Oligomerization of parvovirus B19 nonstructural protein (NS)1 is essentially for viral replication

Parvovirus B19 is a relevant human pathogen whose genome is replicated in the cell nucleus by cellular and viral encoded proteins. Non-structural protein 1 (NS1) has an important role in the process, responsible for nicking and unwinding of the viral genome, as mediated by an N-terminal endonuclease and a central SF3 helicase domain, respectively. In the minute virus of mice (MVM) NS1 and Adeno associated-virus 2/5 (AAV2) Rep68, such domains are connected by an interdomain linker. Up until now, no data regarding B19V NS1 self-association properties are available. Here we combined quantitative confocal laser scanning microscopy (CLSM), protein-fragment complementation assays (PCAs), bioluminescence resonance energy transfer (BRET), molecular modelling and reverse genetics assays to study the importance of B19V NS1 self-interaction in viral replication. We show that NS1 can self-interact in the cytoplasm before nuclear import. Consistently, a structural model of NS1 predicted a heptameric ring closely resembling the one previously reported in AAV2 Rep68, in which each subunit establishes hydrophobic and electrostatic interactions with the adjacent ones via oligomerization and helicase domain residues. Specific amino acid substitutions targeting such residues reduced the self-interaction and abrogated viral genome replication. Notably, a phylogenetic analysis among representative Parvoviridae members revealed that although NS1 orthologs are highly divergent, the common SF3 helicase domains are more conserved, and BRET assays showed their ability to self-interact, consistently with sequence-based structural predictions. Therefore, oligomerization of NS1 is conserved among Parvoviridae members and essential for viral replication, representing a potential target for therapeutic intervention.