Functional Analysis of Oligoadenylate Synthetase in the Emu (Dromaius novaehollandiae)

Abstract

2'-5'-oligoadenylate synthetase (OAS) is one of the proteins that act as a defense mechanism against foreign RNA in cells. OAS has two functions: an antiviral effect against a wide range of virus species via the OAS/RNase L pathway with synthesized oligoadenylates and inhibition of viral replication specific to viruses of the genus Flavivirus, which is independent of enzymatic activity. Several birds have been reported to possess only one type of OAS family member, OASL, which has both enzymatic activity and inhibitory effects on flaviviral replication. However, the ostrich has two types of OASs, OAS1 and OASL, which show different functions-enzymatic and anti-flaviviral activities, respectively. In this study, emu OASs were cloned to investigate their sequence and function and elucidate the role of OASs in emus. The cloning results showed that emus had OAS1 and OASL, suggesting that emu OASs were more closely related to ostrich than to other birds. Functional investigations showed that emu OAS1 and OASL had enzymatic and anti-flaviviral activities, respectively, similar to those of the ostrich. Emus and ostriches are evolutionarily different from most birds and may be more closely related to mammalian OAS diversity.

Keywords: 2′-5′ oligoadenylate synthetase; West Nile virus; emu.

Figure 1

Cloning of the emu OAS sequence was performed to obtain the amino acid sequences of OAS1 (A) and OASL (B). These amino acid sequences were aligned with those of ostriches. Homologous amino acid sequences were marked with * at the top. I–III indicate regions essential for the OAS enzymatic function. (C) A phylogenetic tree of avian OASs with reported nucleotide sequences. The emu OASs of the sequences obtained in this study were put in squares.

Figure 2

Oligoadenylate synthetase activity was determined for emu, ostrich, and mouse OASs. The products after the enzymatic reaction were electrophoresed and detected as oligomerized ATP with ³²P.

Figure 3

Inhibitory effect of emu OASs on WNV replication was measured. (A) Timeline of the antiflavivirus activity experiment. BHK-21 cells were seeded and then sequentially transfected with pOAS plasmid and WNV replicon RNA. The supernatant of the culture medium was collected after 72 h. (B) Inhibitory effect of emu OASs on WNV replication was measured. SEAP, a reporter protein in the culture supernatant, was measured. Error bars indicate standard errors. ** indicates p < 0.01 compared with empty vector.