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Herpes simplex virus type 1 (HSV-1) capsid assembly takes place in the nucleus of infected cells. However, when each of the outer capsid shell proteins, VP5, VP23 and VP26, is expressed in the absence of any other HSV-1 proteins, it does not localize to the nucleus but is distributed throughout the cell. We have previously shown that the HSV-1 capsid scaffolding protein, preVP22a, can relocate VP5 into the nucleus but does not influence the distribution of VP23. We now demonstrate that the outer capsid shell protein, VP19C, is able to relocate both VP5 and VP23 separately into the nucleus. However, nuclear localization of VP26 is only observed when VP5 is present together with either VP19C or preVP22a. Thus, pair-wise interactions involving all of the abundant capsid proteins have now been identified. Electron microscope examination of insect cells coinfected with recombinant baculoviruses expressing VP19C and VP5 reveals the presence of 70 nm diameter 'capsid-like' structures, suggesting that these two proteins can form the basic capsid shell.

The 1250 A diameter herpes simplex virus-1 (HSV-1) capsid shell consists of four major structural proteins, of which VP26 (approximately 12,000 M(r)) is the smallest. Using 400 kV electron cryomicroscopy and computer reconstruction, we have determined the three-dimensional structures of the wild-type capsid and a recombinant baculovirus-generated HSV-1 capsid which lacks VP26. Their difference map demonstrates the presence of VP26 hexamers attached to all the hexons in the wild-type capsid, and reveals that the VP26 molecule consists of a large and a small domain. Although both hexons and pentons are predominantly composed of VP5, VP26 is not present on the penton. Based on the interactions involving VP26 and the hexon subunits, we propose a mechanism for VP26 assembly which would account for its distribution. Possible roles of VP26 in capsid stability and DNA packaging are discussed.

Herpes simplex virus type 1 (HSV-1) polypeptides specified by overlapping genes UL26 and UL26.5 form a scaffold around which the icosahedral capsid shell is assembled. In a series of cleavage events catalysed by the UL26-encoded protease, the full-length UL26 product is processed into capsid proteins VP24 and VP21 and the UL26.5 protein is converted into the capsid protein VP22a by the loss of 25 amino acids from its carboxy terminus. The roles of the UL26 and UL26.5 products were investigated using the baculovirus expression system, focusing on the function of the 25 residues cleaved from the UL26.5 protein. A key conclusion from electron microscopic analysis and protein expression studies is that the 25 amino acids at the carboxy terminus of the full-length UL26.5 protein are required for the interaction of the capsid shell proteins with the scaffold in the formation of intermediate capsids. When cells were multiply infected with baculoviruses expressing a truncated form of the UL26.5 product corresponding to VP22a and the essential components of the capsid shell, no capsids were detected, whereas large numbers of capsids were observed when the full-length UL26.5 product was used as a scaffold. The results are consistent with the proposal that cleavage of the UL26.5 product occurs after capsid assembly or when the UL26.5 protein is in a complex with one or more capsid shell proteins. Expression of VP22a in the absence or presence of capsid shell proteins resulted in the formation of large numbers of 60 nm scaffold-like particles. Since VP22a expressed from baculovirus was unable to participate in capsid assembly, these particles cannot be intermediates in the capsid assembly pathway but may be similar in structure to the protein cores present in HSV-1 immature (B) capsids.

Immature or B capsids of herpes simplex virus type 1 (HSV-1) are composed of seven proteins encoded by six viral genes. The proteins encoded by UL18 (VP23), UL19 (VP5), UL35 (VP26) and UL38 (VP19C) are components of the outer capsid shell whereas those specified by UL26 (VP21 and VP24) and UL26.5 (VP22a), are involved in scaffold formation. We have used a panel of recombinant baculoviruses, each expressing one of the capsid protein genes, to examine the requirements for capsid assembly. Coexpression of the six genes in insect cells resulted in the formation of capsids that were indistinguishable in appearance and protein composition from those made during HSV-1 infection of mammalian cells. This demonstrates that the proteins encoded by the known capsid genes contain all the structural information necessary for capsid assembly and that other virus-encoded proteins are not required for this process. Omission of single recombinant baculoviruses from this system allowed the role of individual HSV-1 proteins in capsid assembly to be determined. Thus, capsid assembly did not take place in the absence of VP23, VP5 or VP19C, whereas lack of VP26 had no discernible effect on capsid formation. Capsids assembled in the absence of the UL26 gene products had a large-cored phenotype resembling that previously described for the HSV-1 mutant ts1201 which has a lesion in this gene. Some apparently intact capsid shells were also made in the absence of the major scaffolding protein, VP22a, whereas the omission of both UL26 and UL26.5 resulted in the appearance of large numbers of partial and deformed capsid shells.