Three-Dimensional Localization of pORF65 in Kaposi's Sarcoma-Associated Herpesvirus Capsid

Abstract

Of the six herpesvirus capsid proteins, the smallest capsid proteins (SCPs) share the least sequence homology among herpesvirus family members and have been implicated in virus specificity during infection. The herpes simplex virus-1 (HSV-1) SCP was shown to be horn shaped and to specifically bind the upper domain of each major capsid protein in hexons but not in pentons. In Kaposi's sarcoma-associated herpesvirus (KSHV), the protein encoded by the ORF65 gene (pORF65) is the putative SCP but its location remains controversial due to the absence of such horn-shaped densities from both the pentons and hexons of the KSHV capsid reconstructions. To directly locate the KSHV SCP, we have used electron cryomicroscopy and threedimensional reconstruction techniques to compare the three-dimensional structure of KSHV capsids to that of anti-pORF65 antibody-labeled capsids. Our difference map shows prominent antibody densities bound to the tips of the hexons but not to pentons, indicating that KSHV SCP is attached to the upper domain of the major capsid protein in hexons but not to that in pentons, similar to HSV-1 SCP. The lack of horn-shaped densities on the hexons indicates that KSHV SCP exhibits structural features that are substantially different from those of HSV-1 SCP. The location of SCP at the outermost regions of the capsid suggests a possible role in mediating capsid interactions with the tegument and cytoskeletal proteins during infection.Herpesvirus virions share a characteristic architecture in which the double-stranded DNA genome is surrounded by an icosahedral protein capsid, a thick tegument layer, and a lipid bilayer envelope (15). The capsid, approximately 1,300 Å in diameter, is a T ϭ 16 icosahedron with 12 pentons forming the vertices, 150 hexons forming the faces and edges, and 320 triplexes interconnecting the pentons and hexons (15,17). These structural features of the capsid are built from four of the six capsid proteins. In Kaposi's sarcoma-associated herpesvirus (KSHV), a gammaherpesvirus, the pentons and hexons are composed of five and six copies, respectively, of the major capsid protein (MCP), pORF25. The triplexes are heterotrimers containing a monomer of the pORF62 protein and a dimer of the pORF26 protein (20, 23). The fourth and smallest capsid protein (SCP), pORF65, is homologous to the SCPs in other herpesviruses, including the structurally well-characterized herpes simplex type 1 (HSV-1) and cytomegalovirus (CMV), representative members of the alpha-and betaherpesvirus subfamilies, respectively. However, of the six capsid proteins, SCPs share the lowest sequence homology between HSV-1, human CMV (HCMV), and KSHV (23). This low sequence homology of SCPs may be related to their virus-specific functional roles. It has been recently shown that the HCMV UL48.5-encoded SCP is essential for HCMV infection in vivo (3), but its HSV-1 counterpart, VP26, is dispensable for HSV-1 infection (5,8). While other proteins making up the capsid shell have been shown to have very similar structures located ...

Unique structures in a tumor herpesvirus revealed by cryo-electron tomography and microscopy

“…The size of the spike densities indicates that the spikes are probably homo-or heterooligomers of the above proteins. However, the direct identification and demonstration of their oligomeric states await future higher resolution cryoET reconstructions, perhaps together with antibody labeling (Lo et al 2003;.…”

CryoEM and mutagenesis reveal that the smallest capsid protein cements and stabilizes Kaposi's sarcoma-associated herpesvirus capsid

“…1 B and D), in contrast to the previous notion that KSHV SCP decorates hexons but not pentons (6). In the hexon, six copies of SCP form a crown-like rim of the hexon ( Fig.…”

“…All herpesviruses share the same architecture with a genome-containing capsid surrounded by a poorly defined tegument layer, which in turn is enclosed in a lipid envelope. The capsids of different herpesviruses are similar in composition and structure (3)(4)(5), each composed of at least five capsid proteins (encoding genes of KSHV given in parentheses): the major capsid protein (MCP, ORF25) in hexameric and pentameric capsomers, triplex proteins 1 and 2 (ORF62 and ORF26, respectively) forming heterotrimers in a 1:2 ratio to connect the capsomers, the smallest capsid protein (SCP, ORF65) previously shown to decorate tips of hexons (6)(7)(8)(9), and the portal protein (ORF43) forming a dodecameric structure for DNA genome packaging at one of the 12 icosahedral vertices (10).…”

Three-Dimensional Localization of the Smallest Capsid Protein in the Human Cytomegalovirus Capsid

“…3D reconstruction has shown that VP26, which is composed mainly of ␤-sheets (20,23), forms a ring of horn-shaped densities around the HSV-1 hexons (19,24). Similarly, we recently used anti-pORF65 antibody labeling to show that pORF65 binds only to hexons in the KSHV capsid (15). Our reconstruction of the antibodylabeled HCMV capsid shows that SCP, like its homologs in other human herpesviruses, binds the tips of MCP hexon subunits.…”

“…Recent biochemical and immunoprecipitation studies by Lai and Britt confirmed that the SCP interacts specifically with the MCP (13). Based on architectural similarities, it was proposed that the HCMV SCP, like the SCPs of HSV-1 and KSHV (15,19,24), binds to MCP hexon subunits only (5, 7). However, these suggestions could not be verified by these structural studies because of the low resolutions (ϳ35 Å) of the HCMV capsid maps.…”