Restriction of XMRV infection Kate Bishop National Institute for Medical Research 7th September 2010 Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA Retroviral restriction factors Cell autonomous inhibitors of retroviral replication Inhibit a wide range of retro (and other) viruses, including MLVs Usually some species specificity - thought to influence zoonotic transmission and pathogenicity of infection Expressed in haematopoietic cells Four families: Fv1 (only in mice) Lilly, 1970; Best et al. 1996 APOBEC proteins Sheehy et al. 2002 TRIM5alpha/TRIMCyp Stremlau et al. 2004 Tetherin Neil et al. 2008; Van Damme et al. 2008 Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA Blocks to the life cycle of a retrovirus 1. Binding 2. Fusion and 10. maturation entry TRIM5 7. Translation 3. Uncoating RTC Reverse transcription APOBEC3G Trafficking Fv1 8. Assembly PIC 4. Nuclear entry 6. Transcription 5. Integration 9. Budding provirus gag pol env Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA APOBEC proteins Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA The human family of APOBEC cytidine deaminases name function APOBEC1 apoB mRNA AID Antibody diversification (SHM & CSR) APOBEC2 APOBEC3A APOBEC3B anti-viral APOBEC3C APOBEC3D/E anti-viral APOBEC3F anti-viral APOBEC3G anti-viral APOBEC3H APOBEC4 cytidine deaminase cytidine deaminase motif motif Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA APOBEC3G/3F proteins Highly expressed in leukocytes (T-cells, B-cells, monocyte), testis and ovary Protein expression upregulated by IFN in macrophage and dendritic cells but not in T-cells Most lentiviruses have a Vif protein that overcomes the APOBEC3G/3F of their host. MLVs do not encode a Vif protein Human APOBEC3G inhibits Mo-MLV Murine Apobec3 only weakly inhibits Mo-MLV. How Mo-MLV escapes inhibition is unclear Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA XMRV is inhibited by human APOBEC3G Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA How do APOBEC proteins inhibit HIV? Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA APOBEC3G induces mainly G to A mutations vif HIV To No APOBEC ACGT A C 2 From G1 T n=6500bp To hA3G ACGT A 1 C 1 From G105 2 T1 n=6500bp Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA Reverse Transcription R U5 PBS PPT U3 R 5’ G AAA 3’ tRNA DNA synthesis R U5 PBS PPT U3 R G AAA RNase H PBS PPT U3 R G AAA RU5 First strand transfer PBS PPT U3 R G AAA DNA synthesis RU5 RNase H PBS G PPT DNA synthesis U3 RU5 RNase H PPT U3 RU5 PBS UC U3 RU5 Second strand transfer DNA synthesis U3 RU5 A U3 RU5 U3 RU5 U U3 RU5 Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA Reverse Transcription R U5 PBS PPT U3 R 5’ G AAA 3’ tRNA DNA synthesis R U5 PBS PPT U3 R AAA RNase H PBS PPT U3 R AAA RU5 First strand transfer PBS PPT U3 R AAA DNA synthesis RU5 RNase H PBS PPT DNA synthesis U3 RU5 RNase H U3 RU5 PBS PPT U3 RU5 Second strand transfer DNA synthesis U3 RU5 A U3 RU5 U3 RU5 U3 RU5 Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA Preferred target sites for different APOBEC proteins Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA XMRV hypermutation in T-cell lines From Paprotka, T. et al. 2010. J. Virol. 84(11):5719-5729 color code: GG to AG in red, GA to AA in cyan, GC to AC in green, GT to AT in magenta, all other mutations in black. Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA Summary (APOBEC) XMRV is inhibited by human APOBEC3G and 3B to a similar extent to Mo-MLV and may be partially susceptible to hA3F (also see Paprotka et al. J. virol 2010; Stieler and Fischer PLoS One 2010) XMRV DNA from T-cell lines (CEM and H9) were extensively mutated, with the formation of stop codons in 11/14 and 12/13 clones respectively (Paprotka et al. J. virol 2010) Human leukocytes express both hA3G and hA3F Human prostate cell lines express little or no hA3G, although the levels in primary prostate tissue vary (Paprotka et al. J. virol 2010; Stieler and Fischer PLoS One 2010; Refsland et al. NAR 2010) XMRV is 10 fold less infectious than Mo-MLV in the presence of mouse Apobec3 Glycosylated Gag may protect against mA3 for some MLVs, but XMRV does not appear to encode a gGag (Kolokithas et al. J virol 2010) Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA Tetherin proteins Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA Tetherin/CD317/BST2 Type II membrane protein of unusual topology Disulfide linked dimer with variable glycosylation Localizes to multiple membrane compartments Expressed in leukocytes (B-cells, T- cells, NK-cells, dendritic cells, macrophage) and several epithelial cell lines Upregulated with cellular activation and IFN treatment Restricts the release of retroviruses, filoviruses, arenaviruses and KSHV Non-specific incorporation into assembling virions Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA Tetherin directly crosslinks nascent virions to the Plasma Membrane See: Perez-Caballero et al (2009) Cell, Kirchhoff (2009) Nat Rev Microbiol. Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA XMRV is restricted by tetherin proteins Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA XMRV env cannot counteract tetherin Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA Summary (tetherin) XMRV is sensitive to human, AGM, Rhesus and mouse tetherins Expression of HIV-1 Vpu rescues XMRV infection in HeLa cells, suggesting XMRV does not have a countermeasure to overcome tetherin restriction XMRV env is not able rescue Vpu-deficient HIV-1 Human PBMCs express tetherin Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA Fv1 and TRIM proteins Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA Similarities between Fv1 and TRIM5alpha Fv1 C. Coil MHR TRIM5alpha RING B-Box C. Coil B30.2 TRIM5CypA RING B-Box C. Coil Cyp A Multimerization Specificity/binding Target the capsid (CA) proteins of retroviruses Ubiquitously expressed (including in T-cells and macrophages). TRIM5alpha induced by IFN TRIM5alpha inhibits viral replication before reverse transcription, whereas Fv1 inhibits after reverse transcription Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA MLV / Fv1 tropism N NB B Fv1n Fv1b Fv1n Fv1b Human TRIM5alpha restricts N-tropic, but not B-tropic, MLV XMRV CA sequence most closely resembles B-tropic MLV at residues critical for restriction (amino acids 109 and 110) Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA XMRV is not restricted by TRIM5 proteins N-MLV B-MLV XMRV Fv1n 1.34 ± 0.01 0.15 ± 0.03 0.12 ± 0.01 Fv1b 0.11 ± 0.01 0.64 ± 0.01 0.18 ± 0.02 Orangutan 0.09 ± 0.003 1.17 ± 0.06 1.09 ± 0.02 Gorilla 0.12 ± 0.01 0.68 ± 0.03 1.04 ± 0.02 Chimpanzee 0.12 ± 0.01 0.18 ± 0.01 1.04 ± 0.09 Human 0.13 ± 0.02 0.34 ± 0.01 1.05 ± 0.02 Sooty mangabey 0.10 ± 0.001 0.26 ± 0.01 1.05 ± 0.04 AGM (tantalus) 0.08 ± 0.01 0.55 ± 0.05 1.06 ± 0.03 Rhesus 0.15 ± 0.01 1.35 ± 0.05 1.06 ± 0.05 Rhesus TrimCyp 1.24 ± 0.01 1.28 ± 0.08 1.11 ± 0.3 Capuchin 0.58 ± 0.001 1.14 ± 0.02 1.09 ± 0.02 Squirrel monkey 1.24 ± 0.01 1.33 ± 0.10 1.10 ± 0.02 Cotton top tamarin 0.07 ± 0.01 1.09 ± 0.03 1.10 ± 0.04 Common marmoset 1.31 ± 0.02 1.32 ± 0.05 1.09 ± 0.03 Owl monkey TrimCyp 1.14 ± 0.03 1.32 ± 0.04 1.25 ± 0.3 Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA XMRV is restricted by endogenous Fv1 Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA MLVs with distinct restriction patterns differ at 18 residues in CA Amino acid Residue position N-MLV B-MLV XMRV Mo-MLV 4LLMA 7NND N 46 T T I I 82 N N N D 94 D D N D 109 Q T T Q 110 R EEA 117 L L L H 147 D D N N 159 E G EE 202 S S S N 214 R R K K 229 V V I I 233 T T I T 242 A A A T 244 E E D D 247 K K R K 249 K K R K * Unique residues Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA Structures of MLV capsid proteins Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA Summary (Fv1 and TRIM5) XMRV is the first reported MLV to be restricted by both Fv1n and Fv1b alleles XMRV is not restricted by any of the TRIM5alpha or TrimCyp proteins tested The CA sequence of XMRV suggests it would be a B-tropic MLV, but it does not have the same restriction profile as the prototype B-tropic MLV Restriction is complicated! Presented at the 1st Intl.