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. 2001 Mar;75(6):2866-78.
doi: 10.1128/JVI.75.6.2866-2878.2001.

Lytic replication of Kaposi's sarcoma-associated herpesvirus results in the formation of multiple capsid species: isolation and molecular characterization of A, B, and C capsids from a gammaherpesvirus

K Nealon  1 , W W Newcomb, T R Pray, C S Craik, J C Brown, D H Kedes
Affiliations

Lytic replication of Kaposi's sarcoma-associated herpesvirus results in the formation of multiple capsid species: isolation and molecular characterization of A, B, and C capsids from a gammaherpesvirus

K Nealon et al. J Virol. 2001 Mar.

Abstract

Despite the discovery of Epstein-Barr virus more than 35 years ago, a thorough understanding of gammaherpesvirus capsid composition and structure has remained elusive. We approached this problem by purifying capsids from Kaposi's sarcoma-associated herpesvirus (KSHV), the only other known human gammaherpesvirus. The results from our biochemical and imaging analyses demonstrate that KSHV capsids possess a typical herpesvirus icosahedral capsid shell composed of four structural proteins. The hexameric and pentameric capsomers are composed of the major capsid protein (MCP) encoded by open reading frame 25. The heterotrimeric complexes, forming the capsid floor between the hexons and pentons, are each composed of one molecule of ORF62 and two molecules of ORF26. Each of these proteins has significant amino acid sequence homology to capsid proteins in alpha- and betaherpesviruses. In contrast, the fourth protein, ORF65, lacks significant sequence homology to its structural counterparts from the other subfamilies. Nevertheless, this small, basic, and highly antigenic protein decorates the surface of the capsids, as does, for example, the even smaller basic capsid protein VP26 of herpes simplex virus type 1. We have also found that, as with the alpha- and betaherpesviruses, lytic replication of KSHV leads to the formation of at least three capsid species, A, B, and C, with masses of approximately 200, 230, and 300 MDa, respectively. A capsids are empty, B capsids contain an inner array of a fifth structural protein, ORF17.5, and C capsids contain the viral genome.

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Figures

FIG. 1
FIG. 1
Transmission electron micrographs of KSHV capsid species at sequential stages of purification. (A and B) Capsid mixtures pelleted from BCBL-1 medium before (A) and after (B) Triton X-100 extraction. Capsids surrounded with material suggestive of a tegument layer are indicated by wide black arrowheads, empty capsids are indicated by arrows, ring-filled capsids are indicated by white arrowheads, and core-containing capsids are indicated by narrow black arrowheads. (C to E) Gradient fractions enriched for each of these last three capsid morphologies (fractions 7, 9, and 12 from the gradient depicted in Fig. 4). Bar, 0.1 μm.
FIG. 2
FIG. 2
Velocity sedimentation of KSHV capsids through a 20 to 50% sucrose gradient. (A) Coomassie blue-stained SDS–10% polyacrylamide gel of eight sequential fractions (numbered at the bottom) containing KSHV capsids sedimented through the gradient (note that fraction 13 was underloaded). (B) Fractions 9 and 11 (from panel A) juxtaposed for comparison and with a slightly lighter exposure. The five protein bands that rose above background and sedimented as components of a particle through the gradient are indicated to the right. Note that the capsids in fraction 9 lack band 3. Molecular mass markers (in Kilodaltons) are indicated to the left.
FIG. 3
FIG. 3
Capsid band 1 is ORF25/MCP. (A) Magnified view of band 1 (arrowhead) stained with Coomassie blue after separation on a 4 to 20% polyacrylamide gel. The band migrates at approximately 150 kDa. (B) Coomassie blue-stained 12% polyacrylamide gel (lane 1) and immunoblot (lane 2) of the capsid mixture prior to gradient purification. The immunoblot was probed with rabbit anti-MCP peptide polyclonal antisera. Molecular mass markers (in kilodaltons) are indicated to the right of each panel.
FIG. 4
FIG. 4
MCP profile across fractions from a sucrose gradient reveals distinct sedimentation velocities of the different KSHV capsid species. The intensity of silver staining of MCP (band 1) is plotted against fraction number (fractions were collected from the top of the gradient as indicated). The first peak (fractions 7 to 9) contains empty and ring-filled capsids, and the second, smaller peak (fractions 12 and 13) contains mainly the less abundant core-filled capsids (see the text). Maximal Coomassie blue staining of MCP (fraction 8) was designated 100%. The silver-stained gels containing fractions 1 to 7 and 8 to 16, respectively, are aligned below the graph, and the position of MCP is indicated by horizontal arrows (a vertical black line marks the break between the two gels).
FIG. 5
FIG. 5
Rabbit antisera raised against a recombinant protein encoded by the 3′ half of orf17 recognizes a KSHV lytic protein in TPA-induced BCBL-1 cells. Overproduced recombinant protein (arrow) from bacterial lysates (lane 1) comigrates with its endogenous counterpart from TPA-induced BCBL-1 cells (lane 2) in Western analyses probed with the rabbit antiserum (anti-ORF17.5). The protein is essentially absent in extracts from uninduced BCBL-1 cells (lane 3). The secondary antibody was HRP-conjugated goat anti-rabbit IgG. Molecular mass markers are shown to the left. Vertical lines indicate where separate lanes from the single gel were juxtaposed for optimal comparison.
FIG. 6
FIG. 6
The 34-kDa capsid band 3 reacts with anti-ORF17.5 antiserum. KSHV capsids from gradient fractions 9 and 11 (Fig. 2B) were subjected to SDS-PAGE and then either stained with Coomassie blue (lanes 1 and 2) or immunoblotted and probed with anti-ORF17.5 antiserum (lanes 3 and 4), as in Fig. 5. The 34-kDa ORF17.5 capsid protein (arrow) was present in the gradient fraction containing ring-filled capsids (lanes 2 and 4) but absent in the fraction containing empty capsids (lanes 1 and 3). Molecular mass markers (in kilodaltons) are indicated to the left. Vertical lines between lanes indicate where separate lanes from the gel and Western blot were juxtaposed for direct comparison.
FIG. 7
FIG. 7
Results of MS of KSHV capsid bands 2, 4, and 5. Coomassie blue-stained capsid bands 2, 4, and 5 (Fig. 2B) were subjected to MS (see Materials and Methods). The resultant peptides from each band and their respective sequences (oval shaded regions) are shown superimposed on the deduced amino acid sequences of ORF62, ORF26, and ORF65, respectively.
FIG. 8
FIG. 8
Immuno-EM reveals the presence of ORF65 on the surface of purified KSHV B capsids. (A and B) Purified KSHV B capsids were incubated with either anti-ORF65 rabbit antiserum (A) or with unimmunized rabbit serum (B) followed by colloidal gold-conjugated goat anti-rabbit IgG antibodies and then subjected to EM (see Materials and Methods). (C) Purified HSV-1 B capsids were also incubated with the anti-ORF65 rabbit antiserum and then secondary antibody as above. Arrows indicate the single capsids magnified approximately fivefold to the right of each panel in the electron micrographs. Colloidal gold appears as dark, uniformly sized circles. Bar, 0.1 μm.
FIG. 9
FIG. 9
Profile of encapsidated DNA from KSHV capsids separated by velocity sedimentation through a sucrose gradient. Relative amounts of KSHV-specific DNA (solid squares) in each fraction from the same gradient shown in Fig. 4 were measured by Southern dot blot analysis (see Materials and Methods). For direct comparison, the MCP profile (open diamonds) from Fig. 4 is also shown. The KSHV DNA peaks in fractions 12 and 13, correlating with the second MCP peak, which contains the core-filled capsids (Fig. 1E).
FIG. 10
FIG. 10
STEM analysis of a mixed population of KSHV capsids. KSHV capsids pelleted from the medium of TPA-induced BCBL-1 cells were subjected to Triton X-100 extraction (but no gradient fractionation) and then analyzed by STEM to determine their masses. Each bar in the graph indicates the frequency with which individually identified particles had a specific mass. Downward arrows indicate the modes and corresponding A, B, and C capsid morphologies.
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