Research

Internal Scaffolding protein		Capsid-DNA interactions
CHP2 and øMH2K Gokushoviruses	External Scaffolding Protein	Spike Proteins

INTRODUCTION

The proper assembly of proteins and nucleic acids into biologically active virions involves numerous and diverse macromolecular interactions. While structural proteins must correctly interact, proper morphogenesis is equally dependent on scaffolding proteins, which are transiently associated with nascent protein complexes during virion assembly. Scaffolding proteins can also mediate conformational switches: structural changes that cause the next assembly process to differ from the previous one. With developed genetics and biochemistry, the Microviridae system (øX174, G4 and alpha3) is well suited for the proposed research. At least six assembly intermediates can be purified. The viral assembly pathway is depicted in Figure 1. The atomic structures of the øX174 virion and procapsid, containing two scaffolding proteins, have been solved (McKenna et al., 1992, 1994; Dokland et al., 1997, 1999) . Hence, genetic and biochemical data can be interpreted within a structural context.

Figure 1: Microviridae Morphogenesis

THE INTERNAL SCAFFOLDING PROTEIN

Current Investigator: Min Chen.

Alumni Investigators: Dr. Asako Achiyama, Dr. April Burch-Alden, Chris Novak.

Scaffolding proteins are transiently associated with morphogenetic intermediates but not found in the mature particle (King & Casjens, 1974). Both structural and biochemical data indicate that coat-scaffolding protein interactions are variable and flexible (Dokland et al., 1997, 1999, Burch et al., 1999, 2000a).  Similar results have been obtained with experiments conducted on both the P22 and Herpes scaffolding proteins (Parker et al., 1998; Preston et al., 1997).

øX174 utilizes two scaffolding proteins during morphogenesis, an internal protein (B) and an external protein (D). The B protein induces a conformational change in coat protein pentamers, enabling them to interact with both spike and external scaffolding proteins. While functions of the carboxyl terminus of protein B have been defined, the functions of the amino terminus remain obscure. To investigate the morphogenetic functions of the amino terminus, several 5' deleted genes were constructed and the proteins expressed in vivo. The results of the biochemical, genetic and second-site genetic analyses indicate that the amino terminus induces conformational changes in the viral coat protein and facilitates minor spike protein incorporation. Defects in conformational switching can be suppressed by substitutions in the external scaffolding protein, suggesting some redundancy of function between the two proteins. By successive rounds of genetic selection, mutant virions that can assemble without the internal scaffolding protein have been isolated (B-free øX174). Mutations reside in the external scaffolding, minor spike, coat and DNA packaging proteins. In addition, two promoter mutations that over-express the external scaffolding protein were recovered. Thus the internal scaffolding protein may be best regarded as an efficiency protein, aiding several steps in the morphogenetic pathway but not absolutely essential for any one reaction. Its main function appears to be lowering the critical concentration of the external scaffolding protein required to nucleate procapsid morphogenesis. The results of this evolutionary study also illustrate the ability of even the smallest organism to adapt to very large selective pressures (Chen et al., 2007).

THE EXTERNAL SCAFFOLDING PROTEIN

Current Investigators: James Cherwa

Alumnus Investigator: Dr. Asako Uchiyama. Dr. April D. Burch-Alden.

The atomic structure of the external scaffolding protein, protein D, suggests that it also shares many features with molecular chaperones (Dokland et al., 1997, 1999). The four D proteins per asymmetric unit (Figures 3 and 4) have different structures (D1, D2, D3, and D4) and are arranged as two similar, but not identical, dimers (D1D2 and D3D4). The D1D2 dimer makes contact with the major spike protein, protein G. The D3D4 dimer makes contact with the major coat protein, protein F. Like a molecular chaperone (Hartl and Martin, 1995) protein D can bind to other proteins in many different ways. Its structure elucidates the conformational requirements needed for the pliable and diverse interactions of chaperone-like molecules with their substrates. Recently the atomic structure  of the  D protein before interactions  with other viral structure proteins has been solved (Morais et al., 2004). This structure  combined with the results of biochemical analyses (Burch and Fane, 2000b) provide insights into  how the protein assumes the different conformations required to mediate assembly.

Figure 3: The Four D subunits per asymmetric unit. Each subunit sits in a different environment and has a different structure. The D4 subunit (blue) is the most structurally unique.

Figure 4: Cross section of the two-fold axis of symmetry. The viral coat protein is depicted in gray. The external scaffolding proteins are depicted in green (D1), yellow (D2), red (D3) and blue (D4)

To further elucidate structure-function relationships in the external scaffolding, we have constructed the chimeric external scaffolding protein genes between the related Microviruses G4, øX174 and alpha3 and then select for mutant virions capable of productively utilizing these proteins for morphogenesis. The results of these analyses have identified the a substrate-specificty domain within the external scaffolding protein. This domain specifies the coat protein with which the scaffolding protein must interact to initiate assembly. The contact point in the underlying coat protein was also identified (Uchiyama et al. 2005). However, the expression of a cloned gene could lead to protein concentrations higher than those found in typical infections. Moreover, its induction before infection could alter the timing of the protein’s accumulation. Both of these factors could drive or facilitate reactions that may not occur under physiological conditions or before programmed cell lysis.  In order to elucidate a more detailed mechanistic model, a chimeric external scaffolding gene was placed directly in the øX174 genome, under wild-type transcriptional and translational control, and the chimeric virus, which was not viable on the level of plaque formation, was characterized.  The results of the genetic and biochemical analyses indicate that a-helix 1 most likely mediates the nucleation reaction for the formation of the first assembly intermediate containing the external scaffolding protein. Mutants that can more efficiently use the chimeric scaffolding protein were isolated. These second-site mutations appear to act on a kinetic level, shortening the lag phase before virion production, perhaps lowering the critical concentration of the chimeric protein required for a nucleation reaction (Uchiyama et al. 2007).

Confirmational switching  is currently being investigated by designing inhibitory external scaffolding proteins. The ability of a particular a-helix to assume two different conformations appears to be critical to asymmetric dimer and procapsid lattice formation. To achieve this arrangement, a 30º bend must occur at glycine residue 61 in subunits D1 and D3. Without this kink, all subunits would have an identical structure and perhaps polymerize into an indefinitely growing helical bundle. A substitution for G61 may allow the protein to fold into only one conformation. This protein would interact with WT subunits but might inhibit assembly. To test this hypothesis, mutations designed to lock this helix into one conformation were introduced into a cloned gene. The mutant proteins were expressed in vivo and assayed for the ability to inhibit WT plaque formation. Inhibition appears to be a function of the size of the substituted amino acid side chain. Substitutions with large side chains (V, K, D) inhibit, while those with small side chains (A, S) do not.

The wild-type assembly pathway has been characterized in the presence of the lethal dominant proteins at various levels of induction. Under low induction, virions form but at least one half of the resulting particles sediment like procapsids. SDS-PAGE analysis of these particles indicates that all procapsid proteins are present. When the lethal dominant gene is expressed at high levels, the largest soluble assembly intermediate found in cell extracts is the 9S* particle, pentamers of coat protein. This unexpected result rules out the simplest model in which interactions between lethal and wild-type subunits remove all D protein from the assembly pathway. In that model, 12S* particles, containing coat, spike and internal scaffolding proteins would accumulate, as seen in the complete absence of D protein. These results suggest that 12S* particles are aggregating into an insoluble complex with the wild type and lethal external scaffolding proteins. The formation of this hypothesized aggregate would be dependent on both the wild-type and lethal dominant proteins.

Viruses resistant to the inhibitory proteins were isolated and mapped to the coat and internal scaffolding proteins.  They cluster in the C-terminus of the internal scaffolding protein and in regions of the viral coat protein with which it interacts.  Strains with multiple resistance mutations have also been isolated. These strains show optimal fitness only in the presence of the inhibitory protein, demonstrating that the selection for resistance may also co-select for dependence.

THE ROLE OF GENOMIC DNA AS A PSEUDO-SCAFFOLDING DURING THE FINAL STAGES OF VIRION MORPHOGENESIS.

Current Investigators: Min Chen.
Alumni investigators, Dr. Susan Hafenstein, Katie Lux, Patty Rohrschneider, Erica Moore

The Microviridae DNA binding proteins are small very basic proteins that are essential for DNA packaging (Aoyama et al., 1983; Hamatake et al., 1985), which link the packaged genome to the inner surface of the capsid. In addition to genome binding and neutralization (Jennings and Fane, 1997)., the J protein may be involved in other morphogenetic processes such as the genome's arrangement within the capsid's icosahedral symmetry. The final stages of morphogenesis involve the collapse of 12 capsid protein pentamers around the single-stranded DNA genome. As assembly proceeds from procapsid to virion, the radial outward shift of the coat protein decreases by 8.5 Å. In the procapsid, very few contacts are found between neighboring coat protein pentamers. Instead, they are held together by internal and external scaffolding proteins, the B and D proteins, respectively. During packaging, B proteins are extruded from the procapsid and replaced by J proteins. Thus, the stabilization function performed by the B protein in the procapsid may be transiently performed by the viral genome and DNA binding proteins during packaging.

 

There are probably two major, but opposing, forces which govern the conformation of the single-stranded genome within the virion. One force tethers the genome to the inner surface of the capsid, the outwards force. The other force may be governed by the ability of the single-stranded DNA genome to base-pair with itself. Altering either the chemical make-up of the genome or its association with the inner surface of the capsid can have dramatic effects on the virion's biophysical properties (Hafenstein and Fane, 2002; Hafenstein et al., 2004; Bernal et al., 2004). The molecular basis of this phenomenon is currently under investigation.

CHP2 and øMH2K

Current Investigators: B. A. Fane, I. N. Clarke's group (University of Southampton).
Alumnus Investigators: Karie Brentlinger, Susan Hafenstein, Chris Novak.

Microviridae isolated from Chlamydia (CHP2), Bdellovibrio (øMH2K) and Spiroplasma (SPV4) appear are distantly related to  øX174 (Liu et al., 2000, Garner et al., 2004; Brentlinger et al., 2002; Chipman et al., 1997). These viruses, which infect obligate intracellular bacteria,  assemble without an external scaffolding protein (Clarke et al.,  2004).  There exists a limited understanding of the life-cycles of viruses such as CHP2 and øMH2K that infect cells that themselves live within other cells. Current efforts in the two laboratories are focused on elucidating the infection and assembly processes of these newly discovered viruses (Everson et al., 2002, 2003). 

Figure  5: CHP2 Virions (left) and stain penetrated empty procapsids (right).

ACKNOWLEDGMENTS

This research is supported by a grant from the National Science Foundation.