Assembly of herpes simplex virus (HSV) intermediate capsids in insect cells infected with recombinant baculoviruses expressing HSV capsid proteins.
The capsid of herpes simplex virus type 1 (HSV-1) is composed of seven proteins, VP5, VP19C, VP21, VP22a, VP23, VP24, and VP26, which are the products of six HSV-1 genes. Recombinant baculoviruses were used to express the six capsid genes (UL18, UL19, UL26, UL26.5, UL35, and UL38) in insect cells. All constructs expressed the appropriate-size HSV proteins, and insect cells infected with a mixture of the six recombinant baculoviruses contained large numbers of HSV-like capsids. Capsids were purified by sucrose gradient centrifugation, and electron microscopy showed that the capsids made in Sf9 cells had the same size and appearance as authentic HSV B capsids. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that the protein composition of these capsids was nearly identical to that of B capsids isolated from HSV-infected Vero cells. Electron microscopy of thin sections clearly demonstrated that the capsids made in insect cells contained the inner electron-translucent core associated with HSV B capsids. In infections in which single capsid genes were left out, it was found that the UL18 (VP23), UL19 (VP5), UL38 (VP19C), and either the UL26 (VP21 and VP24) or the UL26.5 (VP22a) genes were required for assembly of 100-nm capsids. VP22a was shown to form the inner core of the B capsid, since in infections in which the UL26.5 gene was omitted the 100-nm capsids that formed lacked the inner core. The UL35 (VP26) gene was not required for assembly of 100-nm capsids, although assembly of B capsids was more efficient when it was present. These and other observations indicate that (i) the products of the UL18, UL19, UL35, and UL38 genes self-assemble into structures that form the outer surface (icosahedral shell) of the capsid, (ii) the products of the UL26 and/or UL26.5 genes are required (as scaffolds) for assembly of 100-nm capsids, and (iii) the interaction of the outer surface of the capsid with the scaffolding proteins requires the product of the UL18 gene (VP23).
Thomsen
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Upjohn Company, Kalamazoo, Michigan 49001.
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Homa
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D000818:Animals; D015153:Blotting, Western; D002213:Capsid / Q000096:biosynthesis* / Q000235:genetics / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000254:growth & development* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008954:Models, Biological; D009024:Morphogenesis; D009036:Moths / Q000166:cytology; D016624:Negative Staining; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000648:ultrastructure; D014709:Vero Cells
D011994:Recombinant Proteins