Structure of the Open Reading Frame 49 Protein Encoded by Kaposi's Sarcoma-Associated Herpesvirus.
Herpesviruses alternate between the latent and the lytic life cycle. Switching into the lytic life cycle is important for herpesviral replication and disease pathogenesis. Activation of a transcription factor replication and transcription activator (RTA) has been demonstrated to govern this switch in Kaposi's sarcoma-associated herpesvirus (KSHV). The protein encoded by open reading frame 49 from KSHV (ORF49KSHV) has been shown to upregulate lytic replication in KSHV by enhancing the activities of the RTA. We have solved the crystal structure of the ORF49KSHVprotein to a resolution of 2.4 Å. The ORF49KSHVprotein has a novel fold consisting of 12 alpha-helices bundled into two pseudodomains. Most notably are distinct charged patches on the protein surface, which are possible protein-protein interaction sites. Homologs of the ORF49KSHVprotein in the gammaherpesvirus subfamily have low sequence similarities. Conserved residues are mainly located in the hydrophobic regions, suggesting that they are more likely to play important structural roles than functional ones. Based on the identification and position of three sulfates binding to the positive areas, we performed some initial protein-DNA binding studies by analyzing the thermal stabilization of the protein in the presence of DNA. The ORF49KSHVprotein is stabilized in a dose-responsive manner by double-stranded oligonucleotides, suggesting actual DNA interaction and binding. Biolayer interferometry studies also demonstrated that the ORF49KSHVprotein binds these oligonucleotides.


OBJECTIVE
Kaposi's sarcoma-associated herpesvirus (KSHV) is a tumorigenic gammaherpesvirus that causes multiple cancers and lymphoproliferative diseases. The virus exists mainly in the quiescent latent life cycle, but when it is reactivated into the lytic life cycle, new viruses are produced and disease symptoms usually manifest. Several KSHV proteins play important roles in this reactivation, but their exact roles are still largely unknown. In this study, we report the crystal structure of the open reading frame 49 protein encoded by KSHV (ORF49KSHV). Possible regions for protein interaction that could harbor functional importance were found on the surface of the ORF49KSHVprotein. This led to the discovery of novel DNA binding properties of the ORF49KSHVprotein. Evolutionary conserved structural elements with the functional homologs of ORF49KSHVwere also established with the structure.
Division of Structural Biology and Biochemistry, Nanyang Technological University, School of Biological Sciences, Singapore.
Kelly
K
Hew
Division of Structural Biology and Biochemistry, Nanyang Technological University, School of Biological Sciences, Singapore.
Saranya
S
Veerappan
Division of Structural Biology and Biochemistry, Nanyang Technological University, School of Biological Sciences, Singapore.
Daniel
D
Sim
Division of Structural Biology and Biochemistry, Nanyang Technological University, School of Biological Sciences, Singapore.
Tobias
T
Cornvik
Division of Structural Biology and Biochemistry, Nanyang Technological University, School of Biological Sciences, Singapore par.nordlund@ki.se sdahlroth@ntu.edu.sg.|Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden.|Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore.
Pär
P
Nordlund
Division of Structural Biology and Biochemistry, Nanyang Technological University, School of Biological Sciences, Singapore par.nordlund@ki.se sdahlroth@ntu.edu.sg.
Sue-Li
SL
Dahlroth
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D015967:Gene Expression Regulation, Viral; D019288:Herpesvirus 8, Human / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008968:Molecular Conformation; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D055672:Static Electricity; D013816:Thermodynamics; D024363:Transcription Initiation Site; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D004268:DNA-Binding Proteins; D014764:Viral Proteins
Herpesvirus; KSHV; Kaposi's sarcoma-associated herpesvirus; ORF49; structural biology