Type II Restriction of Bacteriophage DNA With 5hmdU-Derived Base Modifications

To counteract bacterial defense systems, bacteriophages (phages) make extensive base modifications (substitutions) to block endonuclease restriction. Here we evaluated Type II restriction of three thymidine (T or 5-methyldeoxyuridine, 5mdU) modified phage genomes: Pseudomonas phage M6 with 5-(2-aminoethyl)deoxyuridine (5-NedU), Salmonella phage ViI (Vi1) with 5-(2-aminoethoxy)methyldeoxyuridine (5-NeOmdU) and Delftia phage phi W-14 (a.k.a. ΦW-14) with α-putrescinylthymidine (putT). Among >200 commercially available restriction endonucleases (REases) tested, phage M6, ViI, and phi W-14 genomic DNAs (gDNA) show resistance against 48.4, 71.0, and 68.8% of Type II restrictions, respectively. Inspection of the resistant sites indicates the presence of conserved dinucleotide TG or TC (TS, S=C, or G), implicating the specificity of TS sequence as the target that is converted to modified base in the genomes. We also tested a number of DNA methyltransferases (MTases) on these phage DNAs and found some MTases can fully or partially modify the DNA to confer more resistance to cleavage by REases. Phage M6 restriction fragments can be efficiently ligated by T4 DNA ligase. Phi W-14 restriction fragments show apparent reduced rate in E. coli exonuclease III degradation. This work extends previous studies that hypermodified T derived from 5hmdU provides additional resistance to host-encoded restrictions, in parallel to modified cytosines, guanine, and adenine in phage genomes. The results reported here provide a general guidance to use REases to map and clone phage DNA with hypermodified thymidine

Arabidopsis thaliana DNA gyrase: expression, characterisation and in vivo insight

DNA gyrase is a type II topoisomerase distinguished by its ability to introduce negative supercoils into double-stranded DNA in a reaction linked to ATP hydrolysis. The essentiality of gyrase in bacteria has permitted its exploitation as an antibacterial target. The unanticipated discovery of gyrase within the nuclear genomes of eukaryotes including Arabidopsis and Plasmodia, was made near to two decades ago. Despite this, our understanding of gyrase within these species remains limited. The work here aimed to heterologously generate eukaryotic gyrases in order to biochemically characterise and better understand their mechanism of actions, gain an insight into their in vivo functions and explore their potential for inhibition. The specific inhibition of gyrase within these species would facilitate the generation of novel herbicidal and antimalarial drugs. In vivo knockdown experiments of A. thaliana gyrase have confirmed the embryo-lethality of GyrA. Arabidopsis plants able to propagate with a knockdown of GyrB1 are dwarfed, chlorotic, have reduced numbers and lengths of lateral roots and altered thylakoid ultrastructure. An increase of GyrB1 transcript mediates a stress response within Arabidopsis. The functional cooperation to achieve supercoiling of a reconstituted gyrase comprising A. thaliana GyrA and E. coli GyrB has been shown. The catalysis of A. thaliana enzyme (GyrA and GyrB2) is differentially mediated by potassium glutamate levels. The A. thaliana DNA gyrase has been determined to be 45-fold more efficient for ATP-independent DNA relaxation than E. coli gyrase. A novel sensitive DNA decatenation substrate, ‘bis-cat’, comprising two singly-linked supercoiled plasmids of disparate sizes has been generated and compared to the current marketed decatenation substrate. The novel substrate determined A. thaliana gyrase to be 35-fold more effective for DNA decatenation than the E. coli enzyme. The herbicidal and bactericidal specificities of novel fluoroquinolone compounds have also been compared

Biology of interleukin-6 production by human natural killer cells

NK cells secrete a variety of immune-regulatory cytokines and chemokines such as interferon - γ (IFN-γ) and tumour necrosis factor- α (TNF-α). However, in some of infections where NK cells have an important role in the defence mechanism, IL-6 appears to play a significant anti-viral role. Considering the role of both IL-6 and NK cells in these infections raises the possibility that IL-6 secretion by NK cells could be a main defence mechanism against them. Morover, NK cell-mediated IL-6 secretion may provide a critical link between the innate and adaptive immune response of the host. Furthermore, changes in this pathway in various autoimmune diseases, for example rheumatoid arthritis (RA) may be relevant for the pathogenesis of these disorders. The results presented in this thesis demonstrated that peripheral blood NK cells from healthy individuals have the ability to secrete IL-6 after co-culture with target cells against which these cells are known to exhibit direct cytotoxicity (either K562 or HeLa cells). The described secretory response was rapid, with IL-6 being significantly higher as early as 1 hour in HeLa co-cultures compared to 6 hours in case of K562 co-cultures. These findings were further confirmed when NK cells were activated alone with high doses of IL-2 or with non specific chemical activators (PMA+ ionomycin). These experiments clearly showed that NK cells have the potential to secrete IL-6 following activation. To analyse whether IL-6 secretion in the co-culture experiments was the result of direct cell-cell interactions between NK cells and the target cells or was induced by the presence of soluble mediators, co-culture experiments were set up where the target cells were separated from NK cells using a 0.4 µm pore size inserts. Separating NK cells and target cells abolished increases in cytokine production proving that direct interaction between NK cells and target cells is necessary for triggering IL-6 production by NK cells, as the semi-permeable membrane of Transwell chambers allows for the free passage of soluble factors but prevents direct cell-cell contact. Investigating the activating pathway which triggers the secretion of IL-6 by NK cells was the next step. This aim was achieved by inducing NK cell activation with immobilized antibodies against NK cell activating receptors and assessing the effect of the engagement of these receptors on peripheral blood NK IL-6 gene expression and protein secretion by quantitative real time PCR and ELISA.The results demonstrated that NKG2D and NKp46 were the two main receptors involved in the IL-6 mRNA expression and secretion by NK cells. The final aim of this thesis was to evaluate the biological significance of this secretion through an in vitro experimental model. We hypothesized that IL-6 secreted by NK cells could contribute to the migration of other inflammatory and immune cells to the site of inflammation. This hypothesis was based on the observations of others that IL-6 could induce direct CD4+ T cell migration. To test this hypothesis, an in vitro transmigration assay using Transwell inserts with 8 and 3 µm pore size were used. Our results demonstrated that CD4+ T cell migration in response to NK cells was inhibited by about 30% in the presence of neutralizing antibody to IL-6. These results signify the relative biological importance of IL-6 induced secretion by NK cells. In conclusion NK cells can contribute to IL-6 secretion. Given that NK cells appear at inflammation sites at the earliest stages of the process, where the number of other cells with a potential to secrete IL-6 is low, it is possible that NK cell-mediated IL-6 secretion is essential in orchestrating and potentiating the later stages of the adaptive immune response

Minicircle DNA Immobilization in Bacterial Ghosts (BGs):

Die vorliegende Arbeit „Minicircle DNA Immobilization in the Bacterial Ghosts: Investigation for the Reduction of Un-recombined Mother Plasmid DNA and Miniplasmid DNA in BGs” diskutiert die verschiedenen Möglichkeiten zur Verbesserung der aktuellen Methodik zur Herstellung von Minicircle DNA (mcDNA) beladenen Bacterial Ghosts (BGs), die frei von restlicher miniplasmid DNA (mpDNA) sowie nicht-rekombinierter mother plasmid DNA (mopDNA) sind. Spezifische Änderungen im Selbstimmobilisierenden Plasmid (SIP) beziehungsweise die Verwendung der Staphylococcal Nuclease A (SNUC) ermöglichten die Produktion von mcDNA-beladenen BGs, die überwiegend frei von mpDNA sowie nicht-rekombinierter mopDNA sind. Die Einführung einer neuen Methode zur Detektion von unterschiedlichen Formen von Plasmid DNA in BGs ermöglicht einen einfacheren, effizienteren und verlässlicheren Quantifizierungsprozess als zuvor. Durch die enzymatische Aktivität von SNUC wurde mpDNA und nicht-rekombinierter mopDNA hydrolysiert: durch quantitative Real Time PCR (qPCR) wurde gezeigt, dass 2.38% der mcDNA (23-59 Plasmidkopien/BG) von der hydrolytischen Aktivität von SNUC nicht betroffen waren. Im Übrigen verfügt dieses System über den Vorteil der Fähigkeit zur Berechnung der Rekombinationseffizienz, ohne auf densitometrische Analysen des Rekombinationsprodukts angewiesen zu sein.This thesis “Minicircle DNA Immobilization in the Bacterial Ghosts: Investigation for the Reduction of Un-recombined Mother Plasmid DNA and Miniplasmid DNA in BGs” discusses the different possibilities for the improvement of current technique for production of minicircle DNA loaded BGs, that are free of residual miniplasmid DNA / un-recombined mother plasmid DNA. Specific changes in the Self Immobilizing Plasmid and use of staphylococcal nuclease A lead to the production of mcDNA loaded BGs that are almost free of miniplasmid DNA / un-recombined mother plasmid DNA. Introduction of new method for detection of plasmid DNA present in BGs in different forms makes the quantification process more easy, efficient and reliable. The mpDNA and un-recombined mopDNA has been hydrolyzed through the enzymatic activity of Staphylococcus aureus nuclease A (SNUC). It has shown through real time quantitative PCR that 2.38% of mcDNA (23-59 plasmid copies / BG) escaped the hydrolysis activity of SNUC. Through introduction of this new technique, mpDNA and mcDNA is quantified efficiently without the involvement of any further assumptions. This system has another advantage over old quantification method due to its ability to calculate the recombination efficiency without the densitometric analysis of recombination product

Impact of DNA sequence on the structure and dynamics of the higher-order chromatin fibre in vitro

DNA in the eukaryotic cell is organised into a complex called chromatin, which protects the DNA from damage and allows the careful regulation of the genome. Precisely how the genome is used by the cell is dependent on the structure and regulation of this complex, and discovering how this is organised is therefore one of the principle challenges of modern biology. DNA is wrapped around histone octamers to form arrays of nucleosomes, which are subsequently folded into a higher-order structure, speculated to be a 30-nm fibre. In vitro studies of higher-order chromatin fibre structure have provided valuable information about this structure, but it is not well understood how changes to the DNA sequence might affect the structure and dynamics of the complex. DNA sequence is known to affect nucleosome binding strength and positioning within an array, and I therefore hypothesised that DNA sequence changes are likely to impact higher-order chromatin fibre structure. Using an in vitro model of chromatin fibre structure, reconstituting purified DNA with purified core histones by salt dialysis, allowed me to isolate the effects of DNA sequence in the absence of confounding factors such as transcription factor binding. I compared the higher-order chromatin structure and dynamics of the well-studied “601” DNA repeat sequence with two novel reconstitution templates which contain biologically-derived nucleosome positioning sequences. Sucrose gradient sedimentation of folded chromatin fibres suggested that non-601 fibres may be as compacted as 601 fibres, but have more heterogeneous structures. However, non-601 fibres were more easily perturbed under tension than 601 fibres, suggesting that such sequences might promote a more accessible chromatin environment. While repetitive 601 fibres were found to have a regular nucleosome repeat length by DFF digestion, non-repetitive, biologically-derived sequences had a more heterogeneous nucleosome repeat length, which I suggest is responsible for their increased accessibility. I also found that the compacted higher-order structure of the 601 fibre is disrupted by the introduction of a single sequence with low affinity for the histone octamer. These structures can be separated by sucrose gradient sedimentation, and I suggest that this could be a useful method to examine the individual effects of a wider range of DNA sequences on higher-order chromatin fibre structure in vitro

Small changes, unexpected consequences: Molecular insights into substrate-dependent adaptation of KsgA/Dim1-dependent ribosomal RNA modifications in archaea

Classically, life is divided into three phylogenetically interconnected groups of organisms, the archaea, bacteria and eukarya. However, they all share a central and universally conserved molecular component of the cell, a ribonucleoprotein complex known as the ribosome. The assembly of this molecular machine is an orchestration of RNA folding/modification events, hierarchical and cooperative incorporation of ribosomal proteins, and the association, dissociation, and interplay of various ribosomal biogenesis factors. Remarkably, this process has become increasingly complex over the course of evolution and has been studied best in bacterial and eukaryotic model organisms. In contrast, the in vivo archaeal ribosome biogenesis pathway(s) lack this detailed insight. Previous in vitro and in silico studies suggested bacterial as well as eukaryotic-like aspects of archaeal ribosome biogenesis. In this work, I have functionally characterized the archaeal homologue of the almost universally conserved ribosomal RNA dimethyltransferase KsgA/Dim1 in vivo. We could show that this ribosome biogenesis factor is dispensable in the Euryarchaeon Haloferax volcanii and Pyrococcus furiosus, as well as in the Crenarchaeon Sulfolobus acidocaldarius. The loss of this ribosome biogenesis factor in H. volcanii is associated to a decreased cellular fitness and stress adaptation, as well as a differential translational landscape which may correlate with altered translation initiation. Moreover, in this and phylogenetically related organism we observed an unusual heterogeneous/ hypomethylated KsgA/Dim1 dependent methylation pattern. Using phylogenetic-based comparison and genetic analysis, we show that the molecular determinant of this unusual methylation status is based on a single nucleotide exchange within the KsgA/Dim1 targeted rRNA substrate. The structural consequences of this variability could be verified with chemical RNA foot-printing and support a model where KsgA/Dim1-dependent modification disrupts local intramolecular interaction and promotes inter-molecular interaction of 16S/18S rRNA helices. Based on these observations and in vitro reconstitution experiments, this study suggests that release competence of KsgA from the 30S subunit is not dependent on full methylation completion but rather on cooperative local and distant rRNA conformational changes controlled by methylation-induced (intra-molecular) destabilization of the KsgA/Dim1 substrate. Finally, and in addition to this functional analysis, I have implemented and applied chemical RNA foot-printing and 4TU pulse (chase) labelling protocol for H. volcanii and S. acidocaldarius. Together, this work contributes to a better understanding of archaeal ribosome biogenesis, KsgA/Dim1 biology, and also expands the archaeal tool-box to study RNA metabolism

Structure, function and subcellular localization of the potato Resistance protein Rx1

Resistance proteins are part of the plant’s immune system and mediate a defence response upon recognizing their cognate pathogens. They are thought to be present in the cell as part of a larger protein complex. The modular architecture of R proteins suggests that they form a scaffold for various interacting proteins, involved in pathogen recognition, downstream signalling or protein stabilization. However, few common interactors have been found for the CC-NB-ARC domains despite extensive screenings for downstream interactors. The objective of thesis was to get new insights in the structure, function and localization of R proteins by using the potato resistance genes Rx1 and Gpa2 as a model system. Initially, a novel T7 phage display method was developed to facilitate high throughput selection of interacting molecules (Chapter 2). However, the use of a T7 cDNA phage library to identify interactors of the CC-NB-ARC domains of Rx1 resulted in the discovery of a large set of highly basic peptides (Chapter 3). In Chapter 4, the functional role of the CC-NB-ARC domain in mediating disease resistance was explored by creating chimeric proteins between Rx1 and Gpa2. This resulted in the observation that the CC-NB-ARC is able to confer both virus and nematode resistance in potato. Furthermore, it was shown that the CC-NB-ARC of Rx1 and the LRR of Gpa2 are incompatible and vice versa. This phenomenon was studied in more detail in Chapter 5, in which a docking model for the interacting surface of these domains was constructed based on the individual structural domains. Finally, the subcellular localisation was investigated to get a better understanding about the R proteins function in the cell (Chapter 6). The lytic T7 phages form a powerful platform for the display of large cDNA libraries and offer the possibility to screen for strong interactions with a variety of substrates. To visualise these interactions directly by fluorescence microscopy, we constructed fluorescent T7 phages by exploiting the flexibility of phages to incorporate modified versions of its capsid protein (Chapter 2). By applying translational frameshift sequences, helper plasmids were constructed that expressed a fixed ratio of both wild-type capsid protein (gp10) and capsid protein fused to enhanced yellow fluorescent protein (EYFP). The frameshift sequences were inserted between the 3’-end of the capsid gene and the sequence encoding EYFP. Fluorescent fusion proteins are only formed when the ribosome makes a -1 shift in reading frame during translation. As far as we know this is the first report of using a translational frameshift for a biotechnological purpose. The phages formed in this way have capsids composed of three different variants of their capsid protein; EYFP-fused versions derived by frameshift translation, non-fused versions derived by regular translation from the helper plasmid, and versions that display peptides encoded in the library ligated in the phage genome. Using standard fluorescence microscopy, we could sensitively monitor the enrichment of specific binders in a cDNA library displayed on fluorescent T7 phages. Closely monitoring the effect of the selection procedure enables fine tuning, and obviates the need for more laborious ELISA or plaque lift assays. Furthermore, with the fast pace of developments in single molecule detection technologies and sorting systems, these fluorescent phages open the way to high throughput platforms for the direct selection of binding molecules. In Chapter 3, cDNA phage display was applied as an alternative method to identify additional downstream Rx1 interactors, which could further resolve the Rx1 signalling pathway. In a pilot experiment the value of T7 phage display to identify specific interactors was demonstrated by using an antibody raised against the PVY coat protein. Screening of a PVY-infected N. benthamiana cDNA phage display library resulted in the selection of peptides harbouring the known PRIKAI epitope. Next, phage display was explored as technique to discover proteins interacting with the potato R protein Rx1. The system turned out to be prone to pick up interactors binding to matrices like Ni-NTA or to fusion proteins like thioredoxine. A possible way to circumvent this weakness was to design the selection procedure in such a way that it alternates between different matrices and to limit the number of selection round. This adapted approach resulted in the identification of a series of highly basic protein fragments and random peptides, for which a specific interaction could be shown. Two cDNA sequences encoded the ribosomal proteins L19 and L36a, which showed a stunted growth phenotype upon gene silencing in N. benthamiana using VIGS and a slightly reduced Rx-mediated HR. The nematode resistance protein Gpa2 and the virus resistance protein Rx1 provide an excellent test system to investigate the exchangeability of recognition and signalling domains and explore the evolutionary flexibility of R proteins, for they confer resistance to completely unrelated pathogens (Globodera pallida and potato virus X, respectively). In Chapter 4, we provide evidence for the hypothesis, that, via intergenic sequence exchanges and various types of mutations, NB-LRR proteins have the potential to alter resistance specificities towards taxonomically unrelated pathogens in relatively short evolutionary time periods. Both the regulatory sequences and CC-NB domains of the paralogs Gpa2 and Rx1 are non-pathogen specific and exchangeable. Remarkably, the genetic fusions of the CC-NB of Rx1 with the LRR of Gpa2 (Rx1CN/Gpa2L) and the reciprocal domain swap (Gpa2CN/Rx1L) were not functional when driven by the endogenous promoters or 35S promoter. Gain of wild type resistance was obtained by re-introducing the first five LRRs of Rx1 in Rx1CN/Gpa2, restoring the compatibility between the N-terminal part of the LRR and the ARC2 domain. Decreasing the expression levels for Gpa2CN/Rx1L resulted in extreme resistance against PVX, indistinguishable from wild type plants. Our results indicate that not only coding sequences, but that also optimizing the expression levels may play a role in generating novel resistances. The CC, NB-ARC, and LRR domains of the Rx1 and Gpa2 proteins interact with each other and recognition of the elicitor mediated by the LRR is translated in an activation of the NB-ARC. The available functional and evolutionary data make Gpa2 a suitable candidate for structural modelling of the individual domains and their interaction (Chapter 5). A structural model of the NB-ARC / LRR interaction could function as a framework for the interpretation of known empirical data and the design of new experiments to test R protein operational mechanisms (Zhang 2009). Therefore, computer aided modelling of the 3D structure domain models for the NB-ARC and the LRR domains were obtained and used as basis for a domain docking study. The functional interaction between the domains was studied via a detailed analysis of their incompatibility in chimeric Gpa2 and Rx1 proteins. A large set of sequence exchanges between the two proteins was created for that purpose. Both in the LRR and in the ARC2 domain small regions could be identified in which the amino acids differing between Gpa2 and Rx1 led to domain incompatibility. Five of the ARC2 positions required for LRR compatibility and three known autoactivating positions from the RX1 LRR were used as constraints in domain docking computation to limit the potential search space. The resulting docking model indicated an important role in the NB-ARC-LRR interaction for electrostatic and hydrophobic interactions. A loop region rich in acidic residues in the ARC2 domain was found close in space to a patch of basic residues grouped together in the LRR. Hydrophobic residues on both the NB and the ARC2 contacted hydrophobic residues on the surface of the LRR. A correlation analysis of the NB-ARC and LRR subdomains detected coevolution between the interacting surfaces, which supports a direct interaction between these two domains. Site-directed mutagenesis and pull-down experiments were used to test the role of surface features that might play an important role in the interdomain docking interface. In Chapter 6, we have made use of the characteristic of the Rx1 protein that it remains functional when its domains are co-expressed as separate polypeptides. This allowed us to create fluorescent constructs, not only of the full length protein, but also of the separate subdomains. Most of these tagged constructs still form functional proteins. C- and N-terminal fusions of Green Fluorescent Protein (GFP) variants to Rx, made it possible to study its subcellular localization in Nicotiana benthamiana cells. Contrary to our expectations we observed the presence of Rx1 in both the cytoplasm and the nucleus. Rx1 does not contain known nuclear localization signals and the size of the protein (140 kDa including GFP) exceeds the limit for passive diffusion through the nuclear pore. Fluorescent fusions of a series of deletion constructs, CC-NB-ARC, NB-ARC, NB-ARC-LRR, CC and LRR showed three distinct patterns of subcellular localization. The NB-ARC-LRR and LRR constructs have a cytoplasmic localization and are mostly absent in the nucleus. The NB-ARC and CC-NB-ARC constructs showed equal fluorescence intensities in both the nucleus and the cytoplasm. The CC alone fused to GFP, however, seems to preferentially accumulate in the nucleus resulting in a three to four times higher fluorescence intensity in the nucleus compared to the cytoplasm. The diffusion behaviour inside the nucleus for both the complete CC and a CC fragment containing the two predicted helices downstream of the central turn, showed that their nuclear accumulation coincides with a significantly reduced nuclear diffusion as compared to unfused GFP and the other CC fragments. This difference might point to a potential interaction between the CC and an unknown nuclear component. Furthermore, SGT1 and Rar1 are thought to function as chaperones involved in stabilizing R proteins. Both the silencing experiments with these two proteins and the P-loop mutation show that the nuclear localisation of Rx1 is probably conformation dependent. Two approaches were followed to see if CP recognition or Rx1 signalling pathway were linked to a certain cellular compartment. At one hand the Rx1 protein itself or its subdomains were directed to either the nucleus or the cytoplasm by fusion to exogenous targeting signals (Nuclear Export Signals or Nuclear Localization Signals). On the other hand the elicitor, the PVX coat protein, was directed to the nucleus or cytoplasm. The PVX coat protein is a much smaller protein and can under normal circumstances diffuse freely between the cytoplasm and the nucleus. The surprising result was that no effect was found for retargeting Rx1, but when the elicitor was targeted to the nucleus, it could not activate Rx1 anymore, indicating that recognition might to take place in the cytoplasm. In the final chapter, the results obtained in this thesis are put into perspective by studying parallels in scientific literature on NB-LRR proteins with similar functions in other organisms. <br/

Relating the structure of the HSV-1 UL25 DNA packaging protein to its function

The herpes simplex virus type 1 (HSV-1) UL25 protein (pUL25) is a minor capsid protein that is essential for packaging the full-length viral genome into preformed precursor capsid. It is also important in virus entry and recently has been implicated in the egress of the virus from the cell (Coller et al., 2007, Preston et al., 2008). The crystallographic structure of an N-terminally truncated form of pUL25 (residues 134-580) has been determined to 2.1 Å, revealing a protein with a novel fold that consists mostly of a-helices and a few minor b-sheets (Bowman et al., 2006). An unusual feature of the protein is the presence of numerous flexible loops extending out from the stable core and its distinctive electrostatic distribution. Five of the extended loops contain unstructured regions, L1-L5, with three additional unstructured amino acids, L6, located at the carboxyl terminus of the protein (Bowman et al., 2006). Four potentially functional clusters of residues, C1-C4, were identified on the surface of the protein using evolutionary trace analysis (Lichtartge and Sowa, 2002). To examine the function of the protein in relation to its structure, site-directed mutations were engineered into the UL25 gene in a protein expression plasmid. A series of mutant proteins was generated, each protein containing a deletion of the unstructured residues in one of the six regions, L1-L6. Another set of mutant proteins were constructed with each member containing substitutions of selected amino acids within one of the four potentially functional clusters, C1-C4, or substitutions of the three disordered amino acids in L6. The amino acid substitutions were generally to alanine, but in one case where the SIFT program predicted alanine would not affect the function of the protein an alternative residue was substituted. To determine the functional significance of the uncrystallised part of pUL25, residues 1-133, three deletion mutant proteins that spanned this region (pUL25D1-45, pUL25D1-59 and pUL25D1-133) were included in the study. Although an existing UL25 null mutant, KUL25NS, was available at the beginning of the project for analysis of the mutant proteins, it had been made by the insertion of multiple stop codons in the UL25 ORF and as a result some UL25 sequences were still present within the virus genome. Consequently, during complementation assays recombination between the UL25 sequences in the KUL25NS genome and the transfected expression plasmid generated low levels of wild-type (wt) progeny virus. To improve the sensitivity of the assay, a new deletion mutant, ΔUL25MO, was created that lacked the entire UL25 gene. This mutant failed to form plaques in non-permissive Vero cells and grew well in the complementing cell line, 8-1. However, contrary to previously published work, electron microscopic (EM) analysis revealed that DNA-containing capsids as well as A- and B-capsids were present in the nuclei of both ΔUL25MO- and KUL25NS-infected cells. As expected, none of the progeny from ΔUL25MO-infected Vero cells expressing the wt pUL25 formed plaques on non-permissive cells. Of the 17 mutant UL25 proteins screened in the complementation assay, nine failed to complement the growth of ΔUL25MO in Vero cells. Three of the non-complementing mutant proteins examined altered the phenotype of ΔUL25MO in a transient DNA packaging assay, allowing the mutant virus to package full-length genomes in U2OS cells co-infected with ΔUL25MO and a mammalian baculovirus vector containing the mutant UL25 gene. These results indicate that viral assembly was disrupted in these cells following DNA packaging. However, five of the mutant proteins did not change the pattern of DNA encapsidation of ΔUL25MO in this system, suggesting that the wild-type residues mutated in these proteins are critical for packaging virus DNA. To determine at which point in the virus growth cycle the post-packaging blocks occurred, EM was used to investigate the pattern of virus assembly in ΔUL25MO-infected cells expressing either of the three packaging-competent mutant proteins. In addition, fluorescent in-situ hybridisation (FISH) analysis was performed to establish the distribution of virus DNA in these cells. The results showed that in ΔUL25MO-infected cells expressing two of the mutant pUL25s the C-capsids failed to exit the nucleus, whereas in cells expressing the third post-packaging mutant protein C-capsids were seen in both the nucleus and the cytoplasm. The FISH data confirmed the EM observations. These studies show that two regions of pUL25 are important for egress of the C-capsids from the nuclei. Since these two regions lie in close proximity to each other on the surface of the molecule they may represent a single functional interface of the protein. In addition, another region of pUL25 was identified that was essential for the interactions required for virus assembly after the C-capsids are released into the cytoplasm. The 62 carboxyl-terminal region of the UL36 gene product (pUL36) has previously been shown to contain a capsid-binding domain (CBD) that interacts with pUL25 (Coller et al., 2007). A GST-pull down assay was used to determine whether the mutations in the post-packaging mutant proteins disrupted the interaction of pUL25 with the CBD of pUL36. However, all of these mutant proteins and the wt pUL25 bound to the pUL36 CBD GST fusion protein. In summary, three different classes of pUL25 mutants, each of which affect a different essential function of pUL25, have been identified, revealing that pUL25 is indeed a versatile viral protein. These mutants provide the first evidence that this DNA packaging protein is crucial for virus assembly at two different stages after DNA encapsidation, one in nuclear egress of C-capsids and the other in the assembly of the virus in the cytoplasm