Bats Possess Unique Variants of the Antiviral Restriction Factor Tetherin

Joshua A. Hayward; Mary Tachedjian; Adam Johnson; Tamsin B. Gordon; Jie Cui; Glenn A. Marsh; Michelle L. Baker; Lin-Fa Wang; Gilda Tachedjian

To validate the capacity of each tetherin plasmid construct to express tetherin protein, mammalian HEK293T (293T) cells, which do not express tetherin in the absence of type I interferon stimulation (17) , were transfected with bat tetherin expression constructs. Cell lysates were extracted using a method specific for GPI-anchored proteins (40) and tetherin expression was evaluated by non-reducing SDS-PAGE and Western blot analysis ( Figure 7 ). P. alecto tetherin isoforms A and B were detected primarily as broad bands at the expected positions ~56-75 kDa while isoform C was present as both dimers and monomers (~32-35 kDa) ( Figure 7A ). The presence of broad/multiple bands in close proximity to each other likely reflects variable levels of tetherin glycosylation as previously reported for human tetherin (22) . M. macropus tetherin A was also detected as a dimer at the expected size of ~55 kDa while tetherin B was only detected as a monomer with a size of ~26 kDa ( Figure 7B ). These data show that all tetherin constructs are capable of expressing tagged tetherin proteins and that these proteins differ in their capacity to form the expected dimerised form of tetherin. Bat tetherin proteins that are homologous to human l-tetherin dimerise as expected, while the structurally unique tetherin proteins behave differently. In this regard, P. alecto tetherin isoform C generates similar amounts of dimers and monomers, while M. macropus tetherin B appears to exist exclusively as a monomer.