RT Journal Article SR Electronic T1 A pentameric protein ring with novel architecture is required for herpesviral packaging JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.07.16.206755 DO 10.1101/2020.07.16.206755 A1 Allison L. Didychuk A1 Stephanie N. Gates A1 Matthew R. Gardner A1 Lisa M. Strong A1 Andreas Martin A1 Britt A. Glaunsinger YR 2020 UL http://biorxiv.org/content/early/2020/07/17/2020.07.16.206755.abstract AB Genome packaging in large double-stranded DNA viruses requires a powerful molecular motor to force the viral genome into nascent capsids. This process appears mechanistically similar in two evolutionarily distant viruses, the herpesviruses and the tailed bacteriophages, which infect different kingdoms of life. While the motor and mechanism as a whole are thought to be conserved, accessory factors that influence packaging are divergent and poorly understood, despite their essential roles. An accessory factor required for herpesviral packaging is encoded by ORF68 in the oncogenic virus Kaposi’s sarcoma-associated herpesvirus (KSHV), whose homolog in Epstein Barr Virus (EBV) is BFLF1. Here, we present structures of both KSHV ORF68 and EBV BFLF1, revealing that these proteins form a highly similar homopentameric ring. The central channel of this ring is positively charged, and we demonstrate that this region of KSHV ORF68 binds double-stranded DNA. Mutation of individual positively charged residues within but not outside the channel ablates DNA binding, and in the context of KSHV infection these mutants fail to package the viral genome or produce progeny virions. Thus, we propose a model in which ORF68 facilitates the transfer of newly replicated viral genomes to the packaging motor.Competing Interest StatementThe authors have declared no competing interest.