VP5, the major capsid protein of herpes simplex virus type 1 (HSV-1), interacts with the C-terminal residues of the scaffold molecules encoded by the overlapping UL26 and UL26.5 open reading frames. Scaffold molecules are cleaved by a UL26 encoded protease (VP24) as part of the normal capsid assembly process. In this study, residues of VP5 have been identified that alter its interaction with the C-terminal residues of the scaffold proteins. A previously isolated virus [KUL26-610/611) was used that encoded a lethal mutation in the UL26 and UL26.5 open reading frames and required a transformed cell line that expresses these proteins for virus growth. The scaffold maturation cleavage site between amino acids 610 and 611 was blocked by changing Ala-Ser to Glu-Phe, which generated a new EcoRI restriction site. Revertant viruses, that formed small plaques on nontransformed cells, were detected at a frequency of 1:3800. Nine revertants were isolated, and all of them retained the EcoRI site and therefore were due to mutations at a second site. The second site mutations were extragenic. Using marker-transfer techniques, the mutation in one of the revertants was mapped to the 5' region of the gene encoding VP5. DNA sequence analysis was performed for the N- terminal 571 codons encoding VP5 for all of the revertant viruses. Six of the nine revertants showed a single base pair change that caused an amino acid substitution between residues 30 and 76 of VP5. Three of these were identical and changed Ata to Val at residue 76. The data provide a partial map of residues of VP5 that alter its interaction with scaffold proteins blocked at their normal cleavage site. The yeast two-hybrid system was used as a measure of the interaction between mutant VP5 and scaffold molecules and varied from 11% to nearly 100%, relative to wild-type VP5. One revertant gave no detectable interaction by this assay. The amount of UL26 encoded protease (VP24) in B capsids for KUL26-610/611 and for revertants was 7% and 25%, respectively, relative to the amount in capsids for wild-type virus. The lack of retention of the viral protease in the mutant virus and a fourfold increase for the revertants suggest an additional essential function for VP24 in capsid maturation, and a role in DNA packaging is indicated.
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