Maintenance of AP-2-Dependent Functional Activities of Nef Restricts Pathways of Immune Escape from CD8 T Lymphocyte Responses

Schouest, Blake; Weiler, Andrea M.; Janaka, Sanath Kumar; Myers, Tereance A.; Das, Arpita; Wilder, Sarah C; Furlott, Jessica; Baddoo, Melody; Flemington, Erik K.; Rakasz, Eva G.; Evans, David T.; Friedrich, Thomas C.; Maness, Nicholas J.

doi:10.1128/jvi.01822-17

Cited by 12 publications

(18 citation statements)

References 87 publications

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“…5D). Similar observations on the role of SIVmac Nef residues His196 and Trp203 were recently reported in the context of MHC escape mutants (32). Thus, H192 in the ExxxLL/M motif plays a key role in the ability of Nef to counteract tetherin and SERINC5 but is dispensable for its ability to remove various immune receptors from the cell surface.…”

Section: Residues Specifically Disrupting Anti-tetherin Activity Of Ssupporting

confidence: 78%

Structural basis for tetherin antagonism as a barrier to zoonotic lentiviral transmission

Buffalo

Stürzel

Heusinger

et al. 2019

Preprint

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Tetherin is a host defense that physically prevents escape of virions from the plasma membrane.Human tetherin lacks the motif DIWK antagonized by SIV, the antecedent of HIV. Here, we reconstituted the AP-2 clathrin adaptor complex with a simian tetherin and SIV Nef and determined its structure by cryo-EM. Nef refolds the first α-helix of the β2 subunit of AP-2 to a β hairpin, creating a binding site for the DIWK sequence. The tetherin binding site in Nef is distinct from those of MHC-I, CD3, and CD4, but overlaps the site for SERINC5 restricting viral infectivity. The structure explains the dependence of SIVs on the host tetherin DIWK sequence and the consequent barrier to human transmission.

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Section: Residues Specifically Disrupting Anti-tetherin Activity Of Ssupporting

confidence: 78%

Structural basis for tetherin antagonism as a barrier to zoonotic lentiviral transmission

Buffalo

Stürzel

Heusinger

et al. 2019

Preprint

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“…It’s possible it arose during replication in cultured cells where selection pressures are undoubtedly different than those the virus experiences in vivo. Given our data suggesting the H 196 Q variant can arise in vivo in SIVmac239 infected macaques as a result of escape from CD8TL responses [30], these data may suggest that SIVmac251 was isolated from an animal that targeted this region with CD8TL, leading to viral escape, and prior to other escape variants becoming dominant, as happened in our previous study [30].…”

Section: Discussionsupporting

confidence: 51%

“…(a) Representative flow cytometric analysis of surface expression of tetherin on primary CD4 T cells infected with wild type SIVmac239 or SIVmac239 harboring the H 196 Q variant alone or in combination with the E 191 R variant. Cells were identified as infected via intracellular Gag p27 staining, top row, as we have described previously [29, 30]. Infected cells (p27+) are orange, while uninfected (p27−) are blue.…”

Section: Resultsmentioning

confidence: 99%

“…We used high throughput next generation sequencing to track evolution in SIV Nef [29, 30], with particular focus on viral escape from antiviral CD8TL responses, including CD8TL targeting the SIV Nef IW9 (IRYPKTFGW 173 , with subscript numbers representing the position in the SIVmac239 Nef protein) and MW9 (MHPAQTSQW 203 , hereafter referred to as MW9) epitopes in rhesus macaques that express Mamu-B*017:01. MW9 overlaps the well-defined “di-leucine” ExxxLM 195 motif and lies immediately upstream of the DD 205 di-acidic motif also important for AP-2 binding [31].…”

Section: Introductionmentioning

confidence: 99%

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

Maness¹,

Schouest²

2019

Preprint

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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 ...

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“…We used high throughput next generation sequencing to track evolution in SIV Nef [ 30 , 31 ], with particular focus on viral escape from antiviral CTL responses, including CTL targeting the SIV Nef IW9 (IRYPKTFGW 173 , with subscript numbers representing the position in the SIVmac239 Nef protein) and MW9 (MHPAQTSQW 203 , hereafter referred to as MW9) epitopes in rhesus macaques that express Mamu-B*017:01. MW9 overlaps the well-defined “di-leucine” ExxxLM 195 motif and lies immediately upstream of the DD 205 di-acidic motif also important for AP-2 binding [ 32 ].…”

Section: Introductionmentioning

confidence: 99%

Tetherin downmodulation by SIVmac Nef lost with the H196Q escape variant is restored by an upstream variant

et al. 2020

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The H 196 residue in SIVmac239 Nef is conserved across the majority of HIV and SIV isolates, lies immediately adjacent to the AP-2 (adaptor protein 2) binding di-leucine domain (ExxxLM 195 ), and is critical for several described AP-2 dependent Nef functions, including the downregulation of tetherin (BST-2/CD317), CD4, and others. Surprisingly, many stocks of the closely related SIVmac251 swarm virus harbor a nef allele encoding a Q 196 . In SIVmac239, this variant is associated with loss of multiple AP-2 dependent functions. Publicly available sequences for SIVmac251 stocks were mined for variants linked to Q 196 that might compensate for functional defects associated with this residue. Variants were engineered into the SIVmac239 backbone and in Nef expression plasmids and flow cytometry was used to examine surface tetherin expression in primary CD4 T cells and surface CD4 expression in SupT1 cells engineered to express rhesus CD4. We found that SIVmac251 stocks that encode a Q 196 residue in Nef uniformly also encode an upstream R 191 residue. We show that R 191 restores the ability of Nef to downregulate tetherin in the presence of Q 196 and has a similar but less pronounced impact on CD4 expression. However, a published report showed Q 196 commonly evolves to H 196 in vivo, suggesting a fitness cost. R 191 may represent compensatory evolution to restore the ability to downregulate tetherin lost in viruses harboring Q 196 .

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Maintenance of AP-2-Dependent Functional Activities of Nef Restricts Pathways of Immune Escape from CD8 T Lymphocyte Responses

Cited by 12 publications

References 87 publications

Structural basis for tetherin antagonism as a barrier to zoonotic lentiviral transmission

Structural basis for tetherin antagonism as a barrier to zoonotic lentiviral transmission

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

Tetherin downmodulation by SIVmac Nef lost with the H196Q escape variant is restored by an upstream variant

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