Insight into the Binding Energetics of Targeted Reversible Covalent Inhibitors of the SARS-CoV-2 Main Protease

The main protease (Mpro) of the SARS-CoV-2 virus is an attractive therapeutic target for developing antivirals to combat COVID-19. Mpro is essential for the replication cycle of the SARS-CoV-2 virus, so inhibiting Mpro blocks a vital piece of the cell replication machinery of the virus. A promising strategy to disrupt the viral replication cycle is to design inhibitors that bind to the active site cysteine (Cys145) of the Mpro. Cysteine targeted covalent inhibitors are gaining traction in drug discovery owing to the benefits of improved potency and extended drug-target engagement. An interesting aspect of these inhibitors is that they can be chemically tuned to form a covalent, but reversible bond, with their targets of interest. Several small-molecule cysteine-targeting covalent inhibitors of the Mpro have been discovered—some of which are currently undergoing evaluation in early phase human clinical trials. Understanding the binding energetics of these inhibitors could provide new insights to facilitate the design of potential drug candidates against COVID-19. Motivated by this, we employed rigorous absolute binding free energy calculations and hybrid quantum mechanical/molecular mechanical (QM/MM) calculations to estimate the energetics of binding of some promising reversible covalent inhibitors of the Mpro. We find that the inclusion of enhanced sampling techniques such as replica-exchange algorithm in binding free energy calculations can improve the convergence of predicted non-covalent binding free energy estimates of inhibitors binding to the Mpro target. In addition, our results indicate that binding free energy calculations coupled with multiscale simulations can be a useful approach to employ in ranking covalent inhibitors to their targets. This approach may be valuable in prioritizing and refining covalent inhibitor compounds for lead discovery efforts against COVID-19 and future coronavirus infections.

Insight into the Binding Energetics of Targeted Reversible Covalent Inhibitors of the SARS-CoV-2 Main Protease

The main protease (Mpro) of the SARS-CoV-2 virus is an attractive therapeutic target for developing antivirals to combat COVID-19. Mpro is essential for the replication cycle of the SARS-CoV-2 virus, so inhibiting Mpro blocks a vital piece of the cell replication machinery of the virus. A promising strategy to disrupt the viral replication cycle is to design inhibitors that bind to the active site cysteine (Cys145) of the Mpro. Cysteine targeted covalent inhibitors are gaining traction in drug discovery owing to the benefits of improved potency and extended drug-target engagement. An interesting aspect of these inhibitors is that they can be chemically tuned to form a covalent, but reversible bond, with their targets of interest. Several small-molecule cysteine-targeting covalent inhibitors of the Mpro have been discovered—some of which are currently undergoing evaluation in early phase human clinical trials. Understanding the binding energetics of these inhibitors could provide new insights to facilitate the design of potential drug candidates against COVID-19. Motivated by this, we employed rigorous absolute binding free energy calculations and hybrid quantum mechanical/molecular mechanical (QM/MM) calculations to estimate the energetics of binding of some promising reversible covalent inhibitors of the Mpro. We find that the inclusion of enhanced sampling techniques such as replica-exchange algorithm in binding free energy calculations can improve the convergence of predicted non-covalent binding free energy estimates of inhibitors binding to the Mpro target. In addition, our results indicate that binding free energy calculations coupled with multiscale simulations can be a useful approach to employ in ranking covalent inhibitors to their targets. This approach may be valuable in prioritizing and refining covalent inhibitor compounds for lead discovery efforts against COVID-19 and future coronavirus infections.

Recent Advances in Bunyavirus Reverse Genetics Research: Systems Development, Applications, and Future Perspectives

Bunyaviruses are members of the Bunyavirales order, which is the largest group of RNA viruses, comprising 12 families, including a large group of emerging and re-emerging viruses. These viruses can infect a wide variety of species worldwide, such as arthropods, protozoans, plants, animals, and humans, and pose substantial threats to the public. In view of the fact that a better understanding of the life cycle of a highly pathogenic virus is often a precondition for developing vaccines and antivirals, it is urgent to develop powerful tools to unravel the molecular basis of the pathogenesis. However, biosafety level −3 or even −4 containment laboratory is considered as a necessary condition for working with a number of bunyaviruses, which has hampered various studies. Reverse genetics systems, including minigenome (MG), infectious virus-like particle (iVLP), and infectious full-length clone (IFLC) systems, are capable of recapitulating some or all steps of the viral replication cycle; among these, the MG and iVLP systems have been very convenient and effective tools, allowing researchers to manipulate the genome segments of pathogenic viruses at lower biocontainment to investigate the viral genome transcription, replication, virus entry, and budding. The IFLC system is generally developed based on the MG or iVLP systems, which have facilitated the generation of recombinant infectious viruses. The MG, iVLP, and IFLC systems have been successfully developed for some important bunyaviruses and have been widely employed as powerful tools to investigate the viral replication cycle, virus–host interactions, virus pathogenesis, and virus evolutionary process. The majority of bunyaviruses is generally enveloped negative-strand RNA viruses with two to six genome segments, of which the viruses with bipartite and tripartite genome segments have mostly been characterized. This review aimed to summarize current knowledge on reverse genetic studies of representative bunyaviruses causing severe diseases in humans and animals, which will contribute to the better understanding of the bunyavirus replication cycle and provide some hints for developing designed antivirals.

In-depth characterization of the chikungunya virus replication cycle

The chikungunya virus has spread globally with a remarkably high attack rate. Infection causes arthralgic sequelae that can last for years. Nevertheless, there are no specific drugs or vaccines to contain the virus. Understanding the biology of the virus, such as its replication cycle, is a powerful tool to identify new drugs and comprehend virus-host interactions. Even though the chikungunya virus has been known for a long time (first described in 1952), many aspects of the replication cycle remain unclear. Furthermore, part of the cycle is based on observations of other alphaviruses. In this study, we used electron and scanning microscopy, as well as biological assays, to analyze and investigate the stages of the chikungunya virus replication cycle. Based on our data, we found other infection cellular activities than those usually described for the chikungunya virus replication cycle, i.e. we show particles enveloping intracellularly without budding in a membrane-delimited morphogenesis area; and we also observed virion release by membrane protrusions. Our work provides novel details regarding the biology of chikungunya virus and fills gaps in our knowledge of its replication cycle. These findings may contribute to a better understanding of virus-host interactions and support the development of antivirals. IMPORTANCE The understanding of virus biology is essential to containing virus dissemination, and exploring the virus replication cycle is a powerful tool to do this. There are many points in the biology of the chikungunya virus that need to be clarified, especially regarding its replication cycle. Our incomplete understanding of chikungunya virus infection stages is based on studies with other alphaviruses. We systematized the chikungunya virus replication cycle using microscopic imaging in the order of infection stages: entry, replication, protein synthesis, assembly/morphogenesis, and release. The imaging evidence shows novel points in the replication cycle of enveloping without budding, as well as particle release by cell membrane protrusion.

A Cytoskeletal Vortex Drives Phage Nucleus Rotation During Jumbo Phage Replication in E. coli

Vortex-like arrays of cytoskeletal filaments that drive cytoplasmic streaming and nucleus rotation have been identified in eukaryotes, but similar structures have not been described in prokaryotes. The only known example of a rotating intracellular body in prokaryotic cells occurs when nucleus-forming jumbo phages infect Pseudomonas. During infection, a bipolar spindle of PhuZ filaments drives intracellular rotation of the phage nucleus, a key aspect of the replication cycle. Here we show the E. coli jumbo phage Goslar assembles a phage nucleus surrounded by an array of PhuZ filaments resembling a vortex instead of a bipolar spindle. Expression of mutant PhuZ strongly reduces Goslar phage nucleus rotation, demonstrating that the PhuZ cytoskeletal vortex is necessary for rotating the phage nucleus. While vortex-like cytoskeletal arrays are important in eukaryotes, this work identifies the first known example of a coherent assembly of filaments into a vortex-like structure driving intracellular rotation within the prokaryotic cytoplasm.

OriCiro® Cell-Free Cloning System/PASS V4.1.2 v1

OriCiro® Cell-Free Cloning System is a rapid and powerful tool replacing cumbersome DNA cloning (plasmid construction) process relying on E. coli. The system consists of two kits. OriCiro Assembly Kit allows seamless assembly of multiple overlapping DNA fragments. The assembly product can be added directly to OriCiro Amp Kit to get selective amplification of your target circular DNA (Figure 1). The amplified product is supercoiled DNA topologically identical to plasmid DNA isolated from E. coli. 1. OriCiro Assembly Kit: Multiple DNA fragments are assembled seamlessly at 42 ̊C for 30 minutes via ~40 bp overlapping ends (Figure 2). DNA fragments generated by PCR or restriction enzyme digestion are available. Our unique enzyme-based annealing mechanism allows powerful assembly up to 50 fragments simultaneously. 2. OriCiro Amp Kit: The reaction consists of 26 purified enzymes involved in chromosome replication of E. coli. The chromosome replication cycle repeats autonomously at around 30 ̊C, enabling exponential amplification of circular DNA having oriC with extremely high fidelity (10-8 error/base/cycle) (Figure 3). The kit yields up to 1 μg circular DNA per 10 μL reaction at 33 ̊C for 6 hr. The maximum amplification size is 50 kb in the current version of the kit. n.b. OriCiro Amp NEEDS oriC Cassette (0.4 kb) which can be inserted into circular DNA using OriCiro assembly kit. References: 1. T. Mukai, T. Yoneji, K. Yamada, H. Fujita, S. Nara, M. Su'etsugu, Overcoming the Challenges of Megabase-Sized Plasmid Construction in Escherichia coli, ACS Synthetic Biology, 2020, 9 (6), 1315- 1327 2. T. Hasebe, K. Narita, S. Hidaka, M. Su'etsugu, Efficient Arrangement of the Replication Fork Trap for In Vitro Propagation of Monomeric Circular DNA in the Chromosome-Replication Cycle Reaction. Life, 2018, 8 (43) 3. M. Su’etsugu, H. Takada, T. Katayama, H. Tsujimoto, Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle, Nucleic Acids Research, 2017, 45 (20), 11525– 11534

The Role of Tricarboxylic Acid Cycle Metabolites in Viral Infections

Host cell metabolism is essential for the viral replication cycle and, therefore, for productive infection. Energy (ATP) is required for the receptor-mediated attachment of viral particles to susceptible cells and for their entry into the cytoplasm. Host cells must synthesize an array of biomolecules and engage in intracellular trafficking processes to enable viruses to complete their replication cycle. The tricarboxylic acid (TCA) cycle has a key role in ATP production as well as in the synthesis of the biomolecules needed for viral replication. The final assembly and budding process of enveloped viruses, for instance, require lipids, and the TCA cycle provides the precursor (citrate) for fatty acid synthesis (FAS). Viral infections may induce host inflammation and TCA cycle metabolic intermediates participate in this process, notably citrate and succinate. On the other hand, viral infections may promote the synthesis of itaconate from TCA cis-aconitate. Itaconate harbors anti-inflammatory, anti-oxidant, and anti-microbial properties. Fumarate is another TCA cycle intermediate with immunoregulatory properties, and its derivatives such as dimethyl fumarate (DMF) are therapeutic candidates for the contention of virus-induced hyper-inflammation and oxidative stress. The TCA cycle is at the core of viral infection and replication as well as viral pathogenesis and anti-viral immunity. This review highlights the role of the TCA cycle in viral infections and explores recent advances in the fast-moving field of virometabolism.

In vitro amplification of whole large plasmids via transposon-mediated oriC insertion

DNA amplification is a fundamental technique in molecular biology. The replication cycle reaction is a new method for amplification of large circular DNA having oriC sequences, which is a replication initiation site of the Escherichia coli chromosome. We here developed a replication cycle reaction-based method useful for amplification of various circular DNAs lacking oriC, even in the absence of any sequence information, via transposon-mediated oriC insertion to the circular DNA template. A 15-kb non- oriC plasmid was amplified from a very small amount of starting DNA (50 fg, 1 fM). The method was also applicable to GC-rich plasmid (69%) or large F-plasmid (230 kb). This method thus provides a powerful tool to amplify various environmental circular DNAs.