RNA Polymerase Complex Research Articles

Characterization of the Conformational Hotspots of the RNA-Dependent RNA Polymerase Complex Identifies a Unique Structural Malleability of nsp8.

Several antiviral therapeutic approaches have been targeted toward the RNA-dependent RNA polymerase (RdRp) complex that is involved in viral genome replication. In SARS-CoV-2, although the RdRp is a multiprotein complex, the focus has been on the ligand binding catalytic core (nonstructural protein nsp12), and not the multiprotein functional dynamics. In this study, we focus on the conformational ensembles of the RdRp complex and their modulation by the presence of RNA, performing comprehensive microsecond-scale atomistic simulations of the apo- and RNA-bound complex. We delineate the differential impact of RNA on the constituent proteins, such as conformational polymorphisms, dominant segment-specific fluctuations, and the switch in dynamical crosstalk within the complex. We distinguish dynamical signatures of nsp7, nsp8, and nsp12 in the apo-state that are reduced in the presence of the RNA and appear to "prime" the complex for activity. Importantly, we identify a unique structural malleability of the nsp8 protein with high conformational heterogeneity in the apo state, especially at three sites (Y71 for nsp8A, and D52 and A66 for nsp8B). Our work highlights the functional implications of the polymorphism of nsp8 structures and reveals possibilities for the development of allosteric inhibitors.

Biochemical simulation of mutation synthesis and repair during SARS-CoV-2 RNA polymerization

We biochemically simulated the mutation synthesis process of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) complex (nsp7/nsp8/nsp12) involving two sequential mechanistic steps that occur during genomic replication: misinsertion (incorporation of incorrect nucleotides) and mismatch extension. Then, we also simulated mismatch repair process catalyzed by the viral nsp10/nsp14 ExoN complex. In these mechanistic simulations, while SARS-CoV-2 RdRp displays efficient mutation synthesis capability, the viral ExoN complex was able to effectively repair the mismatch primers generated during the mutation synthesis. Also, we observed that the delayed RNA synthesis induced by mutation synthesis process was rescued by the viral ExoN activity. Collectively, our biochemical simulations suggest that SARS-CoV-2 ExoN complex may contribute to both maintenance of proper viral genetic diversity levels and successful completion of the viral large RNA genome replication by removing mismatches generated by the viral RdRp.

Characterization of ACTN4 as a novel antiviral target against SARS-CoV-2

The various mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pose a substantial challenge in mitigating the viral infectivity. The identification of novel host factors influencing SARS-CoV-2 replication holds potential for discovering new targets for broad-spectrum antiviral drugs that can combat future viral mutations. In this study, potential host factors regulated by SARS-CoV-2 infection were screened through different high-throughput sequencing techniques and further identified in cells. Subsequent analysis and experiments showed that the reduction of m6A modification level on ACTN4 (Alpha-actinin-4) mRNA leads to a decrease in mRNA stability and translation efficiency, ultimately inhibiting ACTN4 expression. In addition, ACTN4 was demonstrated to target nsp12 for binding and characterized as a competitor for SARS-CoV-2 RNA and the RNA-dependent RNA polymerase complex, thereby impeding viral replication. Furthermore, two ACTN4 agonists, YS-49 and demethyl-coclaurine, were found to dose-dependently inhibit SARS-CoV-2 infection in both Huh7 cells and K18-hACE2 transgenic mice. Collectively, this study unveils the pivotal role of ACTN4 in SARS-CoV-2 infection, offering novel insights into the intricate interplay between the virus and host cells, and reveals two potential candidates for future anti-SARS-CoV-2 drug development.

Preparation and Antigenic Site Identification of Monoclonal Antibodies against PB1 Protein of H9N2 Subtype AIV.

Recently, low pathogenic avian influenza virus (LPAIV), including H9N2 subtype, has been common clinical epidemic strains, and is widely distributed globally. The PB1 protein is a key component of the viral RNA polymerase complex (vRNP), and is vital to viral transcription and translation. In this study, to investigate the antigenic determinants in the PB1 protein, the truncated PB1 sequence (1bp-735bp) from H9N2 subtype AIV was amplified with PCR, and expressed in plasmid pET-28a (+). After purification, the recombinant PB1 protein was used to immunize BALB/c mice. Following immunization, hybridoma cells producing PB1-specific monoclonal antibodies were generated through the fusion of splenic lymphocytes with SP2/0 cells. Then, four stable hybridoma cell lines (5F12, 5B3, 2H9, and 3E6) were screened using indirect ELISA and Western blotting. Furthermore, two antigenic sites, 67NPIDGPLPED76 and 97ESHPGIFENS106, were identified through the construction of truncated overlapping fragments of the PB1 protein. These sites were conserved among 28 AIV strains, and were located on the PB1 protein surface. The findings offer a theoretical reference for the development and improvement of H9N2 vaccines and offer biological materials for virus detection during AIV infection mechanisms.

Harnessing PROTACs to combat H5N1 influenza: A new frontier in viral destruction.

H5N1, a highly pathogenic avian influenza virus, poses an ongoing and significant threat to global public health, primarily due to its potential to cause severe respiratory illness and high mortality rates in humans. Despite extensive efforts in vaccination and antiviral therapy, H5N1 continues to exhibit high mutation rates, resulting in recurrent outbreaks and the emergence of drug-resistant strains. Traditional antiviral therapies, such as neuraminidase inhibitors and M2 ion channel blockers, have demonstrated limited efficacy, necessitating the exploration of innovative therapeutic strategies. Proteolysis-targeting chimeras (PROTACs) emerge as a novel and promising approach, leveraging the ubiquitin-proteasome system to selectively degrade pathogenic proteins. Unlike conventional inhibitors that only block protein function, PROTACs eliminate the target protein, providing a sustained therapeutic effect and potentially reducing the development of resistance. This paper offers a comprehensive examination of the current landscape of H5N1 infections, detailing the pathogenesis and challenges associated with existing treatments. It further explores the mechanism of action, design, and therapeutic potential of PROTACs in inhibiting H5N1. By targeting essential viral proteins, such as hemagglutinin and the RNA-dependent RNA polymerase complex, PROTACs hold the potential to revolutionize the treatment of H5N1 infections, offering a new frontier in antiviral therapy.

Sigma S Involved in Bacterial Survival of Ralstonia pseudosolanacearum

Ralstonia pseudosolanacearum, a plant pathogenic bacterium that can survive for a long time in soil and water, causes lethal wilt in the Solanaceae family. Sigma S is a part of the RNA polymerase complex, which regulates gene expression during bacterial stress response or stationary phase. In this study, we investigated the role of sigma S in R. pseudosolanacearum under stress conditions using a rpoS-defective mutant strain of R. pseudosolanacearum and its wild-type strain. The phenotypes of rpoS-defective mutant were complemented by introducing the original rpoS gene. There were no differences observed in bacterial growth rate and exopolysaccharide production between the wild-type strain and the rpoS mutant. However, the wild-type strain responded more sensitively to nutrient deficiency compared to the mutant strain. Under the nutrient deficiency, the rpoS mutant maintained a high bacterial viability for a longer period, while the viability of the wild-type strain declined rapidly. Furthermore, a significant difference in pH was observed between the culture supernatant of the wild-type strain and the mutant strain. The pH of the culture supernatant for the wild-type strain decreased rapidly during bacterial growth, leading to medium acidification. The rapid decline in the wild-type strain’s viability may be associated with medium acidification and bacterial sensitivity to acidity during transition to the stationary phase. Interestingly, the rpoS mutant strain cannot utilize acetic acid, D-alanine, D-trehalose, and L-histidine. These results suggest that sigma S of R. pseudosolanacearum regulates the production or utilization of organic acids and controls cell death during stationary phase under nutrient deficiency.

Evidence of RNA polymerase III recruitment and transcription at protein-coding gene promoters.

RNA polymerase (Pol) I, II, and III are most commonly described as having distinct roles in synthesizing ribosomal RNA (rRNA), messenger RNA (mRNA), and specific small noncoding (nc)RNAs, respectively. This delineation of transcriptional responsibilities is not definitive, however, as evidenced by instances of Pol II recruitment to genes conventionally transcribed by Pol III, including the co-transcription of RPPH1 - the catalytic RNA component of RNase P. A comprehensive understanding of the interplay between RNA polymerase complexes remains lacking, however, due to limited comparative analyses for all three enzymes. To address this gap, we applied a uniform framework for quantifying global Pol I, II, and III occupancies that integrates currently available human RNA polymerase ChIP-seq datasets. Occupancy maps are combined with a comprehensive multi-class promoter set that includes protein-coding genes, noncoding genes, and repetitive elements. While our genomic survey appropriately identifies recruitment of Pol I, II, and III to canonical target genes, we unexpectedly discover widespread recruitment of the Pol III machinery to promoters of specific protein-coding genes, supported by colocalization patterns observed for several Pol III-specific subunits. We show that Pol III-occupied Pol II promoters are enriched for small, nascent RNA reads terminating in a run of 4 Ts, a unique hallmark of Pol III transcription termination and evidence of active Pol III activity at these sites. Pol III disruption differentially modulates the expression of Pol III-occupied coding genes, which are functionally enriched for ribosomal proteins and genes broadly linked to unfavorable outcomes in cancer. Our map also identifies additional, currently unannotated genomic elements occupied by Pol III with clear signatures of nascent RNA species that are sensitive to disruption of La (SSB) - a Pol III-related RNA chaperone protein. These findings reshape our current understanding of the interplay between Pols II and III and identify potentially novel small ncRNAs with broad implications for gene regulatory paradigms and RNA biology.

Paramutation at the maize pl1 locus is associated with RdDM activity at distal tandem repeats.

Exceptions to Mendelian inheritance often highlight novel chromosomal behaviors. The maize Pl1-Rhoades allele conferring plant pigmentation can display inheritance patterns deviating from Mendelian expectations in a behavior known as paramutation. However, the chromosome features mediating such exceptions remain unknown. Here we show that small RNA production reflecting RNA polymerase IV function within a distal downstream set of five tandem repeats is coincident with meiotically-heritable repression of the Pl1-Rhoades transcription unit. A related pl1 haplotype with three, but not one with two, repeat units also displays the trans-homolog silencing typifying paramutations. 4C interactions, CHD3a-dependent small RNA profiles, nuclease sensitivity, and polyadenylated RNA levels highlight a repeat subregion having regulatory potential. Our comparative and mutant analyses show that transcriptional repression of Pl1-Rhoades correlates with 24-nucleotide RNA production and cytosine methylation at this subregion indicating the action of a specific DNA-dependent RNA polymerase complex. These findings support a working model in which pl1 paramutation depends on trans-chromosomal RNA-directed DNA methylation operating at a discrete cis-linked and copy-number-dependent transcriptional regulatory element.

Identification of B-cell epitopes located on the surface in the PB2 protein of the H9N2 subtype avian influenza virus

ABSTRACT Avian influenza (AI), caused by H9N2 subtype avian influenza virus (AIV), poses a serious threat to poultry farming and public health due to its transmissibility and pathogenicity. The PB2 protein is a major component of the viral RNA polymerase complex. It is of great importance to identify the antigenic determinants of the PB2 protein to explore the function of the PB2 protein. In this study, the PB2 sequence of H9N2 subtype AIV, from 1090 to 1689 bp, was cloned and expressed. The recombinant PB2 protein with cutting gel was used to immunize BALB/c mice. After cell fusion, the hybridoma cell lines secreting monoclonal antibodies (mAbs) targeting the PB2 protein were screened by indirect ELISA and western blotting, and the antigenic epitopes of mAbs were identified by constructing truncated overlapping fragments in the PB2 protein of H9N2 subtype AIV. The results showed that three hybridoma cell lines (4B7, 4D10, and 5H1) that stably secreted mAbs specific to the PB2 protein were screened; the heavy chain of 4B7 was IgG2α, those of 4D10 and 5H1 were IgG1, and all three mAbs had kappa light chain. Also, the minimum B-cell epitope recognized was 475LRGVRVSK482 and 528TITYSSPMMW537. Homology analysis showed that these two epitopes were conserved among the different subtypes of AIV strains and located on the surface of the PB2 protein. The above findings provide an experimental foundation for further investigation of the function of the PB2 protein and developing monoclonal antibody-based diagnostic kits.

Long-term evolution of human seasonal influenza virus A(H3N2) is associated with an increase in polymerase complex activity.

Since the influenza pandemic in 1968, influenza A(H3N2) viruses have become endemic. In this state, H3N2 viruses continuously evolve to overcome immune pressure as a result of prior infection or vaccination, as is evident from the accumulation of mutations in the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). However, phylogenetic studies have also demonstrated ongoing evolution in the influenza A(H3N2) virus RNA polymerase complex genes. The RNA polymerase complex of seasonal influenza A(H3N2) viruses produces mRNA for viral protein synthesis and replicates the negative sense viral RNA genome (vRNA) through a positive sense complementary RNA intermediate (cRNA). Presently, the consequences and selection pressures driving the evolution of the polymerase complex remain largely unknown. Here, we characterize the RNA polymerase complex of seasonal influenza A(H3N2) viruses representative of nearly 50 years of influenza A(H3N2) virus evolution. The H3N2 polymerase complex is a reassortment of human and avian influenza virus genes. We show that since 1968, influenza A(H3N2) viruses have increased the transcriptional activity of the polymerase complex while retaining a close balance between mRNA, vRNA, and cRNA levels. Interestingly, the increased polymerase complex activity did not result in increased replicative ability on differentiated human airway epithelial (HAE) cells. We hypothesize that the evolutionary increase in polymerase complex activity of influenza A(H3N2) viruses may compensate for the reduced HA receptor binding and avidity that is the result of the antigenic evolution of influenza A(H3N2) viruses.

The choreography of chromatin in RNA polymerase III regulation.

Regulation of eukaryotic gene expression involves a dynamic interplay between the core transcriptional machinery, transcription factors, and chromatin organization and modification. While this applies to transcription by all RNA polymerase complexes, RNA polymerase III (RNAPIII) seems to be atypical with respect to its mechanisms of regulation. One distinctive feature of most RNAPIII transcribed genes is that they are devoid of nucleosomes, which relates to the high levels of transcription. Moreover, most of the regulatory sequences are not outside but within the transcribed open chromatin regions. Yet, several lines of evidence suggest that chromatin factors affect RNAPIII dynamics and activity and that gene sequence alone does not explain the observed regulation of RNAPIII. Here we discuss the role of chromatin modification and organization of RNAPIII transcribed genes and how they interact with the core transcriptional RNAPIII machinery and regulatory DNA elements in and around the transcribed genes.

Abstract 1850: From proteomic analysis to early investigations of combining inhibitors targeting PCNA and histone deacetylase

Abstract Proteomic changes were investigated in a neuroblastoma cell line, SK-N-AS, treated with a small molecule inhibitor, AOH1996, designed to specifically bind proliferating cell nuclear antigen (PCNA) in cancer. PCNA is crucial to DNA replication, DNA repair, transcription, cell cycle control, and chromatin organization through interactions with different proteins. Pathway analysis of differential protein expressions between treated and untreated cells revealed less G2/M DNA damage checkpoint regulation and decreased assembly of RNA polymerase complex. Phosphoproteomic analysis by PTM Signature Enrichment Analysis (PTM-SEA) identified signatures of kinase activities, signaling pathways, and perturbations. Specifically, the histone deacetylase inhibitors belinostat and vorinostat were identified as enriched perturbation signatures. Both inhibitors were tested in combination with AOH1996 in SK-N-AS as well as cell lines derived from breast, lung, ovarian, and pancreatic cancer, revealing drug synergies at specific dose combinations. Western blot analysis showed that belinostat decreased protein expression of PCNA, suggestive of mechanistic synergies with AOH1996+belinostat combination therapies. Our proteomic analysis reveals a therapeutic potential of combining AOH1996 and histone deacetylase inhibitors for treating cancer with possibly lower drug dose and toxicities compared to single drug treatment. Citation Format: Caroline M. Li, Krystine Garcia-Mansfield, Robert G. Lingeman, Long Gu, Robert J. Hickey, Patrick Pirrotte, Linda H. Malkas. From proteomic analysis to early investigations of combining inhibitors targeting PCNA and histone deacetylase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1850.

Cytological, Physiological, and Transcriptomic Analyses of the Leaf Color Mutant Yellow Leaf 20 (yl20) in Eggplant (Solanum melongena L.)

Leaf color mutants are ideal materials for studying chlorophyll metabolism, chloroplast development, and photosynthesis in plants. We discovered a novel eggplant (Solanum melongena L.) mutant yl20 (yellow leaf 20) that exhibits yellow leaves. In this study, we compared the leaves of the mutant yl20 and wild type (WT) plants for cytological, physiological, and transcriptomic analyses. The results showed that the mutant yl20 exhibits abnormal chloroplast ultrastructure, reduced chlorophyll and carotenoid contents, and lower photosynthetic efficiency compared to the WT. Transcriptome data indicated 3267 and 478 differentially expressed genes (DEGs) between WT and yl20 lines in the cotyledon and euphylla stages, respectively, where most DEGs were downregulated in the yl20. Gene Ontology (GO) analysis revealed the “plastid-encoded plastid RNA polymerase complex” and the “chloroplast-related” terms were significantly enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that the significantly enriched DEGs were involved in flavone and flavonol biosynthesis, porphyrin and chlorophyll metabolism, etc. We speculated that these DEGs involved in significant terms were closely related to the leaf color development of the mutant yl20. Our results provide a possible explanation for the altered phenotype of leaf color mutants in eggplant and lay a theoretical foundation for plant breeding.

Optimized Recombinant Expression and Purification of the SARS-CoV-2 Polymerase Complex.

An optimized protocol has been developed to express and purify the core RNA-dependent RNA polymerase (RdRP) complex from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The expression and purification of active core SARS-CoV-2 RdRp complex is challenging due to the complex multidomain fold of nsp12, and the assembly of the multimeric complex involving nsp7, nsp8, and nsp12. Our approach adapts a previously published method to express the core SARS-CoV-2 RdRP complex in Escherichia coli and combines it with a procedure to express the nsp12 fusion with maltose-binding protein in insect cells to promote the efficient assembly and purification of an enzymatically active core polymerase complex. The resulting method provides a reliable platform to produce milligram amounts of the polymerase complex with the expected 1:2:1 stoichiometry for nsp7, nsp8, and nsp12, respectively, following the removal of all affinity tags. This approach addresses some of the limitations of previously reported methods to provide a reliable source of the active polymerase complex for structure, function, and inhibition studies of the SARS-CoV-2 RdRP complex using recombinant plasmid constructs that have been deposited in the widely accessible Addgene repository. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and production of SARS-CoV-2 nsp7, nsp8, and nsp12 in E. coli cells Support Protocol: Establishment and maintenance of insect cell cultures Basic Protocol 2: Generation of recombinant baculovirus in Sf9 cells and production of nsp12 fusion protein in T. ni cells Basic Protocol 3: Purification of the SARS-CoV-2 core polymerase complex.

Influenza A virus resistance to 4'-fluorouridine coincides with viral attenuation in vitro and in vivo.

Pre-existing or rapidly emerging resistance of influenza viruses to approved antivirals makes the development of novel therapeutics to mitigate seasonal influenza and improve preparedness against future influenza pandemics an urgent priority. We have recently identified the chain-terminating broad-spectrum nucleoside analog clinical candidate 4'-fluorouridine (4'-FlU) and demonstrated oral efficacy against seasonal, pandemic, and highly pathogenic avian influenza viruses in the mouse and ferret model. Here, we have resistance-profiled 4'-FlU against a pandemic A/CA/07/2009 (H1N1) (CA09). In vitro viral adaptation yielded six independently generated escape lineages with distinct mutations that mediated moderate resistance to 4'-FlU in the genetically controlled background of recombinant CA09 (recCA09). Mutations adhered to three distinct structural clusters that are all predicted to affect the geometry of the active site of the viral RNA-dependent RNA polymerase (RdRP) complex for phosphodiester bond formation. Escape could be achieved through an individual causal mutation, a combination of mutations acting additively, or mutations functioning synergistically. Fitness of all resistant variants was impaired in cell culture, and all were attenuated in the mouse model. Oral 4'-FlU administered at lowest-efficacious (2 mg/kg) or elevated (10 mg/kg) dose overcame moderate resistance when mice were inoculated with 10 LD50 units of parental or resistant recCA09, demonstrated by significantly reduced virus load and complete survival. In the ferret model, invasion of the lower respiratory tract by variants representing four adaptation lineages was impaired. Resistant variants were either transmission-incompetent, or spread to untreated sentinels was fully blocked by therapeutic treatment of source animals with 4'-FlU.

Actin associates with actively elongating genes and binds directly to the Cdk9 subunit of P-TEFb

Nuclear actin has been demonstrated to be essential for optimal transcription, but the molecular mechanisms and direct binding partner for actin in the RNA polymerase complex have remained unknown. By using purified proteins in a variety of biochemical assays, we demonstrate a direct and specific interaction between monomeric actin and Cdk9, the kinase subunit of the positive transcription elongation factor b (P-TEFb) required for RNA polymerase II pause-release. This interaction efficiently prevents actin polymerization, is not dependent on kinase activity of Cdk9 and is not involved with releasing P-TEFb from its inhibitor 7SK snRNP complex. Supporting the specific role for actin in the elongation phase of transcription, chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) reveals that actin interacts with genes only upon their active transcription elongation. This study therefore provides novel insights into the mechanisms by which actin facilitates the transcription process.

A genetic circuit on a single DNA molecule as an autonomous dissipative nanodevice

Realizing genetic circuits on single DNA molecules as self-encoded dissipative nanodevices is a major step toward miniaturization of autonomous biological systems. A circuit operating on a single DNA implies that genetically encoded proteins localize during coupled transcription-translation to DNA, but a single-molecule measurement demonstrating this has remained a challenge. Here, we use a genetically encoded fluorescent reporter system with improved temporal resolution and observe the synthesis of individual proteins tethered to a DNA molecule by transient complexes of RNA polymerase, messenger RNA, and ribosome. Against expectations in dilute cell-free conditions where equilibrium considerations favor dispersion, these nascent proteins linger long enough to regulate cascaded reactions on the same DNA. We rationally design a pulsatile genetic circuit by encoding an activator and repressor in feedback on the same DNA molecule. Driven by the local synthesis of only several proteins per hour and gene, the circuit dynamics exhibit enhanced variability between individual DNA molecules, and fluctuations with a broad power spectrum. Our results demonstrate that co-expressional localization, as a nonequilibrium process, facilitates single-DNA genetic circuits as dissipative nanodevices, with implications for nanobiotechnology applications and artificial cell design.

The E3 ligase TRIM22 restricts SARS-CoV-2 replication by promoting proteasomal degradation of NSP8.

Replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome is mediated by a complex of non-structural proteins (NSPs), of which NSP7 and NSP8 serve as subunits and play a key role in promoting the activity of RNA-dependent RNA polymerase (RdRp) of NSP12. However, the stability of subunits of the RdRp complex has rarely been reported. Here, we found that NSP8 was degraded by the proteasome in host cells, and identified tripartite motif containing 22 (TRIM22) as its E3 ligase. The interferon (IFN) signaling pathway was activated upon viral invasion into host cells, and TRIM22 expression increased. TRIM22 interacted with NSP8 and ubiquitinated it at Lys97 via K48-type ubiquitination. TRIM22 overexpression significantly reduced viral RNA and protein levels. Knockdown of TRIM22 enhanced viral replication. This study provides a new explanation for treating patients suffering from SARS-CoV-2 with IFNs and new possibilities for drug development targeting the interaction between NSP8 and TRIM22.IMPORTANCENon-structural proteins (NSPs) play a crucial role in the replication of severe acute respiratory syndrome coronavirus 2, facilitating virus amplification and propagation. In this study, we conducted a comprehensive investigation into the stability of all subunits comprising the RNA-dependent RNA polymerase complex. Notably, our results reveal for the first time that NSP8 is a relatively unstable protein, which is found to be readily recognized and degraded by the proteasome. This degradation process is mediated by the host E3 ligase tripartite motif containing 22 (TRIM22), which is also a member of the interferon stimulated gene (ISG) family. Our study elucidates a novel mechanism of antiviral effect of TRIM22, which utilizes its own E3 ubiquitin ligase activity to hinder viral replication by inducing ubiquitination and subsequent degradation of NSP8. These findings provide new ideas for the development of novel therapeutic strategies. In addition, the conserved property of NSP8 raises the possibility of developing broad antiviral drugs targeting the TRIM22-NSP8 interaction.

Investigation of protein-protein interactions and hotspot region on the NSP7-NSP8 binding site in NSP12 of SARS-CoV-2.

Background: The RNA-dependent RNA polymerase (RdRp) complex, essential in viral transcription and replication, is a key target for antiviral therapeutics. The core unit of RdRp comprises the nonstructural protein NSP12, with NSP7 and two copies of NSP8 (NSP81 and NSP82) binding to NSP12 to enhance its affinity for viral RNA and polymerase activity. Notably, the interfaces between these subunits are highly conserved, simplifying the design of molecules that can disrupt their interaction. Methods: We conducted a detailed quantum biochemical analysis to characterize the interactions within the NSP12-NSP7, NSP12-NSP81, and NSP12-NSP82 dimers. Our objective was to ascertain the contribution of individual amino acids to these protein-protein interactions, pinpointing hotspot regions crucial for complex stability. Results: The analysis revealed that the NSP12-NSP81 complex possessed the highest total interaction energy (TIE), with 14 pairs of residues demonstrating significant energetic contributions. In contrast, the NSP12-NSP7 complex exhibited substantial interactions in 8 residue pairs, while the NSP12-NSP82 complex had only one pair showing notable interaction. The study highlighted the importance of hydrogen bonds and π-alkyl interactions in maintaining these complexes. Intriguingly, introducing the RNA sequence with Remdesivir into the complex resulted in negligible alterations in both interaction energy and geometric configuration. Conclusion: Our comprehensive analysis of the RdRp complex at the protein-protein interface provides invaluable insights into interaction dynamics and energetics. These findings can guide the design of small molecules or peptide/peptidomimetic ligands to disrupt these critical interactions, offering a strategic pathway for developing effective antiviral drugs.

Type IV secretion system effector sabotages multiple defense systems in a competing bacterium.

Effector proteins secreted by bacteria that infect mammalian and plant cells often subdue eukaryotic host cell defenses by simultaneously affecting multiple targets. However, instances when a bacterial effector injected in the competing bacteria sabotage more than a single target have not been reported. Here, we demonstrate that the effector protein, LtaE, translocated by the type IV secretion system from the soil bacterium Lysobacter enzymogenes into the competing bacterium, Pseudomonas protegens, affects several targets, thus disabling the antibacterial defenses of the competitor. One LtaE target is the transcription factor, LuxR1, that regulates biosynthesis of the antimicrobial compound, orfamide A. Another target is the sigma factor, PvdS, required for biosynthesis of another antimicrobial compound, pyoverdine. Deletion of the genes involved in orfamide A and pyoverdine biosynthesis disabled the antibacterial activity of P. protegens, whereas expression of LtaE in P. protegens resulted in the near-complete loss of the antibacterial activity against L. enzymogenes. Mechanistically, LtaE inhibits the assembly of the RNA polymerase complexes with each of these proteins. The ability of LtaE to bind to LuxR1 and PvdS homologs from several Pseudomonas species suggests that it can sabotage defenses of various competitors present in the soil or on plant matter. Our study thus reveals that the multi-target effectors have evolved to subdue cell defenses not only in eukaryotic hosts but also in bacterial competitors.