Synthetic Virology Research Articles

Leveraging Synthetic Virology for the Rapid Engineering of Vesicular Stomatitis Virus (VSV)

Vesicular stomatitis virus (VSV) is a prototype RNA virus that has been instrumental in advancing our understanding of viral molecular biology and has applications in vaccine development, cancer therapy, antiviral screening, and more. Current VSV genome plasmids for purchase or contract virus services provide limited options for modification, restricted to predefined cloning sites and insert locations. Improved methods and tools to engineer VSV will unlock further insights into long-standing virology questions and new opportunities for innovative therapies. Here, we report the design and construction of a full-length VSV genome. The 11,161 base pair synthetic VSV (synVSV) was assembled from four modularized DNA fragments. Following rescue and titration, phenotypic analysis showed no significant differences between natural and synthetic viruses. To demonstrate the utility of a synthetic virology platform, we then engineered VSV with a foreign glycoprotein, a common use case for studying viral entry and developing anti-virals. To show the freedom of design afforded by this platform, we then modified the genome of VSV by rearranging the gene order, switching the positions of VSV-P and VSV-M genes. This work represents a significant technical advance, providing a flexible, cost-efficient platform for the rapid construction of VSV genomes, facilitating the development of innovative therapies.

Synthetic virology approaches to improve the safety and efficacy of oncolytic virus therapies

The large coding potential of vaccinia virus (VV) vectors is a defining feature. However, limited regulatory switches are available to control viral replication as well as timing and dosing of transgene expression in order to facilitate safe and efficacious payload delivery. Herein, we adapt drug-controlled gene switches to enable control of virally encoded transgene expression, including systems controlled by the FDA-approved rapamycin and doxycycline. Using ribosome profiling to characterize viral promoter strength, we rationally design fusions of the operator element of different drug-inducible systems with VV promoters to produce synthetic promoters yielding robust inducible expression with undetectable baseline levels. We also generate chimeric synthetic promoters facilitating additional regulatory layers for VV-encoded synthetic transgene networks. The switches are applied to enable inducible expression of fusogenic proteins, dose-controlled delivery of toxic cytokines, and chemical regulation of VV replication. This toolbox enables the precise modulation of transgene circuitry in VV-vectored oncolytic virus design.

Abstract 688: Oncolytic VSV with retargeted glycoprotein and genetic ON switch for precise targeting of liver cancer using a synthetic virology platform

Abstract Introduction: Humane Genomics has developed a platform to make artificial oncolytic viruses with unprecedented selectivity and efficiency. The platform is based on Vesicular Stomatitis Virus (VSV), which has several ideal properties (highly oncolytic, non-pathogenic, small genome that’s well understood and easily modified). Here we present the development of HG001, our first therapy for the treatment of liver cancer. Methods: Genome design of HG001 was completed using our proprietary software. The virus was designed with a GPC3 targeting glycoprotein and a genetic ON-switch (aptazyme) with a liver cancer specific aptamer. DNA fragments were synthesized and then assembled to construct the complete viral genome of HG001. Virus was rescued in BHK-21 using reverse genetics and then verified by nanopore sequencing. We evaluated efficacy and safety in cancer- (Huh-7, SNU-449) and healthy- (NIH/3T3) cells. VSV wildtype was used as a control virus. IncuCyte live cell imaging and analysis software was used to quantify efficacy and safety. Cell counts and cell area per well (µm2/well) were measured to evaluate cell health and proliferation. Apoptosis was determined using Annexin V. Student’s t-test was used to assess statistical significance. Results: Our platform enabled us to complete the process from assembly of the fragments to the start of testing of HG001 in under 2 weeks. To determine selectivity and efficacy, we evaluated serial dilutions in Huh7 (high expression of GPC3) and SNU-449 cells (GPC3 negative). Real time monitoring of HG001 showed cell killing in Huh7 cells at MOI=1 (p<0.001) and cytopathic effects (CPE) in MOI=0.1. However, no impact on cell proliferation and viability was found in SNU-449 (p=ns), indicating specificity for the target receptor. To further validate selectivity and demonstrate safety, we assayed normal NIH/3T3 cells with the same titers. We did not observe any indication of cytotoxicity, and kinetic data of cell growth revealed no significant difference between HG001 and mock infection (p=ns). In stark contrast, VSV-WT induced rapid lysis in NIH/3T3 cells at MOI=0.01 (p<0.01). Taken together, HG001 exhibits at least 2 orders of magnitude improvement in safety. Apoptosis assays conducted in these cell lines corroborated these findings. HG001 was well tolerated and showed no adverse effects during initial in vivo studies. Current in vivo studies are ongoing to confirm efficacy. Conclusion: We have shown that a rationally designed synthetic virus can be made efficiently with high specificity and efficacy. Initial testing is showing high promise for future in vivo testing. With further iterations we hope to develop a strong therapeutic for liver cancer. Going forward, we are actively looking for collaborations with oncology centers on other types of cancer we could target using our platform. Citation Format: Chad Moles, Rupsa Basu, Taylor Flaat, Christopher Collado, Peter Weijmarshausen. Oncolytic VSV with retargeted glycoprotein and genetic ON switch for precise targeting of liver cancer using a synthetic virology platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 688.

Synthetic virology: the experts speak.

Even though only a few labs around the world have the means to engineer a purely synthetic virus, debate on the origins of SARS-CoV-2 has resurfaced concerns about the risks and benefits of synthetic virology.

The Future of Virology is Synthetic.

ABSTRACTThe virosphere (i.e., global virome) represents a vast library of unknown genes on the planet. Synthetic biology through engineering principles could be the key to unlocking this massive global gene repository. Synthetic viruses may also be used as tools to understand “the rules of life” in diverse microbial ecosystems. Such insights may be crucial for understanding the assembly, diversity, structure, and scale of virus-mediated function. Viruses directly affect resilience, stability, and microbial community selection via death resistance cycles. Interpreting and clarifying these effects is essential for predicting the system’s ecology, evolution, and ecosystem stability in an increasingly unstable global climate. A “silent looming pandemic” due to multidrug-resistant microbes will directly impact the global economy, and synthetic virology could provide a future strategy of treatment using targeted viral therapy. This commentary will discuss current techniques for manipulating viruses synthetically, contributing to improved human health and sustainable agriculture.

Lectin-Glycan-Mediated Nanoparticle Docking as a Step toward Programmable Membrane Catalysis and Adhesion in Synthetic Protocells

The spontaneous assembly of nanoscale building blocks into continuous semipermeable membranes is a key requirement for the structuration of synthetic protocells. Engineering the functionality and programmability of these building units provides a step toward more complex cell-like entities with adaptive membrane properties. Inspired by the central role of protein (lectin)-carbohydrate interactions in cellular recognition and adhesion, we fabricate semipermeable polysaccharide-polymer microcapsules (polysaccharidosomes) with intrinsic lectin-binding properties. We employ amphiphilic polysaccharide-polymer membrane building blocks endowed with intrinsic bio-orthogonal lectin-glycan recognition sites to facilitate the reversible noncovalent docking of functionalized polymer or zeolitic nanoparticles on the polysaccharidosomes. We show that the programmed attachment of enzyme-loaded nanoparticles gives rise to a membrane-gated spatially localized cascade reaction within the protocells due to the thermoresponsiveness of the polysaccharidosome membrane, and we demonstrate that extended closely packed networks are produced via reversible lectin-mediated adhesion between the protocells. Our results provide a step toward nanoscale engineering of bioinspired cell-like materials and could have longer-term applications in synthetic virology, protobiology, and microbiosensor and microbioreactor technologies.

An Investigation of the Effect of Transfected Defective, Ebola Virus Genomes on Ebola Replication.

As the ongoing outbreak in the Democratic Republic of Congo illustrates, Ebola virus disease continues to pose a significant risk to humankind and this necessitates the continued development of therapeutic options. One option that warrants evaluation is that of defective genomes; these can potentially parasitize resources from the wild-type virus and may even be packaged for repeated co-infection cycles. Deletion and copy-back defective genomes have been identified and reported in the literature. As a crude, mixed preparation these were found to have limiting effects on cytopathology. Here we have used synthetic virology to clone and manufacture two deletion defective genomes. These genomes were tested with Ebola virus using in vitro cell culture and shown to inhibit viral replication; however, and against expectations, the defective genomes were not released in biologically significant numbers. We propose that EBOV might have yet unknown mechanisms to prevent parasitisation by defective interfering particles beyond the known mechanism that prevents sequential infection of the same cell. Understanding this mechanism would be necessary in any development of a defective interfering particle-based therapy.

Cover Image, Volume 12, Issue 2

AbstractThe cover image is based on the Advanced Review From stars to stripes: RNA‐directed shaping of plant viral protein templates ‐ structural synthetic virology for smart biohybrid nanostructures, by Christina Wege and Claudia Koch. https://doi.org/10.1002/wnan.1591. image

From stars to stripes: RNA-directed shaping of plant viral protein templates-structural synthetic virology for smart biohybrid nanostructures.

The self-assembly of viral building blocks bears exciting prospects for fabricating new types of bionanoparticles with multivalent protein shells. These enable a spatially controlled immobilization of functionalities at highest surface densities-an increasing demand worldwide for applications from vaccination to tissue engineering, biocatalysis, and sensing. Certain plant viruses hold particular promise because they are sustainably available, biodegradable, nonpathogenic for mammals, and amenable to in vitro self-organization of virus-like particles. This offers great opportunities for their redesign into novel "green" carrier systems by spatial and structural synthetic biology approaches, as worked out here for the robust nanotubular tobacco mosaic virus (TMV) as prime example. Natural TMV of 300 x 18 nm is built from more than 2,100 identical coat proteins (CPs) helically arranged around a 6,395 nucleotides ssRNA. In vitro, TMV-like particles (TLPs) may self-assemble also from modified CPs and RNAs if the latter contain an Origin of Assembly structure, which initiates a bidirectional encapsidation. By way of tailored RNA, the process can be reprogrammed to yield uncommon shapes such as branched nanoobjects. The nonsymmetric mechanism also proceeds on 3'-terminally immobilized RNA and can integrate distinct CP types in blends or serially. Other emerging plant virus-deduced systems include the usually isometric cowpea chlorotic mottle virus (CCMV) with further strikingly altered structures up to "cherrybombs" with protruding nucleic acids. Cartoon strips and pictorial descriptions of major RNA-based strategies induct the reader into a rare field of nanoconstruction that can give rise to utile soft-matter architectures for complex tasks. This article is categorized under: Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures.

CSV2018: The 2nd Symposium of the Canadian Society for Virology.

The 2nd Symposium of the Canadian Society for Virology (CSV2018) was held in June 2018 in Halifax, Nova Scotia, Canada, as a featured event marking the 200th anniversary of Dalhousie University. CSV2018 attracted 175 attendees from across Canada and around the world, more than double the number that attended the first CSV symposium two years earlier. CSV2018 provided a forum to discuss a wide range of topics in virology including human, veterinary, plant, and microbial pathogens. Invited keynote speakers included David Kelvin (Dalhousie University and Shantou University Medical College) who provided a historical perspective on influenza on the 100th anniversary of the 1918 pandemic; Sylvain Moineau (Université Laval) who described CRISPR-Cas systems and anti-CRISPR proteins in warfare between bacteriophages and their host microbes; and Kate O’Brien (then from Johns Hopkins University, now relocated to the World Health Organization where she is Director of Immunization, Vaccines and Biologicals), who discussed the underlying viral etiology for pneumonia in the developing world, and the evidence for respiratory syncytial virus (RSV) as a primary cause. Reflecting a strong commitment of Canadian virologists to science communication, CSV2018 featured the launch of Halifax’s first annual Soapbox Science event to enable public engagement with female scientists, and the live-taping of the 499th episode of the This Week in Virology (TWIV) podcast, hosted by Vincent Racaniello (Columbia University) and science writer Alan Dove. TWIV featured interviews of CSV co-founders Nathalie Grandvaux (Université de Montréal) and Craig McCormick (Dalhousie University), who discussed the origins and objectives of the new society; Ryan Noyce (University of Alberta), who discussed technical and ethical considerations of synthetic virology; and Kate O’Brien, who discussed vaccines and global health. Finally, because CSV seeks to provide a better future for the next generation of Canadian virologists, the symposium featured a large number of oral and poster presentations from trainees and closed with the awarding of presentation prizes to trainees, followed by a tour of the Halifax Citadel National Historic Site and an evening of entertainment at the historic Alexander Keith’s Brewery.

Physical, chemical, and synthetic virology: Reprogramming viruses as controllable nanodevices.

The fields of physical, chemical, and synthetic virology work in partnership to reprogram viruses as controllable nanodevices. Physical virology provides the fundamental biophysical understanding of how virus capsids assemble, disassemble, display metastability, and assume various configurations. Chemical virology considers the virus capsid as a chemically addressable structure, providing chemical pathways to modify the capsid exterior, interior, and subunit interfaces. Synthetic virology takes an engineering approach, modifying the virus capsid through rational, combinatorial, and bioinformatics-driven design strategies. Advances in these three subfields of virology aim to develop virus-based materials and tools that can be applied to solve critical problems in biomedicine and biotechnology, including applications in gene therapy and drug delivery, diagnostics, and immunotherapy. Examples discussed include mammalian viruses, such as adeno-associated virus (AAV), plant viruses, such as cowpea mosaic virus (CPMV), and bacterial viruses, such as Qβ bacteriophage. Importantly, research efforts in physical, chemical, and synthetic virology have further unraveled the design principles foundational to the form and function of viruses. This article is categorized under: Diagnostic Tools > Diagnostic Nanodevices Biology-Inspired Nanomaterials > Protein and Virus-Based Structures.

Inoculating science against potential pandemics and information hazards.

The recent de novo assembly of horsepox is an instructive example of an information hazard: published methods enabling poxvirus synthesis led to media coverage spelling out the implications, efficiently disseminating true information that might be used to cause harm. Whether or not the benefits justified the risks, the horsepox saga provides ample reason to upgrade the current system for screening synthesized DNA for hazardous sequences, which does not cover the majority of firms and cannot reliably prevent the assembly of potentially pandemic pathogens. An upgraded system might leverage one-way encryption to confidentially scrutinize virtually all commercial production by a cooperative international network of servers whose integrity can be verified by third parties. Funders could support participating institutions to ease the transition or outright subsidize the market to make clean DNA cheaper, while boycotts by journals, institutions, and funders could ensure compliance and require hardware-level locks on future DNA synthesizers. However, the underlying problem is that security and safety discussions among experts typically follow potentially hazardous events rather than anticipating them. Changing norms and incentives to favor preregistration and advisory peer review of planned experiments could test alternatives to the current closeted research model in select areas of science. Because the fields of synthetic mammalian virology and especially gene drive research involve technologies that could be unilaterally deployed and may self-replicate in the wild, they are compelling candidates for initial trials of early-stage peer review.

Molecular biology of Macrobrachium rosenbergii nodavirus infection in giant freshwater prawn.

Macrobrachium rosenbergii nodavirus (MrNV) has been threatening the giant freshwater prawn aquaculture since 1997, causing white tail disease in the prawn species that leads to 100% lethality of the infected postlarvae. Comprehension of the viral infectivity and pathogenesis at molecular biology level has recently resolved the viral capsid protein and evidenced the significant difference in the viral structural protein compared to other nodaviruses that infect fish and insect. Cumulative researches have remarked the proposal to assert MrNV as a member of new genus, gammanodavirus to the Nodaviridae family. The significance of molecular biology in MrNV infection is being highlighted in this current review, revolving the viral life cycle from virus binding and entry into host, virus replication in host cell, to virus assembly and release. The current review also highlights the emerging aptamers technology that is also known as synthetic antibody, its application in disease diagnosis, and its prophylactic and therapeutic properties. The future perspective of synthetic virology technology in understanding viral pathogenesis, as well as its potential in viral vaccine development, is also discussed.

Significant differences in terms of codon usage bias between bacteriophage early and late genes: a comparative genomics analysis

BackgroundViruses undergo extensive evolutionary selection for efficient replication which effects, among others, their codon distribution. In the current study, we aimed at understanding the way evolution shapes the codon distribution in early vs. late viral genes in terms of their expression during different stages in the viral replication cycle. To this end we analyzed 14 bacteriophages and 11 human viruses with available information about the expression phases of their genes.ResultsWe demonstrated evidence of selection for distinct composition of synonymous codons in early and late viral genes in 50% of the analyzed bacteriophages. Among others, this phenomenon may be related to the time specific adaptation of the viral genes to the translation efficiency factors involved at different bacteriophage developmental stages. Specifically, we showed that the differences in codon composition in different temporal gene groups cannot be explained only by phylogenetic proximities between the analyzed bacteriophages, and can be partially explained by differences in the adaptation to the host tRNA pool, nucleotide bias, GC content and more.In contrast, no difference in temporal regulation of synonymous codon usage was observed in human viruses, possibly because of a stronger selection pressure due to a larger effective population size in bacteriophages and their bacterial hosts.ConclusionsThe codon distribution in large fractions of bacteriophage genomes tend to be different in early and late genes. This phenomenon seems to be related to various aspects of the viral life cycle, and to various intracellular processes. We believe that the reported results should contribute towards better understanding of viral evolution and may promote the development of relevant procedures in synthetic virology.

Synthetic plant virology for nanobiotechnology and nanomedicine.

Nanotechnology is a rapidly expanding field seeking to utilize nano‐scale structures for a wide range of applications. Biologically derived nanostructures, such as viruses and virus‐like particles (VLPs), provide excellent platforms for functionalization due to their physical and chemical properties. Plant viruses, and VLPs derived from them, have been used extensively in biotechnology. They have been characterized in detail over several decades and have desirable properties including high yields, robustness, and ease of purification. Through modifications to viral surfaces, either interior or exterior, plant‐virus‐derived nanoparticles have been shown to support a range of functions of potential interest to medicine and nano‐technology. In this review we highlight recent and influential achievements in the use of plant virus particles as vehicles for diverse functions: from delivery of anticancer compounds, to targeted bioimaging, vaccine production to nanowire formation. WIREs Nanomed Nanobiotechnol 2017, 9:e1447. doi: 10.1002/wnan.1447For further resources related to this article, please visit the WIREs website.

An impact of synthetic virology: Recoding arboviruses for vaccine development

An impact of synthetic virology: Recoding arboviruses for vaccine development

Plant viral nanoparticles-based HER2 vaccine: Immune response influenced by differential transport, localization and cellular interactions of particulate carriers

Cancer vaccines are designed to elicit an endogenous adaptive immune response that can successfully recognize and eliminate residual or recurring tumors. Such approaches can potentially overcome shortcomings of passive immunotherapies by generating long-lived therapeutic effects and immune memory while limiting systemic toxicities. A critical determinant of vaccine efficacy is efficient transport and delivery of tumor-associated antigens to professional antigen presenting cells (APCs). Plant viral nanoparticles (VNPs) with natural tropism for APCs and a high payload carrying capacity may be particularly effective vaccine carriers. The applicability of VNP platform technologies is governed by stringent structure-function relationships. We compare two distinct VNP platforms: icosahedral cowpea mosaic virus (CPMV) and filamentous potato virus X (PVX). Specifically, we evaluate invivo capabilities of engineered VNPs delivering human epidermal growth factor receptor 2 (HER2) epitopes for therapy and prophylaxis of HER2+ malignancies. Our results corroborate the structure-function relationship where icosahedral CPMV particles showed significantly enhanced lymph node transport and retention, and greater uptake by/activation of APCs compared to filamentous PVX particles. These enhanced immune cell interactions and transport properties resulted in elevated HER2-specific antibody titers raised by CPMV- vs. PVX-based peptide vaccine. The 'synthetic virology' field is rapidly expanding with numerous platforms undergoing development and preclinical testing; our studies highlight the need for systematic studies to define rules guiding the design and rational choice of platform, in the context of peptide-vaccine display technologies.

Synthetic virology: engineering viruses for gene delivery.

The success of gene therapy relies heavily on the performance of vectors that can effectively deliver transgenes to desired cell populations. As viruses have evolved to deliver genetic material into cells, a prolific area of research has emerged over the last several decades to leverage the innate properties of viruses as well as to engineer new features into them. Specifically, the field of synthetic virology aims to capitalize on knowledge accrued from fundamental virology research in order to design functionally enhanced gene delivery vectors. The enhanced viral vectors, or 'bionic' viruses, feature engineered components, or 'parts', that are natural (intrinsic to viruses or from other organisms) and synthetic (such as man-made polymers or inorganic nanoparticles). Various design strategies--rational, combinatorial, and pseudo-rational--have been pursued to create the hybrid viruses. The gene delivery vectors of the future will likely criss-cross the boundaries between natural and synthetic domains to harness the unique strengths afforded by the various functional parts that can be grafted onto virus capsids. Such research endeavors will further expand and enable enhanced control over the functional capacity of these nanoscale devices for biomedicine.