Bentley A Fane

PMID: 18570973;Abstract:

Studying the replication of the chlamydiaphages presents significant challenges. Their host bacteria, chlamydiae, have a unique obligate intracellular developmental cycle. Using qPCR, immunochemistry, and electron microscopy, the life cycle of chlamydiaphage Chp2 was characterised. Chp2 infection has a dramatic inhibitory effect on bacterial cell division. The RB to EB transition is arrested and RBs enlarge without further division. There is a phase of rapid Chp2 genome replication 36 to 48 h post infection that is coincident with the expression of viral proteins and the replication of the host chromosome. The end stage of Chp2 replication is characterised by the appearance of paracrystalline structures followed by bacterial cell lysis. These data indicate that the Chp2 life cycle is closely coordinated with the developmental cycle of its bacterial host. This is a remarkable adaptation by a microvirus to infect and replicate in a bacterial host that has an obligate intracellular developmental cycle. © 2008 Elsevier Inc. All rights reserved.

PMID: 11991963;PMCID: PMC137031;Abstract:

During the final stages of φX174 morphogenesis, there is an 8.5-Å radial collapse of coat proteins around the packaged genome, which is tethered to the capsid's inner surface by the DNA-binding protein. Two approaches were taken to determine whether protein-DNA interactions affect the properties of the mature virion and thus the final stages of morphogenesis. In the first approach, genome-capsid associations were altered with mutant DNA-binding proteins. The resulting particles differed from the wild-type virion in density, native gel migration, and host cell recognition. Differences in native gel migration were especially pronounced. However, no differences in protein stoichiometries were detected. An extragenic second-site suppressor of the mutant DNA-binding protein restores all assayed properties to near wild-type values. In the second approach, φX174 was packaged with foreign, single-stranded, covalently closed, circular DNA molecules identical in length to the φX174 genome. The resulting particles exhibited native gel migration rates that significantly differed from the wild type. The results of these experiments suggest that the structure of the genome and/or its association with the capsid's inner surface may perform a scaffolding-like function during the procapsid-to-virion transition.

PMID: 10729119;PMCID: PMC111853;Abstract:

Comparisons of the proteome of abortifacient Chlamydia psittaci isolates from sheep by two-dimensional gel electrophoresis identified a novel abundant protein with a molecular mass of 61.4 kDa and an isoelectric point of 6.41. C-terminal sequence analysis of this protein yielded a short peptide sequence that had an identical match to the viral coat protein (VP1) of the avian chlamydiaphage Chp1. Electron microscope studies revealed the presence of a 25-nm-diameter bacteriophage (Chp2) with no apparent spike structures. Thin sections of chlamydia-infected cells showed that Chp2 particles were located to membranous structures surrounding reticulate bodies (RBs), suggesting that Chp2 is cytopathic for ovine C. psittaci RBs. Chp2 double-stranded circular replicative-form DNA was purified and used as a template for DNA sequence analysis. The Chp2 genome is 4,567 bp and encodes up to eight open reading frames (ORFs); it is similar in overall organization to the Chp1 genome. Seven of the ORFs (1 to 5, 7, and 8) have sequence homologies with Chp1. However, ORF 6 has a different spatial location and no cognate partner within the Chp1 genome. Chlamydiaphages have three viral structural proteins, VP1, VP2, and VP3, encoded by ORFs 1 to 3, respectively. Amino acid residues in the ΦX174 procapsid known to mediate interactions between the viral coat protein and internal scaffolding proteins are conserved in the Chp2 VP1 and VP3 proteins. We suggest that VP3 performs a scaffolding-like function but has evolved into a structural protein.

Prokaryotic viruses have evolved various mechanisms to transport their genomes across bacterial cell walls. Many bacteriophages use a tail to perform this function, whereas tail-less phages rely on host organelles. However, the tail-less, icosahedral, single-stranded DNA phiX174-like coliphages do not fall into these well-defined infection processes. For these phages, DNA delivery requires a DNA pilot protein. Here we show that the phiX174 pilot protein H oligomerizes to form a tube whose function is most probably to deliver the DNA genome across the host's periplasmic space to the cytoplasm. The 2.4 Angstrom resolution crystal structure of the in vitro assembled H protein's central domain consists of a 170 Angstron-long alpha-helical barrel. The tube is constructed of ten alpha-helices with their amino termini arrayed in a right-handed super-helical coiled-coil and their carboxy termini arrayed in a left-handed super-helical coiled-coil. Genetic and biochemical studies demonstrate that the tube is essential for infectivity but does not affect in vivo virus assembly. Cryo-electron tomograms show that tubes span the periplasmic space and are present while the genome is being delivered into the host cell's cytoplasm. Both ends of the H protein contain transmembrane domains, which anchor the assembled tubes into the inner and outer cell membranes. The central channel of the H-protein tube is lined with amide and guanidinium side chains. This may be a general property of viral DNA conduits and is likely to be critical for efficient genome translocation into the host.