Capsid Structure of Simian Cytomegalovirus from Cryoelectron Microscopy: Evidence for Tegument Attachment Sites

We have used cryoelectron microscopy and image reconstruction to study B-capsids recovered from both the nuclear and the cytoplasmic fractions of cells infected with simian cytomegalovirus (SCMV). SCMV, a representative betaherpesvirus, could thus be compared with the previously described B-capsids of the alphaherpesviruses, herpes simplex virus type 1 (HSV-1) and equine herpesvirus 1 (EHV-1), and of channel catfish virus, an evolutionarily remote herpesvirus. Nuclear B-capsid architecture is generally conserved with SCMV, but it is 4% larger in inner radius than HSV-1, implying that its ∼30% larger genome should be packed more tightly. Isolated SCMV B-capsids retain a relatively well preserved inner shell (or “small core”) of scaffolding-assembly protein, whose radial-density profile indicates that this protein is ∼16-nm long and consists of two domains connected by a low-density linker. As with HSV-1, the hexons but not the pentons of the major capsid protein (151 kDa) bind the smallest capsid protein (∼8 kDa). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed cytoplasmic B-capsid preparations to contain proteins similar in molecular weight to the basic phosphoprotein (∼119 kDa) and the matrix proteins (65 to 70 kDa). Micrographs revealed that these particles had variable amounts of surface-adherent material not present on nuclear B-capsids that we take to be tegument proteins. Cytoplasmic B-capsids were classified accordingly as lightly, moderately, or heavily tegumented. By comparing the three corresponding density maps with each other and with the nuclear B-capsid, two interactions were identified between putative tegument proteins and the capsid surface. One is between the major capsid protein and a protein estimated by electron microscopy to be 50 to 60 kDa; the other involves an elongated molecule estimated to be 100 to 120 kDa that is anchored on the triplexes, most likely on its dimer subunits. Candidates for the proteins bound at these sites are discussed. This first visualization of such linkages makes a step towards understanding the organization and functional rationale of the herpesvirus tegument.Herpesvirus form an extensive family of DNA viruses, whose host range encompasses much of the animal kingdom. They are classified into three subfamilies alpha-, beta-, and gammaherpesviruses, on the basis of biological properties [53]. Despite this diversity, their assembly pathway is closely conserved: the nucleocapsid is formed in the nucleus and follows a pathway that bears a marked resemblance to those of DNA bacteriophages (8, 14). First, a procapsid is assembled, which then releases its morphogenic internal scaffolding protein and becomes filled with DNA, concomitant with a major conformational transition of the capsid shell. Subsequent events, however, are not phage-like. The nucleocapsid exits the nucleus and acquires its tegument (53) and lipoprotein envelope. The latter events remain a focus of active research and some controversy (see, for example, references 13, 26, 51, and 63).Although virion assembly is a continuous sequential process, several kinds of capsids have been identified as representing stable endpoints or long-lived states. They include A-capsids, empty shells thought to arise from abortive attempts to package DNA or its aberrant release; C-capsids, which are filled with DNA; and B-capsids, which contain internal proteins but little or no DNA (for reviews, see references 28, 49 and 62). “B-capsid” refers either to an intranuclear capsid visualized in situ that contains internal material with different staining properties from the DNA in C-capsids (which we interpret as mainly scaffolding-assembly protein) or to a capsid isolated from the nuclear fraction that contains scaffolding proteins but little or no DNA. Here, we relax the requirement for nuclear provenance and refer to nuclear and cytoplasmic B-capsids, respectively. It now appears that there are several kinds of B-capsids. “Large-cored” B-capsids correspond to the normally short-lived and infrequently observed procapsid (42, 47). “Small-cored” B-capsids (49) derive from large-cored B-capsids and have a mature surface shell. It remains unclear whether these particles represent an assembly intermediate or an abortive byproduct (56) or how many subclasses they may comprise.As noted above, herpesvirus capsid assembly is a conservative process. The current paradigm is that the surface shell is a T=16 icosahedron containing three essential proteins. Nine hundred sixty copies of the major capsid protein (M_r ≃ 120,000 to 155,000, depending on the virus) make up 150 hexons and 12 pentons. The other two proteins form the triplexes, complexes that are present on the outer surface at the 320 sites of local threefold symmetry and have an α₂β stoichiometry (43). Typically, the α-protein has a mass of 33 to 35 kDa, and the mass of the β-protein is either similar or in the 50-kDa range (10). Most herpesvirus capsids also contain an additional low-molecular-weight protein that binds around the rims of hexons but not to pentons (11, 65, 72, 73).The above account owes most to observations relating to herpes simplex virus type 1 (HSV-1), the archetypal alphaherpesvirus. However, one property of herpesviruses that does not suggest an immutable capsid structure is their variation in genome size, which ranges from ∼120 to ∼230 kbp (18). Betaherpesviruses account for three of the eight herpesviruses known to cause disease in humans and include the cytomegaloviruses (CMVs), which have the largest of all known herpesvirus genomes (4). Although the overall structure of the herpesvirus capsid appears to be conserved, it has not been clear whether there are features specific to each subfamily. To investigate this possibility, we have used cryoelectron microscopy to study isolated capsids of simian CMV (SCMV), a close and experimentally more tractable relative of human CMV (HCMV) [24].A second motivation for this study was that it might offer insight into tegumentation. Nonenveloped capsids have been observed in significant numbers in the cytoplasms of cells infected with CMV (57) and appear to be at least partly tegumented (24, 30). We have noticed that, late in the infection of cultured cells with SCMV, B-capsids appear in the cytoplasmic fraction, and these cytoplasmic B-capsids differ from nuclear B-capsids in several respects, including their state of tegumentation. Here, we characterize the structures of nuclear and cytoplasmic B-capsids at a resolution of ∼2.2 nm and, by quantitative comparison between them, identify sites of tegument attachment.