Structural basis of superinfection exclusion by bacteriophage T4 Spackle

AbstractA bacterial cell infected with T4 phage rapidly establishes resistance against further infections by the same or closely related T-even-type bacteriophages – a phenomenon called superinfection exclusion. Here we show that one of the T4 early gene products and a periplasmic protein, Spackle, forms a stoichiometric complex with the lysozyme domain of T4 tail spike protein gp5 and potently inhibits its activity. Crystal structure of the Spackle-gp5 lysozyme complex shows that Spackle binds to a horseshoe-shaped basic patch surrounding the oligosaccharide-binding cleft and induces an allosteric conformational change of the active site. In contrast, Spackle does not appreciably inhibit the lysozyme activity of cytoplasmic T4 endolysin responsible for cell lysis to release progeny phage particles at the final step of the lytic cycle. Our work reveals a unique mode of inhibition for lysozymes, a widespread class of enzymes in biology, and provides a mechanistic understanding of the T4 bacteriophage superinfection exclusion.

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Crystal structure of bacteriophage T4 Spackle as determined by native SAD phasing

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798320010979 ◽

2020 ◽

Vol 76 (9) ◽

pp. 899-904

Author(s):

Ke Shi ◽

Fredy Kurniawan ◽

Surajit Banerjee ◽

Nicholas H. Moeller ◽

Hideki Aihara

Keyword(s):

Crystal Structure ◽

Bacteriophage T4 ◽

Early Gene ◽

Size Exclusion ◽

Amino Terminal ◽

Intramolecular Disulfide Bond ◽

Exclusion Chromatography ◽

T4 Bacteriophage ◽

Sad Phasing ◽

T4 Phage

The crystal structure of a bacteriophage T4 early gene product, Spackle, was determined by native sulfur single-wavelength anomalous diffraction (SAD) phasing using synchrotron radiation and was refined to 1.52 Å resolution. The structure shows that Spackle consists of a bundle of five α-helices, forming a relatively flat disc-like overall shape. Although Spackle forms a dimer in the crystal, size-exclusion chromatography with multi-angle light scattering shows that it is monomeric in solution. Mass spectrometry confirms that purified mature Spackle lacks the amino-terminal signal peptide and contains an intramolecular disulfide bond, consistent with its proposed role in the periplasm of T4 phage-infected Escherichia coli cells. The surface electrostatic potential of Spackle shows a strikingly bipolar charge distribution, suggesting a possible mode of membrane association and inhibition of the tail lysozyme activity in T4 bacteriophage superinfection exclusion.

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Overexpression of the Bacteriophage T4 motB Gene Alters H-NS Dependent Repression of Specific Host DNA

Viruses ◽

10.3390/v13010084 ◽

2021 ◽

Vol 13 (1) ◽

pp. 84

Author(s):

Jennifer Patterson-West ◽

Chin-Hsien Tai ◽

Bokyung Son ◽

Meng-Lun Hsieh ◽

James R. Iben ◽

...

Keyword(s):

Bacteriophage T4 ◽

Early Gene ◽

Dna Binding Domain ◽

Host Gene Expression ◽

Rna Seq ◽

Specific Host ◽

Dnase I Footprinting ◽

Ob Fold ◽

Host Genes

The bacteriophage T4 early gene product MotB binds tightly but nonspecifically to DNA, copurifies with the host Nucleoid Associated Protein (NAP) H-NS in the presence of DNA and improves T4 fitness. However, the T4 transcriptome is not significantly affected by a motB knockdown. Here we have investigated the phylogeny of MotB and its predicted domains, how MotB and H-NS together interact with DNA, and how heterologous overexpression of motB impacts host gene expression. We find that motB is highly conserved among Tevenvirinae. Although the MotB sequence has no homology to proteins of known function, predicted structure homology searches suggest that MotB is composed of an N-terminal Kyprides-Onzonis-Woese (KOW) motif and a C-terminal DNA-binding domain of oligonucleotide/oligosaccharide (OB)-fold; either of which could provide MotB’s ability to bind DNA. DNase I footprinting demonstrates that MotB dramatically alters the interaction of H-NS with DNA in vitro. RNA-seq analyses indicate that expression of plasmid-borne motB up-regulates 75 host genes; no host genes are down-regulated. Approximately 1/3 of the up-regulated genes have previously been shown to be part of the H-NS regulon. Our results indicate that MotB provides a conserved function for Tevenvirinae and suggest a model in which MotB functions to alter the host transcriptome, possibly by changing the association of H-NS with the host DNA, which then leads to conditions that are more favorable for infection.

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ESCHERICHIA COLI RHO FACTOR IS INVOLVED IN LYSIS OF BACTERIOPHAGE T4-INFECTED CELLS

Genetics ◽

10.1093/genetics/111.2.197 ◽

1985 ◽

Vol 111 (2) ◽

pp. 197-218

Author(s):

Claës H Linder ◽

Karin Carlson

Keyword(s):

Bacteriophage T4 ◽

Growth Conditions ◽

Plaque Formation ◽

Early Gene ◽

Multicopy Plasmid ◽

Lysis Inhibition ◽

Cold Sensitive ◽

Phage Production ◽

Hydroxylamine Mutagenesis ◽

High Level

ABSTRACT A Rid (Rho interaction deficient) phenotype of bacteriophage T4 mutants was defined by cold-sensitive restriction (lack of plaque formation) on rho + hosts carrying additional polar mutations in unrelated genes, coupled to suppression (plaque formation) in otherwise isogenic strains carrying either a polarity-suppressing rho or a multicopy plasmid expressing the rho + allele. This suggests that the restriction may be due to lower levels of Rho than what is available to T4 in the suppressing strains.—Rid394×4 was isolated upon hydroxylamine mutagenesis and mapped in the t gene; other t mutants (and mot, as well as dda dexA double mutants) also showed a Rid phenotype. In liquid culture in strains that restricted plaque formation Rid394×4 showed strong lysis inhibition (a known t - phenotype) but no prolonged phage production (another well-known t - phenotype). This implies that when Rho is limiting the t mutant shuts off phage production at the normal time. Lysis inhibition was partially relieved, and phage production prolonged to varying extents depending on growth conditions in strains that allowed plaque formation. No significant effects on early gene expression were found. Apparently, both mutant (polarity-suppressing) and wild-type Rho can function in prolonging phage production and partially relieving lysis inhibition of Rid394×4 when present at a sufficiently high level, and Rho may play other role(s) in T4 development than in early gene regulation.

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Direct binding of TFEα opens DNA binding cleft of RNA polymerase

Nature Communications ◽

10.1038/s41467-020-19998-x ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Sung-Hoon Jun ◽

Jaekyung Hyun ◽

Jeong Seok Cha ◽

Hoyoung Kim ◽

Michael S. Bartlett ◽

...

Keyword(s):

Dna Binding ◽

Rna Polymerase ◽

Transcription Initiation ◽

Structural Basis ◽

Transcription Bubble ◽

Cryo Electron Microscopy ◽

Direct Binding ◽

Binding Cleft ◽

Promoter Dna ◽

Winged Helix Domain

AbstractOpening of the DNA binding cleft of cellular RNA polymerase (RNAP) is necessary for transcription initiation but the underlying molecular mechanism is not known. Here, we report on the cryo-electron microscopy structures of the RNAP, RNAP-TFEα binary, and RNAP-TFEα-promoter DNA ternary complexes from archaea, Thermococcus kodakarensis (Tko). The structures reveal that TFEα bridges the RNAP clamp and stalk domains to open the DNA binding cleft. Positioning of promoter DNA into the cleft closes it while maintaining the TFEα interactions with the RNAP mobile modules. The structures and photo-crosslinking results also suggest that the conserved aromatic residue in the extended winged-helix domain of TFEα interacts with promoter DNA to stabilize the transcription bubble. This study provides a structural basis for the functions of TFEα and elucidates the mechanism by which the DNA binding cleft is opened during transcription initiation in the stalk-containing RNAPs, including archaeal and eukaryotic RNAPs.

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Structural basis for carotenoid cleavage by an archaeal carotenoid dioxygenase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2004116117 ◽

2020 ◽

Vol 117 (33) ◽

pp. 19914-19925 ◽

Cited By ~ 1

Author(s):

Anahita Daruwalla ◽

Jianye Zhang ◽

Ho Jun Lee ◽

Nimesh Khadka ◽

Erik R. Farquhar ◽

...

Keyword(s):

Active Site ◽

Molecular Basis ◽

Structural Basis ◽

Reaction Intermediates ◽

Catalytic Activities ◽

Selective Stabilization ◽

Carotenoid Cleavage Dioxygenases ◽

Active Site Cavity ◽

Binding Cleft ◽

Shed Light

Apocarotenoids are important signaling molecules generated from carotenoids through the action of carotenoid cleavage dioxygenases (CCDs). These enzymes have a remarkable ability to cleave carotenoids at specific alkene bonds while leaving chemically similar sites within the polyene intact. Although several bacterial and eukaryotic CCDs have been characterized, the long-standing goal of experimentally visualizing a CCD–carotenoid complex at high resolution to explain this exquisite regioselectivity remains unfulfilled. CCD genes are also present in some archaeal genomes, but the encoded enzymes remain uninvestigated. Here, we address this knowledge gap through analysis of a metazoan-like archaeal CCD fromCandidatusNitrosotalea devanaterra (NdCCD).NdCCD was active toward β-apocarotenoids but did not cleave bicyclic carotenoids. It exhibited an unusual regiospecificity, cleaving apocarotenoids solely at the C14′–C13′ alkene bond to produce β-apo-14′-carotenals. The structure ofNdCCD revealed a tapered active site cavity markedly different from the broad active site observed for the retinal-formingSynechocystisapocarotenoid oxygenase (SynACO) but similar to the vertebrate retinoid isomerase RPE65. The structure ofNdCCD in complex with its apocarotenoid product demonstrated that the site of cleavage is defined by interactions along the substrate binding cleft as well as selective stabilization of reaction intermediates at the scissile alkene. These data on the molecular basis of CCD catalysis shed light on the origins of the varied catalytic activities found in metazoan CCDs, opening the possibility of modifying their activity through rational chemical or genetic approaches.

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Vaccination against Very Virulent Infectious Bursal Disease Virus Using Recombinant T4 Bacteriophage Displaying Viral Protein VP2

Acta Biochimica et Biophysica Sinica ◽

10.1111/j.1745-7270.2005.00101.x ◽

2005 ◽

Vol 37 (10) ◽

pp. 657-664 ◽

Cited By ~ 13

Author(s):

Yong-Chang Cao ◽

Quan-Cheng Shi ◽

Jing-Yun Ma ◽

Qing-Mei Xie ◽

Ying-Zuo Bi

Keyword(s):

Disease Virus ◽

Infectious Bursal Disease Virus ◽

Clinical Signs ◽

Infectious Bursal Disease ◽

Wild Type ◽

Vp2 Protein ◽

Safe Vaccine ◽

Bursal Disease ◽

T4 Bacteriophage ◽

T4 Phage

AbstractIn order to develop a desirable inexpensive, effective and safe vaccine against the very virulent infectious bursal disease virus (vvIBDV), we tried to take advantage of the emerging T4 bacteriophage surface protein display system. The major immunogen protein VP2 from the vvIBDV strain HK46 was fused to the nonessential T4 phage surface capsid protein, a small outer capsid (SOC) protein, resulting in the 49 kDa SOC-VP2 fusion protein, which was verified by sodium dodecylsulfate polyacrylamide gel electrophoresis and Western blot. Immunoelectromicroscopy showed that the recombinant VP2 protein was successfully displayed on the surface of the T4 phage. The recombinant VP2 protein is antigenic and showed reactivities to various monoclonal antibodies (mAbs) against IBDV, whereas the wild-type phage T4 could not react to any mAb. In addition, the recombinant VP2 protein is immunogenic and elicited specific antibodies in immunized specific pathogen free (SPF) chickens. More significantly, immunization of SPF chickens with the recombinant T4-VP2 phage protected them from infection by the vvIBDV strain HK46. When challenged with the vvIBDV strain HK46 at a dose of 100 of 50% lethal dose (LD50) per chicken 4 weeks after the booster was given, the group vaccinated with the T4-VP2 recombinant phage showed no clinical signs of disease or death, whereas the unvaccinated group and the group vaccinated with the wild-type T4 phage exhibited 100% clinical signs of disease and bursal damages, and 30%-40% mortality. Collectively, the data herein showed that the T4-displayed VP2 protein might be an inexpensive, effective and safe vaccine candidate against vvIBDV.

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The BFRF1 Gene of Epstein-Barr Virus Encodes a Novel Protein

Journal of Virology ◽

10.1128/jvi.74.7.3235-3244.2000 ◽

2000 ◽

Vol 74 (7) ◽

pp. 3235-3244 ◽

Cited By ~ 33

Author(s):

Antonella Farina ◽

Roberta Santarelli ◽

Roberta Gonnella ◽

Roberto Bei ◽

Raffaella Muraro ◽

...

Keyword(s):

Cell Lines ◽

Epstein Barr Virus ◽

Lytic Replication ◽

Lytic Cycle ◽

Particle Identification ◽

Early Gene ◽

Cell Fractionation ◽

Barr Virus ◽

F Region ◽

Epstein Barr

ABSTRACT Computer analysis of the Epstein-Barr virus (EBV) genome indicates there are ∼100 open reading frames (ORFs). Thus far about 30 EBV genes divided into the categories latent and lytic have been identified. The BamHI F region of EBV is abundantly transcribed during lytic replication. This region is highly conserved among herpesviruses, thus suggesting that some common function could be retained in the ORFs encompassed within this viral fragment. To identify putative novel proteins and possible new markers for viral replication, we focused our attention on the first rightward ORF in theBamHI F region (BFRF1). Histidine and glutathione S-transferase-tagged BFRF1 fusion proteins were synthesized to produce a mouse monoclonal antibody (MAb). Analysis of human sera revealed a high seroprevalence of antibodies to BFRF1 in patients affected by nasopharyngeal carcinoma or Burkitt's lymphoma, whereas no humoral response to BFRF1 could be detected among healthy donors. An anti-BFRF1 MAb recognizes a doublet migrating at 37 to 38 kDa in cells extracts from EBV-infected cell lines following lytic cycle activation and in an EBV-negative cell line (DG75) transfected with a plasmid expressing the BFRF1 gene. Northern blot analysis allowed the detection of a major transcript of 3.7 kb highly expressed in EBV-positive lytic cycle-induced cell lines. Treatment with inhibitors of viral DNA polymerase, such as phosphonoacetic acid and acyclovir, reduced but did not abolish the transcription ofBFRF1, thus indicating that BFRF1 can be classified as an early gene. Cell fractionation experiments, as well as immunolocalization by immunofluorescence microscopy, immunohistochemistry, and immunoelectron microscopy, showed that BFRF1 is localized on the plasma membrane and nuclear compartments of the cells and is a structural component of the viral particle. Identification of BFRF1 provides a new marker with which to monitor EBV infection and might help us better understand the biology of the virus.

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Bacteriophage T4 multiplication in a glucose-limited Escherichia coli biofilm

Canadian Journal of Microbiology ◽

10.1139/w01-059 ◽

2001 ◽

Vol 47 (7) ◽

pp. 680-684 ◽

Cited By ~ 30

Author(s):

Brian D Corbin ◽

Robert JC McLean ◽

Gary M Aron

Keyword(s):

Escherichia Coli ◽

Bacteriophage T4 ◽

Confocal Laser ◽

Carbon Limitation ◽

Viral Titer ◽

Susceptibility To Infection ◽

Log Reduction ◽

T4 Bacteriophage ◽

Order Of Magnitude ◽

K 12

An Escherichia coli K-12 biofilm was grown at a dilution rate of 0.028 h-1 for 48 h in a glucose-limited chemostat coupled to a modified Robbins' device to determine its susceptibility to infection by bacteriophage T4. Bacteriophage T4 at a multiplicity of infection (MOI) of 10 caused a log reduction in biofilm density (expressed as colony forming units (CFU) per cm2) at 90 min postinfection. After 6 h, a net decrease and equilibrium in viral titer was seen. When biofilms were exposed to T4 phage at a MOI of 100, viral titer doubled after 90 min. After 6 h, viral titers (expressed as plaque forming units (PFU) per cm2) stabilized at levels approximately one order of magnitude higher than seen at a MOI of 10. Scanning confocal laser microscopy images also indicated disruption of biofilm morphology following T4 infection with the effects being more pronounced at a MOI of 100 than at a MOI of 10. These results imply that biofilms under carbon limitation can act as natural reservoirs for bacteriophage and that bacteriophage can have some influence on biofilm morphology.Key words: bacteriophage T4, biofilm, biofilm morphology, bacteriophage ecology, carbon limitation.

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Identification of Protein Tyrosine Kinases Required for B-Cell- Receptor-Mediated Activation of an Epstein-Barr Virus Immediate-Early Gene Promoter

Journal of Virology ◽

10.1128/jvi.78.16.8543-8551.2004 ◽

2004 ◽

Vol 78 (16) ◽

pp. 8543-8551 ◽

Cited By ~ 7

Author(s):

Sandra Lavens ◽

Emmanuel A. Faust ◽

Fang Lu ◽

Michele Jacob ◽

Messele Leta ◽

...

Keyword(s):

Signal Transduction ◽

B Cell ◽

Tyrosine Kinases ◽

Epstein Barr Virus ◽

Cell Receptor ◽

Lytic Cycle ◽

Protein Tyrosine Kinases ◽

Early Gene ◽

Barr Virus ◽

Epstein Barr

ABSTRACT Epstein-Barr Virus (EBV) is a potentially oncogenic herpesvirus that infects >90% of the world's population. EBV exists predominantly as a latent infection in B lymphocytes, with periodic lytic-cycle reactivation essential for cellular and host transmission. Viral reactivation can be stimulated by ligand-induced activation of B-cell-receptor (BCR)-coupled signaling pathways. The critical first step in the transition from latency to the lytic cycle is the expression of the viral immediate-early gene BZLF1 through the transcription activation of its promoter, Zp. However, the BCR-coupled signal transduction cascade(s) leading to the induction of Zp and the expression of the BZLF1 gene product, Zta, is currently unclear. A major obstacle to delineating the relevant signal transduction events has been the lack of a model of EBV infection that is amenable to genetic manipulation. The use of the avian B-cell line DT40 has proven to be a powerful tool for delineating BCR-mediated signal transduction pathways that appear to be highly conserved between avian and mammalian systems. We demonstrate that the DT40 cell line is a robust and genetically tractable system for the study of BCR-mediated signaling pathways leading to transcriptional activation of BZLF1. Using this system, we demonstrate that activation of Zp requires the BCR-coupled protein tyrosine kinases Syk and Btk and that it is positively regulated by Lyn. Thus, the use of DT40 cells has allowed us to delineate the early signaling components required for BCR-dependent reactivation of latent EBV, and this system is likely to prove useful for further dissection of the downstream signaling cascades involved.

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Structural basis of RNA recognition by the SARS-CoV-2 nucleocapsid phosphoprotein

PLoS Pathogens ◽

10.1371/journal.ppat.1009100 ◽

2020 ◽

Vol 16 (12) ◽

pp. e1009100 ◽

Cited By ~ 1

Author(s):

Dhurvas Chandrasekaran Dinesh ◽

Dominika Chalupska ◽

Jan Silhan ◽

Eliska Koutna ◽

Radim Nencka ◽

...

Keyword(s):

Rna Binding ◽

Mutational Analysis ◽

Rna Virus ◽

Structural Basis ◽

Rna Recognition ◽

Nonstructural Proteins ◽

Viral Membrane ◽

Intrinsically Disordered ◽

Binding Cleft ◽

Rna Complex

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19). SARS-CoV-2 is a single-stranded positive-sense RNA virus. Like other coronaviruses, SARS-CoV-2 has an unusually large genome that encodes four structural proteins and sixteen nonstructural proteins. The structural nucleocapsid phosphoprotein N is essential for linking the viral genome to the viral membrane. Both N-terminal RNA binding (N-NTD) and C-terminal dimerization domains are involved in capturing the RNA genome and, the intrinsically disordered region between these domains anchors the ribonucleoprotein complex to the viral membrane. Here, we characterized the structure of the N-NTD and its interaction with RNA using NMR spectroscopy. We observed a positively charged canyon on the surface of the N-NTD that might serve as a putative RNA binding site similarly to other coronaviruses. The subsequent NMR titrations using single-stranded and double-stranded RNA revealed a much more extensive U-shaped RNA-binding cleft lined with regularly distributed arginines and lysines. The NMR data supported by mutational analysis allowed us to construct hybrid atomic models of the N-NTD/RNA complex that provided detailed insight into RNA recognition.

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