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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 163 records · Page 9
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

CRL4A DTL degrades DNA-PKcs to modulate NHEJ repair and induce genomic instability and subsequent malignant transformation

Genomic instability induced by DNA damage and improper DNA damage repair is one of the main causes of malignant transformation and tumorigenesis. DNA double strand breaks (DSBs) are the most detrimental form of DNA damage, and nonhomologous end-joining (NHEJ) mechanisms play dominant and priority roles in initiating DSB repair. A well-studied oncogene, the ubiquitin ligase Cullin 4A (CUL4A), is reported to be recruited to DSB sites in genomic DNA, but whether it regulates NHEJ mechanisms of DSB repair is unclear. Here, we discovered that the CUL4A-DTL ligase complex targeted the DNA-PKcs protein in the NHEJ repair pathway for nuclear degradation. Overexpression of either CUL4A or DTL reduced NHEJ repair efficiency and subsequently increased the accumulation of DSBs. Moreover, we demonstrated that overexpression of either CUL4A or DTL in normal cells led to genomic instability and malignant proliferation. Consistent with the in vitro findings, in human precancerous lesions, CUL4A expression gradually increased with increasing malignant tendency and was negatively correlated with DNA-PKcs and positively correlated with γ-H2AX expression. Collectively, this study provided strong evidence that the CUL4A-DTL axis increases genomic instability and enhances the subsequent malignant transformation of normal cells by inhibiting NHEJ repair. These results also suggested that CUL4A may be a prognostic marker of precancerous lesions and a potential therapeutic target in cancer.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Active conformation of the p97-p47 unfoldase complex

The p97 AAA+ATPase is an essential and abundant regulator of protein homeostasis that plays a central role in unfolding ubiquitylated substrates. Here we report two cryo-EM structures of human p97 in complex with its p47 adaptor. One of the conformations is six-fold symmetric, corresponds to previously reported structures of p97, and lacks bound substrate. The other structure adopts a helical conformation, displays substrate running in an extended conformation through the pore of the p97 hexamer, and resembles structures reported for other AAA unfoldases. These findings support the model that p97 utilizes a “hand-over-hand” mechanism in which two residues of the substrate are translocated for hydrolysis of two ATPs, one in each of the two p97 AAA ATPase rings. Proteomics analysis supports the model that one p97 complex can bind multiple substrate adaptors or binding partners, and can process substrates with multiple types of ubiquitin modification.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Potent and selective covalent inhibition of the papain-like protease from SARS-CoV-2

Abstract Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we design a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibits PLpro with k inact /K I = 9,600 M −1 s −1 , achieves sub-μM EC 50 values against three SARS-CoV-2 variants in mammalian cell lines, and does not inhibit a panel of human deubiquitinases (DUBs) at >30 μM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validates our design strategy and establishes the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Entire expressed peripheral blood transcriptome in pediatric severe malarial anemia

Abstract This study on severe malarial anemia (SMA: Hb < 6.0 g/dL), a leading global cause of childhood morbidity and mortality, compares the entire expressed whole blood host transcriptome between Kenyan children (3-48 mos.) with non-SMA (Hb ≥ 6.0 g/dL, n = 39) and SMA ( n = 18). Differential expression analyses reveal 1403 up-regulated and 279 down-regulated transcripts in SMA, signifying impairments in host inflammasome activation, cell death, and innate immune and cellular stress responses. Immune cell profiling shows decreased memory responses, antigen presentation, and immediate pathogen clearance, suggesting an immature/improperly regulated immune response in SMA. Module repertoire analysis of blood-specific gene signatures identifies up-regulation of erythroid genes, enhanced neutrophil activation, and impaired inflammatory responses in SMA. Enrichment analyses converge on disruptions in cellular homeostasis and regulatory pathways for the ubiquitin-proteasome system, autophagy, and heme metabolism. Pathway analyses highlight activation in response to hypoxic conditions [Hypoxia Inducible Factor (HIF)−1 target and Reactive Oxygen Species (ROS) signaling] as a central theme in SMA. These signaling pathways are also top-ranking in protein abundance measures and a Ugandan SMA cohort with available transcriptomic data. Targeted RNA-Seq validation shows strong concordance with our entire expressed transcriptome data. These findings identify key molecular themes in SMA pathogenesis, offering potential targets for new malaria therapies.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

An artificial intelligence accelerated virtual screening platform for drug discovery

Abstract Structure-based virtual screening is a key tool in early drug discovery, with growing interest in the screening of multi-billion chemical compound libraries. However, the success of virtual screening crucially depends on the accuracy of the binding pose and binding affinity predicted by computational docking. Here we develop a highly accurate structure-based virtual screen method, RosettaVS, for predicting docking poses and binding affinities. Our approach outperforms other state-of-the-art methods on a wide range of benchmarks, partially due to our ability to model receptor flexibility. We incorporate this into a new open-source artificial intelligence accelerated virtual screening platform for drug discovery. Using this platform, we screen multi-billion compound libraries against two unrelated targets, a ubiquitin ligase target KLHDC2 and the human voltage-gated sodium channel Na V 1.7. For both targets, we discover hit compounds, including seven hits (14% hit rate) to KLHDC2 and four hits (44% hit rate) to Na V 1.7, all with single digit micromolar binding affinities. Screening in both cases is completed in less than seven days. Finally, a high resolution X-ray crystallographic structure validates the predicted docking pose for the KLHDC2 ligand complex, demonstrating the effectiveness of our method in lead discovery.

Science & Technology - Other Topics↗
DOE OSTI2023

An IRE1-proteasome system signalling cohort controls cell fate determination in unresolved proteotoxic stress of the plant endoplasmic reticulum

Excessive accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress, which is an underlying cause of major crop losses and devastating human conditions. Here, ER proteostasis surveillance is mediated by the conserved master regulator of the unfolded protein response (UPR), Inositol Requiring Enzyme 1 (IRE1), which determines cell fate by controlling pro-life and pro-death outcomes through as yet largely unknown mechanisms. Here we report that Arabidopsis IRE1 determines cell fate in ER stress by balancing the ubiquitin–proteasome system (UPS) and UPR through the plant-unique E3 ligase, PHOSPHATASE TYPE 2CA (PP2CA)-INTERACTING RING FINGER PROTEIN 1 (PIR1). Indeed, PIR1 loss leads to suppression of pro-death UPS and the lethal phenotype of an IRE1 loss-of-function mutant in unresolved ER stress in addition to activating pro-survival UPR. Specifically, in ER stress, PIR1 loss stabilizes ABI5, a basic leucine zipper (bZIP) transcription factor, that directly activates expression of the critical UPR regulator gene, bZIP60, triggering transcriptional cascades enhancing pro-survival UPR. Collectively, our results identify new cell fate effectors in plant ER stress by showing that IRE1’s coordination of cell death and survival hinges on PIR1, a key pro-death component of the UPS, which controls ABI5, a pro-survival transcriptional activator of bZIP60.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Core cysteine residues in the Plasminogen-Apple-Nematode (PAN) domain are critical for HGF/c-MET signaling

The Plasminogen-Apple-Nematode (PAN) domain, with a core of four to six cysteine residues, is found in > 28,000 proteins across 959 genera. Still, its role in protein function is not fully understood. The PAN domain was initially characterized in numerous proteins, including HGF. Dysregulation of HGF-mediated signaling results in multiple deadly cancers. The binding of HGF to its cell surface receptor, c-MET, triggers all biological impacts. Here, we show that mutating four core cysteine residues in the HGF PAN domain reduces c-MET interaction, subsequent c-MET autophosphorylation, and phosphorylation of its downstream targets, perinuclear localization, cellular internalization of HGF, and its receptor, c-MET, and c-MET ubiquitination. Furthermore, transcriptional activation of HGF/c-MET signaling-related genes involved in cancer progression, invasion, metastasis, and cell survival were impaired. Thus, targeting the PAN domain of HGF may represent a mechanism for selectively regulating the binding and activation of the c-MET pathway.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The co-crystal structure of Cbl-b and a small-molecule inhibitor reveals the mechanism of Cbl-b inhibition

Cbl-b is a RING-type E3 ubiquitin ligase that is expressed in several immune cell lineages, where it negatively regulates the activity of immune cells. Cbl-b has specifically been identified as an attractive target for cancer immunotherapy due to its role in promoting an immunosuppressive tumor environment. A Cbl-b inhibitor, Nx-1607, is currently in phase I clinical trials for advanced solid tumor malignancies. Using a suite of biophysical and cellular assays, we confirm potent binding of C7683 (an analogue of Nx-1607) to the full-length Cbl-b and its N-terminal fragment containing the TKBD-LHR-RING domains. To further elucidate its mechanism of inhibition, we determined the co-crystal structure of Cbl-b with C7683, revealing the compound’s interaction with both the TKBD and LHR, but not the RING domain. Here, we provide structural insights into a novel mechanism of Cbl-b inhibition by a small-molecule inhibitor that locks the protein in an inactive conformation by acting as an intramolecular glue.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

A ligand discovery toolbox for the WWE domain family of human E3 ligases

The WWE domain is a relatively under-researched domain found in twelve human proteins and characterized by a conserved tryptophan-tryptophan-glutamate (WWE) sequence motif. Six of these WWE domain-containing proteins also contain domains with E3 ubiquitin ligase activity. The general recognition of poly-ADP-ribosylated substrates by WWE domains suggests a potential avenue for development of Proteolysis-Targeting Chimeras (PROTACs). Here, we present novel crystal structures of the HUWE1, TRIP12, and DTX1 WWE domains in complex with PAR building blocks and their analogs, thus enabling a comprehensive analysis of the PAR binding site structural diversity. Furthermore, we introduce a versatile toolbox of biophysical and biochemical assays for the discovery and characterization of novel WWE domain binders, including fluorescence polarization-based PAR binding and displacement assays, 15 N-NMR-based binding affinity assays and 19 F-NMR-based competition assays. Through these assays, we have characterized the binding of monomeric iso -ADP-ribose ( iso -ADPr) and its nucleotide analogs with the aforementioned WWE proteins. Finally, we have utilized the assay toolbox to screen a small molecule fragment library leading to the successful discovery of novel ligands targeting the HUWE1 WWE domain.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Mechanistic insights into a heterobifunctional degrader-induced PTPN2/N1 complex

PTPN2 (protein tyrosine phosphatase non-receptor type 2, or TC-PTP) and PTPN1 are attractive immuno-oncology targets, with the deletion of Ptpn1 and Ptpn2 improving response to immunotherapy in disease models. Targeted protein degradation has emerged as a promising approach to drug challenging targets including phosphatases. We developed potent PTPN2/N1 dual heterobifunctional degraders (Cmpd-1 and Cmpd-2) which facilitate efficient complex assembly with E3 ubiquitin ligase CRL4 CRBN , and mediate potent PTPN2/N1 degradation in cells and mice. To provide mechanistic insights into the cooperative complex formation introduced by degraders, we employed a combination of structural approaches. Our crystal structure reveals how PTPN2 is recognized by the tri-substituted thiophene moiety of the degrader. We further determined a high-resolution structure of DDB1-CRBN/Cmpd-1/PTPN2 using single-particle cryo-electron microscopy (cryo-EM). This structure reveals that the degrader induces proximity between CRBN and PTPN2, albeit the large conformational heterogeneity of this ternary complex. The molecular dynamic (MD)-simulations constructed based on the cryo-EM structure exhibited a large rigid body movement of PTPN2 and illustrated the dynamic interactions between PTPN2 and CRBN. Together, our study demonstrates the development of PTPN2/N1 heterobifunctional degraders with potential applications in cancer immunotherapy. Furthermore, the developed structural workflow could help to understand the dynamic nature of degrader-induced cooperative ternary complexes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

A single genomic region controls primocane fruiting in tetraploid blackberry

The fresh-market blackberry ( Rubus subgenus Rubus ) industry has expanded dramatically in the past 2 decades, driven in part by improved cultivars. Introgression of the primocane-fruiting (PF; annual flowering) trait into elite germplasm has enabled dual cropping in a single year, season extension, and cultivation in tropical and subtropical regions. Despite its economic performance, the genetic basis of PF is not well understood. It has been proposed that the PF trait is controlled by a major recessive locus, but its genomic location is unclear. Here, a genome-wide association study (GWAS) of 365 tetraploid blackberry genotypes identified a single genomic region on chromosome Ra03 (∼33 Mb) strongly associated with PF. Genetic linkage analysis in a biparental population confirmed that the same interval (32–35 Mb) was linked to the PF phenotype. Ten putative candidate genes were identified in this region. Allele mining using whole-genome resequencing of 17 genotypes highlighted 2 high-priority candidates: a CCCH-type zinc finger gene and an ubiquitin-specific protease gene. Use of an improved Rubus argutus “Hillquist” genome annotation (v1.2) enabled refined variant interpretation, including identification of regulatory 3′ UTR polymorphisms in the zinc finger homolog. Two diagnostic KASP markers (PF1 and PF2), designed from the most significant GWAS SNPs, predicted the PF phenotype with over 96% accuracy in a validation panel of 494 tetraploid blackberries from multiple breeding programs. Together, these results provide the first high-resolution mapping of the PF locus in blackberry, identify candidate genes for flowering regulation in Rubus , and deliver diagnostic markers that can be immediately deployed in breeding programs.

GWAS↗
DOE OSTI2021

Rubisco production in maize mesophyll cells through ectopic expression of subunits and chaperones

Abstract C4 plants, such as maize, strictly compartmentalize Rubisco to bundle sheath chloroplasts. The molecular basis for the restriction of Rubisco from the more abundant mesophyll chloroplasts is not fully understood. Mesophyll chloroplasts transcribe the Rubisco large subunit gene and, when normally quiescent transcription of the nuclear Rubisco small subunit gene family is overcome by ectopic expression, mesophyll chloroplasts still do not accumulate measurable Rubisco. Here we show that a combination of five ubiquitin promoter-driven nuclear transgenes expressed in maize leads to mesophyll accumulation of assembled Rubisco. These encode the Rubisco large and small subunits, Rubisco assembly factors 1 and 2, and the assembly factor Bundle sheath defective 2. In these plants, Rubisco large subunit accumulates in mesophyll cells, and appears to be assembled into a holoenzyme capable of binding the substrate analog CABP (carboxyarabinitol bisphosphate). Isotope discrimination assays suggest, however, that mesophyll Rubisco is not participating in carbon assimilation in these plants, most probably due to a lack of the substrate ribulose 1,5-bisphosphate and/or Rubisco activase. Overall, this work defines a minimal set of Rubisco assembly factors in planta and may help lead to methods of regulating the C4 pathway.

Plant Sciences↗
DOE OSTI2024

A Sorghum F-Box Protein Induces an Oxidative Burst in the Defense Against Colletotrichum sublineola

The hemibiotrophic fungal pathogen Colletotrichum sublineola is the causal agent of anthracnose in sorghum ( Sorghum bicolor), resulting in leaf blight, stalk rot, and head blight in susceptible genotypes, with yield losses of up to 50%. The development of anthracnose-resistant cultivars can reduce reliance on fungicides and provide a more sustainable and economical means for disease management. A previous genome-wide association study of the sorghum association panel identified the candidate resistance gene Sobic.005G172300 encoding an F-box protein. To better understand the role of this gene in the defense against C. sublineola, gene expression following infection with C. sublineola was monitored by RNA sequencing in seedlings of sorghum accession SC110, which harbored the resistance allele, and three accessions that harbored a susceptible allele. Only in SC110 did the expression of Sobic.005G172300 increase during the biotrophic phase of infection. Subsequent transcriptome analysis, gene co-expression networks, and gene regulatory networks of inoculated and mock-inoculated seedlings of resistant and susceptible accessions suggest that the increase in expression of Sobic.005G172300 induces an oxidative burst by lowering the concentration of ascorbic acid during the biotrophic phase of infection. Based on gene regulatory network analysis, the protein encoded by Sobic.005G172300 is proposed to target proteins involved in the biosynthesis of ascorbic acid for polyubiquitination through the SCF E3 ubiquitin ligase, causing their degradation via the proteasome.

Plant Sciences↗
DOE OSTI2021

Sepsis induces muscle atrophy by inhibiting proliferation and promoting apoptosis via PLK1‐AKT signalling

Abstract Sepsis and sepsis‐induced skeletal muscle atrophy are common in patients in intensive care units with high mortality, while the mechanisms are controversial and complicated. In the present study, the atrophy of skeletal muscle was evaluated in sepsis mouse model as well as the apoptosis of muscle fibres. Sepsis induced atrophy of skeletal muscle and apoptosis of myofibres in vivo and in vitro. In cell‐based in vitro experiments, lipopolysaccharide (LPS) stimulation also inhibited the proliferation of myoblasts. At the molecular level, the expression of polo‐like kinase 1 (PLK1) and phosphorylated protein kinase B (p‐AKT) was decreased. Overexpression of PLK1 partly rescued LPS‐induced apoptosis, proliferation suppression and atrophy in C2C12 cells. Furthermore, inhibiting the AKT pathway deteriorated LPS‐induced atrophy in PLK1‐overexpressing C2C12 myotubes. PLK1 was found to participate in regulating apoptosis and E3 ubiquitin ligase activity in C2C12 cells. Taken together, these results indicate that sepsis induces skeletal muscle atrophy by promoting apoptosis of muscle fibres and inhibiting proliferation of myoblasts via regulation of the PLK1‐AKT pathway. These findings enhance understanding of the mechanism of sepsis‐induced skeletal muscle atrophy.

Cao, Ying‐Ya↗
DOE OSTI2023

The global dynamic of DNA methylation in response to heat stress revealed epigenetic mechanism of heat acclimation in Saccharina japonica

Saccharina japonica is an ecologically and economically important kelp in cold‐temperate regions. When it is cultivated on a large scale in the temperate and even subtropical zones, heat stress is a frequent abiotic stress. This study is the first attempt to reveal the regulatory mechanism of the response to heat stress from the perspective of DNA methylation in S. japonica . We firstly obtained the characteristics of variation in the methylome under heat stress, and observed that heat stress caused a slight increase in the overall methylation level and methylation rate, especially in the non‐coding regions of the genome. Secondly, we noted that methylation was probably one of factors affecting the expression of genes, and that methylation within the gene body was positively correlated with the gene expression (rho = 0.0784). Moreover, it was found that among the differentially expressed genes regulated by methylation, many genes were related to heat stress response, such as HSP gene family, genes of antioxidant enzymes, genes related to proteasome‐ubiquitination pathway, and plant cell signaling pathways. This study demonstrated that DNA methylation is involved in regulating the response to heat stress, laying a foundation for studying the acclimation and adaptation of S. japonica to heat stress from an epigenetic perspective.

Liu, Fuli↗
DOE OSTI2020

Arabidopsis SnRK1 negatively regulates phenylpropanoid metabolism via Kelch domain-containing F-box proteins

Phenylpropanoid metabolism represents a substantial metabolic sink for the photosynthetically fixed carbon. The evolutionarily conserved Sucrose Non-Fermenting Related Kinase 1 (SnRK1) is a major metabolic sensor that reprograms metabolism upon carbon deprivation. However, it is not clear if and how SnRK1-mediated sugar signaling pathway controls phenylpropanoid metabolism. Here, we show that Arabidopsis SnRK1 negatively regulates phenylpropanoid biosynthesis via a group of Kelch domain-containing F-Box (KFB) proteins that are responsible for the ubiquitination and degradation of phenylalanine ammonia lyase (PAL). Down-regulation of AtSnRK1 significantly promoted the accumulation of soluble phenolics and lignin polymers and drastically increased PAL cellular accumulation but only slightly altered its transcription level. Co-expression of SnRK1α with PAL in Nicotiana benthamiana leaves resulted in the severe attenuation of the latter's protein level, but protein interaction assays suggested PAL is not a direct substrate of SnRK1. Furthermore, up- or down-regulation of AtSnRK1 positively affected KFB PALs gene expression, and energy starvation upregulated KFB PAL expression, which partially depends on AtSnRK1. Collectively, our study reveals that SnRK1 negatively regulates phenylpropanoid biosynthesis, and KFB PALs act as regulatory components of the SnRK1 signaling network, transcriptionally regulated by SnRK1 and subsequently mediating proteasomal degradation of PAL in response to the cellular carbon availability.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

The expression of genes encoding novel Sesame oleosin variants facilitates enhanced triacylglycerol accumulation in Arabidopsis leaves and seeds

Summary Triacylglycerols (TAG), accumulate within lipid droplets (LD), predominantly surrounded by OLEOSINs (OLE), that protect TAG from hydrolysis. We tested the hypothesis that identifying and removing degradation signals from OLE would promote its abundance, preventing TAG degradation and enhancing TAG accumulation. We tested whether mutating potential ubiquitin‐conjugation sites in a previously reported improved Sesamum indicum OLE ( SiO ) variant, o3‐3 Cys‐OLE ( SiCO herein), would stabilize it and increase its lipogenic potential. SiCOv1 was created by replacing all five lysines in SiCO with arginines. Separately, six cysteine residues within SiCO were deleted to create SiCOv2. SiCOv1 and SiCOv2 mutations were combined to create SiCOv3. Transient expression of SiCOv3 in Nicotiana benthamiana increased TAG by two‐fold relative to SiCO. Constitutive expression of SiCOv3 or SiCOv5 , containing the five predominant TAG‐increasing mutations from SiCOv3 , in Arabidopsis along with mouse DGAT2 ( mD ) increased TAG accumulation by 54% in leaves and 13% in seeds compared with control lines coexpressing SiCO and mD. Lipid synthesis rates increased, consistent with an increase in lipid sink strength that sequesters newly synthesized TAG, thereby relieving the constitutive BADC‐dependent inhibition of ACCase reported for WT Arabidopsis. These OLE variants represent novel factors for potentially increasing TAG accumulation in a variety of oil crops.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Structural and functional analyses of SARS-CoV-2 Nsp3 and its specific interactions with the 5’ UTR of the viral genome

ABSTRACT Non-structural protein 3 (Nsp3) is the largest open reading frame encoded in the SARS-CoV-2 genome, essential for the formation of double-membrane vesicles (DMV) wherein viral RNA replication occurs. We conducted an extensive structure-function analysis of Nsp3 and determined the crystal structures of the ubiquitin-like 1 (Ubl1), nucleic acid binding (NAB), β-coronavirus-specific marker (βSM) domains, and a sub-region of the Y domain of this protein. We show that the Ubl1, ADP-ribose phosphatase (ADRP), human SARS Unique (HSUD), NAB, and Y domains of Nsp3 bind the 5’ UTR of the viral genome and that the Ubl1 and Y domains possess affinity for recognition of this region, suggesting high specificity. The Ubl1-Nucleocapsid (N) protein complex binds the 5’ UTR with greater affinity than the individual proteins alone. Our results suggest that multiple domains of Nsp3, particularly Ubl1 and Y, shepherd the 5’ UTR of the viral genome during translocation through the DMV membrane, priming the Ubl1 domain to load the genome onto N protein. IMPORTANCE The largest protein encoded by the SARS-CoV-2 genome is Nsp3. In infected cells, this multi-domain protein forms a pore structure in the virus-induced double-membrane vesicles (DMV). We have incomplete data on Nsp3 molecular structure, and here, we describe crystal structures for multiple domains of Nsp3. It is thought that newly replicated viral RNA transits through the DMV pore; however, we possess incomplete data on which regions of Nsp3 actually interact with RNA. Here, we present data showing that five domains of Nsp3 interact with the 5’ UTR of the SARS-CoV-2 RNA, including the Y domain for which no function has ever been discovered. These data suggest that the pore structure plays an active role in recognizing the terminal end of the genome, transiting and loading the viral RNA onto the cytoplasmic nucleocapsid protein. These data help expand our knowledge of Nsp3 structure and function and the SARS-CoV-2 replication cycle.

Microbiology↗