Biology subjects

Dowgier, G.

Publications and source records attributed to Dowgier, G..

3 recordsLinked to original sources

bioRxiv2023

One-pot Golden Gate Assembly of an avian infectious bronchitis virus reverse-genetics system

Avian infectious bronchitis is an acute respiratory disease of poultry of particular concern for global food security. Investigation of Infectious Bronchitis Virus (IBV), the causative agent of avian infectious bronchitis, via reverse genetics enables deeper understanding of virus biology and a rapid response to emerging variants. Classic methods of reverse genetics for IBV can be time consuming, rely on recombination for the introduction of mutations, and, depending on the system, can be subject to genome instability and unreliable success rates. In this study, we have applied data-optimized Golden Gate Assembly design to create a rapidly executable, flexible, and faithful reverse genetics system for IBV. The IBV genome was divided into 12 fragments at high-fidelity fusion site breakpoints. All fragments were synthetically produced and propagated in E. coli plasmids, amenable to standard molecular biology techniques for DNA manipulation. The assembly can be carried out in a single reaction, with the products used directly in subsequent viral rescue steps. We demonstrate the use of this system for generation of point mutants and gene replacements. This Golden Gate Assembly-based reverse genetics system will enable rapid response to emerging variants of IBV, particularly important to vaccine development for controlling spread within poultry populations.

genetics↗

bioRxiv2022

Global landscape of the host response to SARS-CoV-2 variants reveals viral evolutionary trajectories

A series of SARS-CoV-2 variants of concern (VOCs) have evolved in humans during the COVID-19 pandemic--Alpha, Beta, Gamma, Delta, and Omicron. Here, we used global proteomic and genomic analyses during infection to understand the molecular responses driving VOC evolution. We discovered VOC-specific differences in viral RNA and protein expression levels, including for N, Orf6, and Orf9b, and pinpointed several viral mutations responsible. An analysis of the host response to VOC infection and comprehensive interrogation of altered virus-host protein-protein interactions revealed conserved and divergent regulation of biological pathways. For example, regulation of host translation was highly conserved, consistent with suppression of VOC replication in mice using the translation inhibitor plitidepsin. Conversely, modulation of the host inflammatory response was most divergent, where we found Alpha and Beta, but not Omicron BA.1, antagonized interferon stimulated genes (ISGs), a phenotype that correlated with differing levels of Orf6. Additionally, Delta more strongly upregulated proinflammatory genes compared to other VOCs. Systematic comparison of Omicron subvariants revealed BA.5 to have evolved enhanced ISG and proinflammatory gene suppression that similarly correlated with Orf6 expression, effects not seen in BA.4 due to a mutation that disrupts the Orf6-nuclear pore interaction. Our findings describe how VOCs have evolved to fine-tune viral protein expression and protein-protein interactions to evade both innate and adaptive immune responses, offering a likely explanation for increased transmission in humans. One sentence summarySystematic proteomic and genomic analyses of SARS-CoV-2 variants of concern reveal how variant-specific mutations alter viral gene expression, virus-host protein complexes, and the host response to infection with applications to therapy and future pandemic preparedness.

systems biology↗

bioRxiv2022

Enhanced innate immune suppression by SARS-CoV-2 Omicron subvariants BA.4 and BA.5

SARS-CoV-2 adaptation to humans is evidenced by the emergence of variants of concern (VOCs) with distinct genotypes and phenotypes that facilitate immune escape and enhance transmission frequency. Most recently Omicron subvariants have emerged with heavily mutated spike proteins which facilitate re-infection of immune populations through extensive antibody escape driving replacement of previously-dominant VOCs Alpha and Delta. Interestingly, Omicron is the first VOC to produce distinct subvariants. Here, we demonstrate that later Omicron subvariants, particularly BA.4 and BA.5, have evolved an enhanced capacity to suppress human innate immunity when compared to earliest subvariants BA.1 and BA.2. We find that, like previously dominant VOCs, later Omicron subvariants tend to increase expression of viral innate immune antagonists Orf6 and nucleocapsid. We show Orf6 to be a key contributor to enhanced innate immune suppression during epithelial replication by BA.5 and Alpha, reducing innate immune signaling through IRF3 and STAT1. Convergent VOC evolution of enhanced innate immune antagonist expression suggests common pathways of adaptation to humans and links VOC, and in particular Omicron subvariant, dominance to improved innate immune evasion.

microbiology↗