Incorporation of the Kaposi's sarcoma-associated herpesvirus capsid vertex-specific component (CVSC) into self-assembled capsids

Abstract

Self-assembly of herpesvirus capsids can be accomplished in heterologous expression systems provided all six capsid proteins are present. We have demonstrated the assembly of icosahedral Kaposi's sarcoma-associated herpesvirus (KSHV) capsids in insect cells using the baculovirus expression system. Using this self-assembly system we investigated whether we could add additional capsid associated proteins and determine their incorporation into the assembled capsid. We chose the capsid vertex-specific component (CVSC) proteins encoded by open reading frames (ORFs) 19 and 32 to test this. This complex sits on the capsid vertex and is important for capsid maturation in herpesvirus-infected cells. Co-immunoprecipitation assays were used to initially confirm a bi-molecular interaction between ORF19 and ORF32. Both proteins also precipitated the triplex proteins of the capsid shell (ORF26 and ORF62) as well as the major capsid protein (ORF25). Capsid immunoprecipitation assays revealed the incorporation of ORF19 as well as ORF32 into assembled capsids. Similar experiments also showed that the incorporation of each protein occurred independent of the other. These studies reveal biochemically how the KSHV CVSC interacts with the capsid shell.

Keywords: Capsid; Capsid incorporation; Capsid vertex-specific component; KSHV; Major capsid protein; ORF19; ORF32; ORF69; Triplex proteins.

Fig. 1

Protein expression of tagged ORF19 and ORF32 polypeptides. Sf21 insect cells (1 X 10⁶) were infected with baculoviruses encoding ORF19 and ORF32 tagged at the C-terminus with V5, HA and GFP sequences. Protein lysates were prepared following 48 h of infection, the proteins separated by SDS-polyacrylamide gel electrophoresis and analyzed by immunoblot methods as previously described (Luitweiler et al., 2013). Different blots were probed with mouse monoclonal antibody to V5 (Invitrogen R960), rat monoclonal antibody to Flu HA (Roche clone 3F10) and mouse monoclonal antibody to GFP (Molecular Probes). Protein standards (kD) are shown in the first lane.

Fig. 2

Interactions of the KSHV CVSC proteins with each other and with proteins of the capsid shell. Infected Sf21 protein lysates were prepared as described by (Desai et al., 2012) and used in co-immunoprecipitation assays to detect protein binding partners of ORF19 and ORF32. (A) Protein lysates containing ORF32CGFP or ORF19CGFP were mixed with extracts containing ORF32CV5, ORF19CV5, ORF69CV5 or ORF65CV5 and immunoprecipitated (IP) using anti-rabbit GFP antibody (Abcam). The precipitated complexes were resolved by 4–12% NuPage gels (Invitrogen) and the proteins following transfer to nitrocellulose were incubated with anti-V5 mouse antibody (Blot). The input lysates for the V5 and GFP tagged proteins were also examined by western blots. (B) Similar immunoprecipitations were performed with lysates containing ORF32CHA or ORF19CHA and lysates containing ORF62NV5 or ORF26NV5 as well as ORF25NV5 (panel C) using mouse anti-HA antibody (12CA5 Babco). ORF65CV5 containing lysates were used as control lysates. Again the precipitated proteins and the input lysate proteins on membranes were probed with anti-V5 or anti-HA. Protein standards (kD) are shown in the leftmost lane. IgG heavy chain (hc) and light chain (lc) are labeled where evident.

Fig. 3

Capsid incorporation of the KSHV CVSC. (A). Sf21 infected cell lysates that contain all six KSHV capsid proteins (BA) and the ORF19 and ORF32 proteins were sedimented through sucrose (20–50%) gradients and the light scattering capsid band visualized was harvested. Control lysates, which did not contain the major capsid/scaffold proteins (-MCP) were similarly sedimented and material from the same position where the capsids band was harvested. These fractions were precipitated with anti-HA agarose (Sigma-Aldrich Clone HA-7) and the precipitated proteins were resolved by 4–12% NuPage gels and transferred to membranes prior to probing with anti-V5 antibodies. Proteins detected with the V5 antibody in the starting lysate, the capsid fraction and the immunoprecipitated material (IP) are labeled. (B) Similar capsid purifications and immunoprecipitations were carried out as described above except that this time lysates contained either both ORF32/ORF19 or just ORF19 or ORF32. Immunoblots carried out using anti-V5 antibody was used to detect the capsid associated proteins. For both panels protein standards are shown in the left lane and the IgG heavy (h) and light (l) chains are evident in all samples.