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The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19 illness, has caused millions of infections worldwide. In SARS coronaviruses, the non-structural protein 16 (nsp16), in conjunction with nsp10, methylates the 5′-end of virally encoded mRNAs to mimic cellular mRNAs, thus protecting the virus from host innate immune restriction. We report here the high-resolution structure of a ternary complex of SARS-CoV-2 nsp16 and nsp10 in the presence of cognate RNA substrate analogue and methyl donor, S-adenosyl methionine (SAM). The nsp16/nsp10 heterodimer is captured in the act of 2′-O methylation of the ribose sugar of the first nucleotide of SARS-CoV-2 mRNA. We observe large conformational changes associated with substrate binding as the enzyme transitions from a binary to a ternary state. This induced fit model provides mechanistic insights into the 2′-O methylation of the viral mRNA cap. We also discover a distant (25 Å) ligand-binding site unique to SARS-CoV-2, which can alternatively be targeted, in addition to RNA cap and SAM pockets, for antiviral development. Specific non-structural proteins (nsp) of SARS coronaviruses are involved in methylation of virally encoded mRNAs to mimic cellular mRNAs for protection against host innate immune restriction. Here, the authors present a high resolution structure of SARS-CoV-2 nsp16/nsp10 ternary complex in the presence of cognate RNA substrate analogue and methyl donor, S-adenosyl methionine, revealing unique ligand-binding sites that may represent alternative targets for antiviral development.

Background RNA-binding proteins (RBPs) function as master regulators of gene expression. Alterations in RBP expression and function are often observed in cancer and influence critical pathways implicated in tumor initiation and growth. Identification and characterization of oncogenic RBPs and their regulatory networks provide new opportunities for targeted therapy. Results We identify the RNA-binding protein SERBP1 as a novel regulator of glioblastoma (GBM) development. High SERBP1 expression is prevalent in GBMs and correlates with poor patient survival and poor response to chemo- and radiotherapy. SERBP1 knockdown causes delay in tumor growth and impacts cancer-relevant phenotypes in GBM and glioma stem cell lines. RNAcompete identifies a GC-rich region as SERBP1-binding motif; subsequent genomic and functional analyses establish SERBP1 regulation role in metabolic routes preferentially used by cancer cells. An important consequence of these functions is SERBP1 impact on methionine production. SERBP1 knockdown decreases methionine levels causing a subsequent reduction in histone methylation as shown for H3K27me3 and upregulation of genes associated with neurogenesis, neuronal differentiation, and function. Further analysis demonstrates that several of these genes are downregulated in GBM, potentially through epigenetic silencing as indicated by the presence of H3K27me3 sites. Conclusions SERBP1 is the first example of an RNA-binding protein functioning as a central regulator of cancer metabolism and indirect modulator of epigenetic regulation in GBM. By bridging these two processes, SERBP1 enhances glioma stem cell phenotypes and contributes to GBM poorly differentiated state.

The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2′-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2′- O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2′- O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain. The SARS-CoV-2 nsp16/nsp10 enzyme complex methylates the 2′-OH of the first nucleotide of the viral mRNA, converting the Cap-0 to Cap-1, which helps the virus to evade immune surveillance in the host cell. Here, the authors present the crystal structure of SARS-CoV-2 nsp16/nsp10 with the bound Cap-1 RNA nucleotide product and a post-release SAH containing structure.

PrimPol is a human DNA polymerase-primase that localizes to mitochondria and nucleus and bypasses the major oxidative lesion 7,8-dihydro-8-oxoguanine (oxoG) via translesion synthesis, in mostly error-free manner. We present structures of PrimPol insertion complexes with a DNA template-primer and correct dCTP or erroneous dATP opposite the lesion, as well as extension complexes with C or A as a 3′−terminal primer base. We show that during the insertion of C and extension from it, the active site is unperturbed, reflecting the readiness of PrimPol to accommodate oxoG (anti) . The misinsertion of A opposite oxoG (syn) also does not alter the active site, and is likely less favorable due to lower thermodynamic stability of the oxoG (syn) •A base - pair. During the extension step, oxoG (syn) induces an opening of its base-pair with A or misalignment of the 3′-A primer terminus. Together, the structures show how PrimPol accurately synthesizes DNA opposite oxidatively damaged DNA in human cells. The human DNA primase and DNA polymerase PrimPol replicates through the major oxidative DNA damage lesion 7,8-dihydro-8-oxoguanine (oxoG) via translesion synthesis in a mostly error-free manner thus suppressing oxoG-induced mutagenesis in mitochondria and the nucleus. Here, the authors present crystal structures of PrimPol in complex with an oxoG lesion in different contexts that provide mechanistic insights into how PrimPol performs predominantly accurate synthesis on oxidative-damaged DNAs in human cells.

Magnaporthe oryzae is the causal agent of rice blast disease, the most devastating disease of cultivated rice (Oryza sativa) and a continuing threat to global food security. To cause disease, the fungus elaborates a specialized infection cell called an appressorium, which breaches the cuticle of the rice leaf, allowing the fungus entry to plant tissue. Here, we show that the exocyst complex localizes to the tips of growing hyphae during vegetative growth, ahead of the Spitzenkörper, and is required for polarized exocytosis. However, during infection-related development, the exocyst specifically assembles in the appressorium at the point of plant infection. The exocyst components Sec3, Sec5, Sec6, Sec8, and Sec15, and exocyst complex proteins Exo70 and Exo84 localize specifically in a ring formation at the appressorium pore. Targeted gene deletion, or conditional mutation, of genes encoding exocyst components leads to impaired plant infection. We demonstrate that organization of the exocyst complex at the appressorium pore is a septin-dependent process, which also requires regulated synthesis of reactive oxygen species by the NoxR-dependent Nox2 NADPH oxidase complex. We conclude that septinmediated assembly of the exocyst is necessary for appressorium repolarization and host cell invasion.

Type III R–M enzymes were identified >40 years ago and yet there is no structural information on these multisubunit enzymes. Here we report the structure of a Type III R–M system, consisting of the entire EcoP15I complex (Mod 2 Res 1 ) bound to DNA. The structure suggests how ATP hydrolysis is coupled to long-range diffusion of a helicase on DNA, and how a dimeric methyltransferase functions to methylate only one of the two DNA strands. We show that the EcoP15I motor domains are specifically adapted to bind double-stranded DNA and to facilitate DNA sliding via a novel ‘Pin’ domain. We also uncover unexpected ‘division of labour’, where one Mod subunit recognizes DNA, while the other Mod subunit methylates the target adenine—a mechanism that may extend to adenine N6 RNA methylation in mammalian cells. Together the structure sheds new light on the mechanisms of both helicases and methyltransferases in DNA and RNA metabolism. Type III restriction–modification enzymes consists of two methylation and one or two restriction subunits. Here the authors report the structure of the full EcoP15I complex bound to DNA, which suggests mechanisms for ATP hydrolysis dependent diffusion along DNA and how a dimeric methyltransferase modifies only one DNA strand.

Ensitrelvir is an antiviral drug that specifically targets the conserved main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, mutations in Mpro could confer resistance to antivirals, including ensitrelvir. Thus, identifying SARS-CoV-2 drug-resistant mutants and elucidating their mechanisms of resistance are critical for guiding the selection of effective antiviral therapies. Here, we utilized a recombinant luminescent attenuated SARS-CoV-2 lacking the open reading frames (ORF) 3a and 7b proteins (Δ3a7b-Nluc WT) to safely identify ensitrelvir drug-resistant mutants (DRM-E) without the need of using virulent forms of SARS-CoV-2. We isolated a DRM-E containing a Mpro G23 deletion (G23del) with high resistance (~1,000-fold) to ensitrelvir, but not to the Mpro inhibitor nirmatrelvir or to the RNA-dependent RNA polymerase (RdRp) inhibitor remdesivir. The contribution of G23del in ensitrelvir resistance was confirmed by generating a Δ3a7b-Nluc containing G23del in Mpro (Δ3a7b-Nluc G23del). Δ3a7b-Nluc G23del exhibited resistance to ensitrelvir in both cultured cells and in K18 hACE2 transgenic mice. Binding affinity revealed that the G23del mutation altered ensitrelvir, but not nirmatrelvir, binding to Mpro. Notably, while Δ3a7b-Nluc G23del was affected in viral fitness, serial passage of Δ3a7b-Nluc G23del in the absence of ensitrelvir resulted in the emergence of substitution L50F in Mpro that restored viral fitness loss caused by G23del without altering resistance to ensitrelvir. Our results demonstrate that G23del in Mpro can confer resistance to ensitrelvir. Positively, G23del in Mpro does not render SARS-CoV-2 resistant to nirmatrelvir or remdesivir, suggesting the feasibility of treating SARS-CoV-2 infections containing G23del Mpro with other approved antivirals.IMPORTANCEThe clinical use of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antiviral drugs is increasingly challenged by the emergence of drug-resistant mutants. Thus, there is a pressing need to identify and characterize antiviral escape SARS-CoV-2 variants, particularly for Food and Drug Administration-approved antivirals. Our study addresses this by employing a luminescent attenuated SARS-CoV-2 platform (Δ3a7b-Nluc wild type [WT]) to safely identify and characterize resistance mutations without the concern of using virulent forms of SARS-CoV-2. Using this safe approach, we identified a G23 deletion (G23del) in SARS-CoV-2 Mpro, which mediates resistance to ensitrelvir in vitro and in vivo. Importantly, while G23del was able to confer ~1,000-fold increased resistance to ensitrelvir, a Δ3a7b-Nluc containing G23del remained sensitive to other Mpro (nirmatrelvir) and RdRp (remdesivir) inhibitors. Altogether, this study demonstrates the feasibility of using Δ3a7b-Nluc WT to safely identify and characterize drug-resistant viruses without the biosafety concern of using virulent WT SARS-CoV-2 and advance the design of next-generation antiviral drugs.

The creation of restriction enzymes with programmable DNA-binding and -cleavage specificities has long been a goal of modern biology. The recently discovered Type IIL MmeI family of restriction-and-modification (RM) enzymes that possess a shared target recognition domain provides a framework for engineering such new specificities. However, a lack of structural information on Type IIL enzymes has limited the repertoire that can be rationally engineered. We report here a crystal structure of MmeI in complex with its DNA substrate and an S-adenosylmethionine analog (Sinefungin). The structure uncovers for the first time the interactions that underlie MmeI-DNA recognition and methylation (5'-TCCRAC-3'; R = purine) and provides a molecular basis for changing specificity at four of the six base pairs of the recognition sequence (5'-TCCRAC-3'). Surprisingly, the enzyme is resilient to specificity changes at the first position of the recognition sequence (5'-TCCRAC-3'). Collectively, the structure provides a basis for engineering further derivatives of MmeI and delineates which base pairs of the recognition sequence are more amenable to alterations than others.

Biotrophic fungal plant pathogens can balance their virulence and form intricate relationships with their hosts. Sometimes, this leads to systemic host colonization over long time scales without macroscopic symptoms. However, how plant-pathogenic endophytes manage to establish their sustained systemic infection remains largely unknown. Here, we present a genomic and transcriptomic analysis of Thecaphora thlaspeos. This relative of the well studied grass smut Ustilago maydis is the only smut fungus adapted to Brassicaceae hosts. Its ability to overwinter with perennial hosts and its systemic plant infection including roots are unique characteristics among smut fungi. The T. thlaspeos genome was assembled to the chromosome level. It is a typical smut genome in terms of size and genome characteristics. In silico prediction of candidate effector genes revealed common smut effector proteins and unique members. For three candidates, we have functionally demonstrated effector activity. One of these, TtTue1, suggests a potential link to cold acclimation. On the plant side, we found evidence for a typical immune response as it is present in other infection systems, despite the absence of any macroscopic symptoms during infection. Our findings suggest that T. thlaspeos distinctly balances its virulence during biotrophic growth ultimately allowing for long-lived infection of its perennial hosts.

To cause plant diseases, pathogenic micro-organisms secrete effector proteins into host tissue to suppress immunity and support pathogen growth. Bacterial pathogens have evolved several distinct secretion systems to target effector proteins, but whether fungi, which cause the major diseases of most crop species, also require different secretory mechanisms is not known. Here we report that the rice blast fungus Magnaporthe oryzae possesses two distinct secretion systems to target effectors during plant infection. Cytoplasmic effectors, which are delivered into host cells, preferentially accumulate in the biotrophic interfacial complex, a novel plant membrane-rich structure associated with invasive hyphae. We show that the biotrophic interfacial complex is associated with a novel form of secretion involving exocyst components and the Sso1 t -SNARE. By contrast, effectors that are secreted from invasive hyphae into the extracellular compartment follow the conventional secretory pathway. We conclude that the blast fungus has evolved distinct secretion systems to facilitate tissue invasion. Understanding how fungal pathogens deliver proteins to diverse plant compartments is critical for developing disease control. Giraldo et al . demonstrate that the blast fungus possesses distinct secretion systems for proteins targeted to compartments inside or outside living rice cells.