A mobile functional region of Kaposi's sarcoma-associated herpesvirus ORF50 protein independently regulates DNA binding and protein abundance.
The protein encoded by open reading frame 50 (ORF50) of Kaposi's sarcoma-associated herpesvirus (KSHV) functions as a transcriptional activator and in lytic viral DNA replication to mediate the switch from latent viral infection to the lytic phase. Here we identify regulatory regions of ORF50 protein that independently control DNA binding and abundance of the protein. One region contains a DNA-binding inhibitory sequence (DBIS) located between amino acids (aa) 490 and 535 of ORF50. A cluster of basic amino acids in this sequence is important in inhibiting DNA binding. The DBIS can function at the N or C terminus or internally in the ORF50 protein. Since the DBIS is functional in ORF50 protein purified from Escherichia coli, it is likely to work through an intramolecular mechanism. The second regulatory region, a protein abundance regulatory signal (PARS), consists of two components. Component I of the PARS overlaps the DBIS but can be differentiated from the DBIS by specific substitution of basic amino acid residues. Component II of PARS is located between aa 590 and 650. Mutation or deletion of either component results in abundant expression of ORF50 protein. When the two-component PARS was fused to a heterologous protein, Glutathione S-transferase, the fusion protein was unstable. Mutations in the DBIS or PARS impair the capacity of ORF50 to activate direct and indirect target viral promoters. Since these overlapping regulatory motifs are located in the C-terminal transactivation domain, they are likely to be important in controlling many actions of ORF50 protein.
Chang
Pey-Jium
PJ
Department Pediatrics, Yale University School of Medicine, 333 Cedar St., Room 420 LSOG, New Haven, CT 06520, USA.
Shedd
Duane
D
Miller
George
G
D020816:Amino Acid Motifs; D002460:Cell Line; D004247:DNA / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D015967:Gene Expression Regulation, Viral; D019288:Herpesvirus 8, Human / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D009154:Mutation; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics*
D011994:Recombinant Proteins; D014764:Viral Proteins; D004247:DNA