Biochemical and Biophysical Properties of a Putative Hub Protein Expressed by Vaccinia Virus
Abstract
H5 is a constitutively expressed, phosphorylated vaccinia virus protein that has been implicated in viral DNA replication, post-replicative gene expression, and virus assembly. For the purpose of understanding the role of H5 in vaccinia biology, we have characterized its biochemical and biophysical properties. Previously, we have demonstrated that H5 is associated with an endoribonucleolytic activity. In this study, we have shown that this cleavage results in a 3'OH end suitable for polyadenylation of the nascent transcript, corroborating a role for H5 in vaccinia transcription termination. Furthermore, we have shown that H5 is intrinsically disordered, with an elongated rod-shaped structure that preferentially binds double-stranded nucleic acids in a sequence non-specific manner. The dynamic phosphorylation status of H5 influences this structure and has implications for the role of H5 in multiple processes during virus replication.
... H5 is an abundant multifunctional protein that has been reported to interact with components of the DNA replication machinery as well as the postreplicative transcriptional machinery (24,25,28,30). In addition, the H5 protein copurifies with and may be associated with an enzymatic activity capable of processing the 3= termini of a subset of postreplicative viral mRNAs (53). In support of H5's contribution to transcription, it has also been shown to stimulate late transcription in a semipurified in vitro assay (54). ...
... In support of H5's contribution to transcription, it has also been shown to stimulate late transcription in a semipurified in vitro assay (54). Germane to each of these findings, the work of D'Costa, Condit, and colleagues has confirmed that the purified H5 protein exhibits nucleic acid binding activity, with the highest affinity being observed for dsDNA and double-stranded RNA (dsRNA) (K d s [dissociation constants] ϭ 12 and 14 nM, respectively) (53). ...
... Alternatively, the high-affinity association of H5 with the viral genome might help to organize the genomes within the factory so that they are readily accessible to the replication machinery. H5 is highly abundant, and we know that it forms higher-order structures, with the tetrameric form being the most stable form in vitro (53). H5 might in fact form a lattice that serves as a scaffold on which the genomes and/or the holoenzyme might become arrayed for efficient synthesis. ...
Unlabelled:
The duplication of the poxvirus double-stranded DNA genome occurs in cytoplasmic membrane-delimited factories. This physical autonomy from the host nucleus suggests that poxvirus genomes encode the full repertoire of proteins committed for genome replication. Biochemical and genetic analyses have confirmed that six viral proteins are required for efficient DNA synthesis; indirect evidence has suggested that the multifunctional H5 protein may also have a role. Here we show that H5 localizes to replication factories, as visualized by immunofluorescence and immunoelectron microscopy, and can be retrieved upon purification of the viral polymerase holoenzyme complex. The temperature-sensitive (ts) mutant Dts57, which was generated by chemical mutagenesis and has a lesion in H5, exhibits defects in DNA replication and morphogenesis under nonpermissive conditions, depending upon the experimental protocol. The H5 variant encoded by the genome of this mutant is ts for function but not stability. For a more precise investigation of how H5 contributes to DNA synthesis, we placed the ts57 H5 allele in an otherwise wild-type viral background and also performed small interfering RNA-mediated depletion of H5. Finally, we generated a complementing cell line, CV-1-H5, which allowed us to generate a viral recombinant in which the H5 open reading frame was deleted and replaced with mCherry (vΔH5). Analysis of vΔH5 allowed us to demonstrate conclusively that viral DNA replication is abrogated in the absence of H5. The loss of H5 does not compromise the accumulation of other early viral replication proteins or the uncoating of the virion core, suggesting that H5 plays a direct and essential role in facilitating DNA synthesis.
Importance:
Variola virus, the causative agent of smallpox, is the most notorious member of the Poxviridae family. Poxviruses are unique among DNA viruses that infect mammalian cells, in that their replication is restricted to the cytoplasm of the cell. This physical autonomy from the nucleus has both cell biological and genetic ramifications. Poxviruses must establish cytoplasmic niches that support replication, and the genomes must encode the repertoire of proteins necessary for genome synthesis. Here we focus on H5, a multifunctional and abundant viral protein. We confirm that H5 associates with the DNA polymerase holoenzyme and localizes to the sites of DNA synthesis. By generating an H5-expressing cell line, we were able to isolate a deletion virus that lacks the H5 gene and show definitively that genome synthesis does not occur in the absence of H5. These data support the hypothesis that H5 is a crucial participant in cytoplasmic poxvirus genome replication.
... In Poxviridae, H5 was predicted as the sole candidate to be a scaffold protein for PS. H5 is an essential protein for the infectious cycle of vaccinia virus [37] and was coined as a hub protein due to its importance in DNA replication, transcription and virion morphogenesis [38] . All those phenotypes correlate with a scaffolding function for PS. ...
... All those phenotypes correlate with a scaffolding function for PS. Moreover, H5 binds DNA [38] (function which is modulated by phosphorylation [39] ) and localizes as puncta upon heterologous expression in mammalian cells (indicating spontaneous PS of the protein) [37] . Interestingly, when vaccinia virus uncoating occurs, early viral proteins associated with DNA replication localize to cytoplasmic puncta, including H5 [40] [41] . ...
Phase separation is a common mechanism utilized by viruses to achieve replication, host manipulation and virion morphogenesis. The newly defined phylum _Nucleocytoviricota_ encompass ubiquitous and diverse viruses including _Poxviridae,_ the climate-modulating _Emiliania huxleyi_ virus and the previously termed Nucleocytoplasmic large DNA viruses (NCLDV). Cytoplasmic members of this phylum form viral factories but their nature remains unknow. Here we show that these viral factories are formed by phase separation. We demonstrate that mimivirus viral factories are formed by multilayered phase separation using at least two scaffold proteins. We also generate a pipeline to bioinformatically identify putative scaffold proteins in all other _Nucleocytoviricota_ despite major primary sequence variability. Such predictions were based on a conserved molecular grammar governed by electrostatic interactions. Scaffold candidates were validated for the family _Marseilleviridae_ and highlighted a role of H5 as a scaffold protein in poxviruses. Finally, we provide a repertoire of client proteins of the nucleus-like viral factory of mimivirus and demonstrate important sub-compartmentalization of functions including the central dogma. Overall, we reveal a new mechanism for the acquisition of nuclear-like functions entirely based on phase separation and re-classified phylum _Nucleocytoviricota_ viral factories as biomolecular condensates.
... In addition to the polymerase holoenzyme, the helicase-primase D5, the single-stranded DNA binding protein I3 and the phosphoprotein H5 [10] are required for DNA replication [6]. H5 forms tetramers, binds to DNA and RNA [11] and is supposed to form a hub for other interactors [11]. Roles in RNA processing, viral morphogenesis [12] and very recently as processivity factor of the DNA polymerase [13] have been shown. ...
... In addition to the polymerase holoenzyme, the helicase-primase D5, the single-stranded DNA binding protein I3 and the phosphoprotein H5 [10] are required for DNA replication [6]. H5 forms tetramers, binds to DNA and RNA [11] and is supposed to form a hub for other interactors [11]. Roles in RNA processing, viral morphogenesis [12] and very recently as processivity factor of the DNA polymerase [13] have been shown. ...
The year 2022 was marked by the mpox outbreak caused by the human monkeypox virus (MPXV), which is approximately 98% identical to the vaccinia virus (VACV) at the sequence level with regard to the proteins involved in DNA replication. We present the production in the baculovirus-insect cell system of the VACV DNA polymerase holoenzyme, which consists of the E9 polymerase in combination with its co-factor, the A20-D4 heterodimer. This led to the 3.8 Å cryo-electron microscopy (cryo-EM) structure of the DNA-free form of the holoenzyme. The model of the holoenzyme was constructed from high-resolution structures of the components of the complex and the A20 structure predicted by AlphaFold 2. The structures do not change in the context of the holoenzyme compared to the previously determined crystal and NMR structures, but the E9 thumb domain became disordered. The E9-A20-D4 structure shows the same compact arrangement with D4 folded back on E9 as observed for the recently solved MPXV holoenzyme structures in the presence and the absence of bound DNA. A conserved interface between E9 and D4 is formed by a cluster of hydrophobic residues. Small-angle X-ray scattering data show that other, more open conformations of E9-A20-D4 without the E9-D4 contact exist in solution using the flexibility of two hinge regions in A20. Biolayer interferometry (BLI) showed that the E9-D4 interaction is indeed weak and transient in the absence of DNA although it is very important, as it has not been possible to obtain viable viruses carrying mutations of key residues within the E9-D4 interface.
... D5, a AAA1 ATPase, is the putative primase/helicase that also plays an important role in the uncoating step that releases the genome from the intracellular virion core prior to replication (10-12). H5 has the hallmarks of a replication scaffold, or hub protein, and is essential for viral DNA replication (13,14). The A50 protein is a DNA ligase; in tissue culture cells, the loss of A50 can be complemented by the presence of the cellular DNA ligase I (15). ...
Poxviruses remain of significant interest because of their continuing clinical relevance, their utility for the development of vaccines and oncolytic therapies, and their illustration of fundamental principles of viral replication and virus/cell interactions. These viruses are unique in that they replicate exclusively in the cytoplasm of infected mammalian cells, providing novel challenges for DNA viruses.
... In addition, the virus encodes the D5 protein, a hexameric AAAϩ ATPase that has all of the sequence hallmarks of an SFIII helicase and has an N=-terminal primase domain (6,7). The H5 protein is an abundant phosphoprotein that interacts with numerous binding partners, including other replication proteins, DNA and RNA, and membranes (8,9); we have recently reported that the deletion virus vΔH5 shows a profound block to DNA replication in noncomplementing cells (10). The G5 protein is a member of the FEN-1-like family of structure-specific nucleases, and in its absence, the integrity of replication products is compromised. ...
Vaccinia virus is unusual among DNA viruses in replicating exclusively in the cytoplasm of infected cells. The single-stranded DNA (ssDNA) binding protein (SSB) I3 is among the replication machinery encoded by the 195-kb genome, although direct genetic analysis of I3 has been lacking. Herein, we describe a complementing cell line (CV1-I3) that fully supports the replication of a null virus (vΔI3) lacking the I3 open reading frame (ORF). In noncomplementing CV1-CAT cells, vΔI3 shows a severe defect in the production of infectious virus (≥200-fold reduction). Early protein synthesis and core disassembly occur normally. However, DNA replication is profoundly impaired (≤0.2% of wild-type [WT] levels), and late proteins do not accumulate. When several other noncomplementing cell lines are infected with vΔI3, the yield of infectious virus is also dramatically reduced (168- to 1,776-fold reduction). Surprisingly, the residual levels of DNA accumulation vary from 1 to 12% in the different cell lines (CV1-CAT < A549 < BSC40 < HeLa); however, any nascent DNA that can be detected is subgenomic in size. Although this subgenomic DNA supports late protein expression, it does not support the production of infectious virions. Electron microscopy (EM) analysis of vΔI3-infected BSC40 cells reveals that immature virions are abundant but no mature virions are observed. Aberrant virions characteristic of a block to genome encapsidation are seen instead. Finally, we demonstrate that a CV1 cell line encoding a previously described I3 variant with impaired ssDNA binding activity is unable to complement vΔI3. This report provides definitive evidence that the vaccinia virus I3 protein is the replicative SSB and is essential for productive viral replication.
IMPORTANCE
Poxviruses are of historical and contemporary importance as infectious agents, vaccines, and oncolytic therapeutics. The cytoplasmic replication of poxviruses is unique among DNA viruses of mammalian cells and necessitates that the double-stranded DNA (dsDNA) genome encode the viral replication machinery. This study focuses on the I3 protein. As a ssDNA binding protein (SSB), I3 has been presumed to play essential roles in genome replication, recombination, and repair, although genetic analysis has been lacking. Herein, we report the characterization of an I3 deletion virus. In the absence of I3 expression, DNA replication is severely compromised and viral yield profoundly decreased. The production of infectious virus can be restored in a cell line expressing WT I3 but not in a cell line expressing an I3 mutant that is defective in ssDNA binding activity. These data show conclusively that I3 is an essential viral protein and functions as the viral replicative SSB.
Multiple omics analyzes of Vaccinia virus (VACV) infection have defined molecular characteristics of poxvirus biology. However, little is known about the monkeypox (mpox) virus (MPXV) in humans, which has a different disease manifestation despite its high sequence similarity to VACV. Here, we perform an in-depth multi-omics analysis of the transcriptome, proteome, and phosphoproteome signatures of MPXV-infected primary human fibroblasts to gain insights into the virus-host interplay. In addition to expected perturbations of immune-related pathways, we uncover regulation of the HIPPO and TGF-β pathways. We identify dynamic phosphorylation of both host and viral proteins, which suggests that MAPKs are key regulators of differential phosphorylation in MPXV-infected cells. Among the viral proteins, we find dynamic phosphorylation of H5 that influenced the binding of H5 to dsDNA. Our extensive dataset highlights signaling events and hotspots perturbed by MPXV, extending the current knowledge on poxviruses. We use integrated pathway analysis and drug-target prediction approaches to identify potential drug targets that affect virus growth. Functionally, we exemplify the utility of this approach by identifying inhibitors of MTOR, CHUK/IKBKB, and splicing factor kinases with potent antiviral efficacy against MPXV and VACV.
Poxviruses, of which vaccinia virus is the prototype, are a large family of double-stranded DNA viruses that replicate exclusively in the cytoplasm of infected cells. This physical and genetic autonomy from the host cell nucleus necessitates that these viruses encode most, if not all, of the proteins required for replication in the cytoplasm. In this review, we follow the life of the viral genome through space and time to address some of the unique challenges that arise from replicating a 195-kb DNA genome in the cytoplasm. We focus on how the genome is released from the incoming virion and deposited into the cytoplasm; how the endoplasmic reticulum is reorganized to form a replication factory, thereby compartmentalizing and helping to protect the replicating genome from immune sensors; how the cellular milieu is tailored to support high-fidelity replication of the genome; and finally, how newly synthesized genomes are faithfully and specifically encapsidated into new virions.
Expected final online publication date for the Annual Review of Virology, Volume 9 is September 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
The vaccinia virus I3L gene encodes a single-stranded DNA binding protein (SSB) that is essential for virus DNA replication and is conserved in all Chordopoxviruses. The I3 protein contains a negatively charged C-terminal tail that is a common feature of SSBs. Such acidic tails are critical for SSB-dependent replication, recombination and repair. We cloned and purified variants of the I3 protein, along with a homolog from molluscum contagiosum virus, and tested how the acidic tail affected DNA–protein interactions. Deleting the C terminus of I3 enhanced the affinity for single-stranded DNA cellulose and gel shift analyses showed that it also altered the migration of I3-DNA complexes in agarose gels. Microinjecting an antibody against I3 into vaccinia-infected cells also selectively inhibited virus replication. We suggest that this domain promotes cooperative binding of I3 to DNA in a way that would maintain an open DNA configuration around a replication site.
Prior biochemical analysis of the heterodimeric vaccinia virus mRNA capping enzyme suggests roles not only in mRNA capping but also in early viral gene transcription termination and intermediate viral gene transcription initiation. Prior phenotypic characterization of Dts36, a temperature sensitive virus mutant affecting the large subunit of the capping enzyme was consistent with the multifunctional roles of the capping enzyme in vivo. We report a biochemical analysis of the capping enzyme encoded by Dts36. Of the three enzymatic activities required for mRNA capping, the guanylyltransferase and methyltransferase activities are compromised while the triphosphatase activity and the D12 subunit interaction are unaffected. The mutant enzyme is also defective in stimulating early gene transcription termination and intermediate gene transcription initiation in vitro. These results confirm that the vaccinia virus mRNA capping enzyme functions not only in mRNA capping but also early gene transcription termination and intermediate gene transcription initiation in vivo.
Swinholide A, isolated from the marine sponge Theonella swinhoei, is a 44-carbon ring dimeric dilactone macrolide with a 2-fold axis of symmetry. Recent studies have elucidated its unusual
structure and shown that it has potent cytotoxic activity. We now report that swinholide A disrupts the actin cytoskeleton
of cells grown in culture, sequesters actin dimers in vitro in both polymerizing and non-polymerizing buffers with a binding stoichiometry of one swinholide A molecule per actin dimer,
and rapidly severs F-actin in vitro with high cooperativity. These unique properties are sufficient to explain the cytotoxicity of swinholide A. They also suggest
that swinholide A might be a model for studies of the mechanism of action of F-actin severing proteins and be therapeutically
useful in conditions where filamentous actin contributes to pathologically high viscosities.
Poxviruses are large DNA viruses that replicate within the cytoplasm and encode a complete transcription system, including a multisubunit RNA polymerase, stage-specific transcription factors, capping and methylating enzymes, and a poly(A) polymerase. Expression of the more than 200 open reading frames by vaccinia virus, the prototype poxvirus, is temporally regulated: early mRNAs are synthesized immediately after infection, whereas intermediate and late mRNAs are synthesized following genome replication. The postreplicative transcripts are heterogeneous in length and overlap the entire genome, which pose obstacles for high resolution mapping. We used tag-based methods in conjunction with high throughput cDNA sequencing to determine the precise 5'-capped and 3'-polyadenylated ends of postreplicative RNAs. Polymerase slippage during initiation of intermediate and late RNA synthesis results in a 5'-poly(A) leader that allowed the unambiguous identification of true transcription start sites. Ninety RNA start sites were located just upstream of intermediate and late open reading frames, but many more appeared anomalous, occurring within coding and non-coding regions, indicating pervasive transcription initiation. We confirmed the presence of functional promoter sequences upstream of representative anomalous start sites and demonstrated that alternative start sites within open reading frames could generate truncated isoforms of proteins. In an analogous manner, poly(A) sequences allowed accurate mapping of the numerous 3'-ends of postreplicative RNAs, which were preceded by a pyrimidine-rich sequence in the DNA coding strand. The distribution of postreplicative promoter sequences throughout the genome provides enormous transcriptional complexity, and the large number of previously unmapped RNAs may have novel functions.
A fully open source software program for automated two-dimensional and one-dimensional data reduction and preliminary analysis of isotropic small-angle X-ray scattering (SAXS) data is presented. The program is freely distributed, following the open-source philosophy, and does not rely on any commercial software packages. BioXTAS RAW is a fully automated program that, via an online feature, reads raw two-dimensional SAXS detector output files and processes and plots data as the data files are created during measurement sessions. The software handles all steps in the data reduction. This includes mask creation, radial averaging, error bar calculation, artifact removal, normalization and q calibration. Further data reduction such as background subtraction and absolute intensity scaling is fast and easy via the graphical user interface. BioXTAS RAW also provides preliminary analysis of one-dimensional data in terms of the indirect Fourier transform using the objective Bayesian approach to obtain the pair-distance distribution function, PDDF, and is thereby a free and open-source alternative to existing PDDF estimation software. Apart from the TIFF input format, the program also accepts ASCII-format input files and is currently compatible with one-dimensional data files from SAXS beamlines at a number of synchrotron facilities. BioXTAS RAW is written in Python with C++ extensions.
Intrinsically disordered proteins and intrinsically disordered protein regions are highly abundant in nature. However, the quantitative and qualitative measures of protein intrinsic disorder in species with known genomes are still not available. Furthermore, although the correlation between high fraction of disordered residues and advanced species has been reported, the details of this correlation and the connection between the disorder content and proteome complexity have not been reported as of yet. To fill this gap, we analysed entire proteomes of 3484 species from three domains of life (archaea, bacteria and eukaryotes) and from viruses. Our analysis revealed that the evolution process is characterized by distinctive patterns of changes in the protein intrinsic disorder content. We are showing here that viruses are characterized by the widest spread of the proteome disorder content (the percentage of disordered residues ranges from 7.3% in human coronavirus NL63 to 77.3% in Avian carcinoma virus). For several organisms, a clear correlation is seen between their disorder contents and habitats. In multicellular eukaryotes, there is a weak correlation between the complexity of an organism (evaluated as a number of different cell types) and its overall disorder content. For both the prokaryotes and eukaryotes, the disorder content is generally independent of the proteome size. However, disorder shows a sharp increase associated with the transition from prokaryotic to eukaryotic cells. This suggests that the increased disorder content in eukaryotic proteomes might be used by nature to deal with the increased cell complexity due to the appearance of the various cellular compartments.
Coilin is known as the marker protein for Cajal bodies (CBs), subnuclear domains important for the biogenesis of small nuclear ribonucleoproteins (snRNPs) which function in pre-mRNA splicing. CBs associate non-randomly with U1 and U2 gene loci, which produce the small nuclear RNA (snRNA) component of the respective snRNP. Despite recognition as the CB marker protein, coilin is primarily nucleoplasmic, and the function of this fraction is not fully characterized. Here we show that coilin binds double stranded DNA and has RNase activity in vitro. U1 and U2 snRNAs undergo a processing event of the primary transcript prior to incorporation in the snRNP. We find that coilin displays RNase activity within the CU region of the U2 snRNA primary transcript in vitro, and that coilin knockdown results in accumulation of the 3' pre-processed U1 and U2 snRNA. These findings present new characteristics of coilin in vitro, and suggest additional functions of the protein in vivo.
The telomeres of orthopoxvirus DNAs consist largely of short repeated sequences organized into at least two separate sets. Although the sequence composition of the orthopoxvirus telomeres is highly conserved, these regions do not appear to encode any proteins. At late times during infection, the telomeres of vaccinia virus are transcribed. A promoter in the region between the two sets of repeats directs transcription towards the hairpin-loop end of the viral DNA. This promoter resembles the promoters of other poxvirus late genes, and directs the synthesis of RNAs whose structure is consistent with the presence of 5′ poly(A) sequences typical of late RNAs. The lengths of these late transcripts suggest that some transcription extends through the hairpin-loop region. This might occur either when the genome is in a monomeric form or when the genome is in the concatemeric form of the DNA replication intermediate. The function of late transcription of the telomeres is unclear, but similar transcription of the telomeres of vaccinia virus, cowpox virus, and raccoonpox virus suggests that such transcription may have a role in viral replication.
Deng and Shuman (J. Biol Chem.271, 29386 (1996)) reported that an ATPase different from the known viral termination factor, VTF, is required for vaccinia virus early gene transcription termination. Properties of this ATPase were similar to those of a known vaccinia virus enzyme, nucleoside triphosphate phosphohydrolase I (NPH I) the product of gene D11L. Transcription-competent cell-free extracts were prepared from A549 cells infected with wild-type or mutant vaccinia virus harboring ts mutations in gene D11L. These extracts were employed to investigate the role of NPH I in early gene transcription termination. Extracts prepared under nonpermissive conditions from both wild-type virus and ts mutant virus-infected cells exhibited high levels of early and intermediate gene transcription activity but were incapable of supporting late gene transcription. ts mutant extracts lacked signal-dependent early gene transcription termination activity, which was restored by the addition of either free NPH I or a GST-NPH I fusion protein. A comparison of the NPH I amino acid sequence to the protein databases revealed the presence of a set of sequences characteristic of nucleic acid helicase superfamily II members. A series of site-specific mutations in the helicase motifs and N-terminal and C-terminal deletion mutations were expressed as GST fusion proteins and their activities assessed. Of the mutations in helicase motifs I to VI, alteration of all but motif III reduced the ATPase activity. Removal of as few as 24 amino acids from the N-terminal end eliminated ATPase activity, while deletion of 68 C-terminal amino acids exhibited only a modest decrease in ATP hydrolysis. Larger C-terminal deletions eliminated ATPase activity. Each deletion mutation, and site-specific mutations other than the motif III mutation, failed to support transcription terminationin vitro.Mutations in motifs I, II, V, and VI inhibit wild-type NPH I transcription termination activity. However, deletion of up to 68 amino acids from the C-terminal end eliminates this inhibitory property. This observation is particularly interesting since these C-terminal deletions retain both ATPase activity and single-stranded DNA binding activity. Their failure to inhibit transcription termination suggests that these C-terminal deletion mutations eliminate a site required for a function other than from DNA binding or ATP hydrolysis.