A hydrophobic domain within the small capsid protein of Kaposi's sarcoma-associated herpesvirus is required for assembly.
Kaposi's sarcoma-associated herpesvirus (KSHV) capsids can be produced in insect cells using recombinant baculoviruses for protein expression. All six capsid proteins are required for this process to occur and, unlike for alphaherpesviruses, the small capsid protein (SCP) ORF65 is essential for this process. This protein decorates the capsid shell by virtue of its interaction with the capsomeres. In this study, we have explored the SCP interaction with the major capsid protein (MCP) using GFP fusions. The assembly site within the nucleus of infected cells was visualized by light microscopy using fluorescence produced by the SCP-GFP polypeptide, and the relocalization of the SCP to these sites was evident only when the MCP and the scaffold protein were also present - indicative of an interaction between these proteins that ensures delivery of the SCP to assembly sites. Biochemical assays demonstrated a physical interaction between the SCP and MCP, and also between this complex and the scaffold protein. Self-assembly of capsids with the SCP-GFP polypeptide was evident. Potentially, this result can be used to engineer fluorescent KSHV particles. A similar SCP-His6 polypeptide was used to purify capsids from infected cell lysates using immobilized affinity chromatography and to directly label this protein in capsids using chemically derivatized gold particles. Additional studies with SCP-GFP polypeptide truncation mutants identified a domain residing between aa 50 and 60 of ORF65 that was required for the relocalization of SCP-GFP to nuclear assembly sites. Substitution of residues in this region and specifically at residue 54 with a polar amino acid (lysine) disrupted or abolished this localization as well as capsid assembly, whereas substitution with non-polar residues did not affect the interaction. Thus, this study identified a small conserved hydrophobic domain that is important for the SCP-MCP interaction.
Viral Oncology Program, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA.
Christopher M
CM
Capuano
Viral Oncology Program, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA.
Peter
P
Grzesik
Viral Oncology Program, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA.
Dale
D
Kreitler
Integrated Imaging Center, Department of Biology, Johns Hopkins University, Baltimore, MD, USA.
Erin N
EN
Pryce
Viral Oncology Program, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA.
Keshal V
KV
Desai
Integrated Imaging Center, Department of Biology, Johns Hopkins University, Baltimore, MD, USA.
Gavin
G
Coombs
Integrated Imaging Center, Department of Biology, Johns Hopkins University, Baltimore, MD, USA.
J Michael
JM
McCaffery
Viral Oncology Program, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA.
Prashant J
PJ
Desai
D000818:Animals; D036022:Capsid Proteins / Q000378:metabolism*; D002460:Cell Line; D004252:DNA Mutational Analysis; D019288:Herpesvirus 8, Human / Q000502:physiology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D018411:Spodoptera; D014764:Viral Proteins / Q000378:metabolism*; D019065:Virus Assembly
D036022:Capsid Proteins; C119019:ORF65 protein, human herpesvirus 8; D014764:Viral Proteins