides located at the right end of the HindIII L fragment of the BHV-1 genome, from map units 0.712 to 0.734 revealed four open reading frames (ORFs) designated as UL1, UL2, UL3, and UL3.5 based on their homology with proteins of other alphaherpesviruses. The UL1 ORF of 158 aminoacids exhibited limited homology with the UL 1 (glycoprotein gL) homolog of herpes simplex virus (HSV-1) and pseudorabies virus (PRV). The UL2 ORF of 204 amino acids showed significant homology to the UL2 (uracil-DNA glycosylase) homolog of HSV-1 and PRV. The UL3 ORF of 204 amino acids showed significant homology to UL3 (nuclear phosphoprotein) of HSV-1 and PRV. The UL3.5 ORF of 126 amino acids showed limited homology to the UL3.5 ORF of PRV. The homolog of this gene is absent in HSV-1. Nucleotide sequence analyses also revealed potential TATA boxes located upstream of each ORF. However, only one polyadenylation signal was detected downstream of the UL3.5 ORF. Northern (RNA) blot analyses revealed four transcripts of 2.4, 1.9, 1.3, and 0.7 kb which are transcribed in the same direction and are 3'- coterminal transcripts. These mRNAs appear to yield proteins encoded by UL1 (2.4 kb), UL2 (1.9 kb), UL3 (1.3 kb) and UL3.5 (0.7kb) ORFs. Previously, DNA sequence analysis of the BHV-1 genome revealed the presence of an open reading frame homologous to the UL10 gene of herpes simplex virus-1 (Vlcek et al., 1995). To identify the UL10 product of BHV-1, rabbit antiserum was raised against a UL10-GST fusion protein, constructed by fusing the C-terminal 80 amino acids of UL10 protein with the gene encoding the GST protein. Serum against the UL10-GST fusion protein immunoprecipitated a 36-37 kDa protein from in-vitro translated in-vitro transcribed mRNA, and a 43-44 kDa protein from BHV-1 infected MDBK cells and purified virions. In addition, a second protein of 88-90 kDa, which could represent a dimeric form was observed. Glucosamine labelling and enzymatic deglycosylation assays confirmed that the UL10 protein is glycosylated and was designated BHV-1 glycoprotein gM in accordance with the current herpesvirus glycoprotein nomenclature. (Abstract shortened by UMI.