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

The cells from each well were resuspended in 500 μl of PBS (-) and transferred to 1.5 ml tubes and centrifuged at 200 x g for 5 min. Supernatants were discarded and cell pellets were lysed in 100 μl of NP-40 lysis buffer. Following lysis samples were centrifuged at 20,000 x g for 5 min, supernatants were transferred to a new 1.5 ml tubes and cell debris pellets were discarded. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.08.031203 doi: bioRxiv preprint Figure 1 . Bat Tetherin structure and sequence diversity. A. The consensus bat tetherin amino acid sequence generated through the multiple sequence alignment of tetherin from 27 bat species (SI Data 1) and compared against human tetherin. Strong positive selection of bat tetherin is revealed by the ratio of non-synonymous to synonymous mutations (Table 2) , and is represented as evolutionary hotspots. The YxY dual tyrosine motif, the alternative start site for the short isoform of tetherin, and the GPI anchor attachment site positions are indicated by arrows. The red asterisk (*) indicates the location of the inserted molecular tags in the P. alecto and M. macropus tetherin expression constructs. CD, cytoplasmic domain; TM, transmembrane domain; ED, extracellular domain; GPI, glycophosphatidylinositol; GSP, GPI signal peptide. B. Significant sequence diversity exists among bat tetherin proteins, indicated by the percentage of amino acid sequence conservation at each site of the consensus bat tetherin. Amino acids conserved in all 27 sequences are represented by their letters. Amino acids represented by X are variable and are included to indicate the sequence distance between closely positioned conserved residues. C. The consensus bat tetherin sequence. Amino acid residues represented in > 50% of bat tetherin sequences are indicated with their letter. Positions at which no amino acid residue is represented in > 50% of bat tetherin sequences are indicated with 'X'. The red line indicates the position of the dual tyrosine motif. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.08.031203 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.08.031203 doi: bioRxiv preprint Figure 5 . Pairwise comparisons between human and bat tetherin variants. A. Myotis macropus tetherin A, C, and D. B. M. macropus tetherin B and E analysed separately to A, C and D to account for the inclusion of 60 AA in M. macropus tetherin A, C, and D that is otherwise removed following gap treatment of the alignment of M. macropus B and E. Protein sequences of human and bat tetherins were aligned and gaps were removed. The horizontal axis represents percentage identity between pairs of amino acid sequences, coloured with a white to red gradient indicating increasing similarity as red intensity increases. The vertical axis represents the number of amino acid residue identity differences between sequence pairs, coloured with a white to blue gradient indicating increasing difference as blue intensity increases. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.08.031203 doi: bioRxiv preprint Tables . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://