Brief report: Mutations in SIV Nef that disrupt and restore tetherin downregulation

Abstract

The H196 residue in SIVmac239 Nef is conserved across nearly all HIV and SIV isolates, lies immediately adjacent to the AP-2 (adaptor protein 2) interacting domain (ExxxLM195), and is critical for several described AP-2 dependent Nef functions, including the downregulation of tetherin (BST-2/CD317). Surprisingly, many stocks of the closely related SIVmac251 swarm virus harbor a nef allele encoding a Q196, which is associated with loss of multiple AP-2 dependent functions in SIVmac239. Publicly available sequences for SIVmac251 stocks were mined for variants linked to Q196 that might compensate for functional defects associated with this mutation. Variants were engineered into the SIVmac239 parental plasmid and mutant viruses were used to test tetherin downregulatory capacity in primary CD4 T cells using flow cytometry. SIVmac251 stocks that encode a Q196 residue in Nef uniformly also encode an upstream R191 residue. We show that R191 restores the ability of Nef to downregulate tetherin in the presence of Q196. However, a published report showed Q196 commonly evolves to H196 in vivo, suggesting a fitness cost. R191 may represent compensatory evolution to restore the ability to downregulate tetherin lost in viruses harboring Q196.