AbstractInfection with Hepatitis B virus (HBV) is a major cause of liver disease and cancer in humans. HBVs (family Hepadnaviridae) have been associated with mammals for millions of years. Recently, the Smc5/6 complex, known for its essential housekeeping functions in genome maintenance, was identified as an antiviral restriction factor of human HBV. The virus has however developed a counteraction mechanism by degrading the complex via its regulatory HBx protein. Whether the antiviral activity of the Smc5/6 complex against hepadnaviruses is an important and evolutionary-conserved function is unknown. Here, we used a combined evolutionary and functional approach to address this question. We first performed phylogenetic and positive selection analyses of the six Smc5/6 complex subunits and found that they have been highly conserved in primates and mammals. Yet, the Smc6 subunit showed marks of adaptive evolution, potentially reminiscent of virus-host “arms-race” We then functionally tested the HBx from six very divergent hepadnaviruses now naturally infecting primates, rodents, and bats. Despite little sequence homology, we demonstrate that these HBx efficiently degraded mammalian Smc5/6 complexes, independently of the host species and of the sites under positive selection. Importantly, all also rescued the replication of an HBx-deficient HBV in primary human hepatocytes. These findings point to an evolutionary-conserved requirement for Smc5/6 inactivation by HBx, showing that the Smc5/6 antiviral activity has been an important defense mechanism against hepadnaviruses in mammals. Interestingly, Smc5/6 may further be a restriction factor of other yet unidentified viruses that have driven some of its adaptation.ImportanceInfection with hepatitis B virus (HBV) led to 887000 human deaths in 2015. HBV has been co-evolving with mammals for millions of years. Recently, the Smc5/6 complex, known for its essential housekeeping functions, was identified as a restriction factor of human HBV antagonized by the regulatory HBx protein. Here, we address whether the antiviral activity of Smc5/6 is an important evolutionary-conserved function. We found that all six subunits of Smc5/6 have been conserved in primates with only Smc6 showing signatures of “evolutionary arms-race” Using evolutionary-guided functional assays that include infections of primary human hepatocytes, we demonstrate that HBx from very divergent mammalian HBVs could all efficiently antagonize Smc5/6, independently of the host species and sites under positive selection. These findings show that the Smc5/6 antiviral activity against HBV is an important function in mammals. It also raises the intriguing possibility that Smc5/6 restricts other, yet unidentified viruses.
Hepatitis B Virus Evasion From Cyclic Guanosine Monophosphate–Adenosine Monophosphate Synthase Sensing in Human Hepatocytes