Intracellular parasitism of chlamydiae: specific infectivity of chlamydiaphage Chp2 in Chlamydophila abortus.

The obligate intracellular nature of chlamydiae presents challenges to the characterization of its phages, which are potential tools for a genetic transfer system. An assay for phage infectivity is described, and the infectious properties of phage Chp2 were determined.

Host range of chlamydiaphages phiCPAR39 and Chp3.

The host range of phiCPAR39 is limited to four Chlamydophila species: C. abortus, C. caviae, C. pecorum, and C. pneumoniae. Chp3 (a newly discovered bacteriophage isolated from C. pecorum) shares three of these hosts (C. abortus, C. caviae, and C. pecorum) but can additionally infect Chlamydophila felis. The ability to support replication was directly correlated with the binding properties of the respective bacteriophages with their host species. Binding studies also show that phiCPAR39 and Chp3 use different host receptors to infect the same host cells: cell binding is sensitive to proteinase K treatment, confirming that the chlamydiaphage receptors are proteinaceous in nature.

Spatial abilities following prenatal androgen abnormality: targeting and mental rotations performance in individuals with congenital adrenal hyperplasia.

In most mammals, behaviors that show sex differences are influenced by androgen during early life. In the current study, the hypothesis that androgen influences the development of human spatial abilities was investigated. Participants included 40 females and 29 males with congenital adrenal hyperplasia (CAH), a genetic disorder that causes overproduction of adrenal androgens beginning prenatally, and 29 unaffected female and 30 unaffected male relatives of individuals with CAH. Participants ranged in age from 12-45 years. Measures of spatial abilities included two mental rotations tasks and two targeting tasks, all of which showed large sex differences favoring males in the unaffected relative controls. Females with CAH (exposed to higher than normal levels of androgen prenatally) performed better than unaffected females on the targeting tasks, and resembled unaffected males and males with CAH in this respect. However, females with CAH did not perform better than unaffected females on the measures of mental rotations abilities. Males with CAH showed unaltered performance on the targeting tasks, and impaired performance on the mental rotations tasks. Results are discussed in terms of differences in experiential and hormonal contributions to different spatial abilities, as well as in terms of possible differences in critical periods for hormonal influences on targeting versus mental rotations abilities. Specifically, we speculate that, although androgen may influence targeting abilities prenatally, if hormones influence the development of mental rotations ability, they do so at some other time, perhaps during the first six months of postnatal life.

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.

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.

The role of scaffolding proteins in the assembly of the small, single-stranded DNA virus phiX174.

An empty precursor particle called the procapsid is formed during assembly of the single-stranded DNA bacteriophage phiX174. Assembly of the phiX174 procapsid requires the presence of the two scaffolding proteins, D and B, which are structural components of the procapsid, but are not found in the mature virion. The X-ray crystallographic structure of a "closed" procapsid particle has been determined to 3.5 A resolution. This structure has an external scaffold made from 240 copies of protein D, 60 copies of the internally located B protein, and contains 60 copies of each of the viral structural proteins F and G, which comprise the shell and the 5-fold spikes, respectively. The F capsid protein has a similar conformation to that seen in the mature virion, and differs from the previously determined 25 A resolution electron microscopic reconstruction of the "open" procapsid, in which the F protein has a different conformation. The D scaffolding protein has a predominantly alpha-helical fold and displays remarkable conformational variability. We report here an improved and refined structure of the closed procapsid and describe in some detail the differences between the four independent D scaffolding proteins per icosahedral asymmetric unit, as well as their interaction with the F capsid protein. We re-analyze and correct the comparison of the closed procapsid with the previously determined cryo-electron microscopic image reconstruction of the open procapsid and discuss the major structural rearrangements that must occur during assembly. A model is proposed in which the D proteins direct the assembly process by sequential binding and conformational switching.

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.

Structure of a viral procapsid with molecular scaffolding.

The assembly of a macromolecular structure proceeds along an ordered morphogenetic pathway, and is accomplished by the switching of proteins between discrete conformations as they are added to the nascent assembly. Scaffolding proteins often play a catalytic role in the assembly process, rather like molecular chaperones. Although macromolecular assembly processes are fundamental to all biological systems, they have been characterized most thoroughly in viral systems, such as the icosahedral Escherichia coli bacteriophage phiX174. The phiX174 virion contains the proteins F, G, H and J. During assembly, two scaffoldingproteins B and D are required for the formation of a 108S, 360-A-diameter procapsid from pentameric precursors containing the F, G and H proteins. The procapsid contains 240 copies of protein D, forming an external scaffold, and 60 copies each of the internal scaffolding protein B, the capsid protein F, and the spike protein G. Maturation involves packaging of DNA and J proteins and loss of protein B, producing a 132S intermediate. Subsequent removal of the external scaffold yields the mature virion. Both the F and G proteins have the eight-stranded antiparallel beta-sandwich motif common to many plant and animal viruses. Here we describe the structure of a procapsid-like particle at 3.5-A resolution, showing how the scaffolding proteins coordinate assembly of the virus by interactions with the F and G proteins, and showing that the F protein undergoes conformational changes during capsid maturation.

Genetic analysis of the phi X174 DNA binding protein.

The phi X174 J protein is 37 amino acids in length and contains 12 basic residues. There are no acidic amino acids in the protein. The basic residues are concentrated in two clusters in the N-terminus which are separated by a proline-rich region. To investigate the morphogenetic functions of the J protein and possible mechanisms by which it may bind DNA, a genetic analysis was conducted. Lysine --> leucine and arginine --> leucine substitutions were generated within the basic amino acid clusters. At least three substitutions were required to eliminate viability in vivo. Lethal mutants with three or four substitutions exhibit dominant lethal phenotypes, indicating that the mutant proteins retain enough function to interfere with productive assembly. In cells infected with a dominant lethal mutant, noninfectious packaged particles were produced. Infectivity can be restored by second-site suppressors in the viral coat protein which disrupt polar interactions atop the threefold axis of symmetry in the capsid. The viability of strains containing compensating frameshift mutations within the proline-rich region suggests that only the proline residues in this segment are critical for efficient function.

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.

Host and phi X 174 mutations affecting the morphogenesis or stabilization of the 50S complex, a single-stranded DNA synthesizing intermediate.

The morphogenetic pathway of bacteriophage phi X 174 was investigated in rep mutant hosts that specifically block stage III single-stranded DNA synthesis. The defects conferred by the mutant rep protein most likely affect the formation or stabilization of the 50S complex, a single-stranded DNA synthesizing intermediate, which consists of a viral prohead and a DNA replicating intermediate (preinitiation complex). phi X 174 mutants, ogr (rep), which restore the ability to propagate in the mutant rep hosts, were isolated. The org (rep) mutations confer amino acid substitutions in the viral coat protein, a constituent of the prohead, and the viral A protein, a constituent of the preinitiation complex. Four of the six coat protein substitutions are localized on or near the twofold axis of symmetry in the atomic structure of the mature virion.

Characterization of the morphogenetic defects conferred by cold-sensitive prohead accessory and scaffolding proteins of phi X174.

The morphogenetic defects conferred by the cold-sensitive prohead accessory and scaffolding proteins of phi X174 were determined in vivo. The results suggest that the cold-sensitive prohead accessory protein blocks the formation of the 12S assembly intermediate. The cold-sensitive scaffolding protein most likely affects the stability of the prohead.

Second-site suppressors of a cold-sensitive external scaffolding protein of bacteriophage phi X174.

This report describes the isolation and characterization of second-site suppressors of a cold-sensitive (cs) external scaffolding protein, gpD, of bacteriophage phi X174. Seven genetically distinct suppressors were isolated. Six of them are located in gene F which encodes the major coat protein of the virus. The seventh is located in gene J which encodes the DNA-binding protein. A subset of the suppressors are trans-acting. These second-site suppressors do not exhibit allele specificity; they are able to suppress defects associated with a csD protein for which they were not selected. The initial characterization of the second-site suppressors and their locations within the major coat protein suggest that the mechanism of suppression may involve both structural and stoichiometric phenomena.

Proteolysis of bacteriophage phi X174 prohead accessory protein gpB by Escherichia coli OmpT protease is not essential for phage maturation in vivo.

To examine whether cleavage of the phi X174 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 delta ompT strain were constructed. The results of burst size experiments suggest that neither the cleavage site nor the OmpT protein is required for phi X174 development.

Functional relationship between the J proteins of bacteriophages phi X174 and G4 during phage morphogenesis.

The functions of the small DNA-binding protein, gpJ, of bacteriophages phi X174 and G4 were examined by in vivo cross-complementation and sucrose gradient sedimentation. The morphogenetic roles of the two proteins may differ. The phi X174 J protein may be required for the formation or stabilization of the phi X174 prohead.

Second-site suppressors of a cold-sensitive prohead accessory protein of bacteriophage phi X174.

This study describes the isolation of second-site suppressors which correct for the defects associated with cold-sensitive (cs) prohead accessory proteins of bacteriophage phi X174. Five phenotypically different suppressors were isolated. Three of these suppressors confer novel temperature-sensitive (ts) phenotypes. They were unable to complement a ts mutation in gene F which encodes the major coat protein of the phage. All five suppressor mutations confer nucleotide changes in the gene F DNA sequence. These changes define four amino acid sites in the gene F protein. Three suppressor mutations placed into an otherwise wild-type background display a cold resistant phenotype in liquid culture infections when compared to a wild-type phi X174 control.