Viral avoidance and exploitation of the ubiquitin system Felix Randow and Paul J. Lehner NATURE CELL BIOLOGY May 2009

overview Introduction Ubiquitin Ubiquitylation (Enzymes, Steps) Ubiquitin chain types Main part examples of viral avoidance/ exploitation of the ubiquitin system

Ubiquitin Highly conserved regulatory protein Ubiquitously expressed in eukaryotes Can be covalently attached to a protein via an isopeptide bond has a role in regulating many pathways: protein degradation, protein trafficking, transcription, cell cycle control, cell signaling C terminal tail 7 Lysine residues Lys 63 Lys 48

Ubiquitiylation (Enzymes) Requires the action of 3 enzymes: E1: ubiquitin activating enzymes (2) E2: ubiquitin conjugating enzymes (40) E3: ubiquitin ligases (400): RING (really interesting new gene)cullin- RING complexes HECT (homologous to E6AP carboxy terminus) U-box (UFD2 homology) DUBs: deubiquitylating enzymes (90)

Ubiquitylation (Steps) E1 forms a thiol ester with the C-terminal Gly of Ubiquitin in an ATP dependent reaction (activation for nucleophilic attack) Activated ubiquitin is transferred onto a ubiquitin conjugating enzyme (E2) by transesterification Attachment to the substrate protein through an isopeptide bond through one of the ubiquitin ligases (E3) Proteolytic removal of covalently attached ubiquitin is catalyzed by deubiquitylating enzymes

Ubiquitin chain types Incoming ubiquitin can react with the N-terminal Meth or one of the seven Lys of the acceptor ubiquitin E2s determine linkage specificity in conjunction with associated proteins Lys 48 and Lys 63 chains mark proteins for proteasomal degradation Met-1 chains contribute to NF-κB signalling

Ubiquitin as a target for viruses

Ubiquitin encoded in viral genomes Baculoviruses: encode their own ubiquitin gene : most distantly related, (58/76 residues from animal ubiquitin) additional Lys residue virions incorporate their own and cellular ubiquitin into their membranes in a phospholipid modified form

Ubiquitin encoded in viral genomes Bovine viral diarrhoea viruses: Ubiquitin and Ubiquitin-like genes (UBL) Cytopathic strains have a partial duplication of their genome with in-frame insertions of UBLs upstream of the viral NS3 gene The polyprotein requires proteolytic processing =>the insertion of the UBL leads to release of NS3 (missing in noncytopathic strains)

E3 ligases encoded in viral genomes Viruses can encode their own E3 ligase or use adaptor proteins to recruit cellular E3 ligases Most viral E3 belong to the RING family, some don‘t have a RING nor a HECT motif (Replication Transactivation Activator of HHV8) Other viral genes encoding E3 ligase activity : viral RING-CH family and the ICP0 family

E3 ligases encoded in viral genomes Herpes simplex virus type 1: Encodes ICP0 : RING domain with E3 ligase acivity Transactivates viral and cellular genes for reactivation from latency and suppression of innate immunity Induces ubiquitilation and proteasome dependent degradation of Promyelocytic leukemia protein (PML) => destruction of PML nuclear bodies

The RING-CH family of viral ligases E3 ligases with a RING containing a C4HC3 Cys- His configuration Promote viral evasion Function: ubiquitilation and downregulation of receptors (clathrin dependent sorting to an endosomal compartment/ proteasome-mediated degradation using the ERAD pathway

The RING-CH family of viral ligases Human and murine γ-herspesviruses Encode the canonical viral RING-CH ligase K3 Associates and ubiquitilates MHC class I in a post ER compartment=> internalization, ESCRT (Endosomal sorting complex required for transport) depentent sorting and lysosomal degradation Endolysosomal sorting requires Lys 63-linked polyubiquitin chains

The RING-CH family of viral ligases Karposi‘s sarcoma associated herpesvirus: Encodes K3 RING-CH ligase K3 binds and polyubiquitylates MHC class I ->Clathrin-mediated internalization -> endolysosomal degradation

Non-canonical viral E3 ligases Karposi‘s sarcoma associated herpesvirus: Encodes a replication and transcription activator (RTA) with intrinsic E3 ligase activity Triggers the transition from latency to lytic cycle replication Can be suppressed by cellular factors (IRF7) Blocks IRF7 mediated interferon production by promoting its ubiquitylation and degradation

Viral adaptors recruiting cellular E3 ligases Human papillomavirus 16 and 18: Encode the E6 oncoprotein Redirects host cell E3 ligases to ubiquitylate host proteins => degradation E6 induces degradation of p53 by recruiting a cellular E3 ligase (E6AP)

Viral use of cullin-RING E3 ligases Human immunodeficiency virus: Viral infectivity factor (Vif): Induces polyubiquitylation and proteasomal degradation of apolipoprotein B editing compelx (APOBEC) Vif acts as an adaptor to recruit APOBECG to the CUl5 elongin B/C-Rbx SCF ligase Depletes the pool of cytosolic APOBEC3G Cell Host & Microbe 5, June

Viral use of cullin-RING E3 ligases Apolipoprotein B editing complex (APOBEK): Deamination of the non-coding strand of the viral genome from deoxyC to deoxyU => G to A hypermutaion in the coding strand restricts the propagation of the virus If Vif fails to neutralize all APOBEC3F/G activity, Vpr forms a complex with Cul1 and Cul4 to target cellular uracil DNA glycosylase for ubiquitylation and proteasomal degradation

Viral use of cullin-RING E3 ligases Several paramyxoviruses: Limit the activity of Interferons by targeting STATs for ubiquitilation and degradation V proteins bind directly to STATs and recruit them to DNA Damage Binding Protein 1 (DDB1) a substrate adaptor of the Cul4A E3 ligase

Viral genes target cellular substrates for destruction through the ERAD pathway stringent quality control in the ER: proteins failing to assemble => translocation back to the cytosol for proteasome- mediated degradation Subversion of this pathway: viruses target host proteins in the secretory pathway for rapid degradation

Viral genes target cellular substrates for destruction through the ERAD pathway Human cytomegalovirus: Encodes the proteins US2 and US11: Catalyse the translocation of MHC class I from the ER back to the cytosol Derlin 1 and SEL1L are required for US11-mediated dislocation Signal peptide peptidase is required for US2- mediated translocation

Viral genes target cellular substrates for destruction through the ERAD pathway Human immunodeficiency virus: Vpu acts as viral adaptor: Induces the proteosomal degradation of the HIV cell surface receptor CD4: Binds to cytoplasmic tail of CD4 Recruits ß-transducin repeat- containing protein, the substrate adaptor for the cullin RING- ßTrCP ligase

Viruses encode DUBs Herpes simplex virus 1: Encodes UL36 a ubiquitin specific Cys protease Physiological substrate and role in viral life cycle unclear Viruses with inactive UL36 show slight replication impairment and less pathogenicity

Do viruses avoid self-ubiquitylation by encoding Lys-free proteins? Most substrates are ubiquitylated on an aceptor Lys residue => Lys-free proteins could be advantageous to viruses HSV1 (68%GC content/genome) harbours 8 Lys-free proteins

Do viruses avoid self-ubiquitylation by encoding Lys-free proteins? Epstein-Barr-Virus: Lys free protein BHRF1 (Bcl2 family member) Controls apoptosis by regulating cytochrome C release from mitochondria Removal of Lys residues from Bcl2 inhibits degradation and apoptosis Possibly escapes ubiquitylation under pro- apoptotic conditions

A role for ubiquitin in viral egress Cellular machinery for budding virions equals the formation of multivescular bodies Viral L-domain motifs interact with components of the ESCRT pathway and recruit NEDD4 E3 ligases for membrane fission Essential role of ubiquitin in the release of enveloped viruses from the plasma membrane Oncogene , 1972–1984

Conclusions Identification of viral ineraction with the ubiquitin system provides insights into viral pathogenesis and into basic cellular prosesses (Endoplasmatic Reticulum Associated protein Degradation, receptor internalization) More information about non Lys-48 /Lys-63 chains and their manipulation by viruses is needed

Thank you for your attention! For further reading: Isaacson, MK and Hiddle, LP (2009) Ubiquitination, Ubiquitin-like Modifiers, and Deubiquitination in Viral Infection Cell Host & Microbe 5, June 18