A novel spliced gene in alcelaphine herpesvirus 1 encodes a glycoprotein which is secreted in vitro

Abstract

Herpesviruses often contain cryptic, spliced genes that are not obvious from the initial in silico annotation. Alcelaphine herpesvirus 1 (AlHV-1) contains 72 annotated ORFs but there are also a number of gaps between these that may have protein-coding potential. Comparative analysis of coding potential between AlHV-1 and the related ovine herpesvirus 2 (OvHV-2) revealed a putative novel spliced gene that we have termed A9.5. Analysis of cDNA clones from AlHV-1-infected cells revealed three overlapping clones corresponding to A9.5 and the coding sequence was confirmed by reverse transcription PCR of RNA from AlHV-1-infected cattle tissues. The A9.5 gene was predicted to encode a secreted glycoprotein with molecular mass 19 kDa. Empirical analysis showed that a recombinant haemagglutinin-tagged A9.5 fusion protein was secreted from transfected cells and had a molecular mass of 45 kDa, which was reduced to 20 kDa by endoglycosidase F treatment, confirming that A9.5 was a secreted glycoprotein. In situ RNA hybridization showed that A9.5 was expressed in cells associated with malignant catarrhal fever (MCF) lesions in infected cattle. Detailed analysis of the available OvHV-2 sequences revealed an homologous gene (Ov9.5) with conserved splicing signals and predicted amino acid sequence features in both sequenced isolates of this related virus. We have therefore identified a novel spliced gene in two related macaviruses that is expressed in MCF lesions. Future work will determine its importance for the pathogenesis of disease.

Alcelaphine Herpesvirus-1 (Malignant Catarrhal Fever Virus) in Wildebeest Placenta: Genetic Variation of ORF50 and A9.5 Alleles

“…Each set of assays was controlled by the inclusion of known AlHV-1 positive and negative bovine genomic DNA samples and potential cross-contamination between reactions was controlled by the inclusion of template-free reactions. To study viral variation in positive samples, segments of the ORF50 gene, encoding the lytic cycle trans-activator protein, and of the spliced A9.5 gene [ 24 ] were sequenced and analysed as described previously for OvHV-2 [ 25 ]. ORF50 encodes a transcription factor expected to be highly conserved, whilst A9.5 encodes a predicted glycoprotein of unknown function that has been demonstrated to be polymorphic in both AlHV-1 and OvHV-2 [ 25 ],[ 24 ].…”

“…Primers for nested PCR of the A9.5 gene were designed to target conserved areas flanking the predicted coding region, based on the available sequence of AlHV-1 [ 31 ],[ 24 ]. Primers for amplification of ORF50 (AHVorf50_F, GCC AGG CAG AGG TAT GTG TT and AHVorf50_R, GGC CGT TGT GGG TAC TGT AT) were chosen within exon 2 of the ORF50 gene, to amplify a fragment of 543 base pairs for analysis of sequence variation.…”

“…The novel spliced gene A9.5 is predicted to encode a secreted glycoprotein that has no homologue outside the MCF viruses [ 24 ]. A9.5 does have a detectable homologue in OvHV-2 (Ov9.5) that has been shown to be both polymorphic and highly variable with nine alleles showing as little as 50% predicted amino acid sequence identity [ 25 ].…”

“…A9.5 does have a detectable homologue in OvHV-2 (Ov9.5) that has been shown to be both polymorphic and highly variable with nine alleles showing as little as 50% predicted amino acid sequence identity [ 25 ]. So far, only two alleles of A9.5 have been identified: one (A9.5*0101] [ 24 ] in the C500 strain of AlHV-1, that has been passaged in vivo in the UK for almost 50 years [ 26 ], and the other (A9.5*0201) [ 27 ] in a diagnostic sample taken from an Ankole cow with MCF. The A9.5 alleles share about 80% amino acid sequence identity and retain overall similarity to the Ov9.5 alleles.…”

Alcelaphine herpesvirus 1 glycoprotein B: recombinant expression and antibody recognition

“…The aims of this work were to confirm the interaction of mAb 12B5 with AlHV-1 gB by expression of a recombinant, epitope tagged polypeptide and to characterise the expression and antigenicity of the recombinant gB polypeptide. Human embryonic kidney 293T (HEK293T) cells were used for transfection studies following previous experiments that had shown that this cell line provided both a high frequency of transfection and a high level of expression of MCF virus genes 17,18 . For transfection and subsequent protein expression, cells were grown in IMDM with 10 % FBS and 2 % L-glutamine.…”