Periplaneta fuliginosa densovirus nonstructural protein NS1 contains an endonuclease activity that is regulated by its phosphorylation

Periplaneta fuliginosa densovirus (PfDNV) is a single-stranded DNA virus, belonging to Densovirinae subfamily, Parvoviridae family. Parvovirus nonstructural protein 1 (NS1) contains various activities required for parvoviral DNA replication, like endonuclease, helicase and ATPase, which are regulated by serine/threonine phosphorylation. However, for PfDNV, NS1 endonuclease activity has not been determined. Moreover, for densoviruses, whether NS1 is phosphorylated, and if so, phosphorylation pattern and impact on NS1 activities have not been investigated. Here, we demonstrated that PfDNV NS1 possesses endonuclease activity, covalently attaches to 5′-end of nicking site, and includes an active-site tyrosine (Y178). Moreover, using different phosphatases, we uncovered that both serine/threonine and tyrosine phosphorylations are critical for NS1 endonuclease and helicase activities. Further mass-spec and mutational analyses revealed that Y345 is phosphorylated and functions as a critical regulatory site for NS1 activities. This study should foster our understanding of NS1 activities and regulations in PfDNV and other densoviruses.

► Determine and characterize the endonuclease activity of PfDNV NS1 in detail. ► For Densovirinae subfamily, first report the phosphorylation of NS1. ► Phosphorylation of densovirus NS1 is critical for NS1 activities. ► For Parvoviridae family, first report tyrosine phosphorylation. ►Tyrosine phosphorylation is critical for regulating PfDNV NS1 activities.