Structural insight into tanapoxvirus‐mediated inhibition of apoptosis

Abstract

Premature programmed cell death or apoptosis of cells is a strategy utilized by multicellular organisms to counter microbial threats. Tanapoxvirus (TANV) is a large double‐stranded DNA virus belonging to the poxviridae that causes mild monkeypox‐like infections in humans and primates. TANV encodes for a putative apoptosis inhibitory protein 16L. We show that TANV16L is able to bind to a range of peptides spanning the BH3 motif of human proapoptotic Bcl‐2 proteins and is able to counter growth arrest of yeast induced by human Bak and Bax. We then determined the crystal structures of TANV16L bound to three identified interactors, Bax, Bim and Puma BH3. TANV16L adopts a globular Bcl‐2 fold comprising 7 α‐helices and utilizes the canonical Bcl‐2 binding groove to engage proapoptotic host cell Bcl‐2 proteins. Unexpectedly, TANV16L is able to adopt both a monomeric and a domain‐swapped dimeric topology where the α1 helix from one protomer is swapped into a neighbouring unit. Despite adopting two different oligomeric forms, the canonical ligand binding groove in TANV16L remains unchanged from monomer to domain‐swapped dimer. Our results provide a structural and mechanistic basis for tanapoxvirus‐mediated inhibition of host cell apoptosis and reveal the capacity of Bcl‐2 proteins to adopt differential oligomeric states whilst maintaining the canonical ligand binding groove in an unchanged state. Database Structural data are available in the Protein Data Bank (PDB) under the accession numbers http://6TPQ, http://6TQQ and http://6TRR.

Structural Investigation of Orf Virus Bcl-2 Homolog ORFV125 Interactions with BH3-Motifs from BH3-Only Proteins Puma and Hrk

“…For ORFV125:Puma BH3 complex a clear and continuous electron density map was observed for ORFV125 residues 5–61 and 65–133, and Puma BH3 motif residues 132–155 ( Figure 1 A,C), with the remainder of the residues presumed to be disordered. ORFV125 adopts the conserved Bcl-2 fold with seven α-helices in a domain-swapped dimeric configuration ( Figure 2 A) where the α1 helix of one protomer is swapped with the neighbouring protomer to form the dimer interface as previously seen in other poxvirus genera such as VACV F1L [ 27 , 28 ] ( Figure 2 C), VARV F1L [ 26 ], DPV022 [ 29 ] and TANV16L [ 30 ]. A structural similarity analysis was performed using DALI [ 41 ], which identified DPV022 (PDB ID 4UF1) [ 29 ] as the closest viral Bcl-2 homolog of ORFV125 with an rmsd of 2.6 Å over 119 Cα atoms and human Mcl-1 (PDB ID 3PK1) [ 42 ] as the closest cellular Bcl-2 homolog with an rmsd of 2.7 Å over 97 Cα atoms.…”

“…Bcl-2 homologs are widely used amongst large DNA viruses to ensure viral proliferation and/or survival [ 2 , 13 , 43 ]. Amongst the poxviridae, the majority of genera have been shown to encode apoptosis inhibiting Bcl-2 homologs including the orthopoxviridae vaccinia virus F1L [ 27 , 28 ], variola F1L [ 26 ] and ectromelia viruses EMV025 [ 44 ], leporipoxviridae myxomavirus M11L [ 45 ], cervidpoxviridae deerpox virus DPV022 [ 29 , 46 ], capripoxviridae sheeppox virus SPPV14 [ 47 , 48 ], avipoxviridae fowlpoxvirus FPV039 [ 49 ] and canarypox virus CNP058 [ 50 ], yatapoxviridae tanapox virus TANV16L [ 30 ] and parapoxviridae orf virus ORFV125 [ 23 , 24 , 25 ]. Whilst many poxviridae encode for sequence, structural or functional pro-survival Bcl-2 homologs, considerable diversity exists amongst these proteins [ 2 ].…”

“…Detailed analysis of the interaction between different poxvirus-encoded Bcl-2 proteins and host pro-apoptotic Bcl-2 reveals that whilst the BH1 motif is frequently not conserved in viral protein residues that occupy the structurally equivalent positions of the NWGR motif in the BH1 region of, for example, human Mcl-1 and Bcl-x L , it still performs important roles in the sequence divergent virus encoded Bcl-2 proteins. A comparison of ORFV125:Puma BH3 complex with TANV16L: Puma BH3 [ 30 ] reveals that the mode of interaction is nearly identical, despite the ORFV125:Puma interaction being approximately four-fold lower in affinity [ 25 ]. The hallmark interaction between the Arg residue from the NWGR motif and the Asp from the BH3 motif peptide is present in both complexes; the equivalent region of the BH1 motif in ORFV125 is formed by the sequence ‘SPGR’ whereas in TANV16L it is ‘NDNR’, with both contributing to binding and specificity [ 30 ] ( Figure 3 C).…”

“…ORFV125 supresses UV-induced DNA fragmentation, cytochrome c release and subsequent activation of caspases [ 23 ]. Structural and biochemical studies of ORFV125 revealed that it adopts a domain-swapped dimeric topology in solution [ 25 ] similar to that observed in some other poxviral Bcl-2 proteins such as Vaccinia virus (VACV) and variola virus (VARV) F1L [ 26 , 27 , 28 ], deerpox virus DPV022 [ 29 ] and tanapoxvirus TANV16L [ 30 ], and only interacts with four BH3 motifs of cellular pro-apoptotic Bcl-2 proteins: Bax, Bak, Puma and Harakiri (Hrk) [ 25 ]. Interestingly, ORFV125 did not show any detectable affinity towards Bim BH3 motif peptide, the universal human Bcl-2 interactor [ 25 ].…”

Designing BH3-mimetic Peptide Inhibitors for the Viral Bcl-2 Homologs A179L and BHRF1: Importance of long-range electrostatic interactions

“…However, they exhibit little or no sequence similarity with the anti-apoptotic Bcl-2 proteins. Examples include F1L and N1 from vaccinia virus (PDB ID: 2VTY and 2UXE) 17–18 , M11L from myxoma virus (PDB ID: 2O42) 19 , BHRF1 from Epstein-Barr virus (EBV) (PDB ID: 2WH6) 20 , KsBcl-2 from Kaposi sarcoma herpesvirus (PDB ID: 1K3K) 21 , F1L from Variola virus (PDB ID: 5AJJ) 22 , M11 from y-herpesvirus 68 (PDB ID: 2ABO) 23 , ORFV125 from ORF virus (PDB ID: 7ADS) 24 , GIV66 from grouper iridovirus, (PDB ID: 5VMN) 25 , CNP058 from canarypox virus (PDB ID: 5WOS) 26 , FPV039 from fowlpox virus (PDB ID: 5TZP) 27 , 16L protein from tanapoxvirus (PDB ID: 6TQP) 28 and A179L from African swine fever virus (PDB ID: 5UA4) 29 . These viruses cause diseases such as canarypox (song birds), smallpox (humans), myxomatosis (rabbits), Burkitt’s lymphoma, Hodgkin and non-Hodgkin lymphoma and other cancers (humans), Castleman’s disease (humans), contagious ecthyma (sheep and goats), sleepy grouper disease (fish), fowlpox (chicken) and African swine fever (pigs and warthogs) 30–37 .…”