Higgs, Martin Robert
The role of the herpes simplex virus type 1 UL33 protein in DNA packaging.
PhD thesis, University of Glasgow.
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The UL33 gene of herpes simplex virus type 1 (HSV-1) encodes a 130 amino acid (aa) protein that is essential for the cleavage of concatemeric viral DNA into monomeric genomes and their packaging into preformed capsids. Several lines of evidence have suggested that UL33, along with the UL15 and UL28 gene products, forms part of a terminase enzyme responsible for catalysing this process.
This thesis describes the creation and characterisation of a number of UL33 insertion mutants in an effort to examine structure-function relationships within this protein and gain further insights into its function. Sixteen distinct mutants, encoding polypeptides with 5 aa insertions located at 14 separate positions throughout the protein, were generated. The abilities of these mutants to complement the DNA packaging and growth defects of viruses lacking functional copies of UL33 (the null mutant dlUL33 and the temperature sensitive mutant ts1233) were examined. Nine of the mutants were defective in both assays, and the capacity of all 16 mutants to support DNA packaging correlated precisely with their ability to complement virus growth. Regions of UL33 sensitive to insertion displayed a high degree of sequence conservation with UL33 homologues of other herpesviruses.
In agreement with previous reports, a direct interaction between UL33 and UL28 was demonstrated in immunofluorescence and immunoprecipitation assays. Although all sixteen mutants appeared to interact with UL28 in co-immunoprecipitation experiments, four of the insertion mutants were defective in co-localisation with UL28 in immunofluorescence assays. Interestingly, of these four mutants, three supported DNA packaging to wt levels. Similar experiments confirmed that UL33 interacts directly with UL15, and immunofluorescence assays indicated that none of the mutants was impaired in this interaction.
Novel interactions were also demonstrated between UL33 and the HSV-1 DNA packaging proteins UL6 and UL25. UL6 forms a portal vertex through which DNA is inserted into capsids, whilst UL25 is thought to play a structural role in stabilising capsids upon addition of DNA and is required only during the latter stages of encapsidation. All sixteen UL33 mutants were again able to interact with both partners in immunofluorescence assays. Of the remaining HSV-1 proteins necessary for genome encapsidation, neither UL17 nor UL32 interacted with UL33.
Immunofluorescence studies of virally infected cells revealed that UL15 was necessary for the localisation of the remaining terminase components (UL28 and UL33) to nuclear sites of viral DNA replication, where packaging occurs. This is consistent with a model originally proposed by Yang et al. (J. Virol. 81:6419-6433, 2007), who suggested that a nuclear localisation signal within UL15 was necessary for the nuclear import of the terminase complex. Similar experiments revealed that, in the absence of UL6, none of the terminase components localised to replication compartments (RCs), suggesting that UL6 might be required for retaining the terminase at sites of DNA packaging.
Together, the data presented in this thesis are consistent with UL33 forming part of the HSV-1 terminase via its interactions with UL15 and UL28. It is also possible that UL33 contributes to the transient interaction of terminase with the portal protein, UL6, during packaging. Although the interaction between UL33 and UL25 warrants further examination, it could be relevant to the mechanism by which UL25 is recruited to capsids and functions at the late stages of the head-filling process.
Surprisingly, no clear evidence was obtained that any of the 16 mutants was defective in interactions with UL6, UL15, UL25 or UL28. It is therefore not yet possible to conclude whether the observed interactions of UL33 with these four proteins are essential for viral DNA packaging. By the same token, the reason(s) why nine of the 16 mutants are defective in DNA packaging remains unclear, but does not appear to be associated with their ability to form known protein-protein interactions or to localise to sites of DNA packaging. The development of cell free systems and biochemical assays will be an important step in further characterising these proteins.
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