RESUMO
The small hydrophobic (SH) protein of mumps virus has been reported to interfere with innate immunity by inhibiting tumour necrosis factor alpha-mediated apoptosis. In a yeast two-hybrid screen we have identified the ataxin-1 ubiquitin-like interacting protein (A1Up) as a cellular target of the SH protein. A1Up contains an amino-terminal ubiquitin-like (UbL) domain, a carboxy-terminal ubiquitin-associated (UbA) domain and two stress-inducible heat shock chaperonin-binding (Sti1) motifs. This places it within the ubiquitin-like protein family that is involved in proteasome-mediated activities. Co-immunoprecipitation confirmed the binding of SH and A1Up and demonstrates that a truncated protein fragment corresponding to aa 136-270 of A1Up, which represents the first Sti1-like repeat and an adjacent hydrophobic region, was sufficient for interaction, whereas neither the UbL nor the UbA domains were required for interaction. The ectopic expression of A1Up leads to a redistribution of SH to punctate structures that co-localize with the 20S proteasome in transfected or infected mammalian cells.
Assuntos
Proteínas de Transporte/metabolismo , Interações Hospedeiro-Patógeno , Vírus da Caxumba/patogenicidade , Proteínas Nucleares/metabolismo , Mapeamento de Interação de Proteínas , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Transporte/genética , Linhagem Celular , Chlorocebus aethiops , Humanos , Imunoprecipitação , Microscopia Confocal , Proteínas Nucleares/genética , Ligação Proteica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Porcine circovirus type 2 (PCV2) is an important pathogen in swine, whereas porcine circovirus type 1 (PCV1) is apathogenic. To analyze the interactions between PCV and its host, we have used a yeast two-hybrid assay to identify cellular proteins interacting with Cap and Rep proteins of both PCV genotypes. Six cellular proteins were found to interact with Cap (MKRN1, gC1qR, Par-4, NAP1, NPM1 and Hsp40) and three with Rep (ZNF265, TDG and VG5Q). These interactions were confirmed by co-immunoprecipitation. Investigation of the localisation of the proteins by immunofluorescence revealed in some cases only limited spatial overlapping with Cap, while in others a clear co-localisation and prominent protein redistribution was observed. The nine cellular proteins are associated with distinct aspects of viral lifecycle and our data is likely to support future research in the field of PCV2 pathogenesis.