Fig 1.

KSHV ORF57 promotes preferentially nuclear accumulation of KSHV ORF47 RNA. (A) The KSHV ORF47-FLAG fusion vector pVM80 contains the ORF47 coding region (nt 69415 to 69930; GenBank accession no. KSU75698 [28]) from Bac36-wt DNA (15) inserted into pFLAG-CMV-5.1 (Sigma, St. Louis, MO). (B and C) HEK293 cells (5 × 10⁵ cells/well in a 6-well plate) were cotransfected with 1 μg of pVM80 and 0.5 μg of empty pFLAG vector (negative control) or ORF57-FLAG (pVM7) expression vector and harvested 24 h later in SDS-protein sample buffer with 10% 2-mercaptoethanol. The expression of ORF47-FLAG protein in the absence or presence of ORF57-FLAG was detected by Western blot analysis using anti-FLAG antibody (B). Cellular β-tubulin was used as a loading control. Northern blot analysis was used to examine ORF47 RNA expression in the absence and presence of ORF57 (C). Total RNA (5 μg/lane) isolated by TRIzol reagent (Invitrogen) was separated on an agarose gel, and ORF47 RNA was detected with the ³²P-labeled oligoprobes oVM190 (nt 69770 to 69790; 5′-GCGAAGTGGATAGAGTGGACC-3′) for KSHV ORF47, oZMZ270 (5′-TGAGTCCTTCCACGATACCAAA-3′) for GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and oST197 (5′-AAAATATGGAACGCTTCACGA-3′) for U6 snRNA, and fold values were normalized to GAPDH. (D) Subcellular distribution of ORF47 RNA in the presence or absence of ORF57 expression. The cytoplasmic and nuclear RNA fractions were isolated using buffer A or the Ambion PARIS kit, respectively. Five micrograms of each RNA preparation was analyzed in the Northern blot for the expression of ORF47 RNA. GAPDH served as a loading control. The fractionation efficiency was determined by the presence of U6 snRNP in the Northern blot, and 45S and 32S rRNA precursors were visualized by ethidium bromide staining.