The Contribution of Kaposi’s Sarcoma-Associated Herpesvirus ORF7 and Its Zinc-Finger Motif to Viral Genome Cleavage and Capsid Formation

During Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic infection, lytic-related proteins are synthesized, viral genomes are replicated as a tandemly repeated form, and subsequently, capsids are assembled. The herpesvirus terminase complex is proposed to package an appropriate genome unit into an immature capsid, by cleavage of terminal repeats (TRs) flanking tandemly linked viral genomes. Although the mechanism of capsid formation in alpha- and betaherpesviruses are well-studied, in KSHV, it remains largely unknown. It has been proposed that KSHV ORF7 is a terminase subunit, and ORF7 harbors a zinc-finger motif, which is conserved among other herpesviral terminases. However, the biological significance of ORF7 is unknown. We previously reported that KSHV ORF17 is essential for the cleavage of inner scaffold proteins in capsid maturation, and ORF17 knockout (KO) induced capsid formation arrest between the procapsid and B-capsid stages. However, it remains unknown if ORF7-mediated viral DNA cleavage occurs before or after ORF17-mediated scaffold collapse. We analyzed the role of ORF7 during capsid formation using ORF7-KO-, ORF7&17-double-KO (DKO)-, and ORF7-zinc-finger motif mutant-KSHVs. We found that ORF7 acted after ORF17 in the capsid formation process, and ORF7-KO-KSHV produced incomplete capsids harboring nonspherical internal structures, which resembled soccer balls. This soccer ball-like capsid was formed after ORF17-mediated B-capsid formation. Moreover, ORF7-KO- and zinc-finger motif KO-KSHV failed to appropriately cleave the TR on replicated genome and had a defect in virion production. Interestingly, ORF17 function was also necessary for TR cleavage. Thus, our data revealed ORF7 contributes to terminase-mediated viral genome cleavage and capsid formation. IMPORTANCE In herpesviral capsid formation, the viral terminase complex cleaves the TR sites on newly synthesized tandemly repeating genomes and inserts an appropriate genomic unit into an immature capsid. Herpes simplex virus 1 (HSV-1) UL28 is a subunit of the terminase complex that cleaves the replicated viral genome. However, the physiological importance of the UL28 homolog, KSHV ORF7, remains poorly understood. Here, using several ORF7-deficient KSHVs, we found that ORF7 acted after ORF17-mediated scaffold collapse in the capsid maturation process. Moreover, ORF7 and its zinc-finger motif were essential for both cleavage of TR sites on the KSHV genome and virus production. ORF7-deficient KSHVs produced incomplete capsids that resembled a soccer ball. To our knowledge, this is the first report showing ORF7-KO-induced soccer ball-like capsids production and ORF7 function in the KSHV capsid assembly process. Our findings provide insights into the role of ORF7 in KSHV capsid formation.