The COVID-19 pandemic has caused catastrophic damage to public health and economies all over the world for nearly four years. While the pandemic is under control now, the SARS-CoV-2 virus continues to mutate and inflict unprecedented suffering for millions in the form of myriad lingering symptoms that are collectively termed ‘long COVID’.
Identifying the underlying causes and therapies for long COVID symptoms and devising strategies to protect against known and emerging coronavirus family members remains a top priority in biomedical research.
While the fundamental machinery and mechanisms of how the SARS-CoV-2 virus multiplies are now relatively well-documented, mechanistic aspects of how the virus disrupts the function of a multitude of cells and organs remain a mystery.
Researchers at Baylor College of Medicine, Texas Children’s Hospital, and the University of California at San Diego have now developed a comprehensive toolkit of Drosophila melanogaster (fruit fly) COVID-19 Resources (DCR) to study viral and human proteins interact, with the ultimate goal of developing therapies to counteract symptoms caused by existing and new strains.
The study, published in Cell Reports, was led by Dr. Hugo J. Bellen, distinguished service professor and a March of Dimes professor at Baylor College of Medicine, as well as the Chair of Neurogenetics at the Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital (Duncan NRI); Drs. Shinya Yamamoto and Oguz Kanca, assistant professors at Baylor College and investigators at the Duncan NRI; and Dr. Ethan Bier, a distinguished professor at the University of California, San Diego (UCSD).
Collection of reagents
“By harnessing the powerful genetic tools available in the fruit fly model system, we have created a large collection of reagents that will be freely available to all researchers,” Dr. Bellen said.
“We hope these tools will aid in the systematic global analysis of in vivo interactions between the SARS-CoV-2 virus and human cells at the molecular, tissue, and organ level and help in the development of new therapeutic strategies to meet current and future health challenges that may arise from SARS-Co-V2 virus and related family members.”
“In this study, we generated transgenic fly lines that encode each of the 29 viral proteins and 234 of their most critical human targets as well as 313 transgenic lines that can be used to evaluate the fly versions of the human genes that are targeted by the SARS-CoV-2 virus,” Dr. Shinya Yamamoto said.
“Moreover, we conducted several proof-of-concept experiments using the new DCR lines to validate and functionally assess SARS-CoV-2 genes and their candidate human binding partners.”
The newly-generated DCR toolkit is versatile and can be used in a variety of ways to identify and study interactions between viral and human proteins.
“We found expression of viral proteins disrupted many biological processes in flies. For instance, several viral proteins when expressed in flies were lethal in early stages and nine out of ten viral proteins caused wing defects in adult flies,” added Shenzhao Lu, a postdoctoral fellow in the Bellen lab and one of the first co-authors.
“Expressing viral proteins in flies and testing their effects using survival or phenotypic assays can help delineate the function of individual or pairs of viral factors in different tissues and when combined with human proteins, can be used to understand how they affect human tissues.”
“Second, such experimental paradigms can also be used to screen for therapeutic drugs that reduce adverse cellular effects and improve related symptoms.” Dr. Oguz Kanca added.
Systematic functional analysis
A third way to use this multifunctional DCR toolkit would be to employ them in systematic functional analysis of viral-human protein interactions. The team illustrated this with a compelling example of how they characterized interactions between early-acting viral proteins, particularly the NSP8 protein with ATE1 (Arginyltransferase 1), one of its many candidate human interacting proteins.
“ATE1 is an enzyme that adds the amino acid arginine to other proteins to alter their functions,” said Annabel Guichard from UCSD and another first co-author of the study.
“One such target of ATE1 is actin, a key cytoskeletal protein that is present in all of our cells. We found much higher levels of arginine-modified actin than normal in fly cells when NSP8 and ATE1 were produced together. Intriguingly, abnormal ring-like structures coated with actin formed in these fly cells, and these were reminiscent of similar structures observed in human cells infected with the SARS-CoV-2 virus.
”Moreover, when flies were fed drugs that inhibit the activity of the human ATE1 enzyme, the effects of NSP8 were considerably reduced.” she elaborated.
“A defining feature of viruses is their ability to rapidly evolve - a characteristic that has proven particularly challenging in controlling the SARS-CoV-2 virus,” Dr. Ethan Bier said. “We envision the new resources we have generated will offer researchers the ability easily and quickly assess the functional effects of naturally occurring variants as well as to test the biological effects of specific amino acid residues using synthetically produced mutations that could help predict molecular behaviours of potential new variants.”