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

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 Sheehy et al. 2002

 TRIM5alpha/TRIMCyp Stremlau et al. 2004

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

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

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 , 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. Workshop on XMRV 7-8 September 2010, Bethesda USA Conclusions

 XMRV is restricted by factors expressed in human PBMC: APOBEC3G, Tetherin but not TRIM5alpha

 How might XMRV evade these factors in a natural infection?  The levels of restriction factor may be too low in vivo  It may replicate in cells that do not express these factors

 Could partial inhibition be beneficial for the virus?  Reduce pathogenicity  Increase sequence diversity

 XMRV is susceptible to proteins expressed in mouse: Apobec3, Tetherin, Fv1n and Fv1b

Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA NIMR Jonathan Stoye Melvyn Yap Ian Taylor Dave Goldstone

King’s College London Stuart Neil Darren Wight Rui Pedro Galao Harriet Groom Mirella Nader Virginie Boucherit

Presented at the 1st Intl. Workshop on XMRV 7-8 September 2010, Bethesda USA