Mechanism Of Scaffolding-Assisted Viral Assembly

https://doi.org/10.1016/S0065-3233(03)01007-6Get rights and content

Introduction

The existence of a class of molecules that came to be known as “scaffolding proteins” was recognized in 1974 by Jonathan King and Sherwood Casjens on the basis of their studies of the morphogenetic pathway of the Salmonella bacteriophage P22 (Casjens 1974, King 1974), and those of other groups on bacteriophage T4 and T7 (Kellenberger 1968, Studier 1969, Showe 1973). Three characteristics of scaffolding protein were articulated: hundreds of molecules are necessary for the formation of one product, the catalyst molecule becomes a stable component of the immediate product, and dissociation of the complex is a complicated triggered reaction. Perhaps most importantly, their results indicated that proteins critical for supramolecular assembly may not be found as part of the mature structure. In subsequent years, the existence of scaffolding proteins has been documented in a variety of both bacterial and animal viruses. The critical functions of scaffolding proteins in virus assembly have become clear, and scaffolding-like functions have been identified in assembly-related proteins that may not meet the strict definition of scaffolding proteins.

There appear to be two essential functions of scaffolding protein during viral morphogenesis: (1) facilitating the nucleation of assembly and (2) subsequently mediating the reaction through completion. The assembly of supramolecular structures generally involves an unfavorable nucleation step, after which continued assembly becomes more favorable (Erickson and Pantaloni, 1981). There are two factors that may contribute to the existence of the nucleation barrier. The first is the statistical improbability of getting a sufficient number of molecules together in space and time to promote assembly (Erickson and Pantaloni, 1981). The second is the requirement for an effector molecule to promote a conformational change from an un-associable to an associable conformation (Caspar, 1980). One role of scaffolding proteins may be to increase the effective concentration of the assembly active molecules and thereby lower the nucleation barrier. Lowering the nucleation barrier is frequently coupled to an induced conformational change in the protein subunit. It is worth noting that the existence of a nucleation barrier, and the use of an auxiliary molecule to lower that barrier, provide a convenient biological mechanism to control assembly both temporally and spatially. After nucleating assembly, the second role of scaffolding proteins is to provide form-determining information. There is ample evidence that viral coat proteins are capable of assembling into a variety of morphological forms ranging from sheets and cylinders to icosahedral capsids of altered T number to octahedra (Earnshaw 1978, Salunke 1989, Thuman-Commike 1998). These structures are thought to maintain similar local bonding interactions but to arise from subtle alterations in the relative disposition of the subunits. As is discussed in this review, the presence or absence of scaffolding protein can alter the disposition of the subunits within the lattice, and thereby ensure the proper form of the assembled particle.

In this review, we select two well-described phage systems, the Escherichia coli phage øX174 and the Salmonella phage P22, to illustrate the key functions of scaffolding proteins while drawing parallels to other viral systems. At this time, the functions of scaffolding proteins are best described in phage systems because of the well-developed genetics of phage biology. However, lessons learned from these simple systems readily translate to other, less tractable, viral systems.

Section snippets

øX174 Morphogenesis

With developed genetics, biochemistry, and biophysics the Microviridae system is well suited for the study of virion morphogenesis. Six assembly intermediates can be purified (Farber 1976, Hayashi 1988, Ekechukwu 1995) and the atomic structures of the øX174 virion and procapsid, containing both the internal and external scaffolding proteins, have been solved (McKenna 1992, McKenna 1994, McKenna 1996, Dokland 1997, Dokland 1999). While crystal structures provide a wealth of information, allowing

Prescaffolding Stages: Coat Proteins and Chaperones

As illustrated in Fig. 1, the first øX174 morphogenetic intermediates are the 9S and 6S particles, respective pentamers of viral coat and spike proteins. Several lines of evidence suggest that these particles self-assemble without the aid of scaffolding or host cell proteins, such as groEL and groES (Sternberg 1973, Georgopoulos 1978, Hendrix 1978). In cells infected with nonsense and temperature-sensitive alleles of the internal scaffolding protein, protein B, 9S, and 6S particles accumulate (

The øX174 Internal Scaffolding Protein

In cells infected with null, temperature-sensitive, or cold-sensitive mutations of the øX174 B gene, coat protein pentamers (9S particles) accumulate. Pentamers formed in the absence of functional scaffolding protein do not differ from pentamers formed in its presence. On shift to permissive temperatures in tsB- and csB-infected cells, 9S particles are efficiently chased into virions (Siden 1974, Ekechukwu 1995). However, pentamers formed in the absence of B protein will self-associate, forming

Genetic Data for Scaffolding Protein Flexibility: øX174 and Herpesviridae

Genetic studies of scaffolding function have, in general, been impeded by a dearth of scaffolding protein missense mutations that confer defects in morphogenesis. Of course, in eukaryotic systems, genetic analyses are much more difficult to conduct. However, with advances in recombinant DNA technologies, excellent forays in this area are being made (Desai 1999, Warner 2000). However, the dearth of such mutations in phage systems requires an explanation. If most substitutions are detrimental,

Structural Data for Scaffolding Protein Flexibility: øX174, P22, and Herpesviridae

Evidence for inherent flexibility comes from the øX174 procapsid atomic structure. In the procapsid, the internal scaffolding protein binds to a cleft formed between α-helix 2 and the β barrel of the coat protein (McKenna 1996, Dokland 1997, Dokland 1999). Binding appears to be mediated by the last 24 amino acids of the scaffolding protein, which is the only region of the protein exhibiting a high degree of conservation among the related phages used in the cross-complementation studies (see

The øX174 Internal Scaffolding Protein

Data from three diverse systems, øX174, P22, and HSV-1, suggest that the C termini of the scaffolding proteins play the most critical role in coat protein recognition. The importance of this region in the øX174 system is reinforced by both genetic and biochemical data. As stated above, the scaffolding proteins of the related phages G4, øX174, and α3 are able to cross-complement (Burch and Fane, 2000b). However, there was one instance in which cross-complementation was not observed. The øX174

Internal Scaffolding Protein Function in One and Two Scaffolding Protein Systems: øX174 versus P22 and Herpesviruses

Although the øX174 internal scaffolding protein appears to be mechanistically similar to HSV-1 and P22 proteins in coat protein recognition, there are some critical differences that affect later morphogenetic stages. First, øX174 assembles via coat protein pentamers and internal scaffolding monomers. In contrast, the active forms of the P22 and HSV-1 scaffolding proteins are oligomers (Parker 1997a, Newcomb 1999). In addition, capsomers are a structural, not morphogenetic, phenomenon in these

The Assembly Pathway of Bacteriophage P22

The assembly pathway of the Salmonella typhimurium bacteriophage P22 is typical of the dsDNA-containing bacteriophages (Fig. 2). In a series of genetic and biochemical experiments, the laboratories of Jonathan King and David Botstein demonstrated that the first identifiable structural intermediate is a “procapsid” (Botstein 1973, King 1973). The P22 procapsid is composed of an outer shell of 420 molecules of the 47-kDa coat protein (the product of gene 5) arranged with T=7 symmetry (Casjens 1979

The Role of the P22 Scaffolding Protein

In wild-type P22 infections, approximately 30 min postinfection, the cells burst and release on average 500 newly formed virus particles. In cells infected with mutants that block DNA packaging (e.g., those with mutations in the terminase proteins), the cells accumulate large numbers of procapsids within this time frame. In contrast, when cells are infected with mutants that exclusively prevent the synthesis of functional scaffolding protein, few particles are produced (Casjens and King, 1974).

Functional Domains of the P22 Scaffolding Protein

The scaffolding protein is a multifunctional protein, and a number of domains have been identified by mutational analysis (Fig. 3). The N-terminal region of the protein appears to be involved in translational autoregulation, the central region involved in oligomerization and exit during packaging, and the C-terminal region is involved in coat protein binding.

Secondary Structure and Unfolding Studies

To date the only internal scaffolding proteins for which there are high-resolution structural data is the N-terminal domain of the CMV scaffolding protein (Qiu 1996, Shieh 1996, Tong 1996), the C-terminal domain of the bacteriophage P22 scaffolding protein (Sun et al., 2000), and the øX174 B protein (Dokland 1997, Dokland 1999). However, the secondary structure of P22 scaffolding protein has been examined by both circular dichroism (CD) (Teschke et al., 1993) and Raman spectroscopy (Thomas et al

The Mechanism of Scaffolding-Assisted Assembly

Any model for the mechanism of scaffolding-assisted assembly must accommodate its role both in promoting assembly and ensuring proper form determination. In the assembly of the multishelled bluetongue virus, it has been suggested that the inner core forms a template surface on which the outer shell polymerizes (see Section XVIII). Although such a mechanism is attractive it appears not to be the case as no preformed cores of scaffolding protein could be identified under assembly conditions (

External Scaffolding Proteins

If using a strict definition of a scaffolding protein, one found associated with assembly intermediates but not in the mature virion, external scaffolding proteins such as the øX174 D and P4 Sid proteins are rare. However, some structural proteins, such as the T4 Soc, alphavirus glycoproteins, and Herpesviridae triplex proteins, bear a strong functional and⧸or structural resemblance (Steven 1992, Trus 1996, Iwasaki 2000, Zhou 2000, Olson 2001, Pletnev 2001). Although the decoration protein Soc

The øX174 External Scaffolding Protein

The øX174 external scaffolding protein (protein D) performs many of the functions typically associated with internal species in one-scaffolding-protein systems: the organization of assembly precursors into a procapsid and the stabilization of that structure. However, its function is physically and temporally dependent on the internal scaffolding protein, which induces the conformational changes in capsid pentamers to prevent their premature association. In the procapsid crystal structure, 20 D

P4 Sid Protein

The P2⧸P4 bacteriophage system presents a striking example of the reprogramming of coat protein assembly by a scaffolding protein. Bacteriophage P2 encodes coat (gpN) and internal scaffolding proteins (gpO) that assemble into T=7 P4 procapsids. The satellite bacteriophage P4 encodes the external scaffolding protein Sid (size determination). The presence of the P4 Sid protein alters morphogenesis from a T=7 to a T=4 pathway. In essence its actions force the P2 coat and internal scaffolding

Herpesvirus Triplex Proteins

Although the herpesvirus triplex proteins are components of the mature virion, and thus do not meet the classic criterion of a scaffolding protein, they appear to perform a scaffolding-like function, promoting the morphogenetic fidelity of the T=16 capsid. The triplexes are heterogeneous trimers composed of two molecules of Vp23 and one molecule of Vp19C (Newcomb et al., 1993). The triplexes are interdigitated between and connect adjacent capsomers and are required for HSV-1 procapsid assembly (

Scaffolding-Like Functions

Although not all viruses utilize a distinct scaffolding protein, all viruses must solve the fundamental problem of associating large numbers of coat protein subunits with precise geometry. As might be expected, scaffolding-like functions are common in virus assembly even if the players involved do not meet the strict definition of a scaffolding protein.

In the case of the bacteriophage HK97, there is no distinct scaffolding protein (Duda 1995a, Duda 1995c). However, the coat protein first

Acknowledgements

The authors wish to acknowledge the support of the NIH and NSF.

First page preview

First page preview
Click to open first page preview

References (153)

  • D Bashford et al.

    J. Mol. Biol.

    (1987)
  • D Botstein et al.

    J. Mol Biol.

    (1973)
  • J.L Bryant et al.

    J. Mol. Biol.

    (1984)
  • A.D Burch et al.

    Virology

    (2000)
  • A.D Burch et al.

    J. Mol. Biol.

    (1999)
  • S Casjens et al.

    J. Mol. Biol.

    (1988)
  • S Casjens et al.

    J. Mol. Biol.

    (1982)
  • D.L Caspar

    Biophys. J.

    (1980)
  • M.S Chapman et al.

    Virology

    (1993)
  • X.S Chen et al.

    J. Mol. Biol.

    (2001)
  • J.F Conway et al.

    J. Mol. Biol.

    (1995)
  • P Desai et al.

    Virology

    (1999)
  • T Dokland et al.

    J. Mol. Biol.

    (1999)
  • R.L Duda et al.

    J. Mol. Biol.

    (1995)
  • R.L Duda et al.

    FEMS Microbiol. Rev.

    (1995)
  • R.L Duda et al.

    J. Mol. Biol.

    (1995)
  • W Earnshaw et al.

    J. Mol. Biol.

    (1978)
  • W Earnshaw et al.

    J. Mol. Biol.

    (1976)
  • H.P Erickson et al.

    Biophys. J.

    (1981)
  • M.T Fuller et al.

    Virology

    (1981)
  • M.T Fuller et al.

    J. Mol. Biol.

    (1982)
  • M.L Galisteo et al.

    Biophys. J.

    (1993)
  • C.L Gordon et al.

    J. Biol. Chem.

    (1994)
  • B Greene et al.

    Virology

    (1994)
  • B Greene et al.

    Virology

    (1996)
  • B Greene et al.

    J. Biol. Chem.

    (1999)
  • B Greene et al.

    J. Biol. Chem.

    (1999)
  • P.X Guo et al.

    Virology

    (1991)
  • A.L Hanninen et al.

    Virology

    (1997)
  • L.L Ilag et al.

    Structure

    (1995)
  • K Iwasaki et al.

    Virology

    (2000)
  • P.J Jardine et al.

    J. Mol. Biol.

    (1998)
  • B Jennings et al.

    Virology

    (1997)
  • K.J Kim et al.

    Virology

    (2001)
  • J King et al.

    J. Mol. Biol.

    (1973)
  • J King et al.

    Cell

    (1978)
  • K Kodaira et al.

    Biochim. Biophys. Acta

    (1992)
  • H Krebs et al.

    J. Mol. Biol.

    (1983)
  • J Lescar et al.

    Cell

    (2001)
  • O.J Marvik et al.

    J. Mol. Biol.

    (1995)
  • R McKenna et al.

    J. Mol. Biol.

    (1994)
  • R McKenna et al.

    J. Mol. Biol.

    (1996)
  • J.H Miller et al.

    J. Mol. Biol.

    (1979)
  • W.S Nakonechny et al.

    J. Biol. Chem.

    (1998)
  • W.W Newcomb et al.

    J. Mol. Biol.

    (1993)
  • O Nilssen et al.

    Virology

    (1996)
  • N.H Olson et al.

    Virology

    (2001)
  • M.H Parker et al.

    Virology

    (1998)
  • M.H Parker et al.

    J. Mol. Biol.

    (1997)
  • M.H Parker et al.

    J. Mol. Biol.

    (1998)
  • Cited by (0)

    View full text