Herpesviral DNA packaging into nascent capsids requires multiple conserved viral proteins that coordinate genome encapsidation. Here, we investigated the role of the ORF68 protein of Kaposi's sarcoma-associated herpesvirus (KSHV), a protein required for viral DNA encapsidation whose function remains largely unresolved across the herpesviridae. We found that KSHV ORF68 is expressed with early kinetics and localizes predominantly to viral replication compartments, although it is dispensable for viral DNA replication and gene expression. However, in agreement with its proposed role in viral DNA packaging, KSHV-infected cells lacking ORF68 failed to cleave viral DNA concatemers, accumulated exclusively immature B capsids, and released no infectious progeny virions. ORF68 has no predicted domains aside from a series of putative zinc finger motifs. However, biochemical analyses of purified ORF68 protein revealed that it robustly binds DNA and is associated with nuclease activity. These activities provide new insights into the role of KSHV ORF68 in viral genome encapsidation. Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma and several B-cell cancers, causing significant morbidity and mortality in immunocompromised individuals. A critical step in the production of infectious viral progeny is the packaging of the newly replicated viral DNA genome into the capsid, which involves coordination between at least seven herpesviral proteins. While the majority of these packaging factors have been well studied in related herpesviruses, the role of the KSHV ORF68 protein and its homologs remains unresolved. Here, using a KSHV mutant lacking ORF68, we confirm its requirement for viral DNA processing and packaging in infected cells. Furthermore, we show that the purified ORF68 protein directly binds DNA and is associated with a metal-dependent cleavage activity on double-stranded DNA These activities suggest a novel role for ORF68 in herpesviral genome processing and encapsidation.

Genome packaging in large double-stranded DNA viruses requires a powerful molecular motor to force the viral genome into nascent capsids, which involves essential accessory factors that are poorly understood. Here, we present structures of two such accessory factors from the oncogenic herpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV; ORF68) and Epstein-Barr virus (EBV; BFLF1). These homologous proteins form highly similar homopentameric rings with a positively charged central channel that 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.

12Herpesviral DNA packaging into nascent capsids requires multiple conserved viral 13 proteins that coordinate genome encapsidation. Here, we investigated the role of the ORF68 14 protein of Kaposi's sarcoma-associated herpesvirus (KSHV), a protein required for viral DNA 15 encapsidation whose function remains largely unresolved across the herpesviridae. We found 16 that KSHV ORF68 is expressed with early kinetics and localizes predominantly to viral replication 17 compartments, although it is dispensable for viral DNA replication and gene expression. 18However, in agreement with its proposed role in viral DNA packaging, KSHV-infected cells 19 lacking ORF68 failed to cleave viral DNA concatemers, accumulated exclusively immature B-20 capsids, and released no infectious progeny virions. ORF68 has no predicted domains aside 21 from a series of putative zinc finger motifs. However, in vitro biochemical analyses of purified 22 ORF68 protein revealed that it robustly binds DNA and is associated with nuclease activity. 23 Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's 28 sarcoma and several B-cell cancers, causing significant morbidity and mortality in 29 immunocompromised individuals. A critical step in the production of infectious viral progeny is 30 the packaging of the newly replicated viral DNA genome into the capsid, which involves 31 coordination between at least seven herpesviral proteins. While the majority of these 32 packaging factors have been well studied in related herpesviruses, the role of the KSHV ORF68 33 protein and its homologs remains unresolved. Here, using a KSHV mutant lacking ORF68, we 34 confirm its requirement for viral DNA processing and packaging in infected cells. Furthermore, 35 we show that the purified ORF68 protein directly binds DNA and is associated with a metal-36 dependent cleavage activity on double stranded DNA in vitro. These activities suggest a novel 37 role for ORF68 in herpesviral genome processing and encapsidation. 38 39 Introduction 40BY 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint

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.

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