Structural insights in cell-type specific evolution of intra-host diversity by SARS-CoV-2

As the global burden of SARS-CoV-2 infections escalates, so does the evolution of viral variants with increased transmissibility and pathology. In addition to this entrenched diversity, RNA viruses can also display genetic diversity within single infected hosts with co-existing viral variants evolving differently in distinct cell types. The BriSΔ variant, originally identified as a viral subpopulation from SARS-CoV-2 isolate hCoV-19/England/02/2020, comprises in the spike an eight amino-acid deletion encompassing a furin recognition motif and S1/S2 cleavage site. We elucidate the structure, function and molecular dynamics of this spike providing mechanistic insight into how the deletion correlates to viral cell tropism, ACE2 receptor binding and infectivity of this SARS-CoV-2 variant. Our results reveal long-range allosteric communication between functional domains that differ in the wild-type and the deletion variant and support a view of SARS-CoV-2 probing multiple evolutionary trajectories in distinct cell types within the same infected host.

Engineered Exosomes for the Targeted Delivery of a Novel Therapeutic Cargo to Enhance Sorafenib-Mediated Ferroptosis in Hepatocellular Carcinoma.

Background Sorafenib is one of the few effective first-line drugs approved for the treatment of advanced hepatocellular carcinoma (HCC). However, the development of drug resistance is common among individuals with HCC. Thus, there is an urgent need to solve this problem. Results Recent evidence indicated that the anticancer activity of sorafenib mainly relies on the induction of ferroptosis. In our study, genes that suppress ferroptosis, especially GPX4 and DHODH, were enriched in sorafenib-resistant cells and primary tissues and were associated with poor prognosis of HCC patients who received sorafenib treatment. Therefore, silencing GPX4 and DHODH might be a novel and effective strategy to overcome sorafenib resistance. Here, a novel ferroptosis inducer comprising a multiplex small interfering RNA (multi-siRNA) capable of simultaneously silencing GPX4 and DHODH was created. Then, exosomes with high multi-siRNA loading and HCC-specific targeting were established by fusing the SP94 peptide and the N-terminal RNA recognition motif (RRM) of U1-A with the exosomal membrane protein Lamp2b. The results from the in vitro and in vivo experiments indicate that this tumor-targeting nanodelivery system (ExoSP94−lamp2b−RRM-multi-siRNA) could enhance sorafenib-induced ferroptosis and overcome sorafenib resistance, which might open a new avenue for clinically overcoming sorafenib resistance. Conclusions We designed HCC-targeted exosomes (ExoSP94−Lamp2b−RRM) that can deliver a novel ferroptosis inducer. Our data show that ExoSP94−lamp2b−RRM-multi-siRNA could enhance sorafenib-induced ferroptosis by silencing GPX4 and DHODH expression and consequently increase HCC sensitivity to sorafenib. This is the first study to describe the use of engineered exosomes to overcome acquired sorafenib resistance with respect to ferroptosis.

Structural basis of the interaction between SETD2 methyltransferase and hnRNP L paralogs for governing co-transcriptional splicing

The RNA recognition motif (RRM) binds to nucleic acids as well as proteins. More than one such domain is found in the pre-mRNA processing hnRNP proteins. While the mode of RNA recognition by RRMs is known, the molecular basis of their protein interaction remains obscure. Here we describe the mode of interaction between hnRNP L and LL with the methyltransferase SETD2. We demonstrate that for the interaction to occur, a leucine pair within a highly conserved stretch of SETD2 insert their side chains in hydrophobic pockets formed by hnRNP L RRM2. Notably, the structure also highlights that RRM2 can form a ternary complex with SETD2 and RNA. Remarkably, mutating the leucine pair in SETD2 also results in its reduced interaction with other hnRNPs. Importantly, the similarity that the mode of SETD2-hnRNP L interaction shares with other related protein-protein interactions reveals a conserved design by which splicing regulators interact with one another.

Development of a novel bioluminescent activity assay for peptide ligases

In recent years, some peptide ligases have been identified, such as bacterial sortases and certain plant asparaginyl or prolyl endopeptidases. Peptide ligases have wide applications in protein labeling and cyclic peptide synthesis. To characterize known peptide ligases or identify new ones, we propose a novel bioluminescent activity assay via the genetic fusion of a recognition motif of a peptide ligase to the C-terminus of an inactive large NanoLuc fragment (LgBiT) and the chemical introduction of a nucleophilic motif preferred by the peptide ligase to the N-terminus of the low-affinity SmBiT complementation tag. When the inactive ligation version LgBiT protein was ligated with the low-affinity ligation version SmBiT tag by the expected peptide ligase, its luciferase activity would be restored and could be quantified sensitively according to the measured bioluminescence. In the present study, we first validated the novel bioluminescent activity assay using bacterial sortase A and plant butelase-1. Subsequently, we screened novel peptide ligases from crude extracts of selected plants using two LgBiT-SmBiT ligation pairs. Among 80 common higher plants, we identified that five of them likely express asparaginyl endopeptidase-type peptide ligase and four of them likely express prolyl endopeptidase-type peptide ligase, suggesting that peptide ligases are not so rare in higher plants and more of them await discovery. The novel bioluminescent activity assay is ultrasensitive, convenient for use, and resistant to protease interference, and thus would have wide applications for characterizing known peptide ligases or screening new ones from various sources in future studies.

The DNA Recognition Motif of GapR Has an Intrinsic DNA Binding Preference towards AT-rich DNA

The nucleoid-associated protein GapR found in Caulobacter crescentus is crucial for DNA replication, transcription, and cell division. Associated with overtwisted DNA in front of replication forks and the 3′ end of highly-expressed genes, GapR can stimulate gyrase and topo IV to relax (+) supercoils, thus facilitating the movement of the replication and transcription machines. GapR forms a dimer-of-dimers structure in solution that can exist in either an open or a closed conformation. It initially binds DNA through the open conformation and then undergoes structural rearrangement to form a closed tetramer, with DNA wrapped in the central channel. Here, we show that the DNA binding domain of GapR (residues 1–72, GapRΔC17) exists as a dimer in solution and adopts the same fold as the two dimer units in the full-length tetrameric protein. It binds DNA at the minor groove and reads the spatial distribution of DNA phosphate groups through a lysine/arginine network, with a preference towards AT-rich overtwisted DNA. These findings indicate that the dimer unit of GapR has an intrinsic DNA binding preference. Thus, at the initial binding step, the open tetramer of GapR with two relatively independent dimer units can be more efficiently recruited to overtwisted regions.

Sequences in the cytoplasmic tail of SARS-CoV-2 Spike facilitate expression at the cell surface and syncytia formation

The Spike (S) protein of SARS-CoV-2 binds ACE2 to direct fusion with host cells. S comprises a large external domain, a transmembrane domain, and a short cytoplasmic tail. Understanding the intracellular trafficking of S is relevant to SARS-CoV-2 infection, and to vaccines expressing full-length S from mRNA or adenovirus vectors. Here we report a proteomic screen for cellular factors that interact with the cytoplasmic tail of S. We confirm interactions with the COPI and COPII vesicle coats, ERM family actin regulators, and the WIPI3 autophagy component. The COPII binding site promotes exit from the endoplasmic reticulum, and although binding to COPI should retain S in the early Golgi where viral budding occurs, there is a suboptimal histidine residue in the recognition motif. As a result, S leaks to the surface where it accumulates and can direct the formation of multinucleate syncytia. Thus, the trafficking signals in the tail of S indicate that syncytia play a role in the SARS-CoV-2 lifecycle.

Identification and expression analysis of Dazl homologue in Cynops cyanurus

Summary The Dazl (deleted in azoospermia-like) gene encodes an RNA-binding protein containing an RNA recognition motif (RRM) and a DAZ motif. Dazl is essential for gametogenesis in vertebrates. In this study, we report the cloning of Dazl cDNA from Cynops cyanurus. Ccdazl mRNA showed a germline-specific expression pattern as expected. Ccdazl expression gradually decreased during oogenesis, suggesting that it may be involved in oocyte development. Phylogenetic analysis revealed that the Ccdazl protein shares conserved motifs/domains with Dazl proteins from other species. Cloning of Ccdazl provides a new tool to carry out comparative studies of germ cell development in amphibians.

Improving mobilization of foreign DNA into Zymomonas mobilis ZM4 by removal of multiple restriction systems

Zymomonas mobilis has emerged as a promising candidate for production of high value bioproducts from plant biomass. However, a major limitation in equipping Z. mobilis with novel pathways to achieve this goal is restriction of heterologous DNA. Here, we characterized the contribution of several defense systems of Z. mobilis strain ZM4 to impeding heterologous gene transfer from an Escherichia coli donor. Bioinformatic analysis revealed that Z. mobilis ZM4 encodes a previously described mrr -like Type IV Restriction Modification (RM) system, a Type I-F CRISPR system, a chromosomal Type I RM ( hsdMS c ) and a previously uncharacterized Type I RM system, located on an endogenous plasmid ( hsdRMS p ). The DNA recognition motif of HsdRMS p was identified by comparing the methylated DNA sequence pattern of mutants lacking one or both of the hsdMS c and hsdRMS p systems to the parent strain. The conjugation efficiency of synthetic plasmids containing single or combinations of the HsdMS c and HsdRMS p recognition sites indicated that both systems are active and decrease uptake of foreign DNA. In contrast, deletions of mrr and cas3 led to no detectable improvement in conjugation efficiency for the exogenous DNA tested. Thus, the suite of markerless restriction - strains that we constructed, and the knowledge of this new restriction system and its DNA recognition motif provide the necessary platform to flexibly engineer the next generation of Z. mobilis strains for synthesis of valuable products. Importance Zymomonas mobilis is equipped with a number of traits that make it a desirable platform organism for metabolic engineering to produce valuable bioproducts. Engineering strains equipped with synthetic pathways for biosynthesis of new molecules requires integration of foreign genes. In this study we have developed an all-purpose strain, devoid of known host restriction systems and free of any antibiotic resistance markers, which dramatically improves the uptake efficiency of heterologous DNA into Z. mobilis ZM4. We also confirmed the role of a previously known restriction system as well as identified a previously unknown Type I RM system on an endogenous plasmid. Elimination of the barriers to DNA uptake as shown here will allow facile genetic engineering of Z. mobilis .

Receptor switching in newly evolved adeno-associated viruses

Adeno-associated viruses utilize different glycans and the AAV receptor (AAVR) for cellular attachment and entry. Directed evolution has yielded new AAV variants; however, structure-function correlates underlying their improved transduction are generally overlooked. Here, we report that infectious cycling of structurally diverse AAV surface loop libraries yields functionally distinct variants. Newly evolved variants show enhanced cellular binding, uptake and transduction; but through distinct mechanisms. Using glycan-based and genome-wide CRISPR knockout screens, we discover that one AAV variant acquires the ability to recognize sulfated glycosaminoglycans, while another displays receptor switching from AAVR to Integrin β1 (ITGB1). A previously evolved variant, AAVhum.8, preferentially utilizes the ITGB1 receptor over AAVR. Visualization of the AAVhum.8 capsid by cryo-EM at 2.49Å resolution localizes the newly acquired integrin recognition motif adjacent to the AAVR footprint. These observations underscore the new finding that distinct AAV surface epitopes can be evolved to exploit different cellular receptors for enhanced transduction. Importance Understanding how viruses interact with host cells through cell surface receptors is central to discovery and development of antiviral therapeutics, vaccines and gene transfer vectors. Here, we demonstrate that distinct epitopes on the surface of adeno-associated viruses can be evolved by infectious cycling to recognize different cell surface carbohydrates and glycoprotein receptors and solve the 3D structure of one such newly evolved AAV capsid, which provides a roadmap for designing viruses with improved attributes for gene therapy applications.

Gene-free Viral-like particles (VLPs) offer a safer alternative to inactivating or weakening viral strains for traditional vaccines. VLP-based vaccinations without adjuvants have been found to promote humoral and cellular immunity

Nanotechnology and protein engineering helped revolutionize the invention and upgrading of immunization carriers, as well as medicine packaging and delivery systems. Viruses have been considered functioning NPs in the 21st century. This nano-structure may deliver antigens and medications to multiple locations throughout tissues and organs. Gene-free Viral-like particles (VLPs) offer a safer alternative to inactivating or weakening viral strains for traditional vaccines. This allows for the development of VLPs that can contain polyvalent antigenic structures that can also contain antigenic chemicals to target tissues. Also, they're immunogens. VLPs have also been shown to be excellent adjuvants. VLP-based vaccinations without adjuvants have been found to promote humoral and cellular immunity via the MHC class I and II route in some cases.Also used as therapeutic vaccines, presenting patients' own antigens and assisting them in the fight against chronic and metabolic diseases, as well as various types of cancers. Several vaccines created from VLP have been approved or are being tested in the clinical setting. More investigation is needed to fully assess the effectiveness, bad effects, difficulties, and benefits of VLP-based vaccinations when used for different cancers.Despite technical obstacles such as molecules appearing correctly on the particle surface, current research has provided substantial amounts of knowledge that may assist to alleviate these challenges. One option discovered in our lab is to add a sortase recognition motif (LPXTG) to the surface of distinct VLPs that may be exposed. To protect the VLP integrity, it allows proteins to be attached to the VLP surface. Use of VLP external surfaces to transport medications to a specific cell or tissue in order to treat an illness. The VLP's surface can be manipulated in order to optimize the VLP's efficacy. In order to deliver this complex to a specified target, it must be fused with molecules on the surface. Despite the fact that VLP-based vaccinations have been successful in helping to prevent disease, more work is needed to reach the optimum condition.