Despite the availability of a vaccine, chronic hepatitis B virus (HBV) infection remains a leading cause of cirrhosis and liver cancer. The genome of HBV persists as an extrachromosomal DNA circle in infected cells. The family of Hepadnaviridae includes mammalian and avian hepatitis B viruses. Mammalian HBV genomes, but not the avian HBV genomes, encode an X protein that causes degradation of the multi-subunit restriction factor Smc5/6. The Smc5/6 complex can specifically associate with and block transcription from extrachromosomal DNA. Experiments in chicken cells suggest that none of the X proteins tested cause degradation of the avian Smc5/6 complex and that the avian complex lacks restriction activity. These findings where exploited to perform cross-species experiments to identify subunit(s) involved in Smc5/6 restriction activity and/or binding to the Xproteins. The results so far suggest that one chicken Smc5/6 subunit, Nse4, lacks a function essential for the restriction activity.