- Email: [email protected]
SIV-Nef: Pas de trois Choreographies to Evade Immunity
TIMI 1615 No. of Pages 3
Spotlight
HIV/SIV-Nef: Pas de trois Choreographies to Evade Immunity Luis L.P. daSilva1,* and Gonzalo A. Mardones2,3,* Nef is a major pathogenic factor of human and simian immunodeficiency viruses that hijacks protein trafficking through physical interaction with vesicle coats. This alters the subcellular localization of proteins involved in immunity and neutralizes their function. Understanding the structural bases for these interactions could reveal new targets for antiviral intervention. Viruses depend on living cells to replicate and must overcome unfavorable intracellular conditions to be successful. Conversely, the selection pressure exerted by viruses results in sophisticated strategies to restrict viral replication and dissemination. As a result of this evolutionary arms race, complex primate lentiviruses, such as the human and simian immunodeficiency viruses (HIV-1, HIV2, and SIVs), acquired so-called accessory genes. One such gene is nef, which encodes a cytosolic protein (Nef) that is crucial for robust viral production, promoting disease progression to full-blown AIDS [1,2]. The ability of Nef to engage in promiscuous protein interactions makes it a multifunctional protein [1,2] that (i) prevents superinfection and facilitates virion release by removing CD4, the primary HIV and SIV receptor, from the cell surface; (ii) prevents the destruction of virally infected cells by cytotoxic T cells, precluding cell surface delivery of MHC-I molecules; and (iii) ensures the production of fully infectious HIV particles, displacing SERINC5 from budding sites to prevent viral
incorporation of this restriction factor. The capacity to downregulate CD4, MHC-I, and SERINC5 is conserved among Nef molecules of HIVs and SIVs [1]. In addition, Nef from many SIVs also counteracts tetherin, a broad-spectrum antiviral protein induced by interferon that blocks the release of enveloped viruses by forming physical cross-links between virions and the plasma membrane (PM) [1]. Interestingly, most HIV Nef variants do not effectively antagonize human tetherin. Instead, this function is better executed by other comrades: the accessory protein Vpu in pandemic HIV-1 M strains and the structural protein Env in HIV-2 [1]. All functions of Nef mentioned above depend on its capacity to associate with cellular membranes via an N terminal myristoyl group at sites of vesicle formation, coated by the cytosolic protein complexes AP-1 or AP-2 [1,2]. These heterotetrameric complexes select specific transmembrane protein cargos by binding to sorting motifs in their cytosolic tail (CT), for subsequent incorporation into clathrin-coated vesicles at the trans-Golgi network (TGN) (AP-1) or the PM (AP-2). AP-1 is composed of a g subunit (isoforms g1 or g2), a b1 subunit, a m1 subunit (isoforms m1A or m1B), and a s1 subunit (isoforms s1A, s1B, or s1C). AP-1-m1 and AP-1-s1 contain independent sites that interact with different types of sorting motifs. In one remarkable move, Nef binds the CT of MHC-I or tetherin to accommodate their otherwise suboptimal sorting motifs, forming tripartite complexes with AP-1 [3–5]. In T cells, such binding impairs the constitutive transport of MHC-I or tetherin to the PM, leading to targeting of MHC-I to lysosomes for degradation, or to the accumulation of tetherin at the TGN (Figure 1) [6,7]. An outstanding question was how Nef’s usage of AP-1 yields puzzling, distinct fates for MHC-I and tetherin. A recent study, published in Cell by Morris and
colleagues from Jim Hurley’s group at UC-Berkeley, provides new insights into this issue [8]. Initially, several years ago, the same group reported that AP-1 could be found in a ‘locked’ conformation, not apt for cargo binding, and that Arf1, a small GTPase that attaches itself to the TGN during vesicle formation, promotes the ‘unlocking’ of AP-1 [2,9]. The first clue to understanding the intriguing Nef maneuvers was also provided by the same group, which demonstrated the ability of Nef to induce trimerization of unlocked AP-1 in the presence of Arf1 and cargo, and that, depending on the type of cargo, AP-1 forms either ‘open’ or ‘closed’ trimers [3]. Open trimers can then form higher-order lattices that symmetrically match those of clathrin, promoting clathrin basket polymerization in vitro [3]. When bound to MHC-I CT, Nef induces the formation of open trimers, hijacking Arf1-mediated AP-1 trimerization to a conformation that promotes clathrincoated vesicle (CCV) biogenesis and cargo loading. Strikingly, binding of Nef to tetherin CT, in turn, induces Arf1-mediated oligomerization of AP-1 to a closed trimer conformation, which appeared to be incompatible with clathrin lattice formation [3]. However, the structural bases for the cargo-dependent diversity of Nefinduced AP-1 trimerization and the functional relevance of these differences have remained poorly understood until now. The study by Morris and colleagues employed cryoelectron microscopy (cryo-EM) to determine the structure of the tetherin-bound, closed trimer of AP1 to a high resolution of 3.7 Å. They found that the trimer consists of three central Arf1 molecules assembling around three AP-1 monomers, with one copy of Nef at each AP-1:AP-1 interface. Nef molecules stabilize the trimer in its closed state by using one side to bind the cargo recognition site of AP-1-m1, and another to bind the cargo recognition site of AP-1-s1 in the opposite AP-1 monomer.
Trends in Microbiology, Month Year, Vol. xx, No. yy
1
TIMI 1615 No. of Pages 3
Budding vesicle
Plasma membrane
(A)
Cytosol (B)
5
6 8
3 4
2
1
2ʹ
Retained
7
TGN MHC-I Tetherin Nef
Lysosome
Golgi apparatus
Open Closed AP-1 AP-1 trimer trimer
Clathrin triskelion
Figure 1. HIV/SIV-Nef Counteracts Antiviral Activities of MHC-I and Tetherin Using the Clathrin-Coat Protein AP-1 via Alternative Structural Arrangements. MHC-I and tetherin molecules are synthesized in the endoplasmic reticulum and transported to the Golgi apparatus and the trans-Golgi network (TGN), normally reaching the plasma membrane (PM) to exert antiviral functions (1). HIV/SIVs Nef links the cytosolic tail (CT) of these proteins to AP-1 at the TGN with distinct consequences (2 and 20 ). (A) For MHC-I (allotypes HLA-A and HLA-B), Nef stabilizes the otherwise weak interaction between MHC-I CT and the AP-1-m1 subunit, and induces the trimerization of AP-1 into an ‘open’ conformation (2). Open trimers are prompt to interact with clathrin (3) along the TGN membrane (4), forming higher-order lattices that symmetrically match those of clathrin (5), promoting the formation of MHC-I loaded clathrin-coated vesicles (CCVs) (6). These CCVs deliver MHC-I molecules to endosomes, which will eventually fuse with lysosomes (7). (B) In the case of tetherin, SIV and HIV-1 group O Nef (O-Nef) variants link tetherin CT to AP-1, and induce trimerization of AP-1 into a ‘closed’ conformation (20 ), which seems to be incompatible with clathrin lattice formation (8). Such closed conformation likely prevents tetherin from entering TGN-derived carriers destined to the PM, sequestering tetherin in the TGN.
The study by Morris and colleagues [8] also provides an explanation as to why Nef from HIV-1 strains of pandemic group M (M-Nefs) and of epidemic group O (O-Nefs) displays distinct abilities to counteract tetherin [8]. These HIV-1 groups resulted from independent zoonotic transmission of SIV to humans, and, while M-Nefs are inactive against tetherin, O-Nefs are effective at neutralizing its anti-HIV activity [7]. The authors show that M-Nef molecules can be phosphorylated by host casein kinase I (CK1) at a serine residue that is adjacent to the so-called ‘dileucine motif’, which mediates Nef interaction 2
Trends in Microbiology, Month Year, Vol. xx, No. yy
to AP-1-s1. While phosphorylation of this serine in M-Nefs impairs Nef:AP1-s1 interaction, precluding the formation of closed trimers, in O-Nefs this serine is absent, allowing tetherin counteraction (Figure 1). Is this subversive behavior exclusive toward tetherin? Or only via AP-1-s1? The additional targets of Nef and the possibility of AP-1 variants formed by the combination of different isoforms of its subunits indicate otherwise. In fact, we have recently suggested that Nef could use a functional variant of AP-1, comprising the AP-1-g2 subunit isoform, to retain internalized CD4 in endosomes for its
delivery to lysosomes for degradation [10]. It would be essential to test whether AP-1-g2 plays a similar role in Nef-mediated lysosomal delivery of MHC-I. Moreover, the study by Morris and colleagues reminds us how much cell biology can be learned from viruses, since their finding revealed unprecedented regulatory plasticity of the membrane-trafficking machinery. 1
Department of Cell and Molecular Biology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo 14049-900, Brazil 2 Department of Physiology, School of Medicine and Center for Interdisciplinary Studies of the Nervous System (CISNe), Universidad Austral de Chile, Valdivia 5110566, Chile
TIMI 1615 No. of Pages 3
3
Center for Cell Biology and Biomedicine (CEBICEM), School of Medicine and Science, Universidad San Sebastián, Santiago 7510157, Chile
2. Pereira, E.A. and daSilva, L.L. (2016) HIV-1 Nef: taking control of protein trafficking. Traffic 17, 976–996
*Correspondence: [email protected] (Luis L.P. daSilva) and [email protected] (G.A. Mardones).
4. Jia, X. et al. (2012) Structural basis of evasion of cellular adaptive immunity by HIV-1 Nef. Nat. Struct. Mol. Biol. 19, 701–706
https://doi.org/10.1016/j.tim.2018.09.003 References 1. Sauter, D. and Kirchhoff, F. (2018) Multilayered and versatile inhibition of cellular antiviral factors by HIV and SIV accessory proteins. Cytokine Growth Factor Rev. 40, 3–12
3. Shen, Q.T. et al. (2015) HIV-1 Nef hijacks clathrin coats by stabilizing AP-1:Arf1 polygons. Science 350, aac5137
5. Collins, D.R. and Collins, K.L. (2014) HIV-1 accessory proteins adapt cellular adaptors to facilitate immune evasion. PLoS Pathog. 10, e1003851 6. Roeth, J.F. et al. (2004) HIV-1 Nef disrupts MHC-I trafficking by recruiting AP-1 to the MHC-I cytoplasmic tail. J. Cell Biol. 167, 903–913
7. Kluge, S.F. et al. (2014) Nef proteins of epidemic HIV-1 group O strains antagonize human tetherin. Cell Host Microbe 16, 639–650 8. Morris, K.L. et al. (2018) HIV-1 Nefs are cargo-sensitive AP-1 trimerization switches in tetherin downregulation. Cell 174, 659–671 9. Ren, X. et al. (2013) Structural basis for recruitment and activation of the AP-1 clathrin adaptor complex by Arf1. Cell 152, 755–767 10. Tavares, L.A. et al. (2017) CD4 downregulation by the HIV1 protein Nef reveals distinct roles for the gamma1 and gamma2 subunits of the AP-1 complex in protein trafficking. J. Cell Sci. 130, 429–443
Trends in Microbiology, Month Year, Vol. xx, No. yy
3
Spotlight
HIV/SIV-Nef: Pas de trois Choreographies to Evade Immunity Luis L.P. daSilva1,* and Gonzalo A. Mardones2,3,* Nef is a major pathogenic factor of human and simian immunodeficiency viruses that hijacks protein trafficking through physical interaction with vesicle coats. This alters the subcellular localization of proteins involved in immunity and neutralizes their function. Understanding the structural bases for these interactions could reveal new targets for antiviral intervention. Viruses depend on living cells to replicate and must overcome unfavorable intracellular conditions to be successful. Conversely, the selection pressure exerted by viruses results in sophisticated strategies to restrict viral replication and dissemination. As a result of this evolutionary arms race, complex primate lentiviruses, such as the human and simian immunodeficiency viruses (HIV-1, HIV2, and SIVs), acquired so-called accessory genes. One such gene is nef, which encodes a cytosolic protein (Nef) that is crucial for robust viral production, promoting disease progression to full-blown AIDS [1,2]. The ability of Nef to engage in promiscuous protein interactions makes it a multifunctional protein [1,2] that (i) prevents superinfection and facilitates virion release by removing CD4, the primary HIV and SIV receptor, from the cell surface; (ii) prevents the destruction of virally infected cells by cytotoxic T cells, precluding cell surface delivery of MHC-I molecules; and (iii) ensures the production of fully infectious HIV particles, displacing SERINC5 from budding sites to prevent viral
incorporation of this restriction factor. The capacity to downregulate CD4, MHC-I, and SERINC5 is conserved among Nef molecules of HIVs and SIVs [1]. In addition, Nef from many SIVs also counteracts tetherin, a broad-spectrum antiviral protein induced by interferon that blocks the release of enveloped viruses by forming physical cross-links between virions and the plasma membrane (PM) [1]. Interestingly, most HIV Nef variants do not effectively antagonize human tetherin. Instead, this function is better executed by other comrades: the accessory protein Vpu in pandemic HIV-1 M strains and the structural protein Env in HIV-2 [1]. All functions of Nef mentioned above depend on its capacity to associate with cellular membranes via an N terminal myristoyl group at sites of vesicle formation, coated by the cytosolic protein complexes AP-1 or AP-2 [1,2]. These heterotetrameric complexes select specific transmembrane protein cargos by binding to sorting motifs in their cytosolic tail (CT), for subsequent incorporation into clathrin-coated vesicles at the trans-Golgi network (TGN) (AP-1) or the PM (AP-2). AP-1 is composed of a g subunit (isoforms g1 or g2), a b1 subunit, a m1 subunit (isoforms m1A or m1B), and a s1 subunit (isoforms s1A, s1B, or s1C). AP-1-m1 and AP-1-s1 contain independent sites that interact with different types of sorting motifs. In one remarkable move, Nef binds the CT of MHC-I or tetherin to accommodate their otherwise suboptimal sorting motifs, forming tripartite complexes with AP-1 [3–5]. In T cells, such binding impairs the constitutive transport of MHC-I or tetherin to the PM, leading to targeting of MHC-I to lysosomes for degradation, or to the accumulation of tetherin at the TGN (Figure 1) [6,7]. An outstanding question was how Nef’s usage of AP-1 yields puzzling, distinct fates for MHC-I and tetherin. A recent study, published in Cell by Morris and
colleagues from Jim Hurley’s group at UC-Berkeley, provides new insights into this issue [8]. Initially, several years ago, the same group reported that AP-1 could be found in a ‘locked’ conformation, not apt for cargo binding, and that Arf1, a small GTPase that attaches itself to the TGN during vesicle formation, promotes the ‘unlocking’ of AP-1 [2,9]. The first clue to understanding the intriguing Nef maneuvers was also provided by the same group, which demonstrated the ability of Nef to induce trimerization of unlocked AP-1 in the presence of Arf1 and cargo, and that, depending on the type of cargo, AP-1 forms either ‘open’ or ‘closed’ trimers [3]. Open trimers can then form higher-order lattices that symmetrically match those of clathrin, promoting clathrin basket polymerization in vitro [3]. When bound to MHC-I CT, Nef induces the formation of open trimers, hijacking Arf1-mediated AP-1 trimerization to a conformation that promotes clathrincoated vesicle (CCV) biogenesis and cargo loading. Strikingly, binding of Nef to tetherin CT, in turn, induces Arf1-mediated oligomerization of AP-1 to a closed trimer conformation, which appeared to be incompatible with clathrin lattice formation [3]. However, the structural bases for the cargo-dependent diversity of Nefinduced AP-1 trimerization and the functional relevance of these differences have remained poorly understood until now. The study by Morris and colleagues employed cryoelectron microscopy (cryo-EM) to determine the structure of the tetherin-bound, closed trimer of AP1 to a high resolution of 3.7 Å. They found that the trimer consists of three central Arf1 molecules assembling around three AP-1 monomers, with one copy of Nef at each AP-1:AP-1 interface. Nef molecules stabilize the trimer in its closed state by using one side to bind the cargo recognition site of AP-1-m1, and another to bind the cargo recognition site of AP-1-s1 in the opposite AP-1 monomer.
Trends in Microbiology, Month Year, Vol. xx, No. yy
1
TIMI 1615 No. of Pages 3
Budding vesicle
Plasma membrane
(A)
Cytosol (B)
5
6 8
3 4
2
1
2ʹ
Retained
7
TGN MHC-I Tetherin Nef
Lysosome
Golgi apparatus
Open Closed AP-1 AP-1 trimer trimer
Clathrin triskelion
Figure 1. HIV/SIV-Nef Counteracts Antiviral Activities of MHC-I and Tetherin Using the Clathrin-Coat Protein AP-1 via Alternative Structural Arrangements. MHC-I and tetherin molecules are synthesized in the endoplasmic reticulum and transported to the Golgi apparatus and the trans-Golgi network (TGN), normally reaching the plasma membrane (PM) to exert antiviral functions (1). HIV/SIVs Nef links the cytosolic tail (CT) of these proteins to AP-1 at the TGN with distinct consequences (2 and 20 ). (A) For MHC-I (allotypes HLA-A and HLA-B), Nef stabilizes the otherwise weak interaction between MHC-I CT and the AP-1-m1 subunit, and induces the trimerization of AP-1 into an ‘open’ conformation (2). Open trimers are prompt to interact with clathrin (3) along the TGN membrane (4), forming higher-order lattices that symmetrically match those of clathrin (5), promoting the formation of MHC-I loaded clathrin-coated vesicles (CCVs) (6). These CCVs deliver MHC-I molecules to endosomes, which will eventually fuse with lysosomes (7). (B) In the case of tetherin, SIV and HIV-1 group O Nef (O-Nef) variants link tetherin CT to AP-1, and induce trimerization of AP-1 into a ‘closed’ conformation (20 ), which seems to be incompatible with clathrin lattice formation (8). Such closed conformation likely prevents tetherin from entering TGN-derived carriers destined to the PM, sequestering tetherin in the TGN.
The study by Morris and colleagues [8] also provides an explanation as to why Nef from HIV-1 strains of pandemic group M (M-Nefs) and of epidemic group O (O-Nefs) displays distinct abilities to counteract tetherin [8]. These HIV-1 groups resulted from independent zoonotic transmission of SIV to humans, and, while M-Nefs are inactive against tetherin, O-Nefs are effective at neutralizing its anti-HIV activity [7]. The authors show that M-Nef molecules can be phosphorylated by host casein kinase I (CK1) at a serine residue that is adjacent to the so-called ‘dileucine motif’, which mediates Nef interaction 2
Trends in Microbiology, Month Year, Vol. xx, No. yy
to AP-1-s1. While phosphorylation of this serine in M-Nefs impairs Nef:AP1-s1 interaction, precluding the formation of closed trimers, in O-Nefs this serine is absent, allowing tetherin counteraction (Figure 1). Is this subversive behavior exclusive toward tetherin? Or only via AP-1-s1? The additional targets of Nef and the possibility of AP-1 variants formed by the combination of different isoforms of its subunits indicate otherwise. In fact, we have recently suggested that Nef could use a functional variant of AP-1, comprising the AP-1-g2 subunit isoform, to retain internalized CD4 in endosomes for its
delivery to lysosomes for degradation [10]. It would be essential to test whether AP-1-g2 plays a similar role in Nef-mediated lysosomal delivery of MHC-I. Moreover, the study by Morris and colleagues reminds us how much cell biology can be learned from viruses, since their finding revealed unprecedented regulatory plasticity of the membrane-trafficking machinery. 1
Department of Cell and Molecular Biology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo 14049-900, Brazil 2 Department of Physiology, School of Medicine and Center for Interdisciplinary Studies of the Nervous System (CISNe), Universidad Austral de Chile, Valdivia 5110566, Chile
TIMI 1615 No. of Pages 3
3
Center for Cell Biology and Biomedicine (CEBICEM), School of Medicine and Science, Universidad San Sebastián, Santiago 7510157, Chile
2. Pereira, E.A. and daSilva, L.L. (2016) HIV-1 Nef: taking control of protein trafficking. Traffic 17, 976–996
*Correspondence: [email protected] (Luis L.P. daSilva) and [email protected] (G.A. Mardones).
4. Jia, X. et al. (2012) Structural basis of evasion of cellular adaptive immunity by HIV-1 Nef. Nat. Struct. Mol. Biol. 19, 701–706
https://doi.org/10.1016/j.tim.2018.09.003 References 1. Sauter, D. and Kirchhoff, F. (2018) Multilayered and versatile inhibition of cellular antiviral factors by HIV and SIV accessory proteins. Cytokine Growth Factor Rev. 40, 3–12
3. Shen, Q.T. et al. (2015) HIV-1 Nef hijacks clathrin coats by stabilizing AP-1:Arf1 polygons. Science 350, aac5137
5. Collins, D.R. and Collins, K.L. (2014) HIV-1 accessory proteins adapt cellular adaptors to facilitate immune evasion. PLoS Pathog. 10, e1003851 6. Roeth, J.F. et al. (2004) HIV-1 Nef disrupts MHC-I trafficking by recruiting AP-1 to the MHC-I cytoplasmic tail. J. Cell Biol. 167, 903–913
7. Kluge, S.F. et al. (2014) Nef proteins of epidemic HIV-1 group O strains antagonize human tetherin. Cell Host Microbe 16, 639–650 8. Morris, K.L. et al. (2018) HIV-1 Nefs are cargo-sensitive AP-1 trimerization switches in tetherin downregulation. Cell 174, 659–671 9. Ren, X. et al. (2013) Structural basis for recruitment and activation of the AP-1 clathrin adaptor complex by Arf1. Cell 152, 755–767 10. Tavares, L.A. et al. (2017) CD4 downregulation by the HIV1 protein Nef reveals distinct roles for the gamma1 and gamma2 subunits of the AP-1 complex in protein trafficking. J. Cell Sci. 130, 429–443
Trends in Microbiology, Month Year, Vol. xx, No. yy
3