A nucleoprotein complex in bacteria infected with Pf1 filamentous virus: Isolation and biochemical characterization

A nucleoprotein complex has been isolated from Pseudomonas aeruginosa infected with filamentous virus Pf1, and purified to homogeneity. The complex has a sedimentation coefficient of 48 S in low ionic strength and neutral pH. It consists of single-stranded circular viral DNA combined with about 1500 copie of a virus-specific protein weighing about 16,500 daltons. The complex is similar to one containing viral DNA and gene 5 protein of virus fd. However, the Pf1 DNA-binding protein has a mass about 1.7-fold greater than that of the analogous fd protein, and the Pf1 complex is considerably more resistant to dissociation by high ionic strength than the fd complex. Although the amino acid composition of the Pf1 protein is quite different from that of the fd protein, the two proteins have about the same number of aromatic amino acids, which are thought to be involved in binding single-stranded DNA.     

A nucleoprotein complex in bacteria infected with PF1 filamentous virus: identification and electron microscopic analysis

We report the discovery, partial purification, and high-resolution electron microscopic characterization of an intracellular complex from Pseudomonas aeruginosa bacteria infected by Pf1 filamentous virus. The Pf1 complex resembles the virion precursor complex of DNA and viral gene 5 protein formed by fd virus of Escherichia coli, but the two complexes differ in structure. Image reconstruction indicates that both complexes are single-start morphological helices; specimen tilting shows the Pf1 helix to be right-handed. Although the Pf1 and fd complexes contain a similar number of nucleotides per axial unit length, the mean distance between helical turns is 61 Å for Pf1 but 91 Å for fd under the conditions used for our measurements; two turns of the fd nucleoprotein helix contain about as many nucleotides as do three turns of the Pf1 helix. The Pf1 complex is much shorter than are Pf1 virions, in contrast to the similar lengths of the fd virion and complex. The fd complex is extremely flexible, but the Pf1 complex is more highly regular in structure. Most significant, calculations based on our data indicate that the DNA in the Pf1 complex is probably located at a smaller radius than in the bulk of the protein. If the DNA and morphological helices coincide, the DNA in the Pf1 complex must be well inside of (axial to) the outeer protein surfaces of the complex, rather than being wrapped around the protein subunits as proposed by others for fd complex.     

Identification and specificity of pilus adsorption proteins of filamentous bacteriophages infecting Pseudomonas aeruginosa

Filamentous bacteriophages Pf1 and Pf3 infect Pseudomonas aeruginosa strains K and O, respectively. We show here that the capsids of these bacteriophages each contain a few copies of a minor coat protein (designated g3p) of high molecular mass, which serves as a pilus adsorption protein, much like the protein g3p of the Ff bacteriophages which infect Escherichia coli. Bacteriophage Pf1 was observed to interact with the type IV PAK pilus whereas bacteriophage Pf3 interacted with the conjugative RP4 pilus and not with the type IV PAO pilus. The specificity was found to be mediated by their pilus-binding proteins. This is evidence of a conserved pathway of infection among different classes of filamentous bacteriophage. However, there are likely to be subtle differences yet to be discovered in the way these virions effect entry into their targeted bacterial cells.     

Ribonucleoprotein-like structures from coronavirus particles.

The structure of the ribonucleoprotein (RNP) complex of three coronaviruses was investigated. A single-stranded helix of diam. 14 to 16 nm and up to 320 nm in length was released from disrupted particles of human coronavirus strain 229E and mouse hepatitis virus strain 3 after incubation in mild conditions. The helical complexes appeared to be composed of globular subunits with long axes of 5 to 7 nm surrounding a hollow core of diam. 3 to 4 nm. The complexes were shown to be sensitive to both pancreatic RNase and to pronase. No undegraded internal component was obtained from disrupted avian infectious bronchitis virus particles. We conclude that these structures are RNP complexes. The similarity between these RNPs and those of other large lipid containing RNA viruses is discussed.     

Minor protein content of the gene V protein/phage single-stranded DNA complex of the filamentous bacteriophage f1

The gene V protein/phage single-stranded (SS) DNA complex is an intermediate in the assembly of the filamentous bacteriophage f1. The minor protein content of this complex isolated from wild-type and amber mutant phage-infected Escherichia coli bacteria has been analyzed. Other than the gene V protein, none of the proteins found in purified samples of the complex correspond to any known phage gene products. In particular, the minor coat proteins found in the mature phage particle do not appear to be components of the cytoplasmic gene V protein/f1 SS DNA complex. However, approximately 1-3 molecules of E. coli single-stranded DNA binding protein (SSB) copurify with the complex and may be stably associated with this structure in vivo.     

Purification and characterization of vaccinia virus structural protein VP8

A major vaccinia virus core protein, designated VP8, has been purified from virions to homogeneity through DEAE-cellulose, CM-cellulose, and hydroxyapatite chromatography. VP8 migrates as a 25-kDa band in SDS-polyacrylamide gel electrophoresis and sediments as a monomeric species in neutral sucrose gradient centrifugation. This protein is a significant constitutent of the virion, comprising about 6.5% of the total viral polypeptides by mass. Analysis by filter binding and by sucrose gradient centrifugation shows that VP8 binds to double-stranded as well as to single-stranded DNA at low salt concentrations (25 mM NaCl). At higher salt concentrations (100 mM NaCl), the protein binds with a relatively greater affinity to single-stranded DNA. The results from sucrose gradient centrifugation indicate that VP8 probably binds noncooperatively to all structural forms of DNA. The protein is likely to be a component of the viral nucleoprotein complex.     

The M1 matrix protein controls the filamentous phenotype of influenza A virus

We show that most isolates of influenza A induce filamentous changes in infected cells in contrast to A/WSN/33 and A/PR8/34 strains which have undergone extensive laboratory passage and are mouse-adapted. Using reverse genetics, we created recombinant viruses in the naturally filamentous genetic background of A/Victoria/3/75 and established that this property is regulated by the M1 protein sequence, but that the phenotype is complex and several residues are involved. The filamentous phenotype was lost when the amino acid at position 41 was switched from A to V, at the same time, this recombinant virus also became insensitive to the antibody 14C2. On the other hand, the filamentous phenotype could be fully transferred to a virus containing RNA segment 7 of the A/WSN/33 virus by a combination of three mutations in both the amino and carboxy regions of the M1 protein. This observation suggests that an interaction among these regions of M1 may occur during assembly.     

A new filamentous bacteriophage with sex-factor specificity

The bacteriophage IKe is a filamentous bacteriophage 6.6 nm wide, 900–1300 nm long. In neutral CsCl gradients, it bands at a density of 1.286 g.cm−3. The phage contains DNA but no RNA. This DNA is single-stranded and in CsCl has a buoyant density of 1.7218.     

Identification of protein complexes in detergent-resistant membranes of Plasmodium falciparum schizonts

Merozoite surface proteins of the human malaria parasite Plasmodium falciparum are involved in initial contact with target erythrocytes, a process that begins a cascade of events required for successful invasion of these cells. In order to identify complexes that may play a role in invasion we purified detergent-resistant membranes (DRMs), known to be enriched in merozoite surface proteins, and used blue native-polyacrylamide gel electrophoresis (BN-PAGE) to isolate high molecular weight complexes for identification by mass spectrometry. Sixty-two proteins were detected and these mostly belonged to expected DRM proteins classes including GPI-anchored, multi-membrane spanning and rhoptry proteins. Proteins from seven known complexes were identified including MSP-1/7, the low (RAP1/2 and RAP1/3), and high (RhopH1/H2/H3) molecular weight rhoptry complexes, and the invasion motor complex (GAP45/GAP50/myosinA). Remarkably, a large proportion of identified spectra were derived from only 4 proteins: the GPI-anchored proteins MSP-1 and Pf92, the putative GPI-anchored protein Pf113 and RAP-1, the core component of the two RAP complexes. Each of these proteins predominated in high molecular weight species suggesting their aggregation in much larger complexes than anticipated. To demonstrate that the procedure had isolated novel complexes we focussed on MSP-1, which predominated as a distinct species at approximately 500 kDa by BN-PAGE, approximately twice its expected size. Chemical cross-linking supports the existence of a stable MSP-1 oligomer of approximately 500 kDa, probably comprising a highly stable homodimeric species. Our observations also suggests that oligomerization of MSP-1 is likely to occur outside the C-terminal epidermal growth factor (EGF)-like domains. Confirmation of MSP-1 oligomerization, together with the isolation of a number of known complexes by BN-PAGE, makes it highly likely that novel interactions occur amongst members of this proteome.     

Filamentous phage morphogenetic signal sequence and orientation of DNA in the virion and gene-V protein complex

The circular single-stranded viral DNA (ssDNA) of filamentous phage is oriented in the virus with a hairpin forming sequence called mos at the leading end of the virion-the end that emerges from the cell first. In the experiments that originally defined mos it was also found to enhance virion production 100-fold, though in other circumstances it has no such effect. Using a new electron-microscopic method, we have ascertained the orientation of ssDNA in phage having mutations involving mos and other viral functions, and also in the intracellular precursor to the virion-a rod-shaped complex between the ssDNA and the phage-encoded gene-V protein, pV. The results show (1) that the ssDNA is oriented in the complex as in the virion, with mos at one end; (2) that orientation is maintained even if assembly is not mediated by the complex; (3) that orientation is manifested in polyphage--abnormal particles in which many unit-length ssDNA molecules are sheathed in a single, extremely long capsid; (4) that orientation is imposed by mos itself (or something very nearby) since it disappears in a mos deletion; and (5) that a 229-base segment including the minus strand of mos is also effective at imposing orientation. On the basis of our findings, we speculate that mos determines orientation in two stages, one imposing an axis on the ssDNA loop during formation of the pV/ssDNA complex, the other imposing a direction on the loop during initiation of particle assembly. The sequence requirements for the two stages may be different.     

The replication of rodent parvovirus X14.

The replication of rodent parvovirus X14 DNA has been studied in rat embryo tissue culture cells. Virus DNA was isolated from I M-NaCl-SDS-pronase supernatant fluids from 24 h after infection. The majority of this DNA was 1-7 mum in length and double-stranded, indicating that it was an intermediate in the replication cycle of this single-stranded DNA virus. Single-stranded DNA of equivalent length was isolated directly from X14 virions. The buoyant density of this DNA was 1-728 g/ml whereas the double-stranded form banded at 1-714 g/ml in caesium chloride gradients. Difficulties in detecting significant amounts of single-stranded viral DNA directly from infected cells would appear to indicate that progeny single-stranded DNA is rapidly encapsidated after synthesis.     

Nuclease-resistant single-stranded DNA controls for nucleic acid amplification assays

Molecular diagnostic tests based on the PCR or alternative nucleic acid amplification technologies are commonly used for pathogen screening at blood drawing centers. Contrived process surveillance using test-specific external and internal controls is critical for the efficient leverage of PCR power. We describe here novel control constructs for use in nucleic acid amplification assays for pathogens with a single-stranded DNA genome, e.g., parvovirus B19. These controls are derived from a deletion mutant of the filamentous phage fd-tet, fKN16, and consist of single-stranded DNA packaged in a protein coat. They are essentially noninfectious to Escherichia coli and highly resistant to nuclease degradation. fKN16 based controls can be readily manufactured and highly purified. Despite their confirmed filamentous morphology, they can be precisely and accurately diluted over a wide range. Stability studies reveal that the novel control constructs are highly resistant to temperature stress, regardless of whether they are tested as concentrated stocks in storage buffer or diluted in buffer or human plasma. Real-time amplification curves derived from recombinant control constructs containing a parvovirus B19 specific sequence fragment match those derived from native virus. In summary, our data demonstrate the feasibility of novel nuclease-resistant single-stranded DNA controls as surrogates for parvovirus B19 and their applicability in routine molecular diagnostics.     

Characterization of the cord-like structures emerging from the surface of influenza C virus-infected cells

When HMV-II cells (a human malignant melanoma cell line) infected with a newly isolated influenza C strain (Yamagata/1/88) were examined by simple light microscopy, it was found that a large number of cord-like structures which had lengths up to about 500 microns or greater were emerging from the cell surface. The existence of viral glycoproteins (hemagglutinin-esterase, HE) on the surface of these huge structures was confirmed by hemadsorption experiments with erythrocytes from a variety of species as well as by immunofluorescent staining with anti-HE monoclonal antibody. Furthermore, electron microscopy revealed that numerous filamentous particles in the process of budding, each covered with a layer of surface projections approximately 13 nm in length, aggregated with their long axes parallel to form a cord-like structure visible under a light microscope. An electron-dense layer, which presumably consists of membrane protein (M), was seen in cross-sections of all filamentous virions whereas internal nucleocapsids were rarely seen. SDS-polyacrylamide gel electrophoresis of the purified cords also showed that they contained HE and M polypeptides but not nucleoprotein, confirming that long filamentous particles are mostly devoid of nucleocapsids. The emergence of cords on the cell surface was observed in various cell cultures infected with C/Yamagata/1/88 though their number and length varied markedly depending on cell type. The production of cord-like structures was also evident in HMV-II cells infected with any of several different influenza C strains, which suggests that the cord formation is a common feature of influenza C virus group.     

Characterization of Antibody Responses to a Plasmodium falciparum Blood-Stage Antigen Induced by a DNA Prime/Protein Boost Immunization Protocol

The humoral immune responses elicited by priming with a DNA plasmid and boosting with either the plasmid or the corresponding recombinant protein in alum adjuvant were compared. The plasmid DNA encoded a sequence (M3) derived from the Plasmodium falciparum antigen Pf155/RESA, and the recombinant protein consisted of the identical malarial sequence fused to an albumin-binding region (BB) of streptococcal protein G. Mice of different genetic backgrounds (CBA, Balb/c and C57Bl/6) were primed with plasmid DNA and boosted with either plasmid or recombinant protein. In all strains of mice, boosting with protein elicited higher anti-M3 antibody levels than obtained by boosting with plasmid, yet the kinetics and longevity of the secondary responses were comparable. Antiserum obtained after protein boosting displayed an immunoglobulin (Ig)G subclass profile skewed to the IgG1 isotype, regardless of the mouse strain. In contrast, mice receiving a second injection with plasmid responded with a more mixed IgG subclass profile. Inclusion of a P. falciparum circumsporozoite protein-derived T-helper epitope (CS.T3) in the immunization plasmid as well as in the fusion protein, did not significantly change the humoral responses to M3. The results show the potential of DNA vaccination for the purpose of priming an antibody response against the malarial blood-stage antigen Pf155/RESA. When combined with a protein boost, this DNA priming results in high-titred and long-lasting anamnestic responses.     

Structure of the filamentous bacteriophages: Orientation of the DNA molecule within the phage particle

The distribution of pyrimidine oligodeoxyribonucleotides in quarter-length fragments of DNA derived from the adsorbing ends of the filamentous bacteriophages f1 and fd was compared with the distribution of pyrimidine oligonucleotides in the intact phage DNA. No significant differences were found. It is concluded that in these bacteriophages there is no preferred orientation of the single-stranded, circular DNA component relative to the long axis of the virion.     

Nucleic acid-binding properties of the alfalfa mosaic virus movement protein produced in yeast.

The movement protein of alfalfa mosaic virus (P3) was purified from yeasts transformed with an expression vector containing the P3 gene. Its nucleic acid-binding properties were tested by electrophoretic retardation, nitrocellulose retention, and RNA-protein cross-linking. The recombinant protein had a higher affinity for single-stranded RNA and DNA than for double-stranded nucleic acids. Each nucleic acid molecule bound several protein molecules without sequence specificity. The binding was 80% inhibited by 0.2 M NaCl. These properties are qualitatively similar, but not strictly identical, to those of two other viral movement proteins, the 30-kDa protein of tobacco mosaic virus and the gene I product of cauliflower mosaic virus.     

Filamentous bacterial viruses. XV. DNA replication and abortive infection

Productive infection by filamentous virus is not lethal to infected Escherichia coli, but infection under some conditions that prevent viral morphogenesis is lethal. In particular, the host is killed by those conditions that prevent morphogenesis at a stage subsequent to the onset of viral duplex DNA replication (for instance treatment of infected bacteria with chloramphenicol) whereas the host is not killed by those that inhibit viral duplex DNA replication itself (for instance treatment with nalidixic acid). Associated with the onset of lethal abortive infection is a rapid inhibition of the synthesis of viral single-stranded DNA. Killing can be averted by incubation of infected bacteria at 42° before preventing morphogenesis of the virus; this preincubation is accompanied by a reduction in the size of the intracellular pool of single-stranded DNA. A model is presented that links DNA replication, single-stranded DNA pool size, morphogenesis, and killing via the viral gene 5 protein. Evidence which supports some of the predictions of this model is described in an accompanying report (Timmis and Marvin, 1974).     

POT1-interacting protein PIP1: a telomere length regulator that recruits POT1 to the TIN2/TRF1 complex

Human telomere length is controlled by a negative feedback loop based on the binding of TRF1 to double-stranded telomeric DNA. The TRF1 complex recruits POT1, a single-stranded telomeric DNA-binding protein necessary for cis-inhibition of telomerase. By mass spectrometry, we have identified a new telomeric protein, which we have named POT1-interacting protein 1 (PIP1). PIP1 bound both POT1 and the TRF1-interacting factor TIN2 and could tether POT1 to the TRF1 complex. Reduction of PIP1 or POT1 levels with shRNAs led to telomere elongation, indicating that PIP1 contributes to telomere length control through recruitment of POT1.     

Dual importance of positive charge in the C-terminal region of filamentous bacteriophage coat protein for membrane insertion and DNA-protein interaction in virus assembly

Gene VIII encoding the procoat protein of the Class II filamentous bacteriophage Pf1 (infecting Pseudomonas aeruginosa) has been cloned and expressed in Escherichia coli and subjected to site-directed mutagenesis. The two positively charged residues clustered near the C-terminus, arginine-44 and lysine-45, were systematically converted to uncharged residues and serine-41 was converted to an arginine residue. Removal of positive charge in the C-terminal region of the molecule seriously impaired the ability of the procoat molecule to undergo insertion at the E. coli cell inner membrane, as manifested by the diminished processing of the N-terminal leader peptide. The basic amino acids near the C-terminus of the coat protein are also involved in neutralizing the negatively charged viral DNA during virus assembly. However, despite its additional positive charge, the S41R mutant protein was unable to participate in the assembly of Class I bacteriophage fd in E. coli. This dual requirement of positively charged residues in the C-terminal region of the coat protein for membrane processing and insertion and for electrostatic neutralization of the encapsidated DNA poses important constraints on the evolution of filamentous bacteriophages with two different helical symmetries.