ABSTRACT
Epstein-Barr virus (EBV) is an oncogenic human herpesvirus that persists as a multicopy episome in proliferating host cells. Episome maintenance is strictly dependent on EBNA1, a sequence-specific DNA-binding protein with no known enzymatic activities. Here, we show that EBNA1 forms a cell cycle-dependent DNA crosslink with the EBV origin of plasmid replication oriP. EBNA1 tyrosine 518 (Y518) is essential for crosslinking to oriP and functionally required for episome maintenance and generation of EBV-transformed lymphoblastoid cell lines (LCLs). Mechanistically, Y518 is required for replication fork termination at oriP in vivo and for formation of SDS-resistant complexes in vitro. EBNA1-DNA crosslinking corresponds to single-strand endonuclease activity specific to DNA structures enriched at replication-termination sites, such as 4-way junctions. These findings reveal that EBNA1 forms tyrosine-dependent DNA-protein crosslinks and single-strand cleavage at oriP required for replication termination and viral episome maintenance.
Subject(s)
Cell Cycle , Cross-Linking Reagents/chemistry , DNA, Viral/metabolism , Epstein-Barr Virus Nuclear Antigens/metabolism , Plasmids/metabolism , Replication Origin , Virus Replication/physiology , Amino Acid Sequence , B-Lymphocytes/metabolism , Cell Line , DNA Adducts/metabolism , DNA Replication , Endonucleases/metabolism , Epstein-Barr Virus Nuclear Antigens/chemistry , Epstein-Barr Virus Nuclear Antigens/genetics , Humans , Mutation/genetics , Protein Binding , Recombination, Genetic/genetics , Tyrosine/metabolismABSTRACT
Assembly of Kaposi's sarcoma-associated herpesvirus (KSHV) begins at a bacteriophage-like portal complex that nucleates formation of an icosahedral capsid with capsid-associated tegument complexes (CATCs) and facilitates translocation of an â¼150-kb dsDNA genome, followed by acquisition of a pleomorphic tegument and envelope. Because of deviation from icosahedral symmetry, KSHV portal and tegument structures have largely been obscured in previous studies. Using symmetry-relaxed cryo-EM, we determined the in situ structure of the KSHV portal and its interactions with surrounding capsid proteins, CATCs, and the terminal end of KSHV's dsDNA genome. Our atomic models of the portal and capsid/CATC, together with visualization of CATCs' variable occupancy and alternate orientation of CATC-interacting vertex triplexes, suggest a mechanism whereby the portal orchestrates procapsid formation and asymmetric long-range determination of CATC attachment during DNA packaging prior to pleomorphic tegumentation/envelopment. Structure-based mutageneses confirm that a triplex deep binding groove for CATCs is a hotspot that holds promise for antiviral development.
Subject(s)
Capsid Proteins/chemistry , Capsid/metabolism , DNA Packaging , Herpesvirus 8, Human/chemistry , Herpesvirus 8, Human/physiology , Sarcoma, Kaposi/virology , Virus Assembly , Cryoelectron Microscopy/methods , DNA, Viral/metabolism , Genome, Viral , Humans , Models, MolecularABSTRACT
CRISPR-Cas systems confer an adaptive immunity against viruses. Following viral injection, Cas1-Cas2 integrates segments of the viral genome (spacers) into the CRISPR locus. In type I CRISPR-Cas systems, efficient "primed" spacer acquisition and viral degradation (interference) require both the Cascade complex and the Cas3 helicase/nuclease. Here, we present single-molecule characterization of the Thermobifida fusca (Tfu) primed acquisition complex (PAC). We show that TfuCascade rapidly samples non-specific DNA via facilitated one-dimensional diffusion. Cas3 loads at target-bound Cascade and the Cascade/Cas3 complex translocates via a looped DNA intermediate. Cascade/Cas3 complexes stall at diverse protein roadblocks, resulting in a double strand break at the stall site. In contrast, Cas1-Cas2 samples DNA transiently via 3D collisions. Moreover, Cas1-Cas2 associates with Cascade and translocates with Cascade/Cas3, forming the PAC. PACs can displace different protein roadblocks, suggesting a mechanism for long-range spacer acquisition. This work provides a molecular basis for the coordinated steps in CRISPR-based adaptive immunity.
Subject(s)
Actinomycetales/enzymology , CRISPR-Associated Proteins/metabolism , CRISPR-Cas Systems , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , CRISPR-Associated Proteins/chemistry , DNA, Viral/metabolism , Protein Multimerization , Single Molecule ImagingABSTRACT
Self-nonself discrimination is vital for the immune system to mount responses against pathogens while maintaining tolerance toward the host and innocuous commensals during homeostasis. Here, we investigated how indiscriminate DNA sensors, such as cyclic GMP-AMP synthase (cGAS), make this self-nonself distinction. Screening of a small-molecule library revealed that spermine, a well-known DNA condenser associated with viral DNA, markedly elevates cGAS activation. Mechanistically, spermine condenses DNA to enhance and stabilize cGAS-DNA binding, optimizing cGAS and downstream antiviral signaling. Spermine promotes condensation of viral, but not host nucleosome, DNA. Deletion of viral DNA-associated spermine, by propagating virus in spermine-deficient cells, reduced cGAS activation. Spermine depletion subsequently attenuated cGAS-mediated antiviral and anticancer immunity. Collectively, our results reveal a pathogenic DNA-associated molecular pattern that facilitates nonself recognition, linking metabolism and pathogen recognition.
Subject(s)
DNA, Viral , Spermine , DNA, Viral/metabolism , Immunity, Innate , Antiviral Agents , Nucleotidyltransferases/metabolismABSTRACT
The retroviral enzyme integrase plays an essential role in the virus replication cycle by catalyzing the covalent insertion of newly synthesized viral DNA into the host cell chromosome early after infection. Now, Kessl et al. report a second function of integrase: binding to the viral RNA genome in virion particles late in the virus replication cycle to promote particle maturation.
Subject(s)
HIV-1/enzymology , RNA, Viral/genetics , Cell Line , DNA, Viral/metabolism , Virion/genetics , Virus Replication/geneticsABSTRACT
CRISPR-Cas adaptive immune systems protect bacteria and archaea against foreign genetic elements. In Escherichia coli, Cascade (CRISPR-associated complex for antiviral defense) is an RNA-guided surveillance complex that binds foreign DNA and recruits Cas3, a trans-acting nuclease helicase for target degradation. Here, we use single-molecule imaging to visualize Cascade and Cas3 binding to foreign DNA targets. Our analysis reveals two distinct pathways dictated by the presence or absence of a protospacer-adjacent motif (PAM). Binding to a protospacer flanked by a PAM recruits a nuclease-active Cas3 for degradation of short single-stranded regions of target DNA, whereas PAM mutations elicit an alternative pathway that recruits a nuclease-inactive Cas3 through a mechanism that is dependent on the Cas1 and Cas2 proteins. These findings explain how target recognition by Cascade can elicit distinct outcomes and support a model for acquisition of new spacer sequences through a mechanism involving processive, ATP-dependent Cas3 translocation along foreign DNA.
Subject(s)
Bacteriophage lambda/genetics , CRISPR-Associated Proteins/metabolism , CRISPR-Cas Systems , DNA Helicases/metabolism , DNA, Viral/metabolism , Endodeoxyribonucleases/metabolism , Endonucleases/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/virology , Escherichia coli/immunology , Escherichia coli/metabolism , Models, Biological , Repetitive Sequences, Nucleic AcidABSTRACT
It was recently shown that bacteria use, apart from CRISPR-Cas and restriction systems, a considerable diversity of phage resistance systems1-4, but it is largely unknown how phages cope with this multilayered bacterial immunity. Here we analysed groups of closely related Bacillus phages that showed differential sensitivity to bacterial defence systems, and discovered four distinct families of anti-defence proteins that inhibit the Gabija, Thoeris and Hachiman systems. We show that these proteins Gad1, Gad2, Tad2 and Had1 efficiently cancel the defensive activity when co-expressed with the respective defence system or introduced into phage genomes. Homologues of these anti-defence proteins are found in hundreds of phages that infect taxonomically diverse bacterial species. We show that the anti-Gabija protein Gad1 blocks the ability of the Gabija defence complex to cleave phage-derived DNA. Our data further reveal that the anti-Thoeris protein Tad2 is a 'sponge' that sequesters the immune signalling molecules produced by Thoeris TIR-domain proteins in response to phage infection. Our results demonstrate that phages encode an arsenal of anti-defence proteins that can disable a variety of bacterial defence mechanisms.
Subject(s)
Bacillus Phages , Bacteria , Viral Proteins , Bacillus Phages/classification , Bacillus Phages/genetics , Bacillus Phages/immunology , Bacillus Phages/metabolism , Bacteria/classification , Bacteria/genetics , Bacteria/immunology , Bacteria/virology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA, Viral/genetics , DNA, Viral/metabolism , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
Bacteria encode hundreds of diverse defence systems that protect them from viral infection and inhibit phage propagation1-5. Gabija is one of the most prevalent anti-phage defence systems, occurring in more than 15% of all sequenced bacterial and archaeal genomes1,6,7, but the molecular basis of how Gabija defends cells from viral infection remains poorly understood. Here we use X-ray crystallography and cryo-electron microscopy (cryo-EM) to define how Gabija proteins assemble into a supramolecular complex of around 500 kDa that degrades phage DNA. Gabija protein A (GajA) is a DNA endonuclease that tetramerizes to form the core of the anti-phage defence complex. Two sets of Gabija protein B (GajB) dimers dock at opposite sides of the complex and create a 4:4 GajA-GajB assembly (hereafter, GajAB) that is essential for phage resistance in vivo. We show that a phage-encoded protein, Gabija anti-defence 1 (Gad1), directly binds to the Gabija GajAB complex and inactivates defence. A cryo-EM structure of the virally inhibited state shows that Gad1 forms an octameric web that encases the GajAB complex and inhibits DNA recognition and cleavage. Our results reveal the structural basis of assembly of the Gabija anti-phage defence complex and define a unique mechanism of viral immune evasion.
Subject(s)
Bacteria , Bacterial Proteins , Bacteriophages , Immune Evasion , Protein Multimerization , Bacteria/genetics , Bacteria/immunology , Bacteria/metabolism , Bacteria/virology , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/ultrastructure , Bacteriophages/genetics , Bacteriophages/immunology , Bacteriophages/metabolism , Cryoelectron Microscopy , Crystallography, X-Ray , Deoxyribonucleases/chemistry , Deoxyribonucleases/metabolism , Deoxyribonucleases/ultrastructure , DNA, Viral/chemistry , DNA, Viral/metabolism , DNA, Viral/ultrastructureABSTRACT
Cyclic GMP-AMP synthase (cGAS) senses aberrant DNA during infection, cancer and inflammatory disease, and initiates potent innate immune responses through the synthesis of 2'3'-cyclic GMP-AMP (cGAMP)1-7. The indiscriminate activity of cGAS towards DNA demands tight regulatory mechanisms that are necessary to maintain cell and tissue homeostasis under normal conditions. Inside the cell nucleus, anchoring to nucleosomes and competition with chromatin architectural proteins jointly prohibit cGAS activation by genomic DNA8-15. However, the fate of nuclear cGAS and its role in cell physiology remains unclear. Here we show that the ubiquitin proteasomal system (UPS) degrades nuclear cGAS in cycling cells. We identify SPSB3 as the cGAS-targeting substrate receptor that associates with the cullin-RING ubiquitin ligase 5 (CRL5) complex to ligate ubiquitin onto nuclear cGAS. A cryo-electron microscopy structure of nucleosome-bound cGAS in a complex with SPSB3 reveals a highly conserved Asn-Asn (NN) minimal degron motif at the C terminus of cGAS that directs SPSB3 recruitment, ubiquitylation and cGAS protein stability. Interference with SPSB3-regulated nuclear cGAS degradation primes cells for type I interferon signalling, conferring heightened protection against infection by DNA viruses. Our research defines protein degradation as a determinant of cGAS regulation in the nucleus and provides structural insights into an element of cGAS that is amenable to therapeutic exploitation.
Subject(s)
Nuclear Proteins , Nucleosomes , Nucleotidyltransferases , Proteolysis , Ubiquitin-Protein Ligases , Animals , Humans , Mice , Cell Nucleus/metabolism , Cryoelectron Microscopy , Degrons , DNA Virus Infections/immunology , DNA Viruses/immunology , DNA Viruses/metabolism , DNA, Viral/immunology , DNA, Viral/metabolism , Immunity, Innate , Innate Immunity Recognition , Interferon Type I/immunology , Nuclear Proteins/metabolism , Nucleosomes/chemistry , Nucleosomes/metabolism , Nucleosomes/ultrastructure , Nucleotidyltransferases/chemistry , Nucleotidyltransferases/metabolism , Nucleotidyltransferases/ultrastructure , Proteasome Endopeptidase Complex/metabolism , Protein Stability , Substrate Specificity , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/ultrastructure , UbiquitinationABSTRACT
A string of nucleotides confined within a protein capsid contains all the instructions necessary to make a functional virus particle, a virion. Although the structure of the protein capsid is known for many virus species1,2, the three-dimensional organization of viral genomes has mostly eluded experimental probes3,4. Here we report all-atom structural models of an HK97 virion5, including its entire 39,732 base pair genome, obtained through multiresolution simulations. Mimicking the action of a packaging motor6, the genome was gradually loaded into the capsid. The structure of the packaged capsid was then refined through simulations of increasing resolution, which produced a 26 million atom model of the complete virion, including water and ions confined within the capsid. DNA packaging occurs through a loop extrusion mechanism7 that produces globally different configurations of the packaged genome and gives each viral particle individual traits. Multiple microsecond-long all-atom simulations characterized the effect of the packaged genome on capsid structure, internal pressure, electrostatics and diffusion of water, ions and DNA, and revealed the structural imprints of the capsid onto the genome. Our approach can be generalized to obtain complete all-atom structural models of other virus species, thereby potentially revealing new drug targets at the genome-capsid interface.
Subject(s)
Bacteriophages , Capsid , DNA, Viral , Genome, Viral , Virion , Virus Assembly , Bacteriophages/chemistry , Bacteriophages/genetics , Bacteriophages/growth & development , Bacteriophages/metabolism , Capsid/chemistry , Capsid/metabolism , Capsid Proteins/chemistry , Capsid Proteins/metabolism , Diffusion , DNA, Viral/chemistry , DNA, Viral/genetics , DNA, Viral/metabolism , Ions/analysis , Ions/chemistry , Ions/metabolism , Static Electricity , Virion/chemistry , Virion/genetics , Virion/metabolism , Virus Assembly/genetics , Water/analysis , Water/chemistry , Water/metabolismABSTRACT
Multimeric, ring-shaped molecular motors rely on the coordinated action of their subunits to perform crucial biological functions. During these tasks, motors often change their operation in response to regulatory signals. Here, we investigate a viral packaging machine as it fills the capsid with DNA and encounters increasing internal pressure. We find that the motor rotates the DNA during packaging and that the rotation per base pair increases with filling. This change accompanies a reduction in the motor's step size. We propose that these adjustments preserve motor coordination by allowing one subunit to make periodic, specific, and regulatory contacts with the DNA. At high filling, we also observe the downregulation of the ATP-binding rate and the emergence of long-lived pauses, suggesting a throttling-down mechanism employed by the motor near the completion of packaging. This study illustrates how a biological motor adjusts its operation in response to changing conditions, while remaining highly coordinated.
Subject(s)
Bacillus Phages/physiology , Molecular Motor Proteins/metabolism , Viral Proteins/metabolism , Virus Assembly , Adenosine Triphosphate/metabolism , Capsid/chemistry , DNA, Viral/metabolismABSTRACT
Most bacteria in the biosphere are predicted to be polylysogens harbouring multiple prophages1-5. In studied systems, prophage induction from lysogeny to lysis is near-universally driven by DNA-damaging agents6. Thus, how co-residing prophages compete for cell resources if they respond to an identical trigger is unknown. Here we discover regulatory modules that control prophage induction independently of the DNA-damage cue. The modules bear little resemblance at the sequence level but share a regulatory logic by having a transcription factor that activates the expression of a neighbouring gene that encodes a small protein. The small protein inactivates the master repressor of lysis, which leads to induction. Polylysogens that harbour two prophages exposed to DNA damage release mixed populations of phages. Single-cell analyses reveal that this blend is a consequence of discrete subsets of cells producing one, the other or both phages. By contrast, induction through the DNA-damage-independent module results in cells producing only the phage sensitive to that specific cue. Thus, in the polylysogens tested, the stimulus used to induce lysis determines phage productivity. Considering the lack of potent DNA-damaging agents in natural habitats, additional phage-encoded sensory pathways to lysis likely have fundamental roles in phage-host biology and inter-prophage competition.
Subject(s)
Bacteria , Bacteriophages , Lysogeny , Prophages , Viral Proteins , Bacteriophages/genetics , Bacteriophages/metabolism , Lysogeny/genetics , Prophages/genetics , Prophages/metabolism , Viral Proteins/metabolism , Virus Activation/genetics , Bacteria/virology , DNA Damage , DNA, Viral/genetics , DNA, Viral/metabolism , Single-Cell Analysis , Transcription Factors/metabolism , Host-Pathogen InteractionsABSTRACT
Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Seventy years later, our understanding of viral genome delivery in prokaryotes remains limited, especially for short-tailed phages of the Podoviridae family. These viruses expel mysterious ejection proteins found inside the capsid to form a DNA-ejectosome for genome delivery into bacteria. Here, we reconstitute the phage T7 DNA-ejectosome components gp14, gp15, and gp16 and solve the periplasmic tunnel structure at 2.7 Å resolution. We find that gp14 forms an outer membrane pore, gp15 assembles into a 210 Å hexameric DNA tube spanning the host periplasm, and gp16 extends into the host cytoplasm forming a â¼4,200 residue hub. Gp16 promotes gp15 oligomerization, coordinating peptidoglycan hydrolysis, DNA binding, and lipid insertion. The reconstituted gp15:gp16 complex lacks channel-forming activity, suggesting that the pore for DNA passage forms only transiently during genome ejection.
Subject(s)
Bacteriophage T7/genetics , DNA, Viral/chemistry , Periplasm/chemistry , Viral Core Proteins/chemistry , Computational Biology , Cryoelectron Microscopy , Cytoplasm/chemistry , DNA, Viral/metabolism , Lipid Bilayers/metabolism , Periplasm/genetics , Periplasm/metabolism , Podoviridae/chemistry , Podoviridae/genetics , Viral Core Proteins/metabolismABSTRACT
Immune suppression is a crucial component of immunoregulation and a subgroup of nucleotide-binding domain (NBD), leucine-rich repeat (LRR)-containing proteins (NLRs) attenuate innate immunity. How this inhibitory function is controlled is unknown. A key question is whether microbial ligands can regulate this inhibition. NLRC3 is a negative regulator that attenuates type I interferon (IFN-I) response by sequestering and attenuating stimulator of interferon genes (STING) activation. Here, we report that NLRC3 binds viral DNA and other nucleic acids through its LRR domain. DNA binding to NLRC3 increases its ATPase activity, and ATP-binding by NLRC3 diminishes its interaction with STING, thus licensing an IFN-I response. This work uncovers a mechanism wherein viral nucleic acid binding releases an inhibitory innate receptor from its target.
Subject(s)
DNA, Viral/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Interferon Type I/metabolism , Membrane Proteins/metabolism , Animals , Cell Line , Cell Line, Tumor , HEK293 Cells , HeLa Cells , Humans , Immunity, Innate/immunology , Mice , Mice, Inbred C57BL , Nucleic Acids/metabolism , Protein Binding/immunologyABSTRACT
Tubulins are essential for the reproduction of many eukaryotic viruses, but historically, bacteriophage were assumed not to require a cytoskeleton. Here, we identify a tubulin-like protein, PhuZ, from bacteriophage 201φ2-1 and show that it forms filaments in vivo and in vitro. The PhuZ structure has a conserved tubulin fold, with an unusual, extended C terminus that we demonstrate to be critical for polymerization in vitro and in vivo. Longitudinal packing in the crystal lattice mimics packing observed by EM of in-vitro-formed filaments, indicating how interactions between the C terminus and the following monomer drive polymerization. PhuZ forms a filamentous array that is required for positioning phage DNA within the bacterial cell. Correct positioning to the cell center and optimal phage reproduction only occur when the PhuZ filament is dynamic. Thus, we show that PhuZ assembles a spindle-like array that functions analogously to the microtubule-based spindles of eukaryotes.
Subject(s)
Bacteriophages/physiology , Pseudomonas/virology , Tubulin/metabolism , Viral Proteins/metabolism , Amino Acid Sequence , Cytoskeleton/metabolism , DNA, Viral/metabolism , Guanosine Diphosphate/metabolism , Models, Molecular , Molecular Sequence Data , Pseudomonas/cytology , Sequence Alignment , Tubulin/chemistry , Tubulin/genetics , Viral Proteins/chemistry , Viral Proteins/geneticsABSTRACT
Phage-inducible chromosomal islands (PICIs) represent a novel and universal class of mobile genetic elements, which have broad impact on bacterial virulence. In spite of their relevance, how the Gram-negative PICIs hijack the phage machinery for their own specific packaging and how they block phage reproduction remains to be determined. Using genetic and structural analyses, we solve the mystery here by showing that the Gram-negative PICIs encode a protein that simultaneously performs these processes. This protein, which we have named Rpp (for redirecting phage packaging), interacts with the phage terminase small subunit, forming a heterocomplex. This complex is unable to recognize the phage DNA, blocking phage packaging, but specifically binds to the PICI genome, promoting PICI packaging. Our studies reveal the mechanism of action that allows PICI dissemination in nature, introducing a new paradigm in the understanding of the biology of pathogenicity islands and therefore of bacterial pathogen evolution.
Subject(s)
Bacteriophages/physiology , DNA, Viral/metabolism , Escherichia coli/virology , Genomic Islands , Virus Assembly/physiology , DNA, Viral/genetics , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
DNA recognition is critical for assembly of double-stranded DNA viruses, particularly for the initiation of packaging the viral genome into the capsid. The key component that recognizes viral DNA is the small terminase protein. Despite prior studies, the molecular mechanism for DNA recognition remained elusive. Here, we address this question by identifying the minimal site in the bacteriophage HK97 genome specifically recognized by the small terminase and determining the structure of this complex by cryoEM. The circular small terminase employs an entirely unexpected mechanism in which DNA transits through the central tunnel, and sequence-specific recognition takes place as it emerges. This recognition stems from a substructure formed by the N- and C-terminal segments of two adjacent protomers which are unstructured when DNA is absent. Such interaction ensures continuous engagement of the small terminase with DNA, enabling it to slide along the DNA while simultaneously monitoring its sequence. This mechanism allows locating and instigating packaging initiation and termination precisely at the specific cos sequence.
Subject(s)
DNA, Viral , Genome, Viral , DNA, Viral/genetics , DNA, Viral/metabolism , DNA, Viral/chemistry , Cryoelectron Microscopy , Endodeoxyribonucleases/metabolism , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/genetics , Models, Molecular , DNA Packaging , Virus Assembly/genetics , Bacteriophages/genetics , Viral Genome PackagingABSTRACT
Using an immunofluorescence assay based on CRISPR-dCas9-gRNA complexes that selectively bind to the HIV LTR (HIV Cas-FISH), we traced changes in HIV DNA localization in primary effector T cells from early infection until the cells become quiescent as they transition to memory cells. Unintegrated HIV DNA colocalized with CPSF6 and HIV capsid (CA, p24) was found in the cytoplasm and nuclear periphery at days 1 and 3 post infection. From days 3 to 7, most HIV DNA was distributed primarily in the nuclear intermediate euchromatic compartment and was transcribed. By day 21, the cells had entered quiescence, and HIV DNA accumulated in the perinucleolar compartment (PNC). The localization of proviruses to the PNC was blocked by integrase inhibitor Raltegravir, suggesting it was due to chromosomal rearrangements. During the reactivation of latently infected cells through the T cell receptor (TCR), nascent viral mRNA transcripts associated with HIV DNA in the PNC were detected. The viral trans-activator Tat and its regulatory partners, P-TEFb and 7SK snRNA, assembled in large interchromatin granule clusters near the provirus within 2 h of TCR activation. As T cell activation progressed, the HIV DNA shifted away from the PNC. HIV DNA in latently infected memory T cells from patients also accumulated in the PNC and showed identical patterns of nuclear rearrangements after cellular reactivation. Thus, in contrast to transformed cells where proviruses are found primarily at the nuclear periphery, in primary memory T cells, the nuclear architecture undergoes rearrangements that shape the transcriptional silencing and reactivation of proviral HIV.
Subject(s)
Cell Nucleus , HIV Infections , HIV-1 , Proviruses , Virus Activation , Virus Latency , Humans , Proviruses/genetics , Cell Nucleus/metabolism , Cell Nucleus/virology , HIV-1/genetics , HIV-1/physiology , HIV-1/metabolism , HIV Infections/virology , HIV Infections/metabolism , DNA, Viral/genetics , DNA, Viral/metabolism , HIV Long Terminal Repeat/geneticsABSTRACT
Human bocavirus 1 (HBoV1) is a human parvovirus that causes lower respiratory tract infections in young children. It contains a single-stranded (ss) DNA genome of ~5.5 kb that encodes a small noncoding RNA of 140 nucleotides known as bocavirus-encoded small RNA (BocaSR), in addition to viral proteins. Here, we determined the secondary structure of BocaSR in vivo by using DMS-MaPseq. Our findings reveal that BocaSR undergoes N6-methyladenosine (m6A) modification at multiple sites, which is critical for viral DNA replication in both dividing HEK293 cells and nondividing cells of the human airway epithelium. Mechanistically, we found that m6A-modified BocaSR serves as a mediator for recruiting Y-family DNA repair DNA polymerase (Pol) η and Pol κ likely through a direct interaction between BocaSR and the viral DNA replication origin at the right terminus of the viral genome. Thus, this report represents direct involvement of a viral small noncoding RNA in viral DNA replication through m6A modification.
Subject(s)
Adenosine , DNA Replication , DNA, Viral , DNA-Directed DNA Polymerase , RNA, Viral , Virus Replication , Humans , Adenosine/analogs & derivatives , Adenosine/metabolism , Virus Replication/genetics , DNA-Directed DNA Polymerase/metabolism , DNA-Directed DNA Polymerase/genetics , DNA, Viral/genetics , DNA, Viral/metabolism , HEK293 Cells , RNA, Viral/genetics , RNA, Viral/metabolism , Human bocavirus/genetics , Human bocavirus/metabolism , Genome, Viral/genetics , Parvoviridae Infections/virologyABSTRACT
People with HIV-1 (PWH) on antiretroviral therapy (ART) can maintain undetectable virus levels, but a small pool of infected cells persists. This pool is largely comprised of defective proviruses that may produce HIV-1 proteins but are incapable of making infectious virus, with only a fraction (~10%) of these cells harboring intact viral genomes, some of which produce infectious virus following ex vivo stimulation (i.e. inducible intact proviruses). A majority of the inducible proviruses that persist on ART are formed near the time of therapy initiation. Here we compared proviral DNA (assessed here as 3' half genomes amplified from total cellular DNA) and inducible replication competent viruses in the pool of infected cells that persists during ART to determine if the original infection of these cells occurred at comparable times prior to therapy initiation. Overall, the average percent of proviruses that formed late (i.e. around the time of ART initiation, 60%) did not differ from the average percent of replication competent inducible viruses that formed late (69%), and this was also true for proviral DNA that was hypermutated (57%). Further, there was no evidence that entry into the long-lived infected cell pool was impeded by the ability to use the CXCR4 coreceptor, nor was the formation of long-lived infected cells enhanced during primary infection, when viral loads are exceptionally high. We observed that infection of cells that transitioned to be long-lived was enhanced among people with a lower nadir CD4+ T cell count. Together these data suggest that the timing of infection of cells that become long-lived is impacted more by biological processes associated with immunodeficiency before ART than the replication competency and/or cellular tropism of the infecting virus or the intactness of the provirus. Further research is needed to determine the mechanistic link between immunodeficiency and the timing of infected cells transitioning to the long-lived pool, particularly whether this is due to differences in infected cell clearance, turnover rates and/or homeostatic proliferation before and after ART.