Tricking the immune system Peter Mayerhofer

Epstein-Barr virus: BNLF2a

Cowpox virus: CPXV12

Pfeiffersches Drüsenfieber (Mononukleose)

Herpes simplex virus 1: ICP47

Human cytomegalovirus: US6

Bovine herpesvirus 1: UL49.5

Herpesviridae Poxviridae

Viral inhibitors of TAP

Viral inhibitors of TAP

ICP47 blocks

peptide binding

US6 blocks

ATP binding

UL49.5 induces

proteasomal degradation

ATP

http://www.rikenresearch.riken.jp

ER-associated degradation (ERAD)

Viral inhibitors of TAP

ICP47 blocks

peptide binding

US6 blocks

ATP binding

BNLF2a induces

conformational arrest

CPXV12

? UL49.5 induces

proteasomal degradation

ATP

MHC I surface expression

Flow cytometry analysis (FACS)

MHC I

transient transfection of human cells with

pIRES: BNLF2a & EGFP

rela

tive

cell

num

ber

surface MHC I

BNLF2a + -

GFP BNLF2a

mRNA

DNA

Transcription

Translation

Protein

In vitro translation

•  DNA template •  Transcription (RNA polymerase) •  mRNA •  Translation System (Ribosomes, etc.) •  Amino acids, tRNA, membranes, SRP,… •  Detection system •  Proper RNase-free techniques

Eukaryotic translation systems

50 µl reaction: Protein yield

Rabbit Reticulocyte Lysate 50-200 ng Coupled 150-300 ng

Wheat Germ Extract 30-150 ng Coupled 150-300 ng

Insect Cell Extract Coupled 1-4 µg

Human Cell Lysate 1-2 µg?

http://www.ambion.com

ER microsomal membranes

ER microsomal membranes

human cells (Raji)

TAP1/2

BNLF2a

ER lumen

N-linked glycosylation of BNLF2a

ER lumen

=> BNLF2a is a tail-anchored membrane protein

BNFL2a inserts post-translationally into ER membranes

ER microsomes

in vitro transcription/translation

beads coupled with αTAP2 Ab

human cells (Raji)

Co-immunoprecipitation of BNLF2a and TAP

TAP1/2

BNLF2a

solubilization

Site-specific photocrosslinking

amber stop codon (TAG)

lysine

ANB (5-azido-2-nitrobenzoyl-) forms a nitrene

tRNA

In vitro translation using photocrosslinking tRNAs

In vitro translation using photocrosslinking tRNAs

UAG

mRNA

amber stop codon

ANB-Lys-tRNAamb

30 kDa-

14 kDa-

46 kDa-

66 kDa- 97 kDa-

23 24 25 probe position:

anti Sec61α

Site-specific photocrosslinking

Site-specific photocrosslinking of BNLF2a and TAP

IP: α

TA

P1/

2

A

Q

L

E

L

Q S

S A

L C

G P

G

S

S

T E T R

P

S H

P C

P E D

P

D

V

L

L

C

V

L

C

M V H

V

L

E R

R

L

R

L

L

L

V

V

L

F

G

L

L

L

I

ER membrane cytosol

S 54

97

69 55 46 30

12

kDa

IP: α

IgG

coreTAP/ BNLF2aamb54

Photo-adduct

BNLF2a In

put

Tota

l

ICP47 blocks

peptide binding

US6 blocks

ATP binding

BNLF2a induces

conformational arrest

CPXV12

? UL49.5 induces

proteasomal degradation

ATP

Summary / Outlook

Fine-mapping interaction sites Dynamics?

Mechanism ERAD