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Molecular Cell Jan 2022Exon back-splicing-generated circular RNAs, as a group, can suppress double-stranded RNA (dsRNA)-activated protein kinase R (PKR) in cells. We have sought to synthesize... (Comparative Study)
Comparative Study
Exon back-splicing-generated circular RNAs, as a group, can suppress double-stranded RNA (dsRNA)-activated protein kinase R (PKR) in cells. We have sought to synthesize immunogenicity-free, short dsRNA-containing RNA circles as PKR inhibitors. Here, we report that RNA circles synthesized by permuted self-splicing thymidylate synthase (td) introns from T4 bacteriophage or by Anabaena pre-tRNA group I intron could induce an immune response. Autocatalytic splicing introduces ∼74 nt td or ∼186 nt Anabaena extraneous fragments that can distort the folding status of original circular RNAs or form structures themselves to provoke innate immune responses. In contrast, synthesized RNA circles produced by T4 RNA ligase without extraneous fragments exhibit minimized immunogenicity. Importantly, directly ligated circular RNAs that form short dsRNA regions efficiently suppress PKR activation 10- to 10-fold higher than reported chemical compounds C16 and 2-AP, highlighting the future use of circular RNAs as potent inhibitors for diseases related to PKR overreaction.
Topics: A549 Cells; Bacteriophage T4; HEK293 Cells; HeLa Cells; Humans; Immunity, Innate; Introns; Nucleic Acid Conformation; Protein Kinase Inhibitors; RNA Ligase (ATP); RNA Precursors; RNA, Circular; Thymidylate Synthase; Viral Proteins; eIF-2 Kinase
PubMed: 34951963
DOI: 10.1016/j.molcel.2021.11.019 -
Nature May 2022The cyclic oligonucleotide-based antiphage signalling system (CBASS) and the pyrimidine cyclase system for antiphage resistance (Pycsar) are antiphage defence systems in...
The cyclic oligonucleotide-based antiphage signalling system (CBASS) and the pyrimidine cyclase system for antiphage resistance (Pycsar) are antiphage defence systems in diverse bacteria that use cyclic nucleotide signals to induce cell death and prevent viral propagation. Phages use several strategies to defeat host CRISPR and restriction-modification systems, but no mechanisms are known to evade CBASS and Pycsar immunity. Here we show that phages encode anti-CBASS (Acb) and anti-Pycsar (Apyc) proteins that counteract defence by specifically degrading cyclic nucleotide signals that activate host immunity. Using a biochemical screen of 57 phages in Escherichia coli and Bacillus subtilis, we discover Acb1 from phage T4 and Apyc1 from phage SBSphiJ as founding members of distinct families of immune evasion proteins. Crystal structures of Acb1 in complex with 3'3'-cyclic GMP-AMP define a mechanism of metal-independent hydrolysis 3' of adenosine bases, enabling broad recognition and degradation of cyclic dinucleotide and trinucleotide CBASS signals. Structures of Apyc1 reveal a metal-dependent cyclic NMP phosphodiesterase that uses relaxed specificity to target Pycsar cyclic pyrimidine mononucleotide signals. We show that Acb1 and Apyc1 block downstream effector activation and protect from CBASS and Pycsar defence in vivo. Active Acb1 and Apyc1 enzymes are conserved in phylogenetically diverse phages, demonstrating that cleavage of host cyclic nucleotide signals is a key strategy of immune evasion in phage biology.
Topics: Bacteria; Bacterial Proteins; Bacteriophage T4; Bacteriophages; CRISPR-Cas Systems; Endonucleases; Escherichia coli; Nucleotides, Cyclic; Oligonucleotides; Pyrimidines
PubMed: 35395152
DOI: 10.1038/s41586-022-04716-y -
Nature Aug 2023The mechanisms by which viruses hijack the genetic machinery of the cells they infect are of current interest. When bacteriophage T4 infects Escherichia coli, it uses...
The mechanisms by which viruses hijack the genetic machinery of the cells they infect are of current interest. When bacteriophage T4 infects Escherichia coli, it uses three different adenosine diphosphate (ADP)-ribosyltransferases (ARTs) to reprogram the transcriptional and translational apparatus of the host by ADP-ribosylation using nicotinamide adenine dinucleotide (NAD) as a substrate. NAD has previously been identified as a 5' modification of cellular RNAs. Here we report that the T4 ART ModB accepts not only NAD but also NAD-capped RNA (NAD-RNA) as a substrate and attaches entire RNA chains to acceptor proteins in an 'RNAylation' reaction. ModB specifically RNAylates the ribosomal proteins rS1 and rL2 at defined Arg residues, and selected E. coli and T4 phage RNAs are linked to rS1 in vivo. T4 phages that express an inactive mutant of ModB have a decreased burst size and slowed lysis of E. coli. Our findings reveal a distinct biological role for NAD-RNA, namely the activation of the RNA for enzymatic transfer to proteins. The attachment of specific RNAs to ribosomal proteins might provide a strategy for the phage to modulate the host's translation machinery. This work reveals a direct connection between RNA modification and post-translational protein modification. ARTs have important roles far beyond viral infections, so RNAylation may have far-reaching implications.
Topics: ADP Ribose Transferases; Bacteriophage T4; Escherichia coli; NAD; Ribosomal Proteins; Viral Proteins; Escherichia coli Proteins; RNA; Protein Biosynthesis; Gene Expression Regulation, Bacterial; Protein Processing, Post-Translational
PubMed: 37587340
DOI: 10.1038/s41586-023-06429-2 -
Frontiers in Bioengineering and... 2021Circular RNA (circRNA) is a unique type of noncoding RNA molecule. Compared with traditional linear RNA, circRNA is a covalently closed circle produced by a process... (Review)
Review
Circular RNA (circRNA) is a unique type of noncoding RNA molecule. Compared with traditional linear RNA, circRNA is a covalently closed circle produced by a process called backsplicing. CircRNA is abundant in many cells and has rich functions in cells, such as acting as miRNA sponge, protein sponge, protein scaffold, and mRNA regulator. With the continuous development of circRNA study, circRNA has also played an important role in medical applications, including circRNA vaccines and gene therapy. In this review, we illustrate the synthesis of circRNAs . We focus on biological ligation methods, such as enzymatic ligation from the bacteriophage T4 and ribozyme method. In addition, we summarize the current challenges in the design, synthesis, application, and production of circRNAs, and propose possible solutions in the future. CircRNA is expected to play an essential role in basic research and medical applications.
PubMed: 34917603
DOI: 10.3389/fbioe.2021.787881 -
Viruses Feb 2023Bacteriophage (phage) T4 has served as an extraordinary model to elucidate biological structures and mechanisms. Recent discoveries on the T4 head (capsid) structure,... (Review)
Review
Bacteriophage (phage) T4 has served as an extraordinary model to elucidate biological structures and mechanisms. Recent discoveries on the T4 head (capsid) structure, portal vertex, and genome packaging add a significant body of new literature to phage biology. Head structures in unexpanded and expanded conformations show dramatic domain movements, structural remodeling, and a ~70% increase in inner volume while creating high-affinity binding sites for the outer decoration proteins Soc and Hoc. Small changes in intercapsomer interactions modulate angles between capsomer planes, leading to profound alterations in head length. The in situ cryo-EM structure of the symmetry-mismatched portal vertex shows the remarkable structural morphing of local regions of the portal protein, allowing similar interactions with the capsid protein in different structural environments. Conformational changes in these interactions trigger the structural remodeling of capsid protein subunits surrounding the portal vertex, which propagate as a wave of expansion throughout the capsid. A second symmetry mismatch is created when a pentameric packaging motor assembles at the outer "clip" domains of the dodecameric portal vertex. The single-molecule dynamics of the packaging machine suggests a continuous burst mechanism in which the motor subunits adjusted to the shape of the DNA fire ATP hydrolysis, generating speeds as high as 2000 bp/s.
Topics: Bacteriophage T4; Binding Sites; Capsid; Capsid Proteins; Head
PubMed: 36851741
DOI: 10.3390/v15020527 -
MBio Aug 2022The U.S. Food and Drug Administration-authorized mRNA- and adenovirus-based SARS-CoV-2 vaccines are intramuscularly injected in two doses and effective in preventing...
The U.S. Food and Drug Administration-authorized mRNA- and adenovirus-based SARS-CoV-2 vaccines are intramuscularly injected in two doses and effective in preventing COVID-19, but they do not induce efficient mucosal immunity or prevent viral transmission. Here, we report the first noninfectious, bacteriophage T4-based, multicomponent, needle- and adjuvant-free, mucosal vaccine harboring engineered Spike trimers on capsid exterior and nucleocapsid protein in the interior. Intranasal administration of two doses of this T4 SARS-CoV-2 vaccine 21 days apart induced robust mucosal immunity, in addition to strong systemic humoral and cellular immune responses. The intranasal vaccine induced broad virus neutralization antibody titers against multiple variants, Th1-biased cytokine responses, strong CD4 and CD8 T cell immunity, and high secretory IgA titers in sera and bronchoalveolar lavage specimens from vaccinated mice. All of these responses were much stronger in intranasally vaccinated mice than those induced by the injected vaccine. Furthermore, the nasal vaccine provided complete protection and sterilizing immunity against the mouse-adapted SARS-CoV-2 MA10 strain, the ancestral WA-1/2020 strain, and the most lethal Delta variant in both BALB/c and human angiotensin converting enzyme (hACE2) knock-in transgenic mouse models. In addition, the vaccine elicited virus-neutralizing antibodies against SARS-CoV-2 variants in bronchoalveolar lavage specimens, did not affect the gut microbiota, exhibited minimal lung lesions in vaccinated and challenged mice, and is completely stable at ambient temperature. This modular, needle-free, phage T4 mucosal vaccine delivery platform is therefore an excellent candidate for designing efficacious mucosal vaccines against other respiratory infections and for emergency preparedness against emerging epidemic and pandemic pathogens. According to the World Health Organization, COVID-19 may have caused ~15-million deaths across the globe and is still ravaging the world. Another wave of ~100 million infections is predicted in the United States due to the emergence of highly transmissible immune-escaped Omicron variants. The authorized vaccines would not prevent these transmissions since they do not trigger mucosal immunity. We circumvented this limitation by developing a needle-free, bacteriophage T4-based, mucosal vaccine. This intranasally administered vaccine generates superior mucosal immunity in mice, in addition to inducing robust humoral and cell-mediated immune responses, and provides complete protection and sterilizing immunity against SARS-CoV-2 variants. The vaccine is stable, adjuvant-free, and cost-effectively manufactured and distributed, making it a strategically important next-generation COVID vaccine for ending this pandemic.
Topics: Adjuvants, Immunologic; Animals; Antibodies, Neutralizing; Antibodies, Viral; Bacteriophages; COVID-19; COVID-19 Vaccines; Humans; Mice; Mice, Inbred BALB C; SARS-CoV-2; Spike Glycoprotein, Coronavirus
PubMed: 35900097
DOI: 10.1128/mbio.01822-22 -
MBio Jun 2021Bacteria and bacteriophages (phages) have evolved potent defense and counterdefense mechanisms that allowed their survival and greatest abundance on Earth. CRISPR...
Bacteria and bacteriophages (phages) have evolved potent defense and counterdefense mechanisms that allowed their survival and greatest abundance on Earth. CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated) is a bacterial defense system that inactivates the invading phage genome by introducing double-strand breaks at targeted sequences. While the mechanisms of CRISPR defense have been extensively investigated, the counterdefense mechanisms employed by phages are poorly understood. Here, we report a novel counterdefense mechanism by which phage T4 restores the genomes broken by CRISPR cleavages. Catalyzed by the phage-encoded recombinase UvsX, this mechanism pairs very short stretches of sequence identity (minihomology sites), as few as 3 or 4 nucleotides in the flanking regions of the cleaved site, allowing replication, repair, and stitching of genomic fragments. Consequently, a series of deletions are created at the targeted site, making the progeny genomes completely resistant to CRISPR attack. Our results demonstrate that this is a general mechanism operating against both type II (Cas9) and type V (Cas12a) CRISPR-Cas systems. These studies uncovered a new type of counterdefense mechanism evolved by T4 phage where subtle functional tuning of preexisting DNA metabolism leads to profound impact on phage survival. Bacteriophages (phages) are viruses that infect bacteria and use them as replication factories to assemble progeny phages. Bacteria have evolved powerful defense mechanisms to destroy the invading phages by severing their genomes soon after entry into cells. We discovered a counterdefense mechanism evolved by phage T4 to stitch back the broken genomes and restore viral infection. In this process, a small amount of genetic material is deleted or another mutation is introduced, making the phage resistant to future bacterial attack. The mutant virus might also gain survival advantages against other restriction conditions or DNA damaging events. Thus, bacterial attack not only triggers counterdefenses but also provides opportunities to generate more fit phages. Such defense and counterdefense mechanisms over the millennia led to the extraordinary diversity and the greatest abundance of bacteriophages on Earth. Understanding these mechanisms will open new avenues for engineering recombinant phages for biomedical applications.
Topics: Bacteriophage T4; CRISPR-Cas Systems; DNA Repair; Genome, Viral; Recombination, Genetic
PubMed: 34154416
DOI: 10.1128/mBio.01361-21 -
Viruses Mar 2022Viruses are biochemically complex structures and mainly consist of folded proteins that contain nucleic acids. Bacteriophage T4 is one of most prominent examples, having... (Review)
Review
Viruses are biochemically complex structures and mainly consist of folded proteins that contain nucleic acids. Bacteriophage T4 is one of most prominent examples, having a tail structure that contracts during the infection process. Intracellular phage multiplication leads to separate self-directed assembly reactions of proheads, tails and tail fibers. The proheads are packaged with concatemeric DNA produced by tandem replication reactions of the parental DNA molecule. Once DNA packaging is completed, the head is joined with the tail and six long fibers are attached. The mature particles are then released from the cell via lysis, another tightly regulated process. These processes have been studied in molecular detail leading to a fascinating view of the protein-folding dynamics that direct the structural interplay of assembled complexes. Lindsay W. Black dedicated his career to identifying and defining the molecular events required to form the T4 virion. He leaves us with rich insights into the astonishingly precise molecular clockwork that co-ordinates all of the players in T4 assembly, both viral and cellular. Here, we summarize Lindsay's key research contributions that are certain to stimulate our future science for many years to come.
Topics: Bacteriophage T4; Beauty; Capsid; DNA Packaging; DNA, Viral
PubMed: 35458430
DOI: 10.3390/v14040700 -
Viruses Aug 2021Bacteriophages have long been model systems to study the molecular mechanisms of DNA replication. During DNA replication, a DNA helicase and a DNA polymerase... (Review)
Review
Bacteriophages have long been model systems to study the molecular mechanisms of DNA replication. During DNA replication, a DNA helicase and a DNA polymerase cooperatively unwind the parental DNA. By surveying recent data from three bacteriophage replication systems, we summarized the mechanistic basis of DNA replication by helicases and polymerases. Kinetic data have suggested that a polymerase or a helicase alone is a passive motor that is sensitive to the base-pairing energy of the DNA. When coupled together, the helicase-polymerase complex is able to unwind DNA actively. In bacteriophage T7, helicase and polymerase reside right at the replication fork where the parental DNA is separated into two daughter strands. The two motors pull the two daughter strands to opposite directions, while the polymerase provides a separation pin to split the fork. Although independently evolved and containing different replisome components, bacteriophage T4 replisome shares mechanistic features of Hel-Pol coupling that are similar to T7. Interestingly, in bacteriophages with a limited size of genome like Φ29, DNA polymerase itself can form a tunnel-like structure, which encircles the DNA template strand and facilitates strand displacement synthesis in the absence of a helicase. Studies on bacteriophage replication provide implications for the more complicated replication systems in bacteria, archaeal, and eukaryotic systems, as well as the RNA genome replication in RNA viruses.
Topics: Bacteriophage T4; Bacteriophage T7; DNA Helicases; DNA Replication; DNA, Viral; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Kinetics; Models, Molecular; Viral Proteins; Virus Replication
PubMed: 34578319
DOI: 10.3390/v13091739 -
Nature Communications Jul 2023The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome to couple DNA unwinding with RNA primer synthesis for DNA replication. How the primosome is...
The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome to couple DNA unwinding with RNA primer synthesis for DNA replication. How the primosome is assembled and how the primer length is defined are unclear. Here we report a series of cryo-EM structures of T4 primosome assembly intermediates. We show that gp41 alone is an open spiral, and ssDNA binding triggers a large-scale scissor-like conformational change that drives the ring closure and activates the helicase. Helicase activation exposes a cryptic hydrophobic surface to recruit the gp61 primase. The primase binds the helicase in a bipartite mode in which the N-terminal Zn-binding domain and the C-terminal RNA polymerase domain each contain a helicase-interacting motif that bind to separate gp41 N-terminal hairpin dimers, leading to the assembly of one primase on the helicase hexamer. Our study reveals the T4 primosome assembly process and sheds light on the RNA primer synthesis mechanism.
Topics: Bacteriophage T4; DNA Primase; DNA Helicases; DNA Replication; DNA Primers; DNA, Viral
PubMed: 37474605
DOI: 10.1038/s41467-023-40106-2