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At least 19 recordsLinked to original sources

PubMed1999

Cross-functional analysis of the Microviridae internal scaffolding protein.

The assembly of the viral structural proteins into infectious virions is often mediated by scaffolding proteins. These proteins are transiently associated with morphogenetic intermediates but not found in the mature particle. The genes encoding three Microviridae (phiX174, G4 and alpha3) internal scaffolding proteins (B proteins) have been cloned, expressed in vivo and assayed for the ability to complement null mutations of different Microviridae species. Despite divergence as great as 70% in amino acid sequence over the aligned length, cross-complementation was observed, indicating that these proteins are capable of directing the assembly of foreign structural proteins into infectious particles. These results suggest that the Microviridae internal scaffolding proteins may be inherently flexible. There was one condition in which a B protein could not cross-function. The phiX174 B protein cannot productively direct the assembly of the G4 capsid at temperatures above 21 degreesC. Under these conditions, assembly is arrested early in the morphogenetic pathway, before the first B protein mediated reaction. Two G4 mutants, which can productively utilize the phiX174 B protein at elevated temperatures, were isolated. Both mutations confer amino acid substitutions in the viral coat protein but differ in their relative abilities to utilize the foreign scaffolding protein. The more efficient substitution is located in a region where coat-scaffolding interactions have been observed in the atomic structure and may emphasize the importance of interactions in this region.

Amino Acid Sequence↗

PubMed2000

Foreign and chimeric external scaffolding proteins as inhibitors of Microviridae morphogenesis.

Viral assembly is an ideal system in which to investigate the transient recognition and interplay between proteins. During morphogenesis, scaffolding proteins temporarily associate with structural proteins, stimulating conformational changes that promote assembly and inhibit off-pathway reactions. Microviridae morphogenesis is dependent on two scaffolding proteins, an internal and an external species. The external scaffolding protein is the most conserved protein within the Microviridae, whose canonical members are phiX174, G4, and alpha3. However, despite 70% homology on the amino acid level, overexpression of a foreign Microviridae external scaffolding protein is a potent cross-species inhibitor of morphogenesis. Mutants that are resistant to the expression of a foreign scaffolding protein cannot be obtained via one mutational step. To define the requirements for and constraints on scaffolding protein interactions, chimeric external scaffolding proteins have been constructed and analyzed for effects on in vivo assembly. The results of these experiments suggest that at least two cross-species inhibitory domains exist within these proteins; one domain most likely blocks procapsid formation, and the other allows procapsid assembly but blocks DNA packaging. A mutation conferring resistance to the expression of a chimeric protein (chiD(r)) that inhibits DNA packaging was isolated. The mutation maps to gene A, which encodes a protein essential for packaging. The chiD(r) mutation confers resistance only to a chimeric D protein; the mutant is still inhibited by the expression of foreign D proteins. The results presented here demonstrate how closely related proteins could be developed into antiviral agents that specifically target virion morphogenesis.

Amino Acid Sequence↗

PubMed2000

Efficient complementation by chimeric Microviridae internal scaffolding proteins is a function of the COOH-terminus of the encoded protein.

Microviridae morphogenesis is dependent on two scaffolding proteins, an internal and external species. Both structural and genetic analyses suggest that the COOH-terminus of the internal protein is critical for coat protein recognition and specificity. To test this hypothesis, chimeric internal scaffolding genes between Microviridae members phiX174, G4, and alpha3 were constructed and the proteins expressed in vivo. All of the chimeric proteins were functional in complementation assays. However, the efficient complementation was observed only when the viral coat protein and COOH-terminus of internal scaffolding were of the same origin. Genes with 5' deletions of the phiX174 internal scaffolding gene were also constructed and expressed in vivo. Proteins lacking the first 10 amino acids, which self-associate across the twofold axes of symmetry in the atomic structure, efficiently complement phiX174 am(B) mutants at temperatures above 24 degrees C. These results suggest that internal scaffolding protein self-associations across the twofold axes of symmetry are required only at lower temperatures.

Genes, Viral↗

PubMed1998

Structural analysis of the Spiroplasma virus, SpV4: implications for evolutionary variation to obtain host diversity among the Microviridae.

BACKGROUND: Spiroplasma virus, SpV4, is a small, non-enveloped virus that infects the helical mollicute Spiroplasma melliferum. SpV4 exhibits several similarities to the Chlamydia phage, Chp1, and the Coliphages alpha 3, phi K, G4 and phi X174. All of these viruses are members of the Microviridae. These viruses have isometric capsids with T = 1 icosahedral symmetry, cause lytic infections and are the only icosahedral phages that contain single-stranded circular DNA genomes. The aim of this comparative study on these phages was to understand the role of their capsid proteins during host receptor recognition. RESULTS: The three-dimensional structure of SpV4 was determined to 27 A resolution from images of frozen-hydrated particles. Cryo-electron microscopy (cryo-EM) revealed 20, approximately 54 A long, 'mushroom-like' protrusions on the surface of the capsid. Each protrusion comprises a trimeric structure that extends radially along the threefold icosahedral axes of the capsid. A 71 amino acid portion of VP1 (the SpV4 capsid protein) was shown, by structural alignment with the atomic structure of the F capsid protein of phi X174, to represent an insertion sequence between the E and F strands of the eight-stranded antiparallel beta-barrel. Secondary structure prediction of this insertion sequence provided the basis for a probable structural motif, consisting of a six-stranded antiparallel beta sheet connected by small turns. Three such motifs form the rigid stable trimeric structures (mushroom-like protrusions) at the threefold axes, with hydrophobic depressions at their distal surface. CONCLUSIONS: Sequence alignment and structural analysis indicate that distinct genera of the Microviridae might have evolved from a common primordial ancestor, with capsid surface variations, such as the SpV4 protrusions, resulting from gene fusion events that have enabled diverse host ranges. The hydrophobic nature of the cavity at the distal surface of the SpV4 protrusions suggests that this region may function as the receptor-recognition site during host infection.

Amino Acid Sequence↗

PubMed2002

Microviridae, a family divided: isolation, characterization, and genome sequence of phiMH2K, a bacteriophage of the obligate intracellular parasitic bacterium Bdellovibrio bacteriovorus.

A novel single-stranded DNA phage, phiMH2K, of Bdellovibrio bacteriovorus was isolated, characterized, and sequenced. This phage is a member of the Microviridae, a family typified by bacteriophage phiX174. Although B. bacteriovorus and Escherichia coli are both classified as proteobacteria, phiMH2K is only distantly related to phiX174. Instead, phiMH2K exhibits an extremely close relationship to the Microviridae of Chlamydia in both genome organization and encoded proteins. Unlike the double-stranded DNA bacteriophages, for which a wide spectrum of diversity has been observed, the single-stranded icosahedral bacteriophages appear to fall into two distinct subfamilies. These observations suggest that the mechanisms driving single-stranded DNA bacteriophage evolution are inherently different from those driving the evolution of the double-stranded bacteriophages.

Amino Acid Sequence↗

PubMed2003

Genetic analyses of putative conformation switching and cross-species inhibitory domains in Microviridae external scaffolding proteins.

Putative conformational switching and inhibitory regions in the Microviridae external scaffolding protein were investigated. Substitutions for glycine 61, hypothesized to promote a postdimerization conformational switch, have dominant lethal phenotypes. In previous studies, chimeric alpha3/phiX174 proteins for structures alpha-helix 1 and loop 6/alpha-helix 7 inhibited phiX174 morphogenesis when expressed from high copy number plasmids. To determine if inhibition was due to overexpression, chimeric genes were constructed into the phiX174 genome. In coinfections with wild-type, protein ratios would be 1:1. The helix 1 chimera has a recessive lethal phenotype; thus, overexpression confers inhibition. In single infections, the mutant cannot form procapsids, suggesting that helix 1 mediates the initial recognition of structural proteins. The lethal chimeric helix 7 protein has a dominant phenotype. Alone, the mutant forms defective procapsids, suggesting a later morphogenetic defect. The results of second-site genetic analyses indicate that the capsid-external scaffolding protein interface is larger than revealed in the crystal structure.

Amino Acid Sequence↗

PubMed1989

Possible finger structure in gene A protein of Microviridae.

Microvirid phages alpha 3 and phi K encode for A protein which functions in initiation of the viral DNA synthesis. By nucleotide sequencing analysis, we have found that each gene A protein has 'finger motif structure' which conserves two cysteine and histidine residues similar to the consensus sequence deduced from more than thirty finger motifs reported from many eukaryotic regulatory proteins. In closely related phages, phi X174 and G4, we have detected the same structures in their gene A proteins also.

Amino Acid Sequence↗

PubMed2004

Isolation, molecular characterisation and genome sequence of a bacteriophage (Chp3) from Chlamydophila pecorum.

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.

Base Sequence↗

PubMed2025

Effects of Different Feeding Patterns on the Gut Virome of 6-Month-Old Infants.

The gut microbiome is essential for infant health, and in recent years, the impact of enteroviruses on infant health and disease has received increasing attention. The transmission of breast milk phages to the infant gastrointestinal tract contributes to the shaping of the infant gut virome, while breastfeeding regulates the colonization of the infant gut virome. In this study, we collected fecal samples from healthy infants and analyzed the distribution characteristics of infant viral communities by viral metagenomic analysis, and analyzed the differences in infant viral communities under different feeding practices. Our results indicate that the infant intestinal virome consists of phages and eukaryotic viruses. Caudovirales and Microviridae dominated the phage composition, and except for Siphoviridae, which was more predominant in the intestines of formula-fed infants, there were no significant differences in the overall abundance of other Caudovirales and Microviridae in the intestines of infants with different feeding patterns. Breastfeeding can lead to a higher diversity of infant gut viruses through vertical transmission, and a highly diverse gut virome helps maintain the maturation of the gut microbiome. This study informs the shaping of gut virome in healthy infants by breastfeeding and contributes to further research on infant gut virome characteristics and formation processes.

Humans↗

PubMed2004

Chlamydiaphage Chp2, a skeleton in the phiX174 closet: scaffolding protein and procapsid identification.

Chlamydiaphage Chp2 is a member of the family Microviridae, of which bacteriophage phiX174 is the type species. Although grouped in the same family, the relationship between the Microviridae coliphages and the Chp2-like viruses, which infect obligate intracellular parasitic bacteria, is quite distant, with major differences in structural protein content and scaffolding protein dependence. To investigate the morphogenesis of Chp2, large particles were isolated from infected Chlamydophila abortus by equilibrium and rate zonal sedimentation. A monoclonal antibody that recognizes only assembled viral coat proteins was used in these detection assays. Thus, the detected particles represent virions and/or postcapsid formation assembly intermediates. Two distinct particle types were detected, differing in both protein and DNA content. Filled particles lacked VP3, the putative internal scaffolding protein, whereas empty particles contained this protein. These results indicate that VP3 is a scaffolding protein and that the isolated VP3-containing particles most likely represent Chp2 procapsids.

Bacteriophage phi X 174↗

PubMed2006

Horizontal gene transfer and the evolution of microvirid coliphage genomes.

Bacteriophage genomic evolution has been largely characterized by rampant, promiscuous horizontal gene transfer involving both homologous and nonhomologous source DNA. This pattern has emerged through study of the tailed double-stranded DNA (dsDNA) phages and is based upon a sparse sampling of the enormous diversity of these phages. The single-stranded DNA phages of the family Microviridae, including phiX174, appear to evolve through qualitatively different mechanisms, possibly as result of their strictly lytic lifestyle and small genome size. However, this apparent difference could reflect merely a dearth of relevant data. We sought to characterize the forces that contributed to the molecular evolution of the Microviridae and to examine the genetic structure of this single family of bacteriophage by sequencing the genomes of microvirid phage isolated on a single bacterial host. Microvirids comprised 3.5% of the detectable phage in our environmental samples, and sequencing yielded 42 new microvirid genomes. Phylogenetic analysis of the genes contained in these and five previously described microvirid phages identified three distinct clades and revealed at least two horizontal transfer events between clades. All members of one clade have a block of five putative genes that are not present in any member of the other two clades. Our data indicate that horizontal transfer does contribute to the evolution of the microvirids but is both quantitatively and qualitatively different from what has been observed for the dsDNA phages.

Coliphages↗

PubMed1996

Atomic structure of the degraded procapsid particle of the bacteriophage G4: induced structural changes in the presence of calcium ions and functional implications.

Bacteriophage G4 and phiX174 are members of the Microviridae family. The degree of similarity of the structural proteins ranges from 66% identity of the F protein to 40% identity of the G protein. The atomic structure of the phiX174 virion had previously been determined by X-ray crystallography. Bacteriophage G4 procapsids, consisting of the structural proteins F, G, D, B, H, and small traces of J but no DNA, were set up for crystallization. However, the resultant crystals were of degraded procapsid particles, which had lost the assembly scaffolding proteins D and B, resulting in particles that resembled empty virions. The structure of the degraded G4 procapsid has been determined to 3.0 angstrom resolution. The particles crystallized in the hexagonal space group P6(3)22 with unit cell dimensions a=b=414.2(5) angstrom and c=263.0(3) angstrom. The diffraction data were collected at the Cornell High Energy Synchrotron Source (CHESS) on film and image plates using oscillation photography. Packing considerations indicated there were two particles per unit cell. A self-rotation function confirmed that the particles were positioned on 32 point group special positions in the unit cell. Initial phases were calculated to 6 angstrom resolution, based on the known phiX174 virion model. Phase information was then extended in steps to 3.0 angstrom resolution by molecular replacement electron density modification and particle envelope generation. The resulting electron density map was readily interpretable in terms of the F and G polypeptides, as occur in the mature capsid of phiX174. In a few regions of the electron density map there were inconsistencies between the density and the published amino acid sequence. Redetermining the amino acid sequence confirmed that the density was correct. The r.m.s. deviation between the Calpha backbone of the mature capsid of phiX174 and the degraded G4 procapsid was 0.36 angstrom for the F protein and 1.38 angstrom for the G protein. This is consistent with the greater conservation of the F protein compared to the G protein sequences among members of the Microviridae family. Functionally important features between phiX174 and G4 had greater conservation. Calcium ions (Ca2+) were shown to bind to G4 at a general site located near the icosahedral 3-fold axis on the F protein capsid, equivalent to sites found previously in phiX174. Binding of Ca2+ also caused the ordering of the conserved region of the DNA binding protein J, which was present in the degraded procapsid particle in the absence of DNA.

Amino Acid Sequence↗

PubMed2003

Structural studies of bacteriophage alpha3 assembly.

Bacteriophage alpha3 is a member of the Microviridae, a family of small, single-stranded, icosahedral phages that include phiX174. 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 108S empty procapsid that requires the external D and internal B scaffolding proteins for its formation. The alpha3 "open" procapsid structural intermediate was determined to 15A resolution by cryo-electron microscopy (cryo-EM). Unlike the phiX174 "closed" procapsid and the infectious virion, the alpha3 open procapsid has 30A wide pores at the 3-fold vertices and 20A 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 30A wide pores. The structure of the alpha3 mature virion was solved to 3.5A resolution by X-ray crystallography and was used to interpret the open procapsid cryo-EM structure. The main differences between the alpha3 and phiX174 virion structures are in the spike and the DNA-binding proteins. The alpha3 pentameric spikes have a rotation of 3.5 degrees compared to those of phiX174. The alpha3 DNA-binding protein, which is shorter by 13 amino acid residues at its amino end when compared to the phiX174 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 alpha3 compared to phiX174, allowing the building of about 10% of the ribose-phosphate backbone.

Amino Acid Sequence↗

PubMed2002

Biological properties and cell tropism of Chp2, a bacteriophage of the obligate intracellular bacterium Chlamydophila abortus.

A number of bacteriophages belonging to the Microviridae have been described infecting chlamydiae. Phylogenetic studies divide the Chlamydiaceae into two distinct genera, Chlamydia and Chlamydophila, containing three and six different species, respectively. In this work we investigated the biological properties and host range of the recently described bacteriophage Chp2 that was originally discovered in Chlamydophila abortus. The obligate intracellular development cycle of chlamydiae has precluded the development of quantitative approaches to assay bacteriophage infectivity. Thus, we prepared hybridomas secreting monoclonal antibodies (monoclonal antibodies 40 and 55) that were specific for Chp2. We demonstrated that Chp2 binds both C. abortus elementary bodies and reticulate bodies in an enzyme-linked immunosorbent assay. Monoclonal antibodies 40 and 55 also detected bacteriophage Chp2 antigens in chlamydia-infected eukaryotic cells. We used these monoclonal antibodies to monitor the ability of Chp2 to infect all nine species of chlamydiae. Chp2 does not infect members of the genus Chlamydia (C. trachomatis, C. suis, or C. muridarum). Chp2 can infect C. abortus, C. felis, and C. pecorum but is unable to infect other members of this genus, including C. caviae and C. pneumoniae, despite the fact that these chlamydial species support the replication of very closely related bacteriophages.

Animals↗

PubMed2025

Metagenomic Analysis of the Tonsil Virome Highlights Its Diagnostic Potential for Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease whose exact pathogenesis remains unclear, despite links to genetics, environmental factors, and microbial dysbiosis. Recent studies have highlighted the role of the microbiome in RA, yet the contribution of the tonsil virome remains unexplored. This study aims to investigate whether changes in the tonsil virome are associated with RA progression and assess its diagnostic potential. Using metagenomic data from 32 RA patients and 30 healthy controls (HCs), we identified 45 782 viral operational taxonomic units (vOTUs), with 14 341 classified as core vOTUs. RA patients exhibited significantly reduced virome richness and diversity, whereas Siphoviridae and Microviridae dominated both groups. Statistical analysis identified 235 RA-associated viral markers, including 13 enriched in RA and 222 in HCs. RA-enriched markers were primarily bacteriophages infecting Streptococcaceae, whereas HCs displayed more diverse viral-host interactions. Random forest models demonstrated strong discriminatory power of viral markers in distinguishing RA patients from HCs, achieving an AUC of 0.960, outperforming bacterial markers. Correlation analyses further linked viral markers to immune cell subsets, suggesting that tonsil virome alterations may influence immune dysregulation in RA. This study reveals significant changes in the tonsil virome of RA patients, highlighting its potential as a diagnostic tool and offering new insights into RA pathogenesis. These findings pave the way for future research into the virome's role in autoimmune diseases and therapeutic development.

Humans↗

PubMed2004

The phiX174 protein J mediates DNA packaging and viral attachment to host cells.

Packaging of viral genomes into their respective capsids requires partial neutralization of the highly negatively charged RNA or DNA. Many viruses, including the Microviridae bacteriophages phiX174, G4, and alpha3, have solved this problem by coding for a highly positively charged nucleic acid-binding protein that is packaged along with the genome. The phiX174 DNA-binding protein, J, is 13 amino acid residues longer than the alpha3 and G4 J proteins by virtue of an additional nucleic acid-binding domain at the amino terminus. Chimeric phiX174 particles containing the smaller DNA-binding protein cannot be generated due to procapsid instability during DNA packaging. However, chimeric alpha3 and G4 phages, containing the phiX174 DNA-binding protein in place of the endogenous J protein, assemble and are infectious, but are less dense than the respective wild-type species. In addition, host cell attachment and native gel migration assays indicate surface variations of these viruses that are controlled by the nature of the J protein. The structure of alpha3 packaged with phiX174 J protein was determined to 3.5A resolution and compared with the previously determined structures of phiX174 and alpha3. The structures of the capsid and spike proteins in the chimeric particle remain unchanged within experimental error when compared to the wild-type alpha3 virion proteins. The amino-terminal region of the phiX174 J protein, which is missing from wild-type alpha3 virions, is mostly disordered in the alpha3 chimera. The differences observed between solution properties of wild-type phiX174, wild-type alpha3, and alpha3 chimera, including their ability to attach to host cells, correlates with the degree of order in the amino-terminal domain of the J protein. When ordered, this domain binds to the interior of the viral capsid and, thus, might control the flexibility of the capsid. In addition, the properties of the phiX174 J protein in the chimera and the results of mutational analyses suggest that an evolutionary correlation may exist between the size of the J protein and the stoichiometry of the DNA pilot protein H, required in the initial stages of infection. Hence, the function of the J protein is to facilitate DNA packaging, as well as to mediate surface properties such as cell attachment and infection.

Bacteriophages↗

PubMed1989

Analysis of the complete nucleotide sequence of Chp1, a phage which infects avian Chlamydia psittaci.

We report the complete nucleotide sequence of bacteriophage Chp1. The genome was found to be 4877 bases long and it potentially codes for 11 proteins. Open reading frames (ORFs) 6 and 7 lie within ORFs 2 and 1 respectively but are in a second reading frame. No significant DNA homology was found when Chp1 was compared to the EMBL database. The N-terminal amino acid sequences of the three structural proteins VP1, VP2 and VP3 were determined and it was found that they were encoded by ORFs 1, 2 and 3 respectively. Amino acid homology studies revealed that VP1 has homology with the major structural protein of bacteriophages phi X174 and S13, and that the protein inferred from ORF 4 shows homology to the A proteins of phi X174, S13 and G4. The genome of Chp1 has an organization similar to that of phi X174 although it is 509 bases smaller. We propose that Chp1 is a member of the Microviridae but that it is sufficiently different to warrant its own subfamily which we have called the Chlamydiavirinae.

Amino Acid Sequence↗

PubMed1989

Further characterization of a bacteriophage recovered from an avian strain of Chlamydia psittaci.

The genome of a 22 nm icosahedral phage which infects some avian Chlamydia psittaci strains recovered from domestic ducks has been characterized as a ss circular DNA molecule of about 4850 bases. The replicative form of this genome was isolated from purified chlamydial organisms. A restriction endonuclease cleavage site map of the genome was constructed from dsDNA synthesized in vitro from ss phage DNA and EcoRI fragments were then cloned into pUC9. The phage genome was detected only by Southern blot hybridization in C. psittaci which was productively infected with phage; no evidence was found for the integration of phage DNA into the chlamydial chromosome. Three viral polypeptides, of approximate Mr values 75K, 30K and 16.5K were identified when phage was analysed by SDS-PAGE. This virus, which we have designated Chp 1, is either an aberrant member of the Microviridae or the first member of a new bacteriophage family.

Animals↗