Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 ORF50/Rta lytic switch protein functions as a tetramer.
The Kaposi's sarcoma-associated herpesvirus open reading frame 50 (ORF50) protein (called Rta), is necessary and sufficient for reactivation of the virus from latency. We previously demonstrated that a truncated mutant of ORF50 lacking its C-terminal transcriptional activation domain, called ORF50DeltaSTAD, formed mixed multimers with wild-type (WT) ORF50 and functioned as a dominant negative inhibitor of reactivation. For this report, we investigated the requirements for multimerization of ORF50/Rta in transactivation and viral reactivation. We analyzed multimerization of WT, mutant, and chimeric ORF50 proteins, using Blue Native polyacrylamide gel electrophoresis and size exclusion chromatography. WT and mutant ORF50 proteins form tetramers and higher-order multimers, but not monomers, in solution. The proline-rich, N-terminal leucine heptapeptide repeat (LR) of ORF50 (amino acids [aa] 244 to 275) is necessary but not sufficient for oligomer formation and functions in concert with the central portion of ORF50/Rta (aa 245 to 414). The dominant negative mutant ORF50DeltaSTAD requires the LR to form mixed multimers with WT ORF50 and inhibit its function. In the context of the WT ORF50/Rta protein, mutagenesis of the LR, or replacement of the LR by heterologous multimerization domains from the GCN4 or p53 proteins, demonstrates that tetramers of Rta are sufficient for transactivation and viral reactivation. Mutants of Rta that are unable to form tetramers but retain the ability to form higher-order multimers are reduced in function or are nonfunctional. We concluded that the proline content, but not the leucine content, of the LR is critical for determining the oligomeric state of Rta.
Bu
Wei
W
University of Medicine and Dentistry of New Jersey/New Jersey Medical School, Department of Microbiology and Molecular Genetics and Graduate School of Biomedical Sciences, Newark, NJ 07103, USA.
Carroll
Kyla Driscoll
KD
Palmeri
Diana
D
Lukac
David M
DM
D000595:Amino Acid Sequence; D002460:Cell Line; D019288:Herpesvirus 8, Human / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D006801:Humans; D017874:Immediate-Early Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D007930:Leucine / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames / Q000235:genetics; D011392:Proline / Q000737:chemistry / Q000235:genetics; D020836:Protein Structure, Quaternary; D012514:Sarcoma, Kaposi / Q000821:virology*; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D014775:Virus Activation / Q000235:genetics
D017874:Immediate-Early Proteins; C116742:ORF 50 transactivator; C417604:Rta protein, Human herpesvirus 8; D015534:Trans-Activators; D014764:Viral Proteins; D011392:Proline; D007930:Leucine