Ectromelia virus BTB/kelch proteins, EVM150 and EVM167, interact with cullin-3-based ubiquitin ligases

Cellular proteins containing BTB and kelch domains have been shown to function as adapters for the recruitment of substrates to cullin-3-based ubiquitin ligases. Poxviruses are the only family of viruses known to encode multiple BTB/kelch proteins, suggesting that poxviruses may modulate the ubiquitin pathway through interaction with cullin-3. Ectromelia virus encodes four BTB/kelch proteins and one BTB-only protein. Here we demonstrate that two of the ectromelia virus-encoded BTB/kelch proteins, EVM150 and EVM167, interacted with cullin-3. Similar to cellular BTB proteins, the BTB domain of EVM150 and EVM167 was necessary and sufficient for cullin-3 interaction. During infection, EVM150 and EVM167 localized to discrete cytoplasmic regions, which co-localized with cullin-3. Furthermore, EVM150 and EVM167 co-localized and interacted with conjugated ubiquitin, as demonstrated by confocal microscopy and co-immunoprecipitation. Our findings suggest that the ectromelia virus-encoded BTB/kelch proteins, EVM150 and EVM167, interact with cullin-3 potentially functioning to recruit unidentified substrates for ubiquitination.