Protein synthesis following infection of the blue-green alga Plectonema boryanum with the temperate virus SPI and its ts mutants

The structural proteins of the purified phage and proteins synthesized in virus-infected cells were examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and subsequent autoradiography. A total of 24 viral-induced proteins were identified, 12 of which are present in the mature virus. The kinetics of synthesis of the various viral proteins demonstrates the presence of “early” and “late” genes. Specific suppression of host protein synthesis by the infecting phage takes place late in the infection cycle. Analysis of the proteins synthesized at the nonpermissive temperature by phage temperature-sensitive (ts) mutants demonstrates that some of the viral genes have complex pleiotropic effects on viral protein synthesis. The results suggest the presence of positive regulatory genes, and of specific genes involved in the shutoff of host protein synthesis.     

Interactions between modified phage T4 particles and spheroplasts

Two types of contracted phage particles with full heads were compared. Artificially contracted T4 phage particles were produced by treatment with urea (UTφ), and naturally contracted T4 phage particles lacking gene 12 product were recovered after temporary adsorption to bacteria (ads12^?φ). Both types can infect T4-resistant bacteria if the bacterial envelope is altered by penicillin or lysozyme-EDTA treatment. Both are inactivated by cell membrane preparations and by low concentrations of phosphatidylglycerol which cause the particles to release DNA. Both are sensitive to trypsin. Differences in the particles as to the type of spheroplasts they can infect and the degree of their sensitivity to phosphatidylglycerol and DNase are correlated with mode of contraction rather than with phenotype. The properties of these contracted phage are described in terms of the properties expected for particles at intermediate stages of adsorption. Several mutants in other baseplate genes were tested, and it was found that genes 9, 11, and 12 are unnecessary for infection of spheroplasts. We conclude that baseplates, as well as long tail fibers and their corresponding receptors in the bacterial envelope, do not play a part in the infection process of these contracted particles. This suggests to us that these organelles are necessary only during the initial stages of infection.     

DNA replication in bacteriophage φ29: The requirement of a viral-specific product for association of φ29 DNA with the cell membrane of Bacillus amyloliquefaciens

A rapidly sedimenting complex has been isolated from sheared lysates of φ29-infected Bacillus amyloliquefaciens by sedimentation on linear density gradients of Renografin (0–38%) containing an underlayer of 76% Renografin. The complex, previously identified as membrane-bound bacterial DNA, contains parentally labeled viral DNA and is enriched for pulse-labeled viral DNA. DNA synthesized early in infection is a precursor to DNA found free in the cytoplasm and in mature phage particles. The association of parental phage DNA with the complex is detectable near the onset of viral DNA replication. The maximum amount of parental φ29 DNA associating with the cell membrane occurs by the middle of the DNA synthesis period. This association can be inhibited by actinomycin D or rifamycin if added 5 min but not if added 10 min after infection. Chloramphenicol or puromycin added 10 min before virus, reduces the amount of association of parental DNA with the complex fourfold. A mutant of φ29 is unable to replicate its DNA at 43 C and cannot associate with the complex at this temperature unless co-infected with wild-type phage. Since formation of the complex only occurs when viral DNA is being synthesized, the complex may be a necessary intermediate in the replication of φ29 DNA. The viral mutation TS35 appears to affect a protein whose function is to associate infecting viral DNA with the host cell membrane.     

Morphogenesis of filamentous bacteriophage f1: Orientation of extrusion and production of polyphage

Crosslinking reagents were used to interrupt the process of filamentous phage morphogenesis and investigate the orientation in which nascent virions are extruded through the host cell membrane. Infected bacteria with emerging phage particles were crosslinked with glutaraldehyde. Immunoferritin-labeling studies on these emerging phage using anti-A protein IgG suggested that extrusion begins with the C protein end. To confirm this, phage extruding from infected bacteria were frozen using the reversible crosslinker dimethyl 3,3′-dithiobis-propionimidate and fragments of emerging phage were isolated by shearing. Protein analysis of these fragments showed them to be enriched in C protein relative to A protein, as predicted if phage extrusion begins with the C protein end. The production of multiple-length phage particles (polyphage) by nonpermissive bacterial hosts infected with amber mutant phage strains was also studied. Polyphage were produced upon infection with amber mutants in genes III, VI, VII, and IX which code for proteins found at the ends of the mature phage particle. No polyphage were produced by mutants in the other genes tested. Gene III amber mutants produce noninfective polyphage, but those produced by genes VII and IX are infective. Gene VI amber mutants appear to produce unstable, noninfective polyphage particles.     

The host outer membrane proteins OmpA and OmpC are associated with the Shigella phage Sf6 virion

Assembly of dsDNA bacteriophage is a precisely programmed process. Potential roles of host cell components in phage assembly haven't been well understood. It was previously reported that two unidentified proteins were present in bacteriophage Sf6 virion (Casjens et al, 2004, J.Mol.Biol. 339, 379-394, Fig. 2A). Using tandem mass spectrometry, we have identified the two proteins as outer membrane proteins (OMPs) OmpA and OmpC from its host Shigella flexneri. The transmission electron cryo-microscopy structure of Sf6 shows significant density at specific sites at the phage capsid inner surface. This density fit well with the characteristic beta-barrel domains of OMPs, thus may be due to the two host proteins. Locations of this density suggest a role in Sf6 morphogenesis reminiscent of phage-encoded cementing proteins. These data indicate a new, OMP-related phage:host linkage, adding to previous knowledge that some lambdoid bacteriophage genomes contain OmpC-like genes that express phage-encoded porins in the lysogenic state.     

Temperature-dependent compositional changes in the envelope of phi 6.

Bacteriophage φ6 contains lipid in an envelope that covers an icosahedral core. The envelope has six proteins, of which two constitute the attachment apparatus. The latter are designated P3, the host attachment protein, and P6, its anchor in the phage envelope. The number of molecules of P3 and P6 seem to be equivalent in the particle, but the relative amounts of the two proteins compared to other phage proteins vary with temperature. In phage grown above 24° there is about one-fourth as much P3 and P6 in the particles compared to phage grown below 24°. A result of this compositional change is a reduction in the rate of sedimentation of the virus formed at high temperature. A nonsense mutant defective in the synthesis of protein P6, which makes particles that contain lipid and other membrane proteins but lack P3 and P6 in the envelope, sediments at the same rate as wild-type virus grown at high temperatures. The sedimentation rate of such P3, P6-deficient particles is independent of the temperature at which they are formed. The variation in the amount of P3 and P6 inserted into the phage membrane reflects an aspect of assembly other than synthesis, since the relative amount of the two proteins synthesized with respect to other phage proteins does not change much with temperature.     

Lipid-containing bacteriophage PR4: structure and life cycle.

The structure of the purified lipid-containing phage PR4 was studied electron microscopically using thin sectioning, negative staining and freeze-fracturing techniques. The lipid layer was located inside a rigid capsid and had a bilayer structure. The innermost dense core was probably composed solely of DNA since no major structural proteins were missing in empty phage particles lacking DNA. During infection, phages were attached to the cell wall of the host. They apparently inject their DNA through the cell envelope. The lipid layer of the phage might play an active role in the injection process. The maturation of the phage capsid takes place within the nuclear region of the cell, whereas intact phages with DNA were always seen in the cell periphery.     

The role of gene V protein in f1 single-strand synthesis

We demonstrate that, in cells infected with phage f1, only the gene V protein, a DNA-binding protein, is necessary to effect the switch from double- to single-strand synthesis; we specifically show that no host proteins are required for single-strand synthesis beyond those that are already required for double-strand synthesis. f1 temperature-sensitive (ts) gene II-infected cells were allowed to accumulate excess gene V protein in the absence of DNA replication through incubation at restrictive temperature. The infected cells were then shifted to permissive temperature in the presence of chloramphenicol; single-strand synthesis ensued. When, however, the identical experiment was performed with wild-type f1-infected cells, double, rather than single strands were synthesized. Since the principal difference between the two experiments lay in the accumulation of excess gene V protein in the tsII infection, single-strand synthesis must have halted in the wild-type infection because no free gene V protein was available and not because a chloramphenicol-sensitive host protein was being depleted. Chloramphenicol, by preventing protein synthesis, apparently blocks the two sources of gene V protein normally used for single-strand synthesis. No new gene V protein can be synthesized, and all previously synthesized gene V protein remains stably complexed with preexisting single strands rather than being recycled during morphogenesis of the single strands. In the absence of both new and recycled gene V protein, double-strand synthesis resumes.If a failure to recycle gene V protein were indeed the lesion induced by chloramphenicol treatment, then inactivation of specific f1 gene products required for morphogenesis, through use of amber and temperature-sensitive mutant phage, might also lead to replacement of single-strand synthesis by double-strand synthesis. Such cessation of single-strand synthesis was indeed found after infections by mutants in genes I, IV, VII, and VIII but not after infections by mutants in genes III and VI. The gene III and VI proteins therefore act at a later step in morphogenesis than the gene I, IV, VII, and VIII proteins; they appear to function after the single strands and gene V protein have separated. Data obtained from temperature shift experiments indicate that the supply of gene V protein in an infected cell is carefully regulated and that there is never much free gene V protein available.     

Identification of host cell binding peptide from an overlapping peptide library for inhibition of classical swine fever virus infection

The envelope proteins of classical swine fever virus (CSFV) mediate the binding of CSFV to cell surface molecules and allow CSFV subsequent to enter host cells. However, the proteins binding to host cells and their binding sequences are uncertain. The results showed that the protein E1, E2, and Erns were displayed on the surfaces of T7 phages. The E2 and Erns phage clones showed high binding affinity to host cells, in which the E2 phage clone interacted more specifically with host cells than with other cells, while the Erns phage clone interacted with all tested cells. A 30-mer phage displaying peptide library was constructed and screened against immobilized host cells, in which each peptide was overlapped 10aa to another peptide and spanned all amino acid sequences of Erns and E2. Fifty-eight clones with specific binding to host cells were isolated. Amino acid sequence analyses for two phage clones (P2 and P6) demonstrated the strongest binding positions were at 101–130 (S2) in Erns, and 141–170 (S6) in E2, respectively. The synthetic peptides (S2 and S6) could inhibit the binding of phage clones (P2 and P6) and CSFV to cell. About 86.74 and 74.24% inhibition rates of CSFV infection were achieved at 55 μM of the synthetic peptides S2 and S6. The results also indicated that the S2 (LAEGPPVKECAVTCRYDKDADINVVTQARN) and S6 (AVSPTTLRTEVVKTFRRDKPFPHRMDCVTT) from CSFV were host cell binding peptides, and both of them had potential for research of CSFV entering host cells.     

Biogenesis of poxviruses: genetically controlled modifications of structural and functional components of the plasma membrane

Synthesis of plasma membrane proteins and glycoproteins, which were unlike those present in the uninfected host, was found to take place after vaccinia virus infection of HeLa cells. Preexisting host plasma membrane components were shown, both electro-phoretically and immunologically, to remain unmodified, or modified only slightly, following infection. In addition two plasma membrane functions, the Na⁺K⁺-activated ATPase and poliovirus receptors, were found to retain full or partial activity. Thus, the plasma membrane alterations induced after vaccinia infection differ from those found after infection with some so-called budding viruses in that with certain budding agents, host components are replaced with virion structural proteins, whereas in vaccinia infections both preexisting host components and newly synthesized virus-induced nonvirion components coexisted in the plasma membrane.The major plasma membrane glycoprotein synthesized after infection with the hemagglutinating, nonfusing vaccinia IHD-J has been tentatively identified as the glycoprotein component of the vaccinia hemagglutinin. This nonvirion glycoprotein was absent from plasma membranes of cells infected with nonhemagglutinating, fusion-inducing IHD-W, was inhibited by hydroxyurea but not by rifampicin, and was synthesized with late kinetics, all of the characteristics previously shown to be those of the hemagglutinin. Although a polypeptide component(s) corresponding to the IHD-J-specific glycoprotein in acrylamide gels was found in plasma membranes isolated from both IHD-J and -W infected cells, in the case of IHD-W no carbohydrate was associated with this component. The implications of this finding for the nature of the fusion process are discussed.     

T4 gene 40 mutants. II. Phenotypic properties.

Evidence is presented that p40 affects the solubility of p20 inside the T4-infected cell. In the absence of p20 and p40 function at high temperature, p20 is located specifically and exclusively in the cell envelope. It is possible that p40 may interact with p20 before head assembly can be initiated. Cleavage of the major head protein in gene 40 mutant-infected cells is also inhibited. In addition to producing single-layered polyheads and phage at 30°, am restrictive cells infected with gene 40 am mutants produce some empty capsids. It is likely that p40 function is only strictly required for phage growth at high temperature. Electron microscopy of thin sections of crude cell envelopes showed few recognizable structural components, although most phage structural proteins were present upon sodium dodecyl sulfate-gel electrophoresis of envelope preparations. Extraction with sodium chloride selectively removes some of the viral proteins from the crude cell envelope preparation, while extraction with 4 M guanidine · HCl greatly reduces the amount of T4 protein in the cell envelopes. The envelope association of most of these proteins appears to be nonspecific.     

Bacteriophage N4-induced transcribing activities in Escherichia coli. II. Association of the N4 transcriptional apparatus with the cytoplasmic membrane.

Several lines of evidence indicate that the two rifampicin-resistant transcribing activities induced by Escherichia coli phage N4 are associated with the cell membrane. First, under lysis conditions designed to avoid nonspecific DNA-membrane association, the N4 RNA-synthesizing activities (as well as the endogenous N4 DNA) cosediment with the E. coli DNA membrane complex in sucrose gradients. Second, it has not been possible to dissociate the activities from the DNA-membrane complex by any treatment which has been tested; for example, after high salt treatments (up to 4 M NaCl), which do not irreversibly inactivate the activities, the activities remain stably associated with the complex. However, low detergent concentrations inhibit the activities, and irreversible inactivation results from treatment with the anionic detergent, sodium deoxycholate. Third, upon separation of the cell envelope of N4-infected E. coli into inner (cytoplasmic) and outer membrane fractions, the N4-induced RNA-synthesizing activity is found associated with the cytoplasmic membrane. Based on electrophoretic migration in sodium dodecyl sulfate-polyacrylamide gels, the N4-induced polypeptides found associated with the DNA-membrane complex are also found selectively associated with the cytoplasmic membrane.     

DNA replication of bacteriophage phi29. Effect of two viral genes on the association of phage chromosomes with the host cell membrance.

The kinetics of DNA arrest and the maintenance of the association of viral chromosomes with the cell membrane were examined by temperature-shift experiments using temperature-sensitive mutants in two early bacteriophage φ29 genes required for phage DNA replication. φ29 ts2(35), a mutant in cistron 2 whose product (protein P2) is continuously required for associating phage DNA with the Bacillus subtilis membrane, does not stop phage DNA synthesis immediately after a shift to the nonpermissive temperature. In contrast, bacteria infected with φ29 ts3(28), a mutant in cistron 3 (which codes for protein P3), stop synthesizing phage DNA immediately after transfer to the nonpermissive temperature. Parental phage DNA in φ29 ts2(35) infections rapidly dissociates from the cell membrane after a shift to 45°, whereas φ29 ts3(28) DNA remains associated with the membrane after the shift to the nonpermissive temperature and then slowly dissociates. Thus the rapid dissociation of parental phage φ29 chromosomes from the membrane is dependent on a functional protein P3. These findings are discussed in terms of possible modes of action of these two proteins and suggest that protein P2 operates as a linker of phage chromosomes to the membrane, whereas protein P3 participates directly in the initiation or in the polymerization of viral DNA molecules.     

DNA replication and late protein synthesis during SP82 infection of Bacillus subtilis

During infection of Bacillus subtilis by bacteriophage SP82, substantial quantities of at least two different late proteins were synthesized, even in the absence of detectable phage DNA replication. The two proteins were the lytic enzyme (which is possibly a host specific enzyme) and the protein(s) responsible for serum blocking power (which is presumably phage specific). The absence of replication was assured by the use of an SP82 double mutant carrying a mutation in each of two cistrons required for replication, and was verified by density transfer and isotope incorporation experiments. Thus, it appears that phage DNA replication is not required for late protein synthesis during SP82 infection.     

Filamentous phage assembly: morphogenetically defective mutants that do not kill the host

The filamentous phage virion is assembled without killing the host, by extrusion of the DNA through the envelope and concomitant acquisition of coat proteins from the inner membrane. When assembly is blocked, however, intracellular phage DNA and gene products accumulate and the host is killed. This "cell killing" is largely absent in phage fd-tet, which carries a tetracycline-resistance determinant within the origin of minus-strand synthesis; as a result of the replication defect, phage DNA does not accumulate to high levels intracellularly when virion assembly is blocked. This allows morphogenetically defective mutants except those ablating gene V to be freely propagated in tetracycline-containing medium and studied in the absence of the confounding factor of cell morbidity. Because cultures can be initiated by transfection in the complete absence of input virions, extremely low levels of phage production can be assayed. Using this system, I show that genes III, VI, I, and IV are not required to form the complex between viral DNA and gene-V protein that is the intracellular precursor to mature virions; that genes I and/or IV are absolutely (or nearly absolutely) required for assembly; and that mos, a cis-acting sequence previously shown to enhance phage yield in some circumstances, is without such effect in others.     

Molecular make-up of the Plasmodium parasitophorous vacuolar membrane

Plasmodium, the causative agent of malaria, is an obligate, intracellular, eukaryotic cell that invades, replicates, and differentiates within hepatocytes and erythrocytes. Inside a host cell, a second membrane delineates the developing pathogen in addition to the parasite plasma membrane, resulting in a distinct cellular compartment, termed parasitophorous vacuole (PV). The PV membrane (PVM) constitutes the parasite–host cell interface and is likely central to nutrient acquisition, host cell remodeling, waste disposal, environmental sensing, and protection from innate defense. Over the past two decades, a number of parasite-encoded PVM proteins have been identified. They include multigene families and protein complexes, such as early-transcribed membrane proteins (ETRAMPs) and the Plasmodium translocon for exported proteins (PTEX). Nearly all Plasmodium PVM proteins are restricted to this genus and display transient and stage-specific expression. Here, we provide an overview of the PVM proteins of Plasmodium blood and liver stages. Biochemical and experimental genetics data suggest that some PVM proteins are ideal targets for novel anti-malarial intervention strategies.