Polymerase Activity Research Articles

A human isolate of bovine H5N1 is transmissible and lethal in animal models.

The outbreak of clade 2.3.4.4b highly pathogenic avian influenza viruses of the H5N1 subtype (HPAI H5N1) in dairy cows in the US has so far resulted in spillover infections of at least thirteen farm workers1-3, who presented with mild respiratory symptoms or conjunctivitis, and one individual with no known animal exposure who was hospitalized but recovered3,4. Here, we characterized A/Texas/37/2024 (huTX37-H5N1), a virus isolated from the eyes of an infected farm worker who developed conjunctivitis5. huTX37-H5N1 replicated efficiently in primary human alveolar epithelial cells, but less efficiently in corneal epithelial cells. Despite causing mild disease in the infected worker, huTX37-H5N1 was lethal in mice and ferrets and spread systemically with high titres in respiratory and non-respiratory organs. Importantly, in four independent experiments in ferrets, huTX37-H5N1 transmitted via respiratory droplets in 17%-33% of transmission pairs and five of six exposed ferrets that became infected died. PB2-631L (encoded by bovine isolates), promoted influenza polymerase activity in human cells, suggesting a role in mammalian adaptation like that of PB2-627K (encoded by huTX37-H5N1). Additionally, bovine HPAI H5N1 viruses were found to be susceptible to polymerase inhibitors both in vitro and in mice. Thus, HPAI H5N1 virus derived from dairy cattle transmits by respiratory droplets in mammals without prior adaptation and causes lethal disease in animal models.

Modulating Polymerase Activity Through Light-Oxygen-Voltage Domain Insertion.

Biochemical reaction networks adapt to environmental conditions by sensing chemical or physical stimuli and using tightly controlled mechanisms. While most signals come from molecules, many cells can also sense and respond to light. Among the biomolecular structures that enable light sensing, we selected a light-oxygen-voltage (LOV) domain in a previous study that tested the engineering of novel regulatory mechanisms into a nucleic acid polymerase. In this follow-up study, we studied the activities of previously selected variants in kinetic detail, and we generated additional LOV-polymerase fusion variants based on further insertion criteria. Our results provide mechanistic insights into how LOV domain insertion influences polymerase activity in a light-responsive manner: All active and photoresponsive enzyme variants studied by us to date were partially inhibited (i. e., "turned off") after irradiation with blue light at 470 nm, which can be explained by specific obstructions of the polymerase entry or exit structures (substrate entry channels or product exit channels, or both). Although the effects observed are moderate, we anticipate further engineering strategies that could be used to improve the extent of switchability and possibly to develop a "turn-on mode" insertion.

Tetrameric INTS6-SOSS1 complex facilitates DNA:RNA hybrid autoregulation at double-strand breaks.

DNA double-strand breaks (DSBs) represent a lethal form of DNA damage that can trigger cell death or initiate oncogenesis. The activity of RNA polymerase II (RNAPII) at the break site is required for efficient DSB repair. However, the regulatory mechanisms governing the transcription cycle at DSBs are not well understood. Here, we show that Integrator complex subunit 6 (INTS6) associates with the heterotrimeric sensor of ssDNA (SOSS1) complex (comprising INTS3, INIP and hSSB1) to form the tetrameric SOSS1 complex. INTS6 binds to DNA:RNA hybrids and promotes Protein Phosphatase 2A (PP2A) recruitment to DSBs, facilitating the dephosphorylation of RNAPII. Furthermore, INTS6 prevents the accumulation of damage-associated RNA transcripts (DARTs)and the stabilization of DNA:RNA hybrids at DSB sites. INTS6 interacts with and promotes the recruitment of senataxin(SETX) to DSBs, facilitating the resolution of DNA:RNA hybrids/R-loops. Our results underscore the significance of the tetrameric SOSS1 complex in the autoregulation of DNA:RNA hybrids andefficient DNA repair.

DNA supercoiling enhances DNA condensation by ParB proteins.

The ParABS system plays a critical role in bacterial chromosome segregation. The key component of this system, ParB, loads and spreads along DNA to form a local protein-DNA condensate known as a partition complex. As bacterial chromosomes are heavily supercoiled due to the continuous action of RNA polymerases, topoisomerases and nucleoid-associated proteins, it is important to study the impact of DNA supercoiling on the ParB-DNA partition complex formation. Here, we use an in-vitro single-molecule assay to visualize ParB on supercoiled DNA. Unlike most DNA-binding proteins, individual ParB proteins are found to not pin plectonemes on supercoiled DNA, but freely diffuse along supercoiled DNA. We find that DNA supercoiling enhances ParB-DNA condensation, which initiates at lower ParB concentrations than on DNA that is torsionally relaxed. ParB proteins induce a DNA-protein condensate that strikingly absorbs all supercoiling writhe. Our findings provide mechanistic insights that have important implications for our understanding of bacterial chromosome organization and segregation.

Two amino acid residues in the N-terminal region of the polymerase acidic protein determine the virulence of Eurasian avian-like H1N1 swine influenza viruses in mice.

Reassortant Eurasian avian-like H1N1 (rEA H1N1) viruses carrying the internal genes of H1N1/2009 virus have been circulating in pigs for more than 10 years and have caused sporadic human infections. The enhanced virulence phenotype of the rEA H1N1 viruses highlights potential risks to public health. However, the molecular mechanism underlying the viral pathogenicity of the currently circulating rEA H1N1 viruses remains unclear. In this study, we found that two naturally isolated rEA H1N1 swine influenza viruses, A/swine/Liaoning/FX38/2017 (FX38) and A/swine/Liaoning/SY72/2018 (SY72), possessed similar genetic characteristics but exhibited significantly different pathogenicity in a mouse model. Using reverse genetics, we demonstrated that amino acid mutations at positions 100 and 122 in the polymerase acidic (PA) protein had individual and synergistic effects on the polymerase activity and viral replication capacity in vitro, as well as the viral pathogenicity in mice. Furthermore, we revealed that amino acid residue 100 in PA influenced the transcription of viral RNA (vRNA) by altering the endonuclease activity, and amino acid residue 122 affected the synthesis of complementary RNA and messenger RNA by altering the RNA-binding ability and endonuclease activity of the PA protein. Taken together, we identified that two naturally occurring amino acid mutations in PA derived from H1N1/2009 virus are crucial determinants of the virulence of rEA H1N1 viruses and revealed the differential mechanism by which these two mutations affect the transcription and replication of vRNA. These findings will extend our understanding of the roles of PA in the virulence of influenza A viruses.IMPORTANCEMultiple genetic determinants are involved in the virulence of influenza A viruses. In this study, we identified two naturally occurring amino acid mutations, located at residues 100 and 122 in the polymerase acidic (PA) protein, which are associated with viral polymerase activity, replication competence, and pathogenicity in mice. In particular, we clarified the specific mechanism by which the two residues play an important role in viral transcription and replication. These findings will help to improve understanding the functions of amino acid residues in the N-terminal region of the PA protein involved in the pathogenicity of influenza A viruses.

Structures of a DNA Polymerase Caught while Incorporating Responsive Dual-Functional Nucleotide Probes.

Functionalizing nucleic acids using DNA polymerases is essential in biophysical and biotechnology applications. This study focuses on understanding how DNA polymerases recognize and incorporate nucleotides with diverse chemical modifications, aiming to develop advanced nucleotide probes. We present the crystal structures of ternary complexes of Thermus aquaticus DNA polymerase (KlenTaq) with C5-heterocycle-modified environment-sensitive 2'-deoxyuridine-5'-triphosphate (dUTP) probes. These nucleotides include SedUTP, BFdUTP and FBFdUTP, which bear selenophene, benzofuran and fluorobenzofuran, respectively, at the C5 position of uracil, and exhibit high conformational sensitivity. SedUTP and FBFdUTP serve as dual-app probes, combining a fluorophore with X-ray anomalous scattering Se or 19F NMR labels. Our study reveals that the size of the heterocycle influences how DNA polymerase families A and B incorporate these modified nucleotides during single nucleotide incorporation and primer extension reactions. Remarkably, FBFdUTP's responsiveness enabled real-time monitoring of the binary complex formation and polymerase activity through fluorescence and 19F NMR. Comparative analysis of incorporation profiles, fluorescence, 19F NMR data, and crystal structures of ternary complexes highlights the enzyme's plasticity. Key insights are provided into the role of gatekeeper amino acids (Arg660 and Arg587) in accommodating and processing these modified substrates, offering a structural basis for next-generation nucleotide probe development.

Structures of a DNA Polymerase Caught while Incorporating Responsive Dual‐Functional Nucleotide Probes

Functionalizing nucleic acids using DNA polymerases is essential in biophysical and biotechnology applications. This study focuses on understanding how DNA polymerases recognize and incorporate nucleotides with diverse chemical modifications, aiming to develop advanced nucleotide probes. We present the crystal structures of ternary complexes of Thermus aquaticus DNA polymerase (KlenTaq) with C5‐heterocycle‐modified environment‐sensitive 2′‐deoxyuridine‐5′‐triphosphate (dUTP) probes. These nucleotides include SedUTP, BFdUTP and FBFdUTP, which bear selenophene, benzofuran and fluorobenzofuran, respectively, at the C5 position of uracil, and exhibit high conformational sensitivity. SedUTP and FBFdUTP serve as dual‐app probes, combining a fluorophore with X‐ray anomalous scattering Se or 19F NMR labels. Our study reveals that the size of the heterocycle influences how DNA polymerase families A and B incorporate these modified nucleotides during single nucleotide incorporation and primer extension reactions. Remarkably, FBFdUTP's responsiveness enabled real‐time monitoring of the binary complex formation and polymerase activity through fluorescence and 19F NMR. Comparative analysis of incorporation profiles, fluorescence, 19F NMR data, and crystal structures of ternary complexes highlights the enzyme's plasticity. Key insights are provided into the role of gatekeeper amino acids (Arg660 and Arg587) in accommodating and processing these modified substrates, offering a structural basis for next‐generation nucleotide probe development.

Resistance gene Ty-1 restricts TYLCV infection in tomato by increasing RNA silencing

A major antiviral mechanism in plants is mediated by RNA silencing through the action of DICER-like (DCL) proteins, which cleave dsRNA into discrete small RNA fragments, and ARGONAUTE (AGO) proteins, which use the small RNAs to target single-stranded RNA. RNA silencing can also be amplified through the action of RNA-dependent RNA polymerases (RDRs), which use single stranded RNA to generate dsRNA that in turn is targeted by DCL proteins. As a counter-defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that target different components in the RNA silencing pathway. The tomato Ty-1 gene confers resistance to the DNA virus tomato yellow leaf curl virus (TYLCV) and has been reported to encode an RDRγ protein. However, the molecular mechanisms by which Ty-1 controls TYLCV infection, including whether Ty-1 is involved in RNA silencing, are unknown. Here, by using a transient expression assay, we have confirmed that Ty-1 shows antiviral activity against TYLCV in Nicotiana benthamiana. Also, in transient expression-based silencing assays, Ty-1 augmented systemic transgene silencing in GFP transgenic N. benthamiana plants. Furthermore, co-expression of Ty-1 or other RDRγ proteins from N. benthamiana or Arabidopsis with various proteins resulted in lower protein expression. These results are consistent with a model wherein Ty-1-mediated resistance to TYLCV is due, at least in part, to an increase in RNA silencing activity.

Continuing evolution of H5N1 highly pathogenic avian influenza viruses of clade 2.3.2.1a G2 genotype in domestic poultry of Bangladesh during 2018–2021

ABSTRACT We characterized 15 H5N1 HPAI viruses from different small- and medium-scale poultry flocks across Bangladesh during 2018–2021 based on their complete genome sequences. The antigenic relatedness of H5N1 HPAI viruses from different timepoints was analysed. During 2020–2021, 42.11% of the flocks tested positive for at least one of the respiratory infections, with 15.79% showing influenza A virus, of which 8.77% tested positive for HPAIV H5N1. Co-infections with two to four pathogens were detected in 15.8% of flocks. Phylogeny and gene constellation analyses based on complete genome sequences of 15 HPAI viruses revealed the continuing circulation of H5 clade 2.3.2.1a genotype G2 viruses. In the HA protein of the study isolates, functionally meaningful mutations caused the loss of an N-linked glycosylation site (T156A), a modified antigenic site A (S141P), and a mutation in the receptor binding pocket (E193R/K). Consequently, antigenic analysis revealed a significant loss of cross-reactivity between viruses from different host species and periods. Most viruses displayed oseltamivir resistance markers at positions V96, I97, S227, and N275 (N1 numbering) of the NA protein. In addition, for the PB2, M1, and NS1 proteins, significant mutations were noticed that have been associated with polymerase activity and increased virulence for mammals in all study isolates. These results highlight the need for intensified genomic surveillance of HPAI circulating in poultry in Bangladesh and for establishing appropriate control measures to decrease the circulation of these viruses in poultry in the country.

Structure of the RNA-dependent RNA polymerase P2 from the cystovirus φ8

The replication of RNA viruses relies on the activity of RNA-dependent RNA polymerases (RdRps). Despite large variations in their genomic sequences, viral RdRps share a common architecture generally known as a closed right hand. The P2 polymerase of cystovirus φ6 is currently among the best characterized viral RdRps. This polymerase is responsible for carrying out both replication and transcription of the viral double-stranded RNA genome using de novo initiation. Despite the extensive biochemical and structural studies conducted on φ6 P2, further structural information on other cystoviral RdRps is crucial to elucidate the structural and functional diversity of viral RdRps. Here, we have determined the atomic X-ray structure of the RdRp P2 from the φ6-related cystovirus φ8 at 3Å resolution. This structure completes the existing set of structural information on the φ8 polymerase complex and sheds light on the difference and similarities with related cystoviral RdRps.

Navigating directed evolution efficiently: optimizing selection conditions and selection output analysis.

Directed evolution is a powerful tool that can bypass gaps in our understanding of the sequence-function relationship of proteins and still isolate variants with desired activities, properties, and substrate specificities. The rise of directed evolution platforms for polymerase engineering has accelerated the isolation of xenobiotic nucleic acid (XNA) synthetases and reverse transcriptases capable of processing a wide array of unnatural XNAs which have numerous therapeutic and biotechnological applications. Still, the current generation of XNA polymerases functions with significantly lower efficiency than the natural counterparts and retains a significant level of DNA polymerase activity which limits their in vivo applications. Although directed evolution approaches are continuously being developed and implemented to improve XNA polymerase engineering, the field lacks an in-depth analysis of the effect of selection parameters, library construction biases and sampling biases. Focusing on the directed evolution pipeline for DNA and XNA polymerase engineering, this work sets out a method for understanding the impact of selection conditions on selection success and efficiency. We also explore the influence of selection conditions on fidelity at the population and individual mutant level. Additionally, we explore the sequencing coverage requirements in directed evolution experiments, which differ from genome assembly and other -omics approaches. This analysis allowed us to identify the sequencing coverage threshold for the accurate and precise identification of significantly enriched mutants. Overall, this study introduces a robust methodology for optimizing selection protocols, which effectively streamlines selection processes by employing small libraries and cost-effective NGS sequencing. It provides valuable insights into critical considerations, thereby enhancing the overall effectiveness and efficiency of directed evolution strategies applicable to enzymes other than the ones considered here.

Revised mechanism of hydroxyurea-induced cell cycle arrest and an improved alternative

Replication stress describes endogenous and exogenous challenges to DNA replication in the S-phase. Stress during this critical process causes helicase-polymerase decoupling at replication forks, triggering the S-phase checkpoint, which orchestrates global replication fork stalling and delayed entry into G2. The replication stressor most often used to induce the checkpoint response in yeast is hydroxyurea (HU), a clinically used chemotherapeutic. The primary mechanism of S-phase checkpoint activation by HU has thus far been considered to be a reduction of deoxynucleotide triphosphate synthesis by inhibition of ribonucleotide reductase (RNR), leading to helicase-polymerase decoupling and subsequent activation of the checkpoint, facilitated by the replisome-associated mediator Mrc1. In contrast, we observe that HU causes cell cycle arrest in budding yeast independent of both the Mrc1-mediated replication checkpoint response and the Psk1-Mrc1 oxidative signaling pathway. We demonstrate a direct relationship between HU incubation and reactive oxygen species (ROS) production in yeast and human cells and show that antioxidants restore growth of yeast in HU. We further observe that ROS strongly inhibits the in vitro polymerase activity of replicative polymerases (Pols), Pol α, Pol δ, and Pol ε, causing polymerase complex dissociation and subsequent loss of DNA substrate binding, likely through oxidation of their integral iron-sulfur (Fe-S) clusters. Finally, we present "RNR-deg," a genetically engineered alternative to HU in yeast with greatly increased specificity of RNR inhibition, allowing researchers to achieve fast, nontoxic, and more readily reversible checkpoint activation compared to HU, avoiding harmful ROS generation and associated downstream cellular effects that may confound interpretation of results.

Remdesivir triphosphate is a valid substrate to initiate synthesis of DNA primers by human PrimPol

Remdesivir is a broad-spectrum antiviral drug which has been approved to treat COVID-19. Remdesivir is in fact a prodrug, which is metabolized in vivo into the active form remdesivir triphosphate (RTP), an analogue of adenosine triphosphate (ATP) with a cyano group substitution in the carbon 1’ of the ribose (1’-CN). RTP is a substrate for RNA synthesis and can be easily incorporated by viral RNA-dependent RNA polymerases (RdRp). Importantly, once remdesivir is incorporated (now monophosphate), it will act as a delayed chain terminator, thus blocking viral RNA synthesis. It has been reported that mitochondrial Polγ is also blocked in vitro by RTP, but the low impact in vivo on mitochondrial DNA replication stalling is likely due to repriming by the human DNA-directed DNA Primase/Polymerase (HsPrimPol), which also operates in mitochondria. In this work, we have tested if RTP is a valid substrate for both DNA primase and DNA polymerase activities of HsPrimPol, and its impact in the production of mature DNA primers. RTP resulted to be an invalid substrate for elongation, but it can be used to initiate primers at the 5´site, competing with ATP. Nevertheless, RTP-initiated primers are abortive, ocassionally reaching a maximal length of 4–5 nucleotides, and do not support elongation mediated by primer/template distortions. However, considering that the concentration of ATP, the natural substrate, is much higher than the intracellular concentration of RTP, it is unlikely that HsPrimPol would use RTP for primer synthesis during a remdesivir treatment in real patients.

Expression of thermostable MMLV reverse transcriptase in Escherichia coli by directed mutation

The functionality of Moloney murine leukemia virus reverse transcriptase (MMLV RT) will increase with the improvement of its solubility and thermal stability. Introduce directed mutation at specific positions of the MMLV RT sequence and codon optimization is needed to achieve these properties. The two RT coding sequences with (rRT-K) and without directed mutations (rRT-L) were versatility optimized and expressed to analyze the ribonuclease H (RNase H) inactivity and thermostable polymerase activity. For this purpose, the five-point mutations (438–442aa) and three-point mutations (530, 568, and 659 aa) were done at the RT connection domain and RNase H active site, respectively. High expression levels of rRT-L and rRT-K were obtained in E. coli BL21(DE3) and BL21(shuffle) strains, 0.5 mM IPTG concentration at 37 °C, and 8 hours’ post-induction condition. Then, recombinant enzymes were purified and verified by Ni-NTA resin and western blotting. Insilico analysis (IUpred 3.0) showed that the directed mutation in the RNase H domain caused the formation of disorder regions or instability in the RNase H domain of rRT-K compared to rRT-L. The modified RT-PCR and the RT-LAMP reactions proved the RNase H inactivity of rRT-K. In addition, increasing of thermostability of rRT-K compared to rRT-L and commercial RT was evaluated by the RT-PCR and RT-LAMP reactions. The results showed that rRT-K could successfully tolerate 60 ºC in the two methods. This study revealed that the directed mutations and the versatile sequence optimization can promise to produce thermostable commercial enzymes to decrease non-specific one-step RT-PCR and RT-LAMP products.

B-237 Engineered Reverse Transcriptases and Taq DNA Polymerases deliver robust yield in the presence of clinically relevant inhibitors

Abstract Background The past five years has seen explosive growth in molecular diagnostic assays that rely on sensitive and accurate detection of nucleic acids across a wide range of sample types. As the assays are required to become increasingly sensitive, the need for highly functional and purpose-built enzymatic tools has grown. Reverse transcriptase (RT) and Taq DNA Polymerase (Taq DNAP) are commonly used to detect RNA and/or DNA viruses in patient samples for point of care (POC) diagnostics. However, crude patient samples introduce inhibitors that decrease enzyme activity and increase false negative results in POC testing. In addition, there is a need for faster turn-around time for patient diagnosis which can be enabled by engineering more active and sensitive Taq variants. Herein, we present on our engineered RT and Taq DNAP enzymes that were developed to confer greater activity, thermostability, inhibitor tolerance and/or sensitivity. Methods We take a 3-pronged approach to protein engineering: rational design, computational design and directed evolution. Using these techniques, we identified and characterized many RT and Taq DNAP variants. We deeply characterized each RT variant by measuring processivity, inhibitor tolerance and thermostability. First strand synthesis was done at temperatures from 42-65°C in the presence and absence of various clinically relevant inhibitors followed by qPCR to assess cDNA yield. Next, we characterized the inhibitor tolerance of the Taq variants by performing qPCR reactions in the presence of inhibitors. To determine if the Taq variant would be an appropriate fit for fast PCR, the polymerization activity of the Taq DNAP variants were characterized using a primer extension assay. Results Reverse transcriptases termed; StellarScript, StellarScript HT and StellarScript HT+ exhibit high processivity at 42°C. StellarScript HT+ exhibited the highest inhibitor tolerance, processivity and thermostability at temperatures up to 65°C thus making StellarScript HT+ well suited to produce high yields with clinical samples. Taq Pol variants were identified that efficiently amplify DNA in the presence of inhibitors. Conclusions Based on the increased polymerase activity and inhibitor tolerance, WMG Taq variants are well suited to give robust DNA yields with clinical samples. We were successful in engineering RT and Taq DNAP variants that are highly thermostable, active and inhibitor tolerant and thus will enable breakthroughs in existing POC diagnostics.

Effect of mutations in GDNV motif of viral hemorrhagic septicemia virus (VHSV) L protein on polymerase activity, viral growth, and in vivo virulence

Most Mononegavirales viruses have a GDNQ motif within the L protein, whereas Novirhabdovirus species feature a GDNV motif. This study examined the function of the GDNV motif within the L protein of viral hemorrhagic septicemia virus (VHSV) by modifying its amino acid composition. Substituting the aspartic acid (D) with valine (V) completely abolished polymerase activity in a minigenome assay. Replacing GDNV with GDNQ showed no significant difference in luciferase activity. Further characterization using reverse genetically engineered recombinant viruses revealed that rVHSV-LGDNQ exhibited an accelerated replication rate and higher virus titer in EPC cells than rVHSV-wild. Olive flounder infected with rVHSV-LGDNQ experienced higher early-stage mortality but lower overall mortality than those infected with rVHSV-wild. These findings suggest that while the GDNQ motif may positively influence VHSV replication speed, it may not confer an overall advantage for the ultimate viral pathogenicity.

TRiC/CCT Chaperonin Governs RNA Polymerase II Activity in the Nucleus to Support RNA Homeostasis.

The chaperonin TRiC/CCT is a large hetero-oligomeric ringed-structure that is essential in eukaryotes. While present in the nucleus, TRiC/CCT is typically considered to function in the cytosol where it mediates nascent polypeptide folding and the assembly/disassembly of protein complexes. Here, we investigated the nuclear role of TRiC/CCT. Inactivation of TRiC/CCT resulted in a significant increase in the production of nascent RNA leading to the accumulation of noncoding transcripts. The influence on transcription was not due to cytoplasmic TRiC/CCT-activities or other nuclear proteins as the effect was observed when TRiC/CCT was evicted from the nucleus and restricted to the cytoplasm. Rather, our data support a direct role of TRiC/CCT in regulating RNA polymerase II activity, as the chaperonin modulated nascent RNA production both in vivo and in vitro. Overall, our studies reveal a new avenue by which TRiC/CCT contributes to cell homeostasis by regulating the activity of nuclear RNA polymerase II.

Herpes simplex virus 1 inhibits phosphorylation of RNA polymerase II CTD serine-7.

Transcriptional activity of RNA polymerase II (Pol II) is influenced by post-translational modifications of the C-terminal domain (CTD) of the largest Pol II subunit, RPB1. Herpes simplex virus type 1 (HSV-1) usurps the cellular transcriptional machinery during lytic infection to efficiently express viral mRNA and shut down host gene expression. The viral immediate-early protein ICP22 interferes with serine 2 phosphorylation (pS2) by targeting CDK9 and other CDKs, but the full functional implications of this are not well understood. Using Western blotting, we report that HSV-1 also induces a loss of serine 7 phosphorylation (pS7) of the CTD during lytic infection, requiring expression of the two immediate-early proteins ICP22 and ICP27. ICP27 has also been proposed to target RPB1 for degradation, but we show that pS2/S7 loss precedes the drop in total protein levels. Cells with the RPB1 polyubiquitination site mutation K1268R, preventing proteasomal degradation during transcription-coupled DNA repair, displayed loss of pS2/S7 but retained higher overall RPB1 protein levels later in infection, indicating this pathway is not involved in early CTD dysregulation but may mediate bulk protein loss later. Using α-amanitin-resistant CTD mutants, we observed differential requirements for Ser2 and Ser7 for the production of viral proteins, with Ser2 facilitating viral immediate-early genes and Ser7 appearing dispensable. Despite dysregulation of CTD phosphorylation and different requirements for Ser2/7, all CTD modifications tested could be visualized in viral replication compartments with immunofluorescence. These data expand the known means that HSV employs to create pro-viral transcriptional environments at the expense of host responses.IMPORTANCECells rapidly induce changes in the transcription of RNA in response to stress and pathogens. Herpes simplex virus (HSV) disrupts many processes of host mRNA transcription, and it is necessary to separate the actions of viral proteins from cellular responses. Here, we demonstrate that viral proteins inhibit two key phosphorylation patterns on the C-terminal domain (CTD) of cellular RNA polymerase II and that this is separate from the degradation of polymerases later in infection. Furthermore, we show that viral genes do not require the full "CTD code." Together, these data distinguish multiple steps in the remodeling of RNA polymerase during infection and suggest that shared transcriptional phenotypes during stress responses do not revolve around a core disruption of CTD modifications.

Optineurin inhibits IBDV replication via interacting with VP1

Avibirnavirus, specifically Infectious Bursal Disease Virus (IBDV), is a highly contagious pathogen that causes significant economic losses in the poultry industry. The polymerase protein VP1 of IBDV is critical to the viral life cycle, facilitating the synthesis of viral mRNA and the genome. Previous studies have suggested that various host factors influence the regulation of IBDV polymerase activity. In this study, we identified that IBDV infection induces the expression of optineurin (OPTN), a mitophagy receptor and a protein associated with amyotrophic lateral sclerosis (ALS), as well as a negative regulator of interferon I production. The induced expression of OPTN acts as a suppressor of IBDV replication, a function dependent on its ubiquitin-binding domain (UBAN). Furthermore, we demonstrated that OPTN exerts its antiviral effects through direct interactions with VP1 and VP3, which inhibit the polymerase activity of VP1 by preventing K63-linked ubiquitination of VP1. To our knowledge, this study is the first to report that OPTN, upregulated during IBDV infection, functions as a novel antiviral host factor that limits the virus's replicative capacity, offering a potential target for anti-IBDV therapeutic strategies.

Evidence that DNA polymerase δ proofreads errors made by DNA polymerase α across the Saccharomyces cerevisiae nuclear genome

We show that the rates of single base substitutions, additions, and deletions across the nuclear genome are strongly increased in a strain harboring a mutator variant of DNA polymerase α combined with a mutation that inactivates the 3´-5´ exonuclease activity of DNA polymerase δ. Moreover, tetrad dissections attempting to produce a haploid triple mutant lacking Msh6, which is essential for DNA mismatch repair (MMR) of base•base mismatches made during replication, result in tiny colonies that grow very slowly and appear to be aneuploid and/or defective in oxidative metabolism. These observations are consistent with the hypothesis that during initiation of nuclear DNA replication, single-base mismatches made by naturally exonuclease-deficient DNA polymerase α are extrinsically proofread by DNA polymerase δ, such that in the absence of this proofreading, the mutation rate is strongly elevated. Several implications of these data are discussed, including that the mutational signature of defective extrinsic proofreading in yeast could appear in human tumors.