Bentley A Fane

PMID: 19237183;Abstract:

The first α-helices of Microviridae external scaffolding proteins function as coat protein substrate specificity domains. Mutations in this helix can lengthen the lag phase before progeny production. 5′ deletion genes, encoding N-terminal deletion proteins, were constructed on plasmids and in the øX174 genome. Proteins lacking the first seven amino acids were able to rescue a nullD mutant when expressed from a plasmid. However, the lag phase before progeny production was lengthened. The øX174 mutant with the corresponding genomic gene grew very poorly. The molecular basis of the defective phenotype was complex. External scaffolding protein levels were reduced compared to wild-type and most of the viral coat protein in mutant infected cells appears to be siphoned off the assembly pathway. Second-site suppressors of the growth defects were isolated and appear to act via two different mechanisms. One class of suppressors most likely acts by altering mutant external scaffolding protein expression while the second class of suppressors appears to act on the level of protein-protein interactions. © 2009 Elsevier Inc. All rights reserved.

PMID: 1532389;PMCID: PMC205867;Abstract:

To examine whether cleavage of the φX174 prohead accessory protein, gpB, by the OmpT protease is required for phage development in vivo, a phage mutant lacking the OmpT cleavage site and an Escherichia coli C ΔompT strain were constructed. The results of burst size experiments suggest that neither the cleavage site nor the OmpT protein is required for φX174 development.

PMID: 12473449;Abstract:

Bacteriophage α3 is a member of the Microviridae, a family of small, single-stranded, icosahedral phages that include φX174. These viruses have an ssDNA genome associated with approximately 12 copies of an H pilot protein and 60 copies of a small J DNA-binding protein. The surrounding capsid consists of 60 F coat proteins decorated with 12 pentameric spikes of G protein. Assembly proceeds via a 108 S empty procapsid that requires the external D and internal B scaffolding proteins for its formation. The α3 "open" procapsid structural intermediate was determined to 15 Å resolution by cryo-electron microscopy (cryo-EM). Unlike the φX174 "closed" procapsid and the infectious virion, the α3 open procapsid has 30 Å wide pores at the 3-fold vertices and 20 Å wide gaps between F pentamers as a result of the disordering of two helices in the F capsid protein. The large pores are probably used for DNA entry and internal scaffolding protein exit during DNA packaging. Portions of the B scaffolding protein are located at the 5-fold axes under the spike and in the hydrophobic pocket on the inner surface of the capsid. Protein B appears to have autoproteolytic activity that cleaves at an Arg-Phe motif and probably facilitates the removal of the protein through the 30 Å wide pores. The structure of the α3 mature virion was solved to 3.5 Å resolution by X-ray crystallography and was used to interpret the open procapsid cryo-EM structure. The main differences between the α3 and φX174 virion structures are in the spike and the DNA-binding proteins. The α3 pentameric spikes have a rotation of 3.5° compared to those of φX174. The α3 DNA-binding protein, which is shorter by 13 amino acid residues at its amino end when compared to the φX174 J protein, retains its carboxy-terminal-binding site on the internal surface of the capsid protein. The icosahedrally ordered structural component of the ssDNA appears to be substantially increased in α3 compared to φX174, allowing the building of about 10% of the ribose-phosphate backbone. © 2002 Elsevier Science Ltd. All rights reserved.

PMID: 14976421;Abstract:

Chlamydiae are obligate intracellular pathogens that have a unique developmental cycle. Thirty nine viable isolates representing all nine currently recognised chlamydial species were screened by immunofluorescence with a cross-reacting chlamydiaphage monoclonal antibody. A novel chlamydiaphage (Chp3) was detected in C. pecorum, a chlamydial species not previously known to carry bacteriophages. Chp3 belongs to the Microviridae, members of this virus family are characterised by circular, single-stranded DNA genomes and small T = 1 icosahedral capsids. Double-stranded replicative form Chp3 DNA was purified from elementary bodies and used as a template to determine the complete genome sequence. The genome of Chp3 is 4,554 base pairs and encodes eight open reading frames organised in the same genome structure as other chlamydiaphages. An unrooted phylogenetic tree was constructed based on the major coat proteins of 11 members of the Microviridae and Chp3. This showed that the Microviridae are clearly divided into two discrete sub-families; those that infect the Enterobacteriaceae e.g. ØX174 and the bacteriophages that infect obligate intracellular bacteria or mollicutes including SpV4 (Spiroplasma melliferum), ØMH2K (Bdellovibrio bacteriovorus) and the chlamydiaphages. Comparative analyses demonstrate that the chlamydiaphages can be further subdivided into two groupings, one represented by Chp2/Chp3 and the other by ØCPG1/ØCPAR39.

Defective øX174 H protein-mediated DNA piloting indirectly influences the entire viral lifecycle. Faulty piloting can mask the H protein's other functions or inefficient penetration may be used to explain defects in post-piloting phenomena. For example, optimal synthesis of other viral proteins requires de novo H protein biosynthesis. As low protein concentrations affect morphogenesis, protein H's assembly functions remain obscure. An H protein mutant was isolated that allowed morphogenetic effects to be characterized independent of its other functions. The mutant protein aggregates assembly intermediates. Although excess internal scaffolding protein restores capsid assembly, the resulting mutant H protein-containing particles are less infectious. In addition, nonviable phenotypes of am(H) mutants in Su+ hosts, which insert non-wild-type amino acids, do not always correlate with a lack of missense protein function. Phenotypes are highly influenced by host and phage physiology. This phenomenon was unique to am(H) mutants, not observed with amber mutants in other genes.