Studies on the herpes simplex virus type 1 UL32 DNA packaging protein

Palmer, Elizabeth Ann (2010) Studies on the herpes simplex virus type 1 UL32 DNA packaging protein. PhD thesis, University of Glasgow.

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The work presented in this thesis is concerned with the characterisation of the UL32 gene of herpes simplex virus type 1 (HSV-1). UL32 encodes an essential 596 amino acid cysteine-rich, zinc-binding protein that is highly conserved throughout the herpesviruses. The UL32 protein is essential for the cleavage of concatemeric viral DNA into monomeric genomes and their packaging into preformed capsids. Conservation is highest at the C-terminus and three CxxC motifs are present in almost all known herpesvirus UL32 sequences
The UL32 antibodies available in the laboratory at the beginning of the project were incapable of detecting small amounts of UL32 protein and so new rabbit antisera were created. Soluble extracts from insect cells infected with a UL32-expressing baculovirus (AcUL32) were fractionated by anion exchange chromatography and the UL32-containing fractions used to immunise rabbits. The resultant antisera successfully recognised UL32 from transfected, HSV-1 infected and baculovirus infected cells on western blots, and UL32 in transfected cells by immunofluorescence.
I performed random mutagenesis of the UL32 gene in an effort to examine structure-function relationships within this protein, and generated a panel of 37 mutants containing 5 amino acid insertions at distinct positions. The abilities of these mutants to complement the DNA packaging and growth defects of a virus lacking a functional copy of UL32 (the null mutant hr64) were examined and 15 of the mutants retained functionality in both assays. A complete correlation was found between the ability of mutants to support growth and DNA packaging, suggesting that the key functions of UL32 are confined to the DNA packaging pathway. There was also good correlation with the degree of amino acid conservation within UL32, with most of the mutants which abolished functionality being located in the highly conserved regions, and the functional mutants in less conserved regions. A number of site-specific mutants were also created, in which the paired cysteine residues were replaced with serines (i.e. CxxC to SxxS). Mutation of the first and third cysteine pairs (from the N-terminus) completely abrogated growth and packaging, whereas significant functionality was maintained following mutagenesis of the central pair. Finally, removal of the C-terminal 4 amino acids also resulted in generation of a non-functional protein.
Generation of an HA-tagged UL32 construct and the introduction of this into HSV-1 allowed the localisation of UL32 in infected cells to be studied. In contrast to previous reports, I detected UL32 predominantly in the nuclei of infected cells, co-localising with ICP8 in replication compartments. DNA packaging has previously been shown to occur within the replication compartments and a number of the other packaging proteins also localise to these sites. It was previously reported that UL32 played a role in the localisation of capsids to the replication compartments. However, work presented in this thesis shows this not to be the case, and that capsid proteins VP5 and VP19C were correctly localised in replication compartments during infections with the UL32 mutant hr64. I found no evidence of UL32 interaction with the UL6, UL25 or UL17 DNA packaging proteins in HSV-1 infected or transfected cells, or using immunoprecipitation from baculovirus-expressed cells.
Immunofluorescence studies of co-transfected cells showed that UL15 could direct the partial re-localisation of UL32 from the cytoplasm to the nucleus. The addition of the other terminase subunits UL28 and UL33 caused the complete re-localisation of UL32 to the nucleus, suggesting that UL32 might interact with the terminase complex. Fifteen of the insertional mutants were completely re-localised to the nucleus in the presence of UL15, UL33 and UL28, with eleven further mutants showing an intermediate phenotype of partial nuclear localisation. The ability to at least partially co-localise with the terminase complex appeared necessary for the ability of the mutants to support virus growth and DNA packaging, suggesting that this interaction may be essential for the function of UL32. However, no interaction could be demonstrated between UL32 and any of the individual terminase subunits using immunoprecipitation from insect cells.
A series of experiments was undertaken to further characterise the UL32 protein. A new UL32 mutant virus (Δ32EP) was generated by insertion of a kanamycin resistance cassette in place of a large portion of the UL32 gene. This mutant had an indistinguishable phenotype from hr64, confirming that the main function of UL32 is within DNA packaging. The functional conservation between HSV-1 UL32 and the homologues from HCMV and VZV was examined, but neither protein could support the growth of Δ32EP. DNA fragments from replicated concatemeric DNA from Δ32EP infected cells behaved similarly to wt HSV-1 fragments in PFGE, suggesting that UL32 is not involved in the resolution of branched structures within the genome prior to packaging.
UL32 had previously been reported to bind zinc, and this was confirmed using a zinc-release colourimetric assay. The amount of zinc bound to soluble baculovirus-expressed UL32 was quantified, showing that UL32 bound zinc in a 1:1 molar ratio. UL32 does not share all of the characteristics of a zinc finger motif, but the results of the mutagenesis experiments suggest that the outer CxxC/CxxxC motifs may be important for zinc binding.
Because of its zinc-binding properties and potential interaction with the terminase complex, the DNA binding properties of UL32 were also investigated. It was found that UL32 did not bind to dsDNA containing either the minimal packaging sequence (Uc-DR1-Ub) or an unrelated non-HSV-1 sequence using an electrophoretic mobility shift assay (EMSA).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: HSV-1, UL32, DNA packaging
Subjects: Q Science > QR Microbiology
Q Science > QR Microbiology > QR355 Virology
Q Science > Q Science (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
Supervisor's Name: Stow, Dr. Nigel
Date of Award: 2010
Embargo Date: 2 July 2013
Depositing User: Miss Elizabeth Ann Palmer
Unique ID: glathesis:2010-1987
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 05 Jul 2010
Last Modified: 02 Jul 2013 07:31

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