An ectromelia virus profilin homolog interacts with cellular tropomyosin and viral A-type inclusion protein.
BACKGROUND
Profilins are critical to cytoskeletal dynamics in eukaryotes; however, little is known about their viral counterparts. In this study, a poxviral profilin homolog, ectromelia virus strain Moscow gene 141 (ECTV-PH), was investigated by a variety of experimental and bioinformatics techniques to characterize its interactions with cellular and viral proteins.


RESULTS
Profilin-like proteins are encoded by all orthopoxviruses sequenced to date, and share over 90% amino acid (aa) identity. Sequence comparisons show highest similarity to mammalian type 1 profilins; however, a conserved 3 aa deletion in mammalian type 3 and poxviral profilins suggests that these homologs may be more closely related. Structural analysis shows that ECTV-PH can be successfully modelled onto both the profilin 1 crystal structure and profilin 3 homology model, though few of the surface residues thought to be required for binding actin, poly(L-proline), and PIP2 are conserved. Immunoprecipitation and mass spectrometry identified two proteins that interact with ECTV-PH within infected cells: alpha-tropomyosin, a 38 kDa cellular actin-binding protein, and the 84 kDa product of vaccinia virus strain Western Reserve (VACV-WR) 148, which is the truncated VACV counterpart of the orthopoxvirus A-type inclusion (ATI) protein. Western and far-western blots demonstrated that the interaction with alpha-tropomyosin is direct, and immunofluorescence experiments suggest that ECTV-PH and alpha-tropomyosin may colocalize to structures that resemble actin tails and cellular protrusions. Sequence comparisons of the poxviral ATI proteins show that although full-length orthologs are only present in cowpox and ectromelia viruses, an ~ 700 aa truncated ATI protein is conserved in over 90% of sequenced orthopoxviruses. Immunofluorescence studies indicate that ECTV-PH localizes to cytoplasmic inclusion bodies formed by both truncated and full-length versions of the viral ATI protein. Furthermore, colocalization of ECTV-PH and truncated ATI protein to protrusions from the cell surface was observed.


CONCLUSIONS
These results suggest a role for ECTV-PH in intracellular transport of viral proteins or intercellular spread of the virus. Broader implications include better understanding of the virus-host relationship and mechanisms by which cells organize and control the actin cytoskeleton.
Butler-Cole
Christine
C
Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada. chris.butlercole@gmail.com
Wagner
Mary J
MJ
Da Silva
Melissa
M
Brown
Gordon D
GD
Burke
Robert D
RD
Upton
Chris
C
D000199:Actins / Q000737:chemistry; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000737:chemistry; D004481:Ectromelia virus / Q000235:genetics / Q000502:physiology*; D047468:Immunoprecipitation; D007181:Inclusion Bodies, Viral / Q000737:chemistry; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D010802:Phylogeny; D051304:Profilins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014335:Tropomyosin / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
C088596:A-type inclusion protein, poxvirus; D000199:Actins; D051304:Profilins; D014335:Tropomyosin; D014764:Viral Proteins