Polymerase Function Research Articles

MacP bypass variants of Streptococcus pneumoniae PBP2a suggest a conserved mechanism for the activation of bifunctional cell wall synthases.

Class A penicillin-binding proteins (aPBPs) play critical roles in bacterial cell wall biogenesis. As the targets of penicillin, they are among the most important drug targets in history. Although the biochemical activities of these enzymes have been well studied, little is known about how they are regulated in cells to control when and where peptidoglycan is made. In this report, we isolate variants of the Streptococcus pneumoniae enzyme PBP2a that function in cells without MacP, a partner normally required for its activity. The amino acid substitutions activate the cell wall synthase activity of PBP2a, and their location in a model structure suggests an activation mechanism for this enzyme that is shared with aPBPs from distantly related organisms with distinct activators.

Structural Insights into Phosphorylation-Mediated Polymerase Function Loss for DNA Polymerase β Bound to Gapped DNA.

DNA polymerase β is a member of the X-family of DNA polymerases, playing a critical role in the base excision repair (BER) pathway in mammalian cells by implementing the nucleotide gap-filling step. In vitro phosphorylation of DNA polymerase β with PKC on S44 causes loss in the enzyme's DNA polymerase activity but not single-strand DNA binding. Although these studies have shown that single-stranded DNA binding is not affected by phosphorylation, the structural basis behind the mechanism underlying phosphorylation-induced activity loss remains poorly understood. Previous modeling studies suggested phosphorylation of S44 was sufficient to induce structural changes that impact the enzyme's polymerase function. However, the S44 phosphorylated-enzyme/DNA complex has not been modeled so far. To address this knowledge gap, we conducted atomistic molecular dynamics simulations of pol β complexed with gapped DNA. Our simulations, which used explicit solvent and lasted for microseconds, revealed that phosphorylation at the S44 site, in the presence of Mg ions, induced significant conformational changes in the enzyme. Specifically, these changes led to the transformation of the enzyme from a closed to an open structure. Additionally, our simulations identified phosphorylation-induced allosteric coupling between the inter-domain region, suggesting the existence of a putative allosteric site. Taken together, our results provide a mechanistic understanding of the conformational transition observed due to phosphorylation in DNA polymerase β interactions with gapped DNA. Our simulations shed light on the mechanisms of phosphorylation-induced activity loss in DNA polymerase β and reveal potential targets for the development of novel therapeutics aimed at mitigating the effects of this post-translational modification.

Structures and Mechanisms of Nonsegmented, Negative-Strand RNA Virus Polymerases.

The nonsegmented, negative-strand RNA viruses (nsNSVs), also known as the order Mononegavirales, have a genome consisting of a single strand of negative-sense RNA. Integral to the nsNSV replication cycle is the viral polymerase, which is responsible for transcribing the viral genome, to produce an array of capped and polyadenylated messenger RNAs, and replicating it to produce new genomes. To perform the different steps that are necessary for these processes, the nsNSV polymerases undergo a series of coordinated conformational transitions. While much is still to be learned regarding the intersection of nsNSV polymerase dynamics, structure, and function, recently published polymerase structures, combined with a history of biochemical and molecular biology studies, have provided new insights into how nsNSV polymerases function as dynamic machines. In this review, we consider each of the steps involved in nsNSV transcription and replication and suggest how these relate to solved polymerase structures.

Phosphorylation of the PA subunit of influenza polymerase at Y393 prevents binding of the 5′-termini of RNA and polymerase function

The influenza A virus (IAV) polymerase is a multifunctional machine that can adopt alternative configurations to perform transcription and replication of the viral RNA genome in a temporally ordered manner. Although the structure of polymerase is well understood, our knowledge of its regulation by phosphorylation is still incomplete. The heterotrimeric polymerase can be regulated by posttranslational modifications, but the endogenously occurring phosphorylations at the PA and PB2 subunits of the IAV polymerase have not been studied. Mutation of phosphosites in PB2 and PA subunits revealed that PA mutants resembling constitutive phosphorylation have a partial (S395) or complete (Y393) defect in the ability to synthesize mRNA and cRNA. As PA phosphorylation at Y393 prevents binding of the 5′ promoter of the genomic RNA, recombinant viruses harboring such a mutation could not be rescued. These data show the functional relevance of PA phosphorylations to control the activity of viral polymerase during the influenza infectious cycle.

BosR and PlzA reciprocally regulate RpoS function to sustain Borrelia burgdorferi in ticks and mammals.

The RNA polymerase alternative σ factor RpoS in Borrelia burgdorferi (Bb), the Lyme disease pathogen, is responsible for programmatic-positive and -negative gene regulation essential for the spirochete's dual-host enzootic cycle. RpoS is expressed during tick-to-mammal transmission and throughout mammalian infection. Although the mammalian-phase RpoS regulon is well described, its counterpart during the transmission blood meal is unknown. Here, we used Bb-specific transcript enrichment by tick-borne disease capture sequencing (TBDCapSeq) to compare the transcriptomes of WT and ΔrpoS Bb in engorged nymphs and following mammalian host-adaptation within dialysis membrane chambers. TBDCapSeq revealed dramatic changes in the contours of the RpoS regulon within ticks and mammals and further confirmed that RpoS-mediated repression is specific to the mammalian-phase of Bb's enzootic cycle. We also provide evidence that RpoS-dependent gene regulation, including repression of tick-phase genes, is required for persistence in mice. Comparative transcriptomics of engineered Bb strains revealed that the Borrelia oxidative stress response regulator (BosR), a noncanonical Fur family member, and the cyclic diguanosine monophosphate (c-di-GMP) effector PlzA reciprocally regulate the function of RNA polymerase complexed with RpoS. BosR is required for RpoS-mediated transcription activation and repression in addition to its well-defined role promoting transcription of rpoS by the RNA polymerase alternative σ factor RpoN. During transmission, ligand-bound PlzA antagonizes RpoS-mediated repression, presumably acting through BosR.

PRMT5 Facilitates Infectious Bursal Disease Virus Replication through Arginine Methylation of VP1.

The infectious bursal diseases virus (IBDV) polymerase, VP1 protein, is responsible for transcription, initial translation and viral genomic replication. Knowledge about the new kind of post-translational modification of VP1 supports identification of novel drugs against the virus. Because the arginine residue is known to be methylated by protein arginine methyltransferase (PRMT) enzyme, we investigated whether IBDV VP1 is a substrate for known PRMTs. In this study, we show that VP1 is specifically associated with and methylated by PRMT5 at the arginine 426 (R426) residue. IBDV infection causes the accumulation of PRMT5 in the cytoplasm, which colocalizes with VP1 as a punctate structure. In addition, ectopic expression of PRMT5 significantly enhances the viral replication. In the presence of PMRT5, enzyme inhibitor and knockout of PRMT5 remarkably decreased viral replication. The polymerase activity of VP1 was severely damaged when R426 mutated to alanine, resulting in impaired viral replication. Our study reports a novel form of post-translational modification of VP1, which supports its polymerase function to facilitate the viral replication. IMPORTANCE Post-translational modification of infectious bursal disease virus (IBDV) VP1 is important for the regulation of its polymerase activity. Investigation of the significance of specific modification of VP1 can lead to better understanding of viral replication and can probably also help in identifying novel targets for antiviral compounds. Our work demonstrates the molecular mechanism of VP1 methylation mediated by PRMT5, which is critical for viral polymerase activity, as well as viral replication. Our study expands a novel insight into the function of arginine methylation of VP1, which might be useful for limiting the replication of IBDV.

Characterizing the genomic landscape of POLE/POLD1-mutated colorectal adenocarcinoma.

199 Background: The progression of neoplasia in colorectal cancer (CRC) has been well characterized, with the evolution typically involving mutations in APC, KRAS, and p53, among others. POLE/POLD1 genes encode for proteins essential in enzymatic DNA polymerase function, yet POLE/POLD1 mutations in tumors are poorly characterized. Pathogenic mutations in POLE/POLD1 lead to decreased fidelity of DNA replication, resulting in a high tumor mutational burden (TMB-H) independent of deficient mismatch repair (dMMR) and high microsatellite instability (MSI-H). Studies have shown associations between this hypermutated phenotype and susceptibility to immune checkpoint inhibition, which may be attributable to neoantigen creation. As such, these tumors are a clinically relevant phenotype requiring further investigation. Here, we characterized POLE/POLD1 alterations in a large, real-world cohort of patients with CRC. Methods: We retrospectively analyzed de-identified records of 9,136 primary CRC patients profiled with the Tempus xT assay. The assay includes DNA-seq of 595-648 genes at 500x to evaluate single-nucleotide variants, insertions/deletions, and copy number changes. Immunological markers analyzed included tumor mutational burden (TMB), MSI, and dMMR. MSI-H was determined by the assay through assessment of 239 loci. dMMR was determined by immunohistochemistry. Results: A total of 217 patient tumors harbored somatic POLE/POLD1 mutations (n = 203 POLE, n = 13 POLD1, and n = 1 POLE and POLD1; none germline), with no differences noted in baseline demographics including gender and age. Among the POLE/POLD1-mutant cohort, POLE copy number losses accounted for most mutations (n = 127, 59%), with short variants and copy number amplifications accounting for 35% (n = 76). The remainder of tumors exhibited POLD1 copy number changes (n = 14), including one with both POLE and POLD1 copy number loss. POLE/POLD1-mutated tumors presented with a lower frequency of MSI-H compared to wild-type (1.8% vs 6.1%, P= 0.018). Conversely, there was a higher frequency of TMB-high cases ( > 10 mut/Mb) for POLE/POLD1-mutated compared to wild-type tumors (22% vs 9.9%, P< 0.001). Differences between POLE/POLD1-mutated and wild-type tumors were also observed among many co-mutated genes, including APC (80% vs 65%, P< 0.001), ALK (31% vs 11%, P< 0.001), ATM (12% vs 3.6%, P< 0.001), BRCA2 (12% vs. 3.2%), and RET (24% vs 8.9%, P< 0.001). Conclusions: Patients with POLE/POLD1 mutations exhibited significant differences across immunological markers and molecular co-alterations. POLE/POLD1 mutations in CRC have been previously described to present with a hypermutated phenotype; however, 78% of tumors exhibited low TMB despite POLE/POLD1 mutations. Thus, these results have identified POLE/POLD1-mutated tumors as a unique genomic subpopulation, and further studies are needed to better characterize POLE/POLD1 mutations in CRC.

Functional Importance of the Hydrophobic Residue 362 in Influenza A PB1 Subunit.

PB1, acting as the catalytic subunit of the influenza polymerase, has numerous sequentially and structurally conserved regions. It has been observed that the slight modification of residues in PB1 would greatly affect the polymerase activity and even host adaptation ability. Here, we identified a critical residue, 362M, on the polymerase activity and virus replication. By means of the minireplicon assay, we assured the importance of the hydrophobicity of PB1 362, and the possibility that the size and charge of the side chain might directly interfere with the polymerase function. We also proposed a hydrophobic core between the PA-arch and the PB1 β-hairpin motifs and showed the importance of the core to the polymerase function.

Identification of POLR3B biallelic mutations-associated hypomyelinating leukodystrophy-8 in two siblings.

POLR3B gene encodes the 2nd largest catalytic subunit and affects the function of RNA polymerase III enzymes in transcription. Bi-allelic variants in POLR3B pathogenically cause hypomyelinating leukodystrophy-8 (HLD8). Herein, we recruited a family with two patients, who presented clinically with cerebellar atrophy, intellectual disability, hypogonadotropic hypogonadism, and visual problems. We identified the two affected siblings carrying the compound heterozygous variations (c.165_167del; c.1615G>T) in POLR3B by trio-whole-exome sequencing (trio-WES). The qPCR and western blot showed that both transcriptional and translational levels of the mutation (c.165_167del, p.I55_K56delinsM) were sharply attenuated. Following that, a thorough functional examination of a zebrafish line disrupted for human POLR3B validated the pathogenic effects of the two mutations. Our research broadens the spectrum of HLD8-related pathogenic POLR3B mutations and provides new molecular and animal evidence.

Predicting Modifiers of Genotype-Phenotype Correlations in Craniofacial Development.

Most human birth defects are phenotypically variable even when they share a common genetic basis. Our understanding of the mechanisms of this variation is limited, but they are thought to be due to complex gene-environment interactions. Loss of the transcription factor Gata3 associates with the highly variable human birth defects HDR syndrome and microsomia, and can lead to disruption of the neural crest-derived facial skeleton. We have demonstrated that zebrafish gata3 mutants model the variability seen in humans, with genetic background and candidate pathways modifying the resulting phenotype. In this study, we sought to use an unbiased bioinformatic approach to identify environmental modifiers of gata3 mutant craniofacial phenotypes. The LINCs L1000 dataset identifies chemicals that generate differential gene expression that either positively or negatively correlates with an input gene list. These chemicals are predicted to worsen or lessen the mutant phenotype, respectively. We performed RNA-seq on neural crest cells isolated from zebrafish across control, Gata3 loss-of-function, and Gata3 rescue groups. Differential expression analyses revealed 551 potential targets of gata3. We queried the LINCs database with the 100 most upregulated and 100 most downregulated genes. We tested the top eight available chemicals predicted to worsen the mutant phenotype and the top eight predicted to lessen the phenotype. Of these, we found that vinblastine, a microtubule inhibitor, and clofibric acid, a PPAR-alpha agonist, did indeed worsen the gata3 phenotype. The Topoisomerase II and RNA-pol II inhibitors daunorubicin and triptolide, respectively, lessened the phenotype. GO analysis identified Wnt signaling and RNA polymerase function as being enriched in our RNA-seq data, consistent with the mechanism of action of some of the chemicals. Our study illustrates multiple potential pathways for Gata3 function, and demonstrates a systematic, unbiased process to identify modifiers of genotype-phenotype correlations.

Synergistic HA and NS mutations enhanced the virulence of a mouse-adapted H1N1 influenza A virus

H1N1 reassortants between the swine Eurasian avian-like (EA) and H1N1 2009 pandemic (H1N1 pdm/09) viruses have been circulating stably in pig populations for more than ten years, and they may have contributed to increased human infections. Whether these H1N1 viruses acquire adaptive mutations to increase their pathogenicity towards a new host is unknown. To address this problem, mouse-adapted (MA) variants of swine-origin EA H1N1 influenza virus isolated from dogs (A/canine/Guangxi/LZ57/2015[LZ57-MA]) were generated by serial lung-to-lung passages in BALB/c mice. These exhibited greater virulence and replication capability than the wild-type virus (LZ57-WT). Of the six adaptive mutations, two were mapped to the ribonucleoprotein (RNP) complex (PB2-E578D and PA-T97I), two to hemagglutinin (HA-N198D and HA-A227E) and two to the non-structural protein 1 (NS1) and nuclear export protein (NS1-A53D and NEP-R42K, respectively). Reverse genetic substitution of the viral genes and mutation experiments demonstrated that the mutations in PA-T97I could enhance the polymerase activity, but a significant downregulation of activity was seen with PB2-E578D, which was consistent with a decrease in virulence. However, HA and NS, which are genes that act synergistically, were found to be determinants of virulence in mice. The reassortant viruses bearing HA mutations (N198D and A227E) were acquired during adaptation enhanced early-stage viral replication in mammalian cells. The single-point mutations in the NS genes had limited effects on virulence. Furthermore, a combination of HA (N198D and A227E) with NS(A53D) in the rLZ57-WT backbone resulted in efficient replication and a significant increase in virulence. The results suggest that these substitutions could compensate for the polymerase function and contribute to enhanced virulence, which highlights a major role for mutations in the HA and NS genes.

A REVIEW: ZINC AS AN ANTIVIRUS ALTERNATIVE TREATMENT FOR HERPES SIMPLEX VIRUS INFECTION

This study aimed to review zinc's effectiveness as an antivirus in treating herpes simplex virus infection. The authors use international journals published from 2000-2022, and use search engines such as Google Scholar, PubMed, and Science Direct with the keywords "zinc and herpes simplex virus". The herpes simplex virus that often causes symptoms in humans are HSV type 1 and type 2. The lesions appear as vesicles which then rupture into ulcers. Zinc is one of the most abundant nutrients or metals in the human body besides iron. Studies about the effects of zinc on HSV have shown that it has the function of inhibiting the viral life cycle. HSV attaches to the host cells to replicate and synthesize new viral proteins. Zinc can inhibit this process by depositing on the surface of the virion and inactivating the enzymatic function which is required for the attachment to the host cell, disrupting the surface glycoprotein of the viral membrane so it could not adhere and carry out the next life cycle, it can also inhibit the function of DNA polymerase that works for viral replication in the host cell. This article showed that zinc has effectiveness as an antivirus against the herpes simplex virus, therefore, patients infected with HSV can be treated with zinc as an alternative to an antivirus drug.

Progression of Antiviral Agents Targeting Viral Polymerases.

Viral DNA and RNA polymerases are two kinds of very important enzymes that synthesize the genetic materials of the virus itself, and they have become extremely favorable targets for the development of antiviral drugs because of their relatively conserved characteristics. There are many similarities in the structure and function of different viral polymerases, so inhibitors designed for a certain viral polymerase have acted as effective universal inhibitors on other types of viruses. The present review describes the development of classical antiviral drugs targeting polymerases, summarizes a variety of viral polymerase inhibitors from the perspective of chemically synthesized drugs and natural product drugs, describes novel approaches, and proposes promising development strategies for antiviral drugs.

DarT-mediated mtDNA damage induces dynamic reorganization and selective segregation of mitochondria.

Mitochondria are dynamic organelles that play essential roles in cell growth and survival. Processes of fission and fusion are critical for the distribution, segregation, and maintenance of mitochondria and their genomes (mtDNA). While recent work has revealed the significance of mitochondrial organization for mtDNA maintenance, the impact of mtDNA perturbations on mitochondrial dynamics remains less understood. Here, we develop a tool to induce mitochondria-specific DNA damage using a mitochondrial-targeted base modifying bacterial toxin, DarT. Following damage, we observe dynamic reorganization of mitochondrial networks, likely driven by mitochondrial dysfunction. Changes in the organization are associated with the loss of mtDNA, independent of mitophagy. Unexpectedly, perturbation to exonuclease function of mtDNA replicative polymerase, Mip1, results in rapid loss of mtDNA. Our data suggest that, under damage, partitioning of defective mtDNA and organelle are de-coupled, with emphasis on mitochondrial segregation independent of its DNA. Together, our work underscores the importance of genome maintenance on mitochondrial function, which can act as a modulator of organelle organization and segregation.

RdRp-scan: A bioinformatic resource to identify and annotate divergent RNA viruses in metagenomic sequence data.

Despite a rapid expansion in the number of documented viruses following the advent of metagenomic sequencing, the identification and annotation of highly divergent RNA viruses remain challenging, particularly from poorly characterized hosts and environmental samples. Protein structures are more conserved than primary sequence data, such that structure-based comparisons provide an opportunity to reveal the viral 'dusk matter': viral sequences with low, but detectable, levels of sequence identity to known viruses with available protein structures. Here, we present a new open computational resource-RdRp-scan-that contains a standardized bioinformatic toolkit to identify and annotate divergent RNA viruses in metagenomic sequence data based on the detection of RNA-dependent RNA polymerase (RdRp) sequences. By combining RdRp-specific hidden Markov models (HMMs) and structural comparisons, we show that RdRp-scan can efficiently detect RdRp sequences with identity levels as low as 10 per cent to those from known viruses and not identifiable using standard sequence-to-sequence comparisons. In addition, to facilitate the annotation and placement of newly detected and divergent virus-like sequences into the diversity of RNA viruses, RdRp-scan provides new custom and curated databases of viral RdRp sequences and core motifs, as well as pre-built RdRp multiple sequence alignments. In parallel, our analysis of the sequence diversity detected by the RdRp-scan revealed that while most of the taxonomically unassigned RdRps fell into pre-established clusters, some fell into potentially new orders of RNA viruses related to the Wolframvirales and Tolivirales. Finally, a survey of the conserved A, B, and C RdRp motifs within the RdRp-scan sequence database revealed additional variations of both sequence and position that might provide new insights into the structure, function, and evolution of viral polymerases.

RNA Polymerase II Transcription in Pneumocystis: TFIIB from Pneumocystis carinii Can Replace the Transcriptional Functions of Fission Yeast Schizosaccharomyces pombe TFIIB In Vivo and In Vitro.

The Pneumocystis genus is an opportunistic fungal pathogen that infects patients with AIDS and immunocompromised individuals. The study of this fungus has been hampered due to the inability to grow it in a (defined media/pure) culture. However, the use of modern molecular techniques and genomic analysis has helped researchers to understand its complex cell biology. The transcriptional process in the Pneumocystis genus has not been studied yet, although it is assumed that it has conventional transcriptional machinery. In this work, we have characterized the function of the RNA polymerase II (RNAPII) general transcription factor TFIIB from Pneumocystis carinii using the phylogenetically related biological model Schizosaccharomyces pombe. The results of this work show that Pneumocystis carinii TFIIB is able to replace the essential function of S. pombe TFIIB both in in vivo and in vitro assays. The S. pombe strain harboring the P carinii TFIIB grew slower than the parental wild-type S. pombe strain in complete media and in minimal media. The S. pombe cells carrying out the P. carinii TFIIB are larger than the wild-type cells, indicating that the TFIIB gene replacement confers a phenotype, most likely due to defects in transcription. P. carinii TFIIB forms very weak complexes with S. pombe TATA-binding protein on a TATA box promoter but it is able to form stable complexes in vitro when S. pombe TFIIF/RNAPII are added. P. carinii TFIIB can also replace the transcriptional function of S. pombe TFIIB in an in vitro assay. The transcription start sites (TSS) of the endogenous adh gene do not change when P. carinii TFIIB replaces S. pombe TFIIB, and neither does the TSS of the nmt1 gene, although this last gene is poorly transcribed in vivo in the presence of P. carinii TFIIB. Since transcription by RNA polymerase II in Pneumocystis is poorly understood, the results described in this study are promising and indicate that TFIIB from P. carinii can replace the transcriptional functions of S. pombe TFIIB, although the cells expressing the P. carini TFIIB show an altered phenotype. However, performing studies using a heterologous approach, like this one, could be relevant to understanding the basic molecular processes of Pneumocystis such as transcription and replication.

Abstract 5697: Targeting PARP inhibitor resistance with Polθ inhibitors

Abstract To target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining (TMEJ), without targeting Non-Homologous End Joining. Moreover, we show that exposure to ART558 can elicit DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumor cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which are a cause of PARP inhibitor resistance, result in in vitro and in vivo sensitivity to Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells. The inhibition of DNA nucleases that promote end-resection, such as Exo1 or Blm-Dna2 reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance. Citation Format: Diana Zatreanu, Helen Robinson, Omar Alkhatib, Marie Boursier, Harry Finch, Lerin Geo, Diego Grande, Vera Grinkevich, Robert Heald, Sophie Langdon, Jayesh Majithiya, Claire McWhirter, Niall Martin, Shaun Moore, Joana Neves, Eeson Rajendra, Marco Ranzani, Theresia Schaedler, Martin Stockley, Kimberley Wiggins, Rachel Brough, Sandhya Sridhar, Aditi Gulati, Nan Shao, Luted Badder, Daniela Novo, Eleanor Knight, Rebecca Marlow, Syed Haider, Elsa Callen, Graeme Hewitt, Joost Schimmel, Remko Prevo, Christina Alli, Amanda Ferdinand, Cameron Bell, Peter Blencowe, Chris Bot, Mathew Calder, Mark Charles, Jayne Curry, Tennyson Ekwuru, Andre Nussenzweig, Marcel Tijsterman, Andrew N. Tutt, Simon Boulton, Geoff Higgins, Stephen J. Pettitt, Graeme C. Smith, Christopher J. Lord. Targeting PARP inhibitor resistance with Polθ inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5697.

Conserved Viral Transcription Plays a Key Role in Virus-Like Particle Production of the Parasitoid Wasp Venturia canescens.

Nudiviruses are large double-stranded DNA viruses related to baculoviruses known to be endogenized in the genomes of certain parasitic wasp species. These wasp-virus associations allow the production of viral particles or virus-like particles that ensure wasp parasitism success within lepidopteran hosts. Venturia canescens is an ichneumonid wasp belonging to the Campopleginae subfamily that has endogenized nudivirus genes belonging to the Alphanudivirus genus to produce "virus-like particles" (Venturia canescens virus-like particles [VcVLPs]), which package proteic virulence factors. The main aim of this study was to determine whether alphanudivirus gene functions have been conserved following endogenization. The expression dynamics of alphanudivirus genes was monitored by a high throughput transcriptional approach, and the functional role of lef-4 and lef-8 genes predicted to encode viral RNA polymerase components was investigated by RNA interference. As described for baculovirus infections and for endogenized nudivirus genes in braconid wasp species producing bracoviruses, a transcriptional cascade involving early and late expressed alphanudivirus genes could be observed. The expression of lef-4 and lef-8 was also shown to be required for the expression of alphanudivirus late genes allowing correct particle formation. Together with previous literature, the results show that endogenization of nudiviruses in parasitoid wasps has repeatedly led to the conservation of the viral RNA polymerase function, allowing the production of viruses or viral-like particles that differ in composition but enable wasp parasitic success. IMPORTANCE This study shows that endogenization of a nudivirus genome in a Campopleginae parasitoid wasp has led to the conservation, as for endogenized nudiviruses in braconid parasitoid wasps, of the viral RNA polymerase function, required for the transcription of genes encoding viral particles involved in wasp parasitism success. We also showed for the first time that RNA interference (RNAi) can be successfully used to downregulate gene expression in this species, a model in behavioral ecology. This opens the opportunity to investigate the function of genes involved in other traits important for parasitism success, such as reproductive strategies and host choice. Fundamental data acquired on gene function in Venturia canescens are likely to be transferable to other parasitoid wasp species used in biological control programs. This study also renders possible the investigation of other nudivirus gene functions, for which little data are available.

Ubiquitination of Ebola virus VP35 at lysine 309 regulates viral transcription and assembly.

Ebola virus (EBOV) VP35 is a polyfunctional protein involved in viral genome packaging, viral polymerase function, and host immune antagonism. The mechanisms regulating VP35's engagement in different functions are not well-understood. We previously showed that the host E3 ubiquitin ligase TRIM6 ubiquitinates VP35 at lysine 309 (K309) to facilitate virus replication. However, how K309 ubiquitination regulates the function of VP35 as the viral polymerase co-factor and the precise stage(s) of the EBOV replication cycle that require VP35 ubiquitination are not known. Here, we generated recombinant EBOVs encoding glycine (G) or arginine (R) mutations at VP35/K309 (rEBOV-VP35/K309G/-R) and show that both mutations prohibit VP35/K309 ubiquitination. The K309R mutant retains dsRNA binding and efficient type-I Interferon (IFN-I) antagonism due to the basic residue conservation. The rEBOV-VP35/K309G mutant loses the ability to efficiently antagonize the IFN-I response, while the rEBOV-VP35/K309R mutant's suppression is enhanced. The replication of both mutants was significantly attenuated in both IFN-competent and -deficient cells due to impaired interactions with the viral polymerase. The lack of ubiquitination on VP35/K309 or TRIM6 deficiency disrupts viral transcription with increasing severity along the transcriptional gradient. This disruption of the transcriptional gradient results in unbalanced viral protein production, including reduced synthesis of the viral transcription factor VP30. In addition, lack of ubiquitination on K309 results in enhanced interactions with the viral nucleoprotein and premature nucleocapsid packaging, leading to dysregulation of virus assembly. Overall, we identified a novel role of VP35 ubiquitination in coordinating viral transcription and assembly.

Disrupting Transcription and Folate Biosynthesis Leads to Synergistic Suppression of Escherichia coli Growth

The use of synergistic antibiotic combinations has emerged as a viable approach to contain the rapid spread of antibiotic‐resistant pathogens. Here we report the discovery of a new strongly synergistic pair – microcin J25 and sulfamonomethoxine. The former is a lasso peptide that inhibits the function of RNA polymerase and the latter is a sulfonamide antibacterial agent that disrupts the folate pathway. Key to our discovery was a screening strategy that focuses on an antibiotic (microcin J25) that targets a hub (transcription) in the densely interconnected network of cellular pathways. The rationale was that disrupting such a hub likely weakens the entire network, generating weak links that potentiate the growth inhibitory effect of other antibiotics. We found that MccJ25 potentiates five other antibiotics as well. These results showcase the merit of taking a more targeted approach in the search and study of synergistic antibiotic pairs.