Characterisation and expression of ORF2 in genome segment 10 of African horse sickness virus

Abstract

Viral genome segments are often under strong selective pressure to enhance the coding capacity of the genome, allowing expression of multiple proteins from a single mRNA. A second additional open reading frame, ORF2, was identified in African horse sickness virus (AHSV) Seg-10 which encodes the non-structural protein NS3. The ORF2 conservation, size, nature, expression, function and potential subcellular localisation of the putative protein product AHSV is not known and is investigated in this project. A bioinformatic analysis of all available AHSV Seg-10 sequence data was done. ORF2 was maintained in over 400 AHSV Seg-10 sequences, and six size variants were identified ranging from 183 to 252 nucleotides. ORF2 showed high conservation in the central region. AHSV Seg-10 ORF2 shows strong positive selection. The protein encoded by AHSV ORF2 was recombinantly expressed in insect and mammalian cells. Recombinant baculoviruses were confirmed to express ORF2 or ORF2-eGFP. Localisation of ORF2-eGFP was specific within the nucleus and cytoplasm of Sf9 cells. ORF2 transiently expressed in BSR-T7 cells initially localised in the cytoplasm and later moved to the nucleus, and ultimately caused cells to shrivel showing a cytotoxic effect. Antibodies raised against ORF2 was used to investigate the presence and subcellular localisation of the protein during AHSV infection. Using this antibody, ORF2 could be detected in AHSV infected cells, potentially identifying a novel AHSV non-structural protein. This is the first detection of a putative protein product of Seg-10 ORF2 during normal AHSV infection and paves the way for further functional analysis of this protein in the AHSV replication cycle.