A pentameric protein ring with novel architecture is required for herpesviral packaging

Abstract

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.

The viral packaging motor potentiates late gene expression in Kaposi’s sarcoma-associated herpesvirus

“…(1:10,000), rabbit anti-K8.1 (1:10,000), rabbit anti-vinculin (1:1,000; Abcam), streptavidin-HRP (1:500 Thermo Fisher), rabbit anti-XRN1 (1:1,000; Bethyl), mouse anti-CASK (1:1,000; Abcam), rabbit anti-PABPC1 (1:1,000; Cell Signaling Technologies), rabbit anti-ORF6 (1:10,000), rabbit anti-PABPC4 (1:1,000; Bethyl), mouse anti-ORF26(1:500; Novus), rabbit anti-ORF68 (1:5,000), mouse anti-GAPDH (1:1,000; Abcam), rabbit anti-ORF29 (1:500). Rabbit anti-ORF59, K8.1, ORF6, and ORF68 have all been previously described (8,54). Preparation of the rabbit anti-ORF29 antibody is described below.…”

DNA processing by the Kaposi’s sarcoma-associated herpesvirus alkaline exonuclease SOX contributes to viral gene expression and infectious virion production

“…Southern blotting of viral genomes to monitor cleavage that occurs during viral genome packaging was performed as previously described (34). Briefly, 1x10 6 BAC16.iSLK cells were reactivated for 72 hours, washed with PBS, and harvested by scraping.…”