Membrane-containing viruses with icosahedrally symmetric capsids.

Viruses with an icosahedrally symmetric protein capsid and a membrane infect hosts from all three domains of life. Similar architectural principles are shared by different viral families, as exemplified by double-stranded DNA viruses such as PRD1 and STIV. During virus assembly, the membrane lipids are selectively acquired from the host cell. The X-ray structure of bacteriophage PRD1 revealed that the lipids are asymmetrically distributed between the two leaflets and facet length is controlled by a tape-measure protein. In most membrane-containing viruses, viral and host membranes fuse during viral entry. In the best-understood systems of the alphaviruses, flaviviruses and herpes viruses, fusion is mediated by viral glycoproteins. Recent structural advances reveal how very different protein architectures can be used to form trimeric extensions that extend into the target cell membrane and then fold back to mediate fusion of the target and viral membranes.

Reversible pressure dissociation of R17 bacteriophage. The physical individuality of virus particles.

In the absence of urea, pressures up to 2.5 kbar promote only 10% dissociation of the whole particles of R17 bacteriophage. In the presence of concentrations of urea between 1.0 and 5.0 M, pressure promotes complete, reversible dissociation of the virus particles. At the lower urea concentrations reversible dissociation of R17 virus particles shows no dependence on protein concentration indicating a high degree of heterogeneity of the particles, but higher urea concentrations, 2.5 to 5.0 M, result in progressive restoration of the protein concentration dependence of the pressure dissociation. At still higher urea concentrations, 5.0 to 8.0 M, irreversible dissociation of virus takes place at atmospheric pressure. In contrast, the dissociation of the isolated dimers of the capsid protein was dependent on protein concentration to the extent predicted for a stochastic equilibrium, and dimers were much less stable than the whole virus both to dissociation by pressure or urea. In contradistinction, the reversible whole-virus dissociation observed at urea concentrations below 2.5 M appears to be a typical deterministic equilibrium, without appreciable dynamic exchange between whole particle and subunits during the lengthy experiments. The experiments demonstrate that the "thermodynamic individuality" of the virus particles arises in conformational differences in the assembled viruses, and that there is a direct relation between the stability of the particles and their heterogeneity.

Conjugation system of IncC plasmid RA1, and the interaction of RA1 pili with specific RNA phage C-1.

RA1 was the only IncC plasmid that was slightly temperature-sensitive for replication and transfer. At 30 degrees C, RA1 determined constitutive synthesis of conjugative pili and yet was transfer-repressed. Attachment of shaft-adsorbing RNA phage C-1 virions prevented the probable retraction of pili under heat stimulus (55 degrees C). Electron microscopy showed single adsorbed virions at pilus bases where they were thought to have stopped retraction.

[Genetically engineered mutants of the envelope protein of the RNA-containing bacteriophage]. / Gennoinzhenernye mutanty belka obolochki RNK-soderzhashchego bakteriofaga.

Expression of the coat protein gene of RNA bacteriophage fr in Escherichia coli cells leads to the formation of capsid-like structures of ca. 25 nm in diameter, which are immunologically indistinguishable from the native phage fr capsids. The modification strategy of the coat protein gene by gene engineering technique was developed in order to localize coat protein regions, which are exposed on the capsid surface and are capable to include foreign amino acid inserts without an appreciable effect on the capsid self-assembly. The oligonucleotide linkers, coding short amino acid sequences and bearing also convenient restriction sites, were synthesized and inserted into different regions of the coat protein gene. The mutant proteins, containing insertions of 2-12 amino acids in potentially exposed regions, were obtained. It was shown that N- and C-terminal insertions, as well as the insertion into codon 51 in the RNA-binding region, do not prevent the self-assembly. The regions (codons 96 and 112) were also revealed, insertions in them decreased drastically the protein yield as a consequence of a block in the self-assembly.

The capsid of the small RNA phage PRR1 is stabilized by metal ions.

Many nonenveloped virus particles are stabilized by calcium ions bound in the interfaces between the protein subunits. These ions may have a role in the disassembly process. The small RNA phages of the Leviviridae family have T=3 quasi-symmetry and are unique among simple viruses in that they have a coat protein with a translational repressor activity and a fold that has not been observed in other viruses. The crystal structure of phage PRR1 has been determined to 3.5 A resolution. The structure shows a tentative binding site for a calcium ion close to the quasi-3-fold axis. The RNA-binding surface used for repressor activity is mostly conserved. The structure does not show any significant differences between quasi-equivalent subunits, which suggests that the assembly is not controlled by conformational switches as in many other simple viruses.

The three-dimensional structure of genomic RNA in bacteriophage MS2: implications for assembly.

Using cryo-electron microscopy, single particle image processing and three-dimensional reconstruction with icosahedral averaging, we have determined the three-dimensional solution structure of bacteriophage MS2 capsids reassembled from recombinant protein in the presence of short oligonucleotides. We have also significantly extended the resolution of the previously reported structure of the wild-type MS2 virion. The structures of recombinant MS2 capsids reveal clear density for bound RNA beneath the coat protein binding sites on the inner surface of the T=3 MS2 capsid, and show that a short extension of the minimal assembly initiation sequence that promotes an increase in the efficiency of assembly, interacts with the protein capsid forming a network of bound RNA. The structure of the wild-type MS2 virion at approximately 9 A resolution reveals icosahedrally ordered density encompassing approximately 90% of the single-stranded RNA genome. The genome in the wild-type virion is arranged as two concentric shells of density, connected along the 5-fold symmetry axes of the particle. This novel RNA fold provides new constraints for models of viral assembly.

Bacteriophages F0lac h, SR, SF: phages which adsorb to pili encoded by plasmids of the S-complex.

Phage F0lac is an RNA-containing phage which plates only on strains carrying the plasmid EDP208, a pilus derepressed derivative of the unique incompatibility plasmid F0lac. A host range mutant, phage F0lac h, was selected which plated on strains carrying the ungrouped plasmid pPLS::Tn5 and lysed strains carrying another ungrouped plasmid TP224::Tn10 or the Com9 plasmid R71. An RNA-containing phage, SR, was isolated from sewage on bacteria harbouring plasmid pPLS::Tn5. It was antigenically distinct from the above two phages but had the same host range as phage F0lac h. Phages F0lac h and SR adsorbed unevenly to the shafts of the conjugative pili. Another phage, SF, was filamentous and plated or propagated on strains carrying any of the above plasmids as well as on strains harbouring IncD or F-complex plasmids. Plasmids TP224::Tn10 and pPLS::Tn5 were compatible with representative plasmids of all Inc groups also encoding thick flexible pili. The four plasmids EDP208, R71, TP224::Tn10 and pPLS::Tn5 were compatible with one another except for the reaction of TP224::Tn10 in the presence of pPLS::Tn5 which was slightly ambiguous. The host ranges of the bacteriophages, together with the serological relatedness of the thick flexible pili determined by these four compatible plasmids, suggested that they constitute a new complex, here designated S.

Bacteriophage M: an incompatibility group M plasmid-specific phage.

Phage M was specific for bacterial strains, of various genera, harbouring plasmids of the M incompatibility group. It formed turbid plaques which varied from pin point to more than 2 mm in diameter on all hosts where plaques were detected. The phage had an hexagonal outline with a diameter of 27 nm. It contained RNA but differed from other plasmid-dependent RNA phages in being sensitive to chloroform. It adsorbed along the length of shafts of M pili.

Phage t: a group T plasmid-dependent bacteriophage.

Phage t was isolated from sewage from Pretoria. It formed plaques only on Escherichia coli and Salmonella typhimurium strains that carried plasmids belonging to incompatibility group T. Five of six group T plasmids permitted visible lysis of R+ host strains. There was no visible lysis of E. coli J53-2 or S. typhimurium LT2trpA8 carrying the T plasmid Rts1 although the strains supported phage growth as indicated by at least a 10-fold increase in phage titre. The latter strains transferred the plasmid at high frequency to E. coli strain CSH2 and the resulting transconjugants plated the phage. Proteus mirabilis strain PM5006(R402) failed to support phage growth although it transferred the plasmid and concomitant phage sensitivity to E. coli J53-2. The phage was hexagonal in outline, RNA-containing, resistant to chloroform and adsorbed to the shafts of pili determined by T plasmids.

Phage Folac: an Folac plasmid-dependent bacteriophage.

By enriching sewage with Escherichia coli or Salmonella typhimurium strains harbouring the plasmid EDP208, a constitutive pilus-producing derivative of plasmid F olac, a phage was isolated which plated on these two organisms but not on isogenic strains without the plasmid. The phage was named F olac; it has a hexagonal outline with a diameter of 28 nm, contained RNA, was resistant to chloroform, and probably adsorbed preferentially to the sides of EDP208 pili very near the tip. Phage multiplication could be demonstrated on E. coli or S. typhimurium strains carrying the plasmid F olac, but an increase in titre did not occur on E. coli strains carrying plasmids of the F complex. Results of phage multiplication experiments on strains carrying the depressed pilus-producing plasmids R71 or TP224-Tc, which determine pili serologically related to those of EDP208, were inconclusive. Phage F olac was found to be serologically related to phage UA-6, another isolate specific for EDP208.

Altered 3'-terminal RNA structure in phage Qbeta adapted to host factor-less Escherichia coli.

The RNA phage Qbeta requires for the replication of its genome an RNA binding protein called Qbeta host factor or Hfq protein. Our previous results suggested that this protein mediates the access of replicase to the 3'-end of the Qbeta plus strand RNA. Here we report the results of an evolutionary experiment in which phage Qbeta was adapted to an Escherichia coli Q13 host strain with an inactivated host factor (hfq) gene. This strain initially produced phage at a titer approximately 10,000-fold lower than the wild-type strain and with minute plaque morphology, but after 12 growth cycles, phage titer and plaque size had evolved to levels near those of the wild-type host. RNAs isolated from adapted Qbeta mutants were efficient templates for replicase without host factor in vitro. Electron microscopy showed that mutant RNAs, in contrast to wild-type RNA, efficiently interacted with replicase at the 3'-end in the absence of host factor. The same set of four mutations in the 3'-terminal third of the genome was found in several independently evolved phage clones. One mutation disrupts the base pairing of the 3'-terminal CCCOH sequence, suggesting that the host factor stimulates activity of the wild-type RNA template by melting out its 3'-end.

Analysis of RNA phage fr coat protein assembly by insertion, deletion and substitution mutagenesis.

A structure-function analysis of the icosahedral RNA bacteriophage fr coat protein (CP) assembly was undertaken using linker-insertion, deletion and substitution mutagenesis. Mutations were specifically introduced into either pre-existing or artificially created restriction enzyme sites within fr CP gene expressed in Escherichia coli from a recombinant plasmid. This directs synthesis of wild type protein that undergoes self-assembly and forms capsid-like particles indistinguishable morphologically and immunologically from native phage particles. A series of fr CP variants containing sequence alterations in the regions which are (i) exposed on the external surface of capsid or (ii) located on the contacting areas between CP subunits were obtained and their assembly properties investigated. The majority of mutants demonstrated reduction of assembly ability and formed either CP dimers (mutations at residues 2, 10, 63 or 129) or both dimer and capsid structures (residue 2 or 69). The exceptions were variants demonstrating normal assembly and containing insertions at residues 2, 50 or 129 of the fr CP. A third type of assembled structure was formed by a variant with a single amino acid substitution I104T. The alpha A-helix region (residues 97-111) is particularly sensitive to mutation and any alteration in this region decreases accumulation of mutant protein in E. coli. The relative contributions of particular fr CP domains in maintenance of capsid structural integrity as well as the possible capsid assembly mechanism are discussed.