Figure 5. Residues in ORF68 that ablate dsDNA binding in vitro are required for genome cleavage and packaging in vivo.

(a) iSLK cell lines containing ORF68 mutants (68S, K435A, and 3+) and their corresponding mutant rescues (MR) were established using the KSHV BAC16 system. Progeny virion production by these cell lines was assayed by supernatant transfer and flow cytometry of target cells. (b) Western blot of whole cell lysate (25 μg) from ORF68.stop iSLK cell lines. GAPDH was used as a loading control. ORF6 is an early gene and K8.1 is a late gene. (c) Southern blot of DNA isolated from iSLK cell lines using a probe for the terminal repeats. DNA was digested with PstI, which cuts within the genome but not within the terminal repeats and generates a ladder of terminal repeat-containing DNA when successful cleavage and packaging occurs.

Figure 5—figure supplement 1. Construction and validation of mutant viruses.

(a) Schematic of the genomic locus of ORF68, with the location of introduced mutations depicted in detail below. Sanger sequencing traces for the mutants and corresponding mutant rescues are shown to the right. (b) Digestion of recombinant BACs with RsrII and SbfI was used to assess whether large-scale recombination had occurred during mutagenesis. (c) Viral DNA replication was measured by qPCR before and after reactivation. Data are from three independent biological replicates, with statistics being calculated using an unpaired t test. **p<0.01. MR, mutant rescue.