Identification of Cohesive Ends and Genes Encoding the Terminase of Phage 16-3

Cohesive ends of 16-3, a temperate phage of Rhizobium meliloti 41, have been identified as 10-base-long, 3′-protruding complementary G/C-rich sequences. terS and terL encode the two subunits of 16-3 terminase. Significant homologies were detected among the terminase subunits of phage 16-3 and other phages from various ecosystems

Challenging packaging limits and infectivity of phage λ

The terminase motors of bacteriophages have been shown to be among the strongest active machines in the biomolecular world, being able to package several tens of kilobase pairs of viral genome into a capsid within minutes. Yet these motors are hindered at the end of the packaging process by the progressive build-up of a force resisting packaging associated with already packaged DNA. In this experimental work, we raise the issue of what sets the upper limit on the length of the genome that can be packaged by the terminase motor of phage λ and still yield infectious virions, and the conditions under which this can be efficiently performed. Using a packaging strategy developed in our laboratory of building phage λ from scratch, together with plaque assay monitoring, we have been able to show that the terminase motor of phage λ is able to produce infectious particles with up to 110% of the wild-type (WT) λ-DNA length. However, the phage production rate, and thus the infectivity, decreased exponentially with increasing DNA length, and was a factor of 103 lower for the 110% λ-DNA phage. Interestingly, our in vitro strategy was still efficient in fully packaging phages with DNA lengths as high as 114% of the WT length, but these viruses were unable to infect bacterial cells efficiently. Further, we demonstrated that the phage production rate is modulated by the presence of multivalent ionic species. The biological consequences of these finding are discussed

Portal control of viral prohead expansion and DNA packaging

AbstractBacteriophage T4 terminase packages DNA in vitro into empty small or large proheads (esps or elps). In vivo maturation of esps yields the more stable and voluminous elps required to contain the 170 kb T4 genome. Functional proheads can be assembled containing portal–GFP fusion proteins. In the absence of terminase activity these accumulated in esps in vivo, whereas wild-type portals were found in elps. By nuclease protection assay dsDNAs of lengths 0.1, 0.2, 0.5, 5, 11, 20, 40 or 170 kb were efficiently packaged into wild-type elps in vitro, but less so into esps and gp20–GFP elps; particularly with DNAs shorter than 11 kb. However, 0.1 kb substrates were equally efficiently packaged into all types of proheads as judged by fluorescence correlation spectroscopy. These data suggest the portal controls the expansion of the major capsid protein lattice during prohead maturation, and that this expansion is necessary for DNA protection but not for packaging

ATP-Reactive Sites in the Bacteriophage λ Packaging Protein Terminase Lie in the N-Termini of Its Subunits, gpA and gpNu1

AbstractATP-reactive sites in terminase and its subunits have been successfully identified using three different affinity analogs of ATP (2- and 8-azidoATP and FITC). GpA, the larger subunit of terminase, was shown to have a higher affinity for these analogs than gpNu1, the smaller subunit. The suitability of these reagents as affinity analogs of ATP was demonstrated by ATP protection experiments andin vitroassays done with the modified proteins. These analogs were thus shown to modify the ATP-reactive sites. The results obtained from these experiments also indicate the importance of subunit–subunit interactions in the holoenzyme. Terminase, gpA, and gpNu1 were modified with these analogs and the ATP-reactive sites were identified by isolating the modified peptide by reverse-phase chromatography. The sequence analysis of the modified peptides indicates a region including amino acids 18–35 in the N-terminus of gpNu1 and a region including amino acids 59–85 in the N-terminus of gpA as being the ATP-reactive sites

The role of the herpes simplex virus type 1 UL33 protein in DNA packaging

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

The portal protein plays essential roles at different steps of the SPP1 DNA packaging process

AbstractA large number of viruses use a specialized portal for entry of DNA to the viral capsid and for its polarized exit at the beginning of infection. These families of viruses assemble an icosahedral procapsid containing a portal protein oligomer in one of its 12 vertices. The viral ATPase (terminase) interacts with the portal vertex to form a powerful molecular motor that translocates DNA to the procapsid interior against a steep concentration gradient. The portal protein is an essential component of this DNA packaging machine. Characterization of single amino acid substitutions in the portal protein gp6 of bacteriophage SPP1 that block DNA packaging identified sequential steps in the packaging mechanism that require its action. Gp6 is essential at early steps of DNA packaging and for DNA translocation to the capsid interior, it affects the efficiency of DNA packaging, it is a central component of the headful sensor that determines the size of the packaged DNA molecule, and is essential for closure of the portal pore by the head completion proteins to prevent exit of the DNA encapsidated. Functional regions of gp6 necessary at each step are identified within its primary structure. The similarity between the architecture of portal oligomers and between the DNA packaging strategies of viruses using portals strongly suggests that the portal protein plays the same roles in a large number of viruses

Characterizing the Intact Prophage of Mycobacterium chelonae Bergey

Mycobacteriophage (phage), are viruses that infect bacteria. All bacteria can be infected by phage, and each bacterial species has a unique set of phage that infect them, making phage prime candidates for studying viral diversity and evolution. Some phage integrate their genome into the host genome upon infection (prophage), where they may potentially remain indefinitely, coevolving with the host, and providing growth factors and other benefits to the host. The purpose of my research is to characterize a prophage within the genome of the bacterial host Mycobacterium chelonae Bergey to determine if it is still functional and potentially impacting the fitness of the host bacterium. Characterization of this prophage has revealed that multiple genes are conserved with regard to both the DNA and protein sequences. The integrase cassette is highly conserved, complete with integrase and two potential repressors, suggesting the phage may be capable of excising from the host genome. Multiple structural genes including capsid and tail proteins are also conserved, suggesting the prophage may be capable of producing intact virions. At least two prophage genes are transcriptionally active. These include a predicted repressor and transmembrane protein. Expression of these genes suggests that the prophage does indeed have some potential for affecting the biology of the host bacterium. Experiments are currently underway to determine if intact virion particles are being produced during bacterial growth

Genome sequence of bacteriophage ΦAR29: a basis for integrative plasmid vectors

The initial aim of this project was to characterise the integrative recombination mechanism of bacteriophage ΦAR29 , to provide a better understanding for development of the shuttle plasmid pBA as a site-specific Bacteroides integration vector. RT-PCR showed that the previously identified ΦAR29 recombination genes, integrase (Int) and excisionase (Xis), were transcribed from pBA in E. coli SCS110, B. thetaiotaomicron AR29 and B. uniformis AR20. In silico derived amino acid sequences from both genes showed only very low levels of similarity to other known Int and Xis in GenBank. To improve understanding of the phage recombination system, the ΦAR29 genome was sequenced. This revealed a 35,558 bp double-stranded DNA genome with GC content of 39.11%. Bioinformatic analysis identified 53 open reading frames (>30 codons) and gene promoters and terminators that allowed the genome arrangement to be compared with other phages. Comparison of deduced gene products with proteins from other phages identified 6 reading frames, allowed tentative identification of 7 others, but left 40 ORFs unidentified. Those with strong homology to known genes were: large terminase subunit (44.66 kDa), dnaC (27.94 kDa), helix-turn-helix (HTH) transcription regulator (14.69 kDa), cI repressor (26.48 kDa), amidase (18.42kDa) and a novel integrase (54.22 kDa). The integrase gene is located 162 base-pairs downstream of the phage attachment (attP) core site, rather than the previously suggested location upstream of the integration site. The ΦAR29 attP was shown to include a 16-bp att core region, 117 bp upstream of the previously suggested location. Integration of ΦAR29 was found to occur at the 3'end of an arg-tRNA gene on the AR29 genome (attB). Imperfect direct repeats with a consensus sequence (ANGTTGTGCAA) were found surrounding the attP core. A review of pBA sequence showed that only the 5' end (435 bp) of the newly identified Int gene was cloned in pBA. Despite this, PCR analysis revealed integration of pBA into the AR29 genome. Serial subculturing of pBA transformed AR29 was able to cure AR29 of the ΦAR29 prophage, providing an improved host for integrative plasmids, and for detailed studies of AR29 physiology and ΦAR29 life cycles