FIG 1.

Derivation of an MHV68 recombinant that encodes KSHV LANA. (A) Schematic representation of the ORF73 locus and surrounding genes for WT MHV68 and KSHV LANA knock-in (KLKI) MHV68. Locations of recognition sites for specific restriction enzymes are indicated. (B) Restriction fragment length polymorphism analysis of WT MHV68 (lane 1), two independent clones of KLKI MHV68 (lanes 2 and 3), and a KLKI MHV68 WT revertant (KLKI.MR; lane 4). BACs were digested with the indicated restriction enzymes and resolved by agarose-gel electrophoresis. (C) 3T12 cells were mock infected or infected with WT, KLKI, or LANA-null (73.STOP) MHV68 at an MOI of 10 PFU/cell. Lysates were collected 18 h postinfection, and proteins were resolved by SDS-PAGE. Immunoblot analyses were performed using antibodies that recognize the indicated proteins. BCBL-1 lysates were used as a positive control for kLANA expression. (D) MEFs were infected with WT, KLKI, or 73.STOP MHV68 at an MOI of 20 PFU/cell. Cells were fixed 18 h postinfection and stained for indirect immunofluorescence analyses using antibodies that recognize the indicated proteins. DNA was stained with DAPI.