Mutation of single hydrophobic residue I27, L35, F39, L58, L65, L67, or L71 in the N terminus of VP5 abolishes interaction with the scaffold protein and prevents closure of herpes simplex virus type 1 capsid shells.

The construction of a protein shell that encloses the viral genome is an essential step in the morphogenesis of phage and animal viruses. The assembly process of the herpes simplex virus type 1 (HSV-1) particle results in the production of three sedimentable capsid particles from lysates of infected cells. These are designated A, B, and C capsids depending on the distance sedimented in sucrose gradients (13). C capsids, which sediment farthest, contain viral DNA and are the capsids that mature into infectious virions. B capsids do not contain DNA but do contain the scaffold proteins, whereas A capsids are empty, containing neither DNA nor scaffold proteins. These are thought to be capsids that failed in the DNA packaging process (reviewed in references 24, 27, and 31). Another fourth capsid type that cannot be isolated in sucrose gradients but has been observed by ultrastructural analysis of infected cells is the procapsid, which is thought to be the earliest spherical structure formed in HSV-1-infected cells (19, 26). There are seven proteins in B capsids. They and the genes encoding them are VP5 (UL19), VP19C (UL38), 21 (UL26), 22a (UL26.5), VP23 (UL18), VP24 (UL26), and VP26 (UL35). The capsid shell comprises four proteins, VP5, VP19C, VP23, and VP26 (reviewed in references 24, 27, and 31). During the capsid maturation process the scaffold proteins (22a and 21) occupy the internal space of the capsid, and the shell achieves icosahedral symmetry due to the presence of these scaffold proteins (8, 33, 34). A key interaction for assembly of the capsid is that between the scaffold proteins and the major capsid protein VP5. This interaction has been detected in vitro with the yeast-two hybrid assay, coimmunoprecipitation, colocalization, and protein-binding assays (9, 15, 20, 22, 25, 34). Several studies have shown that the C-terminal amino acids of the scaffold protein are necessary and sufficient for the interaction with the major capsid protein (2, 9, 15, 16, 17, 21, 22, 34, 38). A study by Hong et al. (15) showed that the C-terminal tail of pre-22a that interacts with VP5 conforms to an alpha-helical structure. The hydrophobic residues that are exposed on this amphipathic helix are key for the interaction with VP5. In particular, a highly conserved phenylalanine was important for this interaction (15). Before or concurrent with packaging the DNA, the scaffold proteins are released from the capsid. Cleavage of the scaffold proteins at the maturation site by the virally encoded protease (VP24) results in the loss of proteins 22a and 21 from the interior of the capsid. This space is subsequently occupied by the viral genome (reviewed in references 24, 27, and 31). We recently identified a small region at the N terminus of VP5 as being critical for its interaction with pre-22a. These studies utilized a virus in which the maturation cleavage site in the scaffold proteins was blocked by mutation (23). This mutation is lethal because it prevents the release of the scaffold molecules from the capsid structure, and thus these capsids failed to acquire a genome; however, spontaneous revertants of this mutant virus were readily isolated on noncomplementing cells (11). These revertants had second-site mutations, and subsequent mapping and sequence analysis revealed that the majority (21 out of 33) of these mutations were localized at the N terminus of VP5, between residues 30 and 80 (see Fig. ​Fig.1)1) (36). The mutations occurred in two 12-amino-acid stretches; five were at amino acid 34, and 10 were at amino acid 78 (see Fig. ​Fig.1)1) (36). FIG. 1. Amino acid sequence of the N terminus of VP5, which is important for interaction with the scaffold protein. The amino acid sequence of VP5 (strain KOS) from residues 20 to 90 is shown. Displayed above the sequence are the mutations identified in the revertant ... These second-site mutations are suggested to act by weakening the interaction between VP5 and pre-22a once the capsid has formed, thereby allowing the release of the scaffold proteins from the maturing capsid in the absence of cleavage (36). With the in vitro yeast two-hybrid assay, mutants of KOS (wild-type) VP5 that result in loss of activity in this assay were selected following random mutagenesis. Several of the mutations were mapped to residue F39 (see Fig. ​Fig.1)1) (37), indicating the importance of this hydrophobic residue for interaction with pre-22a. The goal of the present study was to target residues of VP5 that directly participate in this interaction for mutagenesis. The study by Hong et al. (15) showed that the interaction is hydrophobic in nature by virtue of the structural data, the mutagenesis experiments, and the biochemical analysis of this interaction. To this end, we mutagenized the hydrophobic residues of VP5 that are found in the N terminus spanning residues 23 to 84. In the study described below, we have identified single amino acids of this large protein that are important for interaction with the scaffold protein, as judged from the yeast two-hybrid assay, and that are essential for the closure of HSV-1 capsid shells in infected cells.