Functional characterization of Kaposi's sarcoma-associated herpesvirus small capsid protein by bacterial artificial chromosome-based mutagenesis.
A systematic investigation of interactions amongst KSHV capsid proteins was undertaken in this study to comprehend lesser known KSHV capsid assembly mechanisms. Interestingly the interaction patterns of the KSHV small capsid protein, ORF65 suggested its plausible role in viral capsid assembly pathways. Towards further understanding this, ORF65-null recombinant mutants (BAC-∆65 and BAC-stop65) employing a bacterial artificial chromosome (BAC) system were generated. No significant difference was found in both overall viral gene expression and lytic DNA replication between stable monolayers of 293T-BAC36 (wild-type) and 293T-BAC-ORF65-null upon induction with 12-O-tetradecanoylphorbol-13-acetate, though the latter released 30-fold fewer virions to the medium than 293T-BAC36 cells. Sedimentation profiles of capsid proteins of ORF65-null recombinant mutants were non-reflective of their organization into the KSHV capsids and were also undetectable in cytoplasmic extracts compared to noticeable levels in nuclear extracts. These observations collectively suggested the pivotal role of ORF65 in the KSHV capsid assembly processes.
Sathish
Narayanan
N
Department of Microbiology, University of Pennsylvania School of Dental Medicine, 240 S. 40th Street, Philadelphia, PA 19104, USA.
Yuan
Yan
Y
D002213:Capsid / Q000378:metabolism; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D019288:Herpesvirus 8, Human / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016296:Mutagenesis; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000235:genetics / Q000378:metabolism; D014773:Virology / Q000379:methods*; D019065:Virus Assembly
D036022:Capsid Proteins; C119019:ORF65 protein, human herpesvirus 8; D014764:Viral Proteins