The assembly domain of the small capsid protein of Kaposi's sarcoma-associated herpesvirus

Abstract

Self-assembly of Kaposi's sarcoma-associated herpesvirus capsids occurs when six proteins are coexpressed in insect cells using recombinant baculoviruses; however, if the small capsid protein (SCP) is omitted from the coinfection, assembly does not occur. Herein we delineate and identify precisely the assembly domain and the residues of SCP required for assembly. Hence, six residues, R14, D18, V25, R46, G66, and R70 in the assembly domain, when changed to alanine, completely abolish or reduce capsid assembly.

Fig 1

Assembly domain of the KSHV SCP. Polypeptide truncation mutants were expressed in Sf21 cells to determine the assembly domain of ORF65. (A) Amino acid sequence of ORF65 and representation of the N- and C-terminal truncation mutants made for this experiment. The ORF65 gene encodes a 170-amino-acid polypeptide. Under CAPSID are the results of the assembly experiments. (B) Western blot analysis of SF21 cells infected with the recombinant baculoviruses expressing the different polypeptide truncation mutants. The protein standards are shown in the first lane, and the membrane was probed with anti-HA antibody (Roche clone 3F10) using previously described methods (3). The 65-amino-acid polypeptide was not detected using the NuPage (Invitrogen) gel system, and the 87-amino-acid polypeptide resolved into multiple protein species, most likely because of posttranslational modifications. (C) Negative-stained images of KSHV capsids harvested from sucrose gradients following sedimentation of infected cell lysates performed using methods described by Perkins et al. (13). Scale bars are all 100 nm except for the second 170 image, for which the bar is 200 nm. A cosedimenting baculovirus particle can also be seen in the 125 panel.

Fig 2

Amino acids R14, D18, V25, R46, G66, and R70 are important for assembly of KSHV capsids. (A) An alignment of the N-terminal 89 amino acids of the gammaherpesvirus SCPs is shown (ClustalW algorithm in MacVector). (B) ORF65 N-terminal 86-amino-acid sequence; all the residues underlined and in bold were changed to alanine. (C) Western blot analysis of infected Sf21 cells (harvested 48 h postinfection) showing stable expression of the ORF65 mutants except for Y58A. Protein standards are shown in the first lane, and the membrane was probed with anti-HA antibody (WT, wild-type; MI, mock-infected). (D) Quantitation of the peak fractions from sucrose gradients. Radiolabeled lysates were sedimented through sucrose gradients and fractionated, and the ³⁵S counts per minute were determined in the peak fraction. The data derived from 2 or 3 independent experiments were plotted and the numbers derived by calculating the percentage of counts relative to the wild-type samples in each experiment. (E) Biochemical analysis of sucrose gradient fractions. Radiolabeled lysates were sedimented through sucrose gradients, and fractions 7, 8, and 9 (F7, F8, and F9, respectively) were examined by SDS-PAGE (7). Capsids normally band in fraction 8, as was observed for the wild-type ORF65 protein. For V25A, the radioactivity detected in the fraction was much lower and there was no evident increase in radioactivity in F8. The mobilities of the capsid proteins in the gel are indicated on the right of the gel. Protein standards are shown in lane M and are 97.4, 66, 46, 30, and 14.3 kDa.

Fig 3

Ultrastructural analysis of coinfected Sf21 cells. Cells were coinfected with viruses FBDORF17.5/25, FBDORF26/62, FBORF17, and the virus expressing the wild-type or mutant ORF65 polypeptide. Cells were processed for TEM 68 h after infection, according to procedures described by Perkins et al. (13). Scale bars: R46A (left), 1,000 nm; R46A (right) and V25A (top), 400 nm; G66A (middle) and D18A, 200 nm; G66A (left), R70A, WT, and V25A (lower), 100 nm.