Functional and structural studies of the vaccinia virus virulence factor N1 reveal a Bcl-2-like anti-apoptotic protein.
Vaccinia virus (VACV) encodes many immunomodulatory proteins, including inhibitors of apoptosis and modulators of innate immune signalling. VACV protein N1 is an intracellular homodimer that contributes to virus virulence and was reported to inhibit nuclear factor (NF)-kappaB signalling. However, analysis of NF-kappaB signalling in cells infected with recombinant viruses with or without the N1L gene showed no difference in NF-kappaB-dependent gene expression. Given that N1 promotes virus virulence, other possible functions of N1 were investigated and this revealed that N1 is an inhibitor of apoptosis in cells transfected with the N1L gene and in the context of VACV infection. In support of this finding virally expressed N1 co-precipitated with endogenous pro-apoptotic Bcl-2 proteins Bid, Bad and Bax as well as with Bad and Bax expressed by transfection. In addition, the crystal structure of N1 was solved to 2.9 A resolution (0.29 nm). Remarkably, although N1 shows no sequence similarity to cellular proteins, its three-dimensional structure closely resembles Bcl-x(L) and other members of the Bcl-2 protein family. The structure also reveals that N1 has a constitutively open surface groove similar to the grooves of other anti-apoptotic Bcl-2 proteins, which bind the BH3 motifs of pro-apoptotic Bcl-2 family members. Molecular modelling of BH3 peptides into the N1 surface groove, together with analysis of their physico-chemical properties, suggests a mechanism for the specificity of peptide recognition. This study illustrates the importance of the evolutionary conservation of structure, rather than sequence, in protein function and reveals a novel anti-apoptotic protein from orthopoxviruses.
Cooray
Samantha
S
Department of Virology, Faculty of Medicine, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG, UK.
Bahar
Mohammad W
MW
The Oxford Protein Production Facility and The Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
Abrescia
Nicola G A
NGA
The Oxford Protein Production Facility and The Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
McVey
Colin E
CE
Department of Virology, Faculty of Medicine, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG, UK.
Bartlett
Nathan W
NW
Department of Virology, Faculty of Medicine, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG, UK.
Chen
Ron A-J
RA
Department of Virology, Faculty of Medicine, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG, UK.
Stuart
David I
DI
The Oxford Protein Production Facility and The Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
Grimes
Jonathan M
JM
The Oxford Protein Production Facility and The Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
Smith
Geoffrey L
GL
Department of Virology, Faculty of Medicine, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG, UK.
D000595:Amino Acid Sequence; D000818:Animals; D051023:BH3 Interacting Domain Death Agonist Protein / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D018360:Crystallography, X-Ray; D006801:Humans; D047468:Immunoprecipitation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014616:Vaccinia virus / Q000276:immunology / Q000472:pathogenicity*; D014764:Viral Proteins / Q000737:chemistry* / Q000502:physiology*; D037521:Virulence Factors / Q000737:chemistry* / Q000502:physiology*; D051028:bcl-2-Associated X Protein / Q000737:chemistry / Q000378:metabolism; D051025:bcl-Associated Death Protein / Q000737:chemistry / Q000378:metabolism; D051020:bcl-X Protein / Q000737:chemistry
D051023:BH3 Interacting Domain Death Agonist Protein; C464479:N1L protein, Vaccinia virus; D016328:NF-kappa B; D014764:Viral Proteins; D037521:Virulence Factors; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein