FIG. 5.

An NLS was critical for the nuclear translocation of ORF 50 protein, an IE gene product of KSHV, which is mainly encoded by ORF50. (A) Diagram of the genomic region containing the ORF50-genomic clone and the ORF50-cDNA clone encoding the ORF 50 gene product. ORFs are shown in open boxes. The direction of the ORF is indicated with an arrow above the box. The numbers indicate nucleotide positions in the KSHV genome (48). The position of the ORF50-cDNA clone used for transfection studies is shown, with the splice site between 71,613 and 72,572 indicated. The two open boxes indicate the fused ORF50-cDNA ORF. The positions of the primers used for PCR amplification of the ORF50-genomic and ORF50-cDNA clones are indicated by arrows below the boxes. (B) Construction of ORF50-genomic, ORF50-cDNA, and β-Gal–NLS-1. The ORF50-genomic clone lacks the N-terminal 60 aa that was added to the ORF50-cDNA clone through a splicing event. This region also includes a putative NLS (NLS-1). To test the function of this NLS, the first 13 aa of ORF50-cDNA was fused to β-Gal and its nuclear localization ability was tested. (C) Immunofluorescence staining results. The ORF50-cDNA clone localized to the nucleus, while most of the ORF50-genomic clone localized to both the nucleus and the cytoplasm. The upper two panels show the localization of the ORF50-genomic and ORF50-cDNA clones. The lower two panels show that β-Gal was able to localize to the nucleus when fused with NLS-1 of KSHV ORF 50 protein.