Yeast Expression and Activity Assay of RVFV Polymerase.

Rift Valley fever (RVF) virus is widespread worldwide and poses a severe threat to human life and property. RVF viral polymerase plays a vital role in the replication and transcription of the virus. Here, we describe how to express and purify this polymerase and perform tests for its in vitro activity assays.

Emerging Topics in Protein Crystallography.

Protein crystallography is the discipline concerned with the determination of the three-dimensional structure of biological macromolecules in a crystalline state [...].

Structural Basis of Nucleic Acid Recognition and 6mA Demethylation by Caenorhabditis elegans NMAD-1A.

N6-methyladenine (6mA) of DNA is an emerging epigenetic mark in the genomes of Chlamydomonas, Caenorhabditis elegans, and mammals recently. Levels of 6mA undergo drastic fluctuation and thus affect fertility during meiosis and early embryogenesis. Here, we showed three complex structures of 6mA demethylase C. elegans NMAD-1A, a canonical isoform of NMAD-1 (F09F7.7). Biochemical results revealed that NMAD-1A prefers 6mA Bubble or Bulge DNAs. Structural studies of NMAD-1A revealed an unexpected "stretch-out" conformation of its Flip2 region, a conserved element that is usually bent over the catalytic center to facilitate substrate base flipping in other DNA demethylases. Moreover, the wide channel between the Flip1 and Flip2 of the NMAD-1A explained the observed preference of NMAD-1A for unpairing substrates, of which the flipped 6mA was primed for catalysis. Structural analysis and mutagenesis studies confirmed that key elements such as carboxy-terminal domain (CTD) and hypothetical zinc finger domain (ZFD) critically contributed to structural integrity, catalytic activity, and nucleosome binding. Collectively, our biochemical and structural studies suggest that NMAD-1A prefers to regulate 6mA in the unpairing regions and is thus possibly associated with dynamic chromosome regulation and meiosis regulation.

Structures of MPND Reveal the Molecular Recognition of Nucleosomes.

Adenine N6 methylation in DNA (6mA) is a well-known epigenetic modification in bacteria, phages, and eukaryotes. Recent research has identified the Mpr1/Pad1 N-terminal (MPN) domain-containing protein (MPND) as a sensor protein that may recognize DNA 6mA modification in eukaryotes. However, the structural details of MPND and the molecular mechanism of their interaction remain unknown. Herein, we report the first crystal structures of the apo-MPND and MPND-DNA complex at resolutions of 2.06 Å and 2.47 Å, respectively. In solution, the assemblies of both apo-MPND and MPND-DNA are dynamic. In addition, MPND was found to possess the ability to bind directly to histones, no matter the N-terminal restriction enzyme-adenine methylase-associated domain or the C-terminal MPN domain. Moreover, the DNA and the two acidic regions of MPND synergistically enhance the interaction between MPND and histones. Therefore, our findings provide the first structural information regarding the MPND-DNA complex and also provide evidence of MPND-nucleosome interactions, thereby laying the foundation for further studies on gene control and transcriptional regulation.

Structures of human TR4LBD-JAZF1 and TR4DBD-DNA complexes reveal the molecular basis of transcriptional regulation.

Testicular nuclear receptor 4 (TR4) modulates the transcriptional activation of genes and plays important roles in many diseases. The regulation of TR4 on target genes involves direct interactions with DNA molecules via the DNA-binding domain (DBD) and recruitment of coregulators by the ligand-binding domain (LBD). However, their regulatory mechanisms are unclear. Here, we report high-resolution crystal structures of TR4DBD, TR4DBD-DNA complexes and the TR4LBD-JAZF1 complex. For DNA recognition, multiple factors come into play, and a specific mutual selectivity between TR4 and target genes is found. The coactivators SRC-1 and CREBBP can bind at the interface of TR4 originally occupied by the TR4 activation function region 2 (AF-2); however, JAZF1 suppresses the binding through a novel mechanism. JAZF1 binds to an unidentified surface of TR4 and stabilizes an α13 helix never reported in the nuclear receptor family. Moreover, the cancer-associated mutations affect the interactions and the transcriptional activation of TR4 in vitro and in vivo, respectively. Overall, our results highlight the crucial role of DNA recognition and a novel mechanism of how JAZF1 reinforces the autorepressed conformation and influences the transcriptional activation of TR4, laying out important structural bases for drug design for a variety of diseases, including diabetes and cancers.

Co-crystalization reveals the interaction between AtYchF1 and ppGpp.

AtYchF1 is an unconventional G-protein in Arabidopsis thaliana that exhibits relaxed nucleotide-binding specificity. The bindings between AtYchF1 and biomolecules including GTP, ATP, and 26S rRNA have been reported. In this study, we demonstrated the binding of AtYchF1 to ppGpp in addition to the above molecules. AtYchF1 is a cytosolic protein previously reported as a negative regulator of both biotic and abiotic stresses while the accumulation of ppGpp in the cytoplasm induces retarded plant growth and development. By co-crystallization, in vitro pull-down experiments, and hydrolytic biochemical assays, we demonstrated the binding and hydrolysis of ppGpp by AtYchF1. ppGpp inhibits the binding of AtYchF1 to ATP, GTP, and 26S rRNA. The ppGpp hydrolyzing activity of AtYchF1 failed to be activated by AtGAP1. The AtYchF1-ppGpp co-crystal structure suggests that ppGpp might prevent His136 from executing nucleotide hydrolysis. In addition, upon the binding of ppGpp, the conformation between the TGS and helical domains of AtYchF1 changes. Such structural changes probably influence the binding between AtYchF1 and other molecules such as 26S rRNA. Since YchF proteins are conserved among different kingdoms of life, the findings advance the knowledge on the role of AtYchF1 in regulating nucleotide signaling as well as hint at the possible involvement of YchF proteins in regulating ppGpp level in other species.

The Receptor Binding Domain of SARS-CoV-2 Lambda Variant Has a Better Chance Than the Delta Variant in Evading BNT162b2 COVID-19 mRNA Vaccine-Induced Humoral Immunity.

The SARS-CoV-2 Delta and Lambda variants had been named variants of concern (VOC) and variants of interest (VOI), respectively, by the World Health Organization (WHO). Both variants have two mutations in the spike receptor binding domain (RBD) region, with L452R and T478K mutations in the Delta variant, and L452Q and F490S mutations in the Lambda variant. We used surface plasmon resonance (SPR)-based technology to evaluate the effect of these mutations on human angiotensin-converting enzyme 2 (ACE2) and Bamlanivimab binding. The affinity for the RBD ligand, ACE2, of the Delta RBD is approximately twice as strong as that of the wild type RBD, an increase that accounts for the increased infectivity of the Delta variant. On the other hand, in spite of its amino acid changes, the Lambda RBD has similar affinity to ACE2 as the wild type RBD. The protective anti-wild type RBD antibody Bamlanivimab binds very poorly to the Delta RBD and not at all to the Lambda RBD. Nevertheless, serum antibodies from individuals immunized with the BNT162b2 vaccine were found to bind well to the Delta RBD, but less efficiently to the Lambda RBD in contrast. As a result, the blocking ability of ACE2 binding by serum antibodies was decreased more by the Lambda than the Delta RBD. Titers of sera from BNT162b2 mRNA vaccinated individuals dropped 3-fold within six months of vaccination regardless of whether the target RBD was wild type, Delta or Lambda. This may account partially for the fall off with time in the protective effect of vaccines against any variant.

Tracking Changes in Students' Online Self-Regulated Learning Behaviors and Achievement Goals Using Trace Clustering and Process Mining.

Success in online and blended courses requires engaging in self-regulated learning (SRL), especially for challenging STEM disciplines, such as physics. This involves students planning how they will navigate course assignments and activities, setting goals for completion, monitoring their progress and content understanding, and reflecting on how they completed each assignment. Based on Winne & Hadwin's COPES model, SRL is a series of events that temporally unfold during learning, impacted by changing internal and external factors, such as goal orientation and content difficulty. Thus, as goal orientation and content difficulty change throughout a course, so might students' use of SRL processes. This paper studies how students' SRL behavior and achievement goal orientation change over time in a large (N = 250) college introductory level physics course taught online. Students' achievement goal orientation was measured by repeated administration of the achievement goals questionnaire-revised (AGQ-R). Students' SRL behavior was measured by analyzing their clickstream event traces interacting with online learning modules via a combination of trace clustering and process mining. Event traces were first divided into groups similar in nature using agglomerative clustering, with similarity between traces determined based on a set of derived characteristics most reflective of students' SRL processes. We then generated causal nets for each cluster of traces via process mining and interpreted the underlying behavior and strategy of each causal net according to the COPES SRL framework. We then measured the frequency at which students adopted each causal net and assessed whether the adoption of different causal nets was associated with responses to the AGQ-R. By repeating the analysis for three sets of online learning modules assigned at the beginning, middle, and end of the semester, we examined how the frequency of each causal net changed over time, and how the change correlated with changes to the AGQ-R responses. Results have implications for measuring the temporal nature of SRL during online learning, as well as the factors impacting the use of SRL processes in an online physics course. Results also provide guidance for developing online instructional materials that foster effective SRL for students with different motivational profiles.

The Novel Protease Activities of JMJD5-JMJD6-JMJD7 and Arginine Methylation Activities of Arginine Methyltransferases Are Likely Coupled.

The surreptitious discoveries of the protease activities on arginine-methylated targets of a subfamily of Jumonji domain-containing family including JMJD5, JMJD6, and JMJD7 pose several questions regarding their authenticity, function, purpose, and relations with others. At the same time, despite several decades of efforts and massive accumulating data regarding the roles of the arginine methyltransferase family (PRMTs), the exact function of this protein family still remains a mystery, though it seems to play critical roles in transcription regulation, including activation and inactivation of a large group of genes, as well as other biological activities. In this review, we aim to elucidate that the function of JMJD5/6/7 and PRMTs are likely coupled. Besides roles in the regulation of the biogenesis of membrane-less organelles in cells, they are major players in regulating stimulating transcription factors to control the activities of RNA Polymerase II in higher eukaryotes, especially in the animal kingdom. Furthermore, we propose that arginine methylation by PRMTs could be a ubiquitous action marked for destruction after missions by a subfamily of the Jumonji protein family.

Structural insights into the interactions and epigenetic functions of human nucleic acid repair protein ALKBH6.

Human AlkB homolog 6, ALKBH6, plays key roles in nucleic acid damage repair and tumor therapy. However, no precise structural and functional information are available for this protein. In this study, we determined atomic resolution crystal structures of human holo-ALKBH6 and its complex with ligands. AlkB members bind nucleic acids by NRLs (nucleotide recognition lids, also called Flips), which can recognize DNA/RNA and flip methylated lesions. We found that ALKBH6 has unusual Flip1 and Flip2 domains, distinct from other AlkB family members both in sequence and conformation. Moreover, we show that its unique Flip3 domain has multiple unreported functions, such as discriminating against double-stranded nucleic acids, blocking the active center, binding other proteins, and in suppressing tumor growth. Structural analyses and substrate screening reveal how ALKBH6 discriminates between different types of nucleic acids and may also function as a nucleic acid demethylase. Structure-based interacting partner screening not only uncovered an unidentified interaction of transcription repressor ZMYND11 and ALKBH6 in tumor suppression but also revealed cross talk between histone modification and nucleic acid modification in epigenetic regulation. Taken together, these results shed light on the molecular mechanism underlying ALKBH6-associated nucleic acid damage repair and tumor therapy.

Structural Insights into the Ligand-Binding and -Releasing Mechanism of Helicoverpa armigera Pheromone-Binding Protein PBP1.

Cotton bollworm (Helicoverpa armigera) is a worldwide agricultural pest in which the transport of pheromones is indispensable and perceived by pheromone-binding proteins (PBPs). However, three-dimensional structure, pheromone binding, and releasing mechanisms of PBPs are not completely illustrated. Here, we solved three structures of the cotton bollworm HarmPBP1 at different pH values and its complex with ligand, Z-9-hexadecenal. Although apo-HarmPBP1 adopts a common PBP scaffold of six α-helices surrounding a predominantly hydrophobic central pocket, the conformation is greatly distinct from other apo-PBPs. The Z-9-hexadecenal is bound mainly by hydrophobic interaction. The pheromone can enter this cavity through an opening between the helices α5 and α6, as well as the loop between α3 and α4. Structural analysis suggests that ligand entry into the pocket is followed by a shift of Lys94 and Lys138, which may act as a lid at the opening of the pocket. Acidic pH will cause a subtle structural change of the lid, which in turn affects its ligand-binding ability, differently from other family proteins. Taken together, this study provides structural bases for the interactions between pheromones and PBPs, the pH-induced conformational switch, and the design of small inhibitors to control cotton bollworms by disrupting male-female chemosensory communication.

Structure of Rift Valley Fever Virus RNA-Dependent RNA Polymerase.

Rift Valley fever virus (RVFV) belongs to the order Bunyavirales and is the type species of genus Phlebovirus, which accounts for over 50% of family Phenuiviridae species. RVFV is mosquito-borne and causes severe diseases in both humans and livestock, and consists of three segments (S, M, L) in the genome. The L segment encodes an RNA-dependent RNA polymerase (RdRp, L protein) that is responsible for facilitating the replication and transcription of the virus. It is essential for the virus and has multiple drug targets. Here, we established an expression system and purification procedures for full-length L protein, which is composed of an endonuclease domain, RdRp domain, and cap-binding domain. A cryo-EM L protein structure was reported at 3.6 Å resolution. In this first L protein structure of genus Phlebovirus, the priming loop of RVFV L protein is distinctly different from those of other L proteins and undergoes large movements related to its replication role. Structural and biochemical analyses indicate that a single template can induce initiation of RNA synthesis, which is notably enhanced by 5' viral RNA. These findings help advance our understanding of the mechanism of RNA synthesis and provide an important basis for developing antiviral inhibitors. IMPORTANCE The zoonosis RVF virus (RVFV) is one of the most serious arbovirus threats to both human and animal health. RNA-dependent RNA polymerase (RdRp) is a multifunctional enzyme catalyzing genome replication as well as viral transcription, so the RdRp is essential for studying the virus and has multiple drug targets. In our study, we report the structure of RVFV L protein at 3.6 Å resolution by cryo-EM. This is the first L protein structure of genus Phlebovirus. Strikingly, a single template can initiate RNA replication. The structure and assays provide a comprehensive and in-depth understanding of the catalytic and substrate recognition mechanism of RdRp.

Structural Basis of Human Parainfluenza Virus 3 Unassembled Nucleoprotein in Complex with Its Viral Chaperone.

Human parainfluenza virus 3 (HPIV3) belongs to the Paramyxoviridae, causing annual worldwide epidemics of respiratory diseases, especially in newborns and infants. The core components consist of just three viral proteins: nucleoprotein (N), phosphoprotein (P), and RNA polymerase (L), playing essential roles in replication and transcription of HPIV3 as well as other paramyxoviruses. Viral genome encapsidated by N is as a template and recognized by RNA-dependent RNA polymerase complex composed of L and P. The offspring RNA also needs to assemble with N to form nucleocapsids. The N is one of the most abundant viral proteins in infected cells and chaperoned in the RNA-free form (N0) by P before encapsidation. In this study, we presented the structure of unassembled HPIV3 N0 in complex with the N-terminal portion of the P, revealing the molecular details of the N0 and the conserved N0-P interaction. Combined with biological experiments, we showed that the P binds to the C-terminal domain of N0 mainly by hydrophobic interaction and maintains the unassembled conformation of N by interfering with the formation of N-RNA oligomers, which might be a target for drug development. Based on the complex structure, we developed a method to obtain the monomeric N0. Furthermore, we designed a P-derived fusion peptide with 10-fold higher affinity, which hijacked the N and interfered with the binding of the N to RNA significantly. Finally, we proposed a model of conformational transition of N from the unassembled state to the assembled state, which helped to further understand viral replication. IMPORTANCE Human parainfluenza virus 3 (HPIV3) causes annual epidemics of respiratory diseases, especially in newborns and infants. For the replication of HPIV3 and other paramyxoviruses, only three viral proteins are required: phosphoprotein (P), RNA polymerase (L), and nucleoprotein (N). Here, we report the crystal structure of the complex of N and its chaperone P. We describe in detail how P acts as a chaperone to maintain the unassembled conformation of N. Our analysis indicated that the interaction between P and N is conserved and mediated by hydrophobicity, which can be used as a target for drug development. We obtained a high-affinity P-derived peptide inhibitor, specifically targeted N, and greatly interfered with the binding of the N to RNA, thereby inhibiting viral encapsidation and replication. In summary, our results provide new insights into the paramyxovirus genome replication and nucleocapsid assembly and lay the basis for drug development.

Structures and Functional Diversities of ASFV Proteins.

African swine fever virus (ASFV), the causative pathogen of the recent ASF epidemic, is a highly contagious double-stranded DNA virus. Its genome is in the range of 170~193 kbp and encodes 68 structural proteins and over 100 non-structural proteins. Its high pathogenicity strains cause nearly 100% mortality in swine. Consisting of four layers of protein shells and an inner genome, its structure is obviously more complicated than many other viruses, and its multi-layered structures play different kinds of roles in ASFV replication and survival. Each layer possesses many proteins, but very few of the proteins have been investigated at a structural level. Here, we concluded all the ASFV proteins whose structures were unveiled, and explained their functions from the view of structures. Those structures include ASFV AP endonuclease, dUTPases (E165R), pS273R protease, core shell proteins p15 and p35, non-structural proteins pA151R, pNP868R (RNA guanylyltransferase), major capsid protein p72 (gene B646L), Bcl-2-like protein A179L, histone-like protein pA104R, sulfhydryl oxidase pB119L, polymerase X and ligase. These novel structural features, diverse functions, and complex molecular mechanisms promote ASFV to escape the host immune system easily and make this large virus difficult to control.

The Lambda variant of SARS-CoV-2 has a better chance than the Delta variant to escape vaccines.

The newly emerging variants of SARS-CoV-2 from India (Delta variant) and South America (Lambda variant) have led to a higher infection rate of either vaccinated or unvaccinated people. We found that sera from Pfizer-BioNTech vaccine remain high reactivity toward the receptor binding domain (RBD) of Delta variant while it drops dramatically toward that of Lambda variant. Interestingly, the overall titer of antibodies of Pfizer-BioNTech vaccinated individuals drops 3-fold after 6 months, which could be one of major reasons for breakthrough infections, emphasizing the importance of potential third boost shot. While a therapeutic antibody, Bamlanivimab, decreases binding affinity to Delta variant by ~20 fold, it fully lost binding to Lambda variant. Structural modeling of complexes of RBD with human receptor, Angiotensin Converting Enzyme 2 (ACE2), and Bamlanivimab suggest the potential basis of the change of binding. The data suggest possible danger and a potential surge of Lambda variant in near future.

Structure and Function of N-Terminal Zinc Finger Domain of SARS-CoV-2 NSP2.

SARS-CoV-2 has become a global pandemic threatening human health and safety. It is urgent to find effective therapeutic agents and targets with the continuous emergence of novel mutant strains. The knowledge of the molecular basis and pathogenesis of SARS-CoV-2 in host cells requires to be understood comprehensively. The unknown structure and function of nsp2 have hindered our understanding of its role in SARS-CoV-2 infection. Here, we report the crystal structure of the N-terminal of SARS-CoV-2 nsp2 to a high resolution of 1.96 Å. This novel structure contains three zinc fingers, belonging to the C2H2, C4, and C2HC types, respectively. Structure analysis suggests that nsp2 may be involved in binding nucleic acids and regulating intracellular signaling pathways. The binding to single or double-stranded nucleic acids was mainly through the large positively charged region on the surface of nsp2, and K111, K112, K113 were key residues. Our findings lay the foundation for a better understanding of the relationship between structure and function for nsp2. It is helpful to make full use of nsp2 as further research and development of antiviral targets and drug design.

The stability of R-spine defines RAF inhibitor resistance: A comprehensive analysis of oncogenic BRAF mutants with in-frame insertion of αC-ß4 loop.

Although targeting BRAF mutants with RAF inhibitors has achieved promising outcomes in cancer therapy, drug resistance remains a remarkable challenge, and underlying molecular mechanisms are not fully understood. Here, we characterized a previously unknown group of oncogenic BRAF mutants with in-frame insertions (LLRins506 or VLRins506) of αC-ß4 loop. Using structure modeling and molecular dynamics simulation, we found that these insertions formed a large hydrophobic network that stabilizes R-spine and thus triggers the catalytic activity of BRAF. Furthermore, these insertions disrupted BRAF dimer interface and impaired dimerization. Unlike BRAF(V600E), these BRAF mutants with low dimer affinity were strongly resistant to all RAF inhibitors in clinic or clinical trials, which arises from their stabilized R-spines. As predicted by molecular docking, the stabilized R-spines in other BRAF mutants also conferred drug resistance. Together, our data indicated that the stability of R-spine but not dimer affinity determines the RAF inhibitor resistance of oncogenic BRAF mutants.

501Y.V2 and 501Y.V3 variants of SARS-CoV-2 lose binding to bamlanivimab in vitro.

The newly emerging variants of SARS-CoV-2 from South Africa (B.1.351/501Y.V2) and Brazil (P.1/501Y.V3) have led to a higher infection rate and reinfection of COVID-19 patients. We found that the mutations K417N, E484K, and N501Y within the receptor-binding domains (RBDs) of the virus could confer ~2-fold higher binding affinity to the human receptor, angiotensin converting enzyme 2 (ACE2), compared to the wildtype RBD. The mutated version of RBD also completely abolishes the binding of bamlanivimab, a therapeutic antibody, in vitro. Detailed analysis shows that the ~10-fold gain of binding affinity between ACE2 and Y501-RBD, which also exits in the high contagious variant B.1.1.7/501Y.V1 from the United Kingdom, is compromised by additional introduction of the K417/N/T mutation. Mutation of E484K leads to the loss of bamlanivimab binding to RBD, although this mutation does not affect the binding between RBD and ACE2.

The Function of the PRRSV-Host Interactions and Their Effects on Viral Replication and Propagation in Antiviral Strategies.

Porcine reproductive and respiratory syndrome virus (PRRSV) affects the global swine industry and causes disastrous economic losses each year. The genome of PRRSV is an enveloped single-stranded positive-sense RNA of approximately 15 kb. The PRRSV replicates primarily in alveolar macrophages of pig lungs and lymphatic organs and causes reproductive problems in sows and respiratory symptoms in piglets. To date, studies on how PRRSV survives in the host, the host immune response against viral infections, and pathogenesis, have been reported. PRRSV vaccines have been developed, including inactive virus, modified live virus, attenuated live vaccine, DNA vaccine, and immune adjuvant vaccines. However, there are certain problems with the durability and effectiveness of the licensed vaccines. Moreover, the high variability and fast-evolving populations of this RNA virus challenge the design of PRRSV vaccines, and thus effective vaccines against PRRSV have not been developed successfully. As is well known, viruses interact with the host to escape the host's immune response and then replicate and propagate in the host, which is the key to virus survival. Here, we review the complex network and the mechanism of PRRSV-host interactions in the processes of virus infection. It is critical to develop novel antiviral strategies against PRRSV by studying these host-virus interactions and structures to better understand the molecular mechanisms of PRRSV immune escape.