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A team of Canadian scientists has recently discovered a broad interaction between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host proteins using proximity-dependent biotinylation approaches. The study is currently available at bioRxiv * prepress server.
SARS-CoV-2, the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, is a deadly virus that has already claimed more than 890,000 lives worldwide. To date, no specific drugs or vaccines have been identified to combat the spread of the virus. To design and develop therapeutic interventions effectively, it is of utmost importance to determine the host virus-protein-protein interaction patterns, which is a prerequisite for virus survival and replication within host cells.
.jpg "Transmission electron micrograph of SARS-CoV-2 virus particles isolated from a patient. Image captured and enhanced in color at the NIAID Integrated Research Facility (IRF) at Fort Detrick, Maryland. Credit: NIAID")
Transmission electron micrograph of SARS-CoV-2 virus particles isolated from a patient. Image captured and enhanced in color at the NIAID Integrated Research Facility (IRF) at Fort Detrick, Maryland. Credit: NIAID
In the current study, scientists performed proximity-dependent biotinylation (BioID) with the miniTurbo enzyme to 27 N- and C-tagged SARS-CoV-2 proteins in a lung adenocarcinoma cell line to characterize the intracellular localization of viral proteins and to map the protein-protein virus-host interaction network.
Why are BioID approaches better than conventional affinity purification approaches?
The SARS-CoV-2 genome contains a large 5 ‘open reading frame (ORF) encoding two large polyproteins, which are subjected to proteolysis mediated by viral proteases to generate non-structural proteins (NSP1-NSP16). In addition, the SARS-CoV2 genome encodes structural proteins, including the spike protein, viral envelope, membrane proteins, and nucleocapsid. All of these proteins together make up the SARS-CoV2 proteome, and interactions between viral proteins and host proteins are the essential requirement for the virus to continue its life cycle within host cells.
Conventional affinity purification approaches are the gold standard for analyzing biochemically stable protein-protein interactions; however, these approaches are not well suited for studying weak or transient interactions and interactions that occur at poorly soluble intracellular locations, such as membranes.
The proximity dependent biotinylation approach involves the fusion of a “bait” protein with a bacterial enzyme (biotin protein ligase) to facilitate covalent biotinylation of lysine residues of any protein in the vicinity of the bait. Due to covalent biotinylation, strong cell lysis conditions can be used so that all cellular components (organelles, membranes, protein complexes) can be solubilized and analyzed to identify interacting proteins. Subsequently, all biotinylated proteins are purified using streptavidin affinity purification in conjunction with mass spectrometry.
The BioID approach used in the current study
Initially, a E. coli In BioID the enzyme BirA with a single mutation was used; however, the catalytic activity of the enzyme was relatively low. To overcome this problem, new variants of BirA have been identified, such as miniTurbo.
In the current study, the scientists performed BioID with miniTurbo, which is an N-terminally truncated BirA protein that contains many additional mutations that significantly enhance the catalytic activity of the enzyme. The scientists expressed miniTurbo-tagged viral bait proteins in a human lung alveolar epithelial cell line using the lentiviral delivery system.
SARS-CoV-2 – host protein interactome
Using the miniTurbo-based BioID approach, the scientists identified proximity interactions of 7,810 viral proteins with 2,242 host proteins. The scientists also found that most of the viral proteins are located in the cytoplasm; however, many proteins are also associated with the endoplasmic reticulum, Golgi apparatus, mitochondria, and other organelle membranes.
Three viral proteins (NSP7, NSP8 and NSP12) were found to form a complex that interacts with the host proteome. Host proteins that interacted with NSP7 / 8 included components of the minichromosome maintenance protein complex, DNA damage checkpoint proteins, histone-binding proteins, ubiquitin system proteins, proteasome components, etc. Similarly, analysis of the interactome of two viral proteins, NSP4 and NSP6, revealed that these proteins could be associated with membrane reorganization and the formation of viral replicating organelles.
Viral ORF9B was found to interact with several host mitochondrial outer membrane proteins, and also with proteins responsible for regulating the host’s innate immune system. Regarding the utilization of host protein synthesis machinery for virion production, several viral proteins were found to interact with host ribosomal components and cytosolic RNA binding proteins. Analysis of the interactome of the viral nucleocapsid protein revealed that this protein could be responsible for inhibiting the formation of stress granules of the host cell, probably by preventing the interaction between several host proteins that are essential for the formation of granules of stress.
Importance of the study
The scientists created a website, called ‘covid19interactome.org’, and uploaded the findings of the current study so that the entire dataset can be widely available to the scientific community. The data set established in the current study classifies a large number of new viral protein-host protein interactions that can be used to study the life cycle of SARS-CoV-2 in more detail, as well as to identify new therapeutic targets.
*Important news
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guide clinical practice / health-related behavior, or be treated as established information.
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