Characterisation of the Herpes Simplex Virus Type 1 (HSV-1) Triplex Proteins

Within the herpes simplex type-1 (HSV-1) capsid the heterotrimeric protein complex, the triplex, plays an essential structural role in both the capsid assembly pathway and subsequent conformational changes involved in the maturation of the procapsid into the WT polyhedral T=16 icosahedral capsid. Cryo-EM has shown that the HSV-1 capsid contains 6 different types of triplexes (Ta-Tf) which contribute to a single asymmetric unit and occupy the local and, with respect to triplex 'Tf', global 3-fold rotational axes. These triplexes in themselves represent unique asymmetrical structures within the HSV-1 capsid that violate the rules of quasi-equivalence. The triplexes have been shown not only to be involved in the interconnection of hexon and penton capsomers but also to interact with, and probably influence the development of, the capsid floor. This thesis analyses the structural relationship between the single copy of VP19c and the two copies of VP23 which make up the triplex and the means by which these unique asymmetric structures form their unusual interactions within the HSV-1 capsid. The interactions between the triplex proteins were analysed by using a genetic approach based on the yeast two-hybrid system, termed the reverse yeast two- hybrid system (described in section 3.1). Using this system, VP23 and VP19c were shown to interact within yeast. Since little is known about the structure of the individual HSV-1 capsid proteins, it was decided to analyse their biophysical properties through the use of size exclusion chromatography, near and far-UV circular dichroism (CD), differential scanning calorimetry (DSC), fluorescence spectroscopy, and 8-anilino-1-naphthalene sulfonate (ANS) binding studies. In order to carry out these experiments both bacterial and baculovirus recombinant expression systems were employed to express the individual triplex proteins VP23 and VP19c, as well as, co-expressing these proteins to form triplexes in vivo. Proteins were engineered to contain a 6xHis epitope tag at their N-termini. Using this tag, purification strategies were developed to maximise the recovery and solubility of the recombinantly expressed proteins. Monoclonal antibodies raised against these proteins were used in their characterisation. Abstract VP23 was shown to exist either as a monomer or a dimer, depending on the purification buffer used during the analysis, and both were shown to be functionally active in capsid assembly. Dimers were shown not to be linked by inter-molecular disulphide bonds. Conserved cysteine residues within VP23 were also shown not to be essential for either triplex formation or capsid assembly. Biophysical experiments were performed on both triplexes and triplex proteins purified to homogeneity through the use of Ni-NTA agarose affinity chromatography and shown to be functionally active via in vitro capsid assembly experiments. Fluorescence and far-UV CD analysis demonstrated that these purified proteins and complexes were folded and contained high levels of secondary structure. DSC and near-UV CD analysis demonstrated that VP23, either as a monomer or dimer, contained little or no stable tertiary structure. This was also demonstrated for purified VP 19c. ANS binding studies demonstrated that these proteins were able to bind ANS and that the organic moiety could become fluorescent following excitation. This indicates that ANS had access to the hydrophobic core of these proteins suggesting that they were able to undergo further conformational rearrangements. Triplexes, in comparison, were shown to contain well-defined tertiary structure while still retaining the ability to bind ANS that could become fluorescent following excitation. Taken together, the results show that the individual triplex proteins exist in a "molten globule" like intermediate state of folding. Molten globule-like proteins typically demonstrate a compact state with a high degree of secondary structure but with little or no rigid tertiary structure. Triplexes were shown to contain well-defined tertiary structure but still retained some of the characteristics of a molten globule. These results suggest that partial folding of the triplex proteins plays an important role in the formation of the triplex and its extensive interactions with other capsid proteins required for procapsid assembly and subsequent maturation. These results are discussed in relation to the known functions of the triplex and their overall importance to capsid structure