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

Abstract

17 The H 196 residue in SIVmac239 Nef is conserved across nearly all HIV and SIV isolates, 18 lies immediately adjacent to the AP-2 (adaptor protein 2) interacting domain (ExxxLM 195 ), 19 and is critical for several described AP-2 dependent Nef functions, including the 20 downregulation of tetherin (BST-2/CD317). Surprisingly, many stocks of the closely 21 related SIVmac251 swarm virus harbor a nef allele encoding a Q 196 , which is associated 22 with loss of multiple AP-2 dependent functions in SIVmac239. Publicly available 23 sequences for SIVmac251 stocks were mined for variants linked to Q 196 that might 24 compensate for functional defects associated with this mutation. Variants were 25 engineered into the SIVmac239 parental plasmid and mutant viruses were used to test 26 tetherin downregulatory capacity in primary CD4 T cells using flow cytometry.27 SIVmac251 stocks that encode a Q 196 residue in Nef uniformly also encode an upstream 28 R 191 residue. We show that R 191 restores the ability of Nef to downregulate tetherin in the 29 presence of Q 196 . However, a published report showed Q 196 commonly evolves to H 196 in 30 vivo, suggesting a fitness cost. R 191 may represent compensatory evolution to restore 31 the ability to downregulate tetherin lost in viruses harboring Q 196 . 34The lentiviral Nef protein is a common target of CD8-T lymphocyte (CD8TL) 35 responses in both HIV-1 infected persons and SIV infected rhesus macaques and 36 readily evolves to evade these responses [1][2][3][4][5][6]. Nef is highly pleiotropic and mediates 37 the downregulation of several cell surface molecules involved in innate and adaptive 38 immune responses against virus infected cells such as TCR-CD3 (in most SIVs but not 39 HIV-1) [7], CD4 [8-10], CD8 [11], CD28 [12], tetherin (BST2 or CD317; in most SIVs 40 and in HIV-1 group O, but not HIV-1 group M) [13-15], MHC-I [16], MHC-II [17], CD1d 41 [18], CD80/CD86 [19] and likely others as well as enhancing viral infectivity by 42 preventing virion incorporation of host serine incorporator 3 (SERINC3) and SERINC543 proteins [20-23]. Nef-mediated modulation of several of these molecules, including CD4, 44 CD8, CD28, tetherin, and SERINC3 and SERINC5 requires interactions between Nef 45 and adaptor protein (AP)-2 complexes [11, 20,[24][25][26][27][28]. 46We used high throughput next generation sequencing to track evolution in SIV 47 Nef [29, 30], with particular focus on viral escape from antiviral CD8TL responses, 48 including CD8TL targeting the SIV Nef IW9 (IRYPKTFGW 173 , with subscript numbers 49 representing the position in the SIVmac239 Nef protein) and MW9 (MHPAQTSQW 203 , 50 hereafter referred to as MW9) epitopes in rhesus macaques that express Mamu-51 B*017:01. MW9 overlaps the well-defined "di-leucine" ExxxLM 195 motif and lies 52 immediately upstream of the DD 205 di-acidic motif also important for AP-2 binding [31]. 53 Though selection eventually favored changes of the first position in MW9, specifically 54 M 195 I or M 195 V, an H 196 Q (second position in ...