Nef-mediated tetherin antagonism

Transcription

1 Nef-mediated tetherin antagonism Frank Kirchhoff Institute of Molecular Virology Ulm Medical Center, Germany 9/11

2 African primates represent a large reservoir for immunodeficiency viruses and chimpanzees, gorillas and mangabeys transmitted the virus to humans Bieniasz & Ho Cell 2008 Some SIV-infected monkey species (AGMs, SMs) do not develop disease

3 HIV-1 is the result of a recombination event and multiple cross-species transmissions (Bailes et al., Science 2003; others) p ( ) Only one of at least four independent transmissions of SIVcpz Only one of at least four independent transmissions of SIVcpz from chimpanzees and SIVgor from gorillas to humans led to a pandemic

4 Humans developed a natural combination therapy long before HAART TRIM5 : destabilization of the viral capsid APOBEC3G: lethal hyper-mutations ti Tetherin: inhibition of virus release Kirchhoff Cell Host & Microbe (2010)

5 Humans developed a natural combination therapy long before HAART TRIM5 : destabilization of the viral capsid APOBEC3G: lethal hyper-mutations ti Tetherin: inhibition of virus release Kirchhoff Cell Host & Microbe (2010)

6 Humans developed a natural combination therapy long before HAART TRIM5 : destabilization of the viral capsid APOBEC3G: lethal hyper-mutations ti Tetherin: inhibition of virus release Kirchhoff Cell Host & Microbe (2010)

7 Humans developed a natural combination therapy long before HAART TRIM5 : destabilization of the viral capsid APOBEC3G: lethal hyper-mutations ti Tetherin: inhibition of virus release Kirchhoff Cell Host & Microbe (2010)

8 Humans developed a natural combination therapy long before HAART TRIM5 : destabilization of the viral capsid APOBEC3G: lethal hyper-mutations ti Tetherin: inhibition of virus release Usually pretty effective: >10% of our genome are of retroviral origin But HIV has developed d effective countermeasures

9 Nature, in press

10 MORE antiviral factors are out there Broadly acting effectors included IRF1, C6orf150 (also known as MB21D1), HPSE, RIG-I (also known as DDX58), MDA5 (also known as IFIH1) and IFITM3, whereas more targeted antiviral specificity was observed with DDX60, IFI44L, IFI6, IFITM2, MAP3K14, MOV10, NAMPT (also known as PBEF1), OASL, RTP4, TREX1 and UNC84B (also known as SUN2).

11 It underscores the paradox of the association between increasing levels of viral load and greater expression of antiviral defense pathways. IFN-stimulated genes encoding intrinsic antiretroviral defense factors have become indicators rather than suppressors of HIV-1 replication.

12 Humans developed a natural combination therapy long before HAART TRIM5 : destabilization of the viral capsid APOBEC3G: lethal hyper-mutations ti Tetherin: inhibition of virus release Usually pretty effective: >10% of our genome are of retroviral origin But HIV has developed d effective countermeasures

13 Why do these antiviral factors not control HIV? High variability and development of resistance Not HIV-1 and HIV-2 Not SIVagm

14 Some host restriction factors target essential viral components in a relatively l unspecific manner TRIM5 : viral capsid APOBEC3G: viral RNA Tth Tetherin: viral membrane Broad antiviral activity & no simple escape

15 HIV and SIV contain several small accessory genes

16 Accessory genes of HIV and SIV Rabbit ~10 million years RELIK: Tat, Rev Lemur: ~7 million years psivgml: Tat, Rev, Vif Monkeys, Apes, Humans: today HIV & SIV: Tat, Rev, Vif, Vpr, Nef, Vpu, Vpx Kirchhoff, Cell Host & Microbe (2010)

17 Vif targets APOBEC3G for proteasomal degradation

18 Tetherin a broad-based inhibitor of virus release MLV SIV HIV HHV-8 XMRV Lassa virus Marburg virus VSV Ebola virus JSRV PERV adapted from Murphy, University of California

19 Vpu antagonizes tetherin, which blocks virus release and induces CD4 degradation Neil et al., Nature 2008; Van Damme et al., Cell Host & Microbe 2008 Courtesy Paul Spearman Kirchhoff, Nat. Rev. Microbiology 2009 Perez-Caballero et al., Cell 2009

20 Vpu antagonizes tetherin, which blocks virus release and induces CD4 degradation Neil et al., Nature 2008; Van Damme et al., Cell Host & Microbe 2008 Courtesy Paul Spearman Kirchhoff, Nat. Rev. Microbiology 2009

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22 Genes encoding host restriction factors, such as TRIM5, APOBEC3G and tetherin, evolve unusually fast (Sawyer et al., 2004; Sawyer et al., 2007; MNtt McNatt et al., 2009). This positive selection for diversification is driven by the need to antagonize new emerging pathogens and to escape viral antagonists over millions of years of virus-host co-evolution. TRIM5, APOBEC3G and tetherin all show a high degree of sequence divergence and constitute barriers to zoonotic viral transmissions because the viral antagonists often act in a species-specific manner.

23 HIV-1 is the result of a recombination event and multiple cross-species transmissions (Bailes et al., Science 2003; others) Adaptation to apes inactivated human TRIM5 and APOBEC3G Only one of at least four independent transmissions of SIVcpz Only one of at least four independent transmissions of SIVcpz from chimpanzees and SIVgor from gorillas to humans led to a pandemic

24 Vpu function of SIVs and non-pandemic HIV-1 O, N & P strains?

25 Downregulation of CD4 surface expression by different lentiviral Vpus FACS analysis of 293T cells cotransfected with a CD4 expression vector (1µg) and different pcgcg-vpus p (5µg, IRES GFP)

26 All Vpu proteins induce CD4 degradation except those from HIV-1 group N Daniel Sauter

27 SIV / / d HIV 1M&N V t t i t th i SIVgsn/mus/mon and HIV-1 M & N use Vpu to antagonize tetherin and SIVcpz & SIVgor use Nef to antagonize tetherin

28 How do SIVcpz, SIVgor and HIV-1 1O antagonize tetherin? th and SIVcpz & SIVgor use Nef to antagonize tetherin Tetherin Vpu Tetherin Nef Some SIVs that don t have Vpu use Nef as tetherin antagonist (Zhang et al., 2009; Jia et al., 2009)

29 Nef is important for efficient persistence and for full virulence of HIV-1 in humans and SIVmac in rhesus macaques (Kestler et al., Cell 1991; Deacon et al., Science 1995; Kirchhoff et al., New Engl J Med. 1995) nef+: saids nef: No disease

30 Nef: structure and function

31 Nef: structure and function CD4 cell membrane myristoylated Nef Uptake globular core AP 2 cellular receptors endosome 2 flexible loops AP Uptake into the cell and degradation degradation lysosome Nef is expressed early and at very high levels

32 Nef: a multi-functional master manipulator of the host cell

33 Nef interacts with the CT of tetherin and may antagonize it by an AP-2 dependent mechanism (Zhang et al., 2011)

34 SIV / / dhiv1m&n V SIVgsn/mus/mon and HIV-1 M & N use Vpu and SIVcpz & SIVgor use Nef to antagonize tetherin

35 SIVgsn/mus/mon / and dhiv1m&n HIV-1 use Vpu and SIVcpz & SIVgor use Nef to antagonize tetherin HIV-1O&PVpus Vpus &Nefsdonot promote virus release

36 Why did HIV-1 M switch from Nef to Vpu to antagonize tetherin? (Jia et al., 2009; Sauter et al., 2009; Zhang et al. 2009) SIVcpz & SIV gor HIV-1 M & N Tetherin Tetherin TM Nef CT Vpu Human tetherin contains a unique deletion that renders it resistent to Nef

37 The Protective Deletion in Tetherin Evolved at Least 1 Mio Years Ago Neanderthal Denisova modern human mya Sauter et al., 2011

38 Switches between Nef- and Vpu-mediated tetherin antagonism preceded ddthe emergence of fhiv-1

39 Tetherin is a significant but not insurmountable barrier to zoonotic transmission of SIVs to humans Sauter et al., Cell Host & Microbe (2009) HIV-1 Vpu function M N O P Tetherin CD Sauter et al., Cell (2010) Only the HIV-1 1MVpu is optimally adapted to humans

40 Adaptive changes of SIVcpz Vpus to humans HIV-1 N: gain of anti-tetherin activity TM domain 86 EK505 MLLLIKLGFI GLAIETLIVI VVWAIVYRIY REVKVEEKIS QLRQRIRDRA EDSGNESDGD AEEL ANLLPPDRI DQDNWV EK505mut......A...A....LL YBF S... A.GAAVS.AV I...LL..E. KKI.LQ...K HI...E.E.....W.DGDEEW LVT..SSSKL..G... AxxxA LL HIV-1 M: gain of anti-tetherin activity TM domain 8 MB897 MEIFIILGLI GIVIELVIAI VVWLKAYECY KALKRQERRD QLIDRIRERA EDSGNESDGD TEELEAILIP EDRHVLVAIR GY MB897mut.QP-..VAIV AL.VAII SIVII NL43.QP-..VAIV AL.VAII......SIVIIE. RKIL..RKI. R...LI......E.E VSA.VEMGVE MGH.APWD.D DL TM domain Helix 1 HIV-1 N: loss of CD4 degradation YBF30 ML---SLGFI ALGAAVSIAV IVWALLYREY KKIKLQEKIK HIRQRIRERE EDSGNESDGD AEWLDGDEEW LVTLLSSSKL DQGNWV YBF30mut VE...S QL EK505..LLIK... G.AIETL.VI V...IV..I. REV.VE...S QL...D.A.....E AN..PPDRI..D...

41 Adaptation of SIVcpz to humans resulted in gain of Vpu and loss of Nef-mediated tetherin antagonism What happens to Vpu and Nef when HIV-1 is reintroduced into chimpanzees?

42 Molecular cloning of HIV-1 strains from chimpanzees (Mwaengo and Novembre, JVI 1998) Nicola Götz Daniel Sauter JC16 & NC7: CPZ adapted molecular HIV-1 clones

43 The CPZ-adapted HIV-1 strain maintains Vpu-mediated tetherin antagonism

44 ... and evolved anti-tetherin activity by Nef

45 The C loop of the JC16 Nef is important for tetherin antagonism

46 Five amino acid changes (ENTSLLHPVS to EDNILLHPMC) Five amino acid changes (ENTSLLHPVS to EDNILLHPMC) were sufficient to render HIV-1 Nefs active against CPZ

47 Five amino acid changes (ENTSLLHPVS to EDNILLHPMC) were sufficient to render HIV-1 Nefs active against CPZ Only three in the case of the SF-2 Nef protein

48 Summary Nef can gain anti-tetherin activity within a single in vivo passage CPZ-adapted HIV-1usesboth Vpu & Nef to antagonize tetherin The C-loop of Nef is relevant for tetherin antagonism Is tetherin antagonism important in vivo? Pro: many viruses evolved specific tetherin antagonists Con: HIV-1 O causes AIDS although it may lack anti-tetherin activity

49 SHIVnefs replicate to high levels and cause fatal disease in about half of infected rhesus macaques (Alexander et al., 1999; Kirchhoff et al., 1999; Mandell et al., 1999) The capability of SHIVnefs to replicate efficiently and to cause disease in rhesus macaques was not associated with the acquisition of anti-tetherin activity by HIV-1 Nef (Goetz et al., in preparation)

50 Humans and other mammals have evolved antiretroviral factors (TRIM5, APOBEC3G, tetherin) As a countermeasure some modern retroviruses, like HIV-1, evolved specific tools (Vif, Vpu, Vpr, Vpx, Nef) to antagonize them Kirchhoff, Cell Host & Microbe (2010)

51 Most primate lentiviruses (including the direct precursors of HIV-1 and HIV-2) use Nef to antagonize tetherin

52 Nef interacts with the CT of tetherin and may antagonize it by an AP-2 dependent mechanism (Zhang et al., 2011)

53 Human tetherin contains a unique deletion that renders it resistent to Nef chimpanzee Tetherin human Tetherin DDIWK

54 Summary: Vpu/tetherin Tth Tetherin is a significant ifi tbarrier against ttransmissions i of fsivs to human Only pandemic HIV-1 strains mastered this hurdle perfectly Tetherin antagonism: more important t for transmission i than for pathogenesis? Nef, Vpu Nef Nef, Env

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56 Characteristics of pandemic HIV-1 M strains Pathogenicity: Vpu, lack of TCR-CD3 down-modulation, CXCR4 tropism Spread: efficient i antagonism of tetherin by Vpu

57 Acknowledgments Beatrice H. Hahn Hui Li Frederic Bibollet-Ruche Matthis Kraus (Alabama, USA) Guido Silvestri (Philadelphia, USA) Paul Sharp Elisabeth Bailes (Nottingham, UK) Michaela Müller-Trutwin (Paris, France) Martine Peeters (Montpellier, France) Ulrich Schubert Jörg Votteler (Erlangen, Germany) Paul Bieniasz Theodora Hatziioannou (New York, USA) Cristian Apetrei Ivona Pandrea (Tulane, USA) Donald Sodora (Seattle, USA) Christiane Stahl-Hennig Ulrike Sauermann (DPZ, Germany)

58 Institute of Molecular Virology, Ulm Funding: DFG, EU, NIH

59 Nef ape Tetherin SIVcpz Nef SIVgor Nef AIDS pandemic human Tetherin HIV 1 M HIV 1 N HIV 1 O HIV 1 P Vpu Nef Vpu??? Sauter et al., 2009

60 Sauter et al., 200 Sauter et al., 201 Sauter et al., 201