Identification of a minimal hydrophobic domain in the herpes simplex virus type 1 scaffolding protein which is required for interaction with the major capsid protein

Abstract

Recent biochemical and genetic studies have demonstrated that an essential step of the herpes simplex virus type 1 capsid assembly pathway involves the interaction of the major capsid protein (VP5) with either the C terminus of the scaffolding protein (VP22a, ICP35) or that of the protease (Pra, product of UL26). To better understand the nature of the interaction and to further map the sequence motif, we expressed the C-terminal 30-amino-acid peptide of ICP35 in Escherichia coli as a glutathione S-transferase fusion protein (GST/CT). Purified GST/CT fusion proteins were then incubated with 35 S-labeled herpes simplex virus type 1-infected cell lysates containing VP5. The interaction between GST/CT and VP5 was determined by coprecipitation of the two proteins with glutathione Sepharose beads. Our results revealed that the GST/CT fusion protein specifically interacts with VP5, suggesting that the C-terminal domain alone is sufficient for interaction with VP5. Deletion analysis of the GST/CT binding domain mapped the interaction to a minimal 12-amino-acid motif. Substitution mutations further revealed that the replacement of hydrophobic residues with charged residues in the core region of the motif abolished the interaction, suggesting that the interaction is a hydrophobic one. A chaotropic detergent, 0.1% Nonidet P-40, also abolished the interaction, further supporting the hydrophobic nature of the interaction. Computer analysis predicted that the minimal binding motif could form a strong ␣-helix structure. Most interestingly, the ␣-helix model maximizes the hydropathicity of the minimal domain so that all of the hydrophobic residues are centered around a Phe residue on one side of the ␣-helix. Mutation analysis revealed that the Phe residue is absolutely critical for the binding, since changes to Ala, Tyr, or Trp abrogated the interaction. Finally, in a peptide competition experiment, the C-terminal 25-amino-acid peptide, as well as a minimal peptide derived from the binding motif, competed with GST/CT for interaction with VP5. In addition, a cyclic analog of the minimal peptide which is designed to stabilize an ␣-helical structure competed more efficiently than the minimal peptide. The evidence suggests that the C-terminal end of ICP35 forms an ␣-helical secondary structure, which may bind specifically to a hydrophobic pocket in VP5.

Electrostatic Interactions Drive Scaffolding/Coat Protein Binding and Procapsid Maturation in Bacteriophage P22

“…For most viruses with T number Ͻ7, all of the information required for coat protein subunit conformational switching is contained within the subunits themselves. However, for viruses with T number Ն7, such as dsDNA phage (Casjens and Hendrix, 1988), herpesviruses (Hong et al, 1996;Wood et al, 1997), and adenoviruses (D'Halluin et al, 1978;Hasson et al, 1989Hasson et al, , 1992) the assistance of a scaffolding protein, which both directs the form determination and accelerates the assembly process, is required. Therefore, understanding of the assembly processes for these viruses requires insight into the mechanisms by which scaffolding proteins interact with the coat or capsid proteins.…”

“…Although herpesviruses such as HSV-1 and CMV have many similarities to P22, the electrostatic model we have presented may not apply to them. While the scaffolding proteins of both HSV-1 and CMV appear to have short amphipathic helical domains at their carboxyl termini which are required for activity Hong et al, 1996), there is no evidence that they form helix-loop-helix motifs. Furthermore, neither of these proteins has the high concentration of charged residues in the carboxyl-terminal region which is found in the P22 scaffolding protein.…”

“…The Salmonella typhimurium bacteriophage P22 assembles by a pathway that is typical of the doublestranded DNA (dsDNA) 2 phage (Hendrix, 1985;Casjens and Hendrix, 1988) and of related viruses such as herpesviruses (Rixon, 1993;Donaghy and Jupp, 1995;Hong et al, 1996;Newcomb et al, 1996;Trus et al, 1996;Wood et al, 1997) and adenoviruses (D'Halluin et al, 1978). The initial step in P22 assembly is the copolymerization of 420 molecules of the coat protein (gp5) into a Tϭ7 icosahedral shell termed a`procapsid'' (Casjens, 1979;Prasad et al, 1993;Thuman-Commike et al, 1996).…”

“…A similar requirement for about 15±25 residues at the carboxyl terminus has been described for the scaffolding proteins from the related viruses herpes simplex type I (HSV-1; Matusick-Kumar et al, 1995; and cytomegalovirus (CMV;Wood et al, 1997). The scaffolding proteins from all three of these species have a pattern of hydrophobic residues at the carboxyl terminus that strongly suggests that they form amphipathic helices (Cohen and Parry, 1990), and helicity in this domain has been shown to be required for scaffolding/major capsid protein interactions in HSV-1 (Hong et al, 1996). For the P22 scaffolding protein, circular dichroism, Raman spectroscopy, and NMR have indicated that when 11 residues are removed from the carboxyl terminus, the protein loses a significant amount of helicity.…”

The protease and the assembly protein of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8)

“…Immature capsids lack DNA but contain an abundant internal polypeptide, the assembly protein (AP), that is not found in the mature, DNA-containing particles (25,27,34,48,55). This protein is known to interact, through its carboxy-terminal domain, with the major capsid protein (32,69). This interaction is required for nuclear transport of the major capsid protein (47) and has also been proposed to act as a scaffold to facilitate the assembly of the capsid shell.…”