A worldwide pandemic requires scientific research to understand the viral pathogen. The focused efforts of global scientists are even more necessary in the face of a novel coronavirus like SARS-CoV-2, the causative agent of COVID-19. However, because SARS-CoV-2 causes human disease, research efforts are restricted by the need for physical laboratories that are equipped to handle the required level of containment and personnel trained to handle pathogens in these facilities. But what if we could bypass the restrictive facility requirements by engineering a synthetic, replication-defective version of SARS-CoV-2 that more researchers could use to study the pandemic coronavirus, expanding the capacity to test and develop methods to attenuate its devastating effect on humans?
The challenge is to develop a derivative of SARS-CoV-2 that reflects how it behaves in the cell but is compromised such that it is unable to infect cells more than a single time. That is, the virus can get into a cell or be introduced into cells and replicate but is unable to produce infectious virus would offer a pathway to expand research capacity without the use of special laboratory facilities. This replication-defective SARS-CoV-2 could be created to encode as much or as little of the genome needed to examine its lifecycle without becoming a fully infectious virus. In fact, this replication-defective version of SARS-CoV-2 could include additional genetic elements that could be used to control its expression, track the virus in cells and measure the level of its replication. This task has been undertaken by Dr. Bill Sudgen’s group at the University of Wisconsin–Madison McArdle Laboratory for Cancer Research, explained by graduate student Rebecca Hutcheson during her presentation “Making the Virus Causing COVID-19 Safe for Research”.
Strategizing the Creation of a Safer Virus
Based on this presentation, the lab’s approach to making SARS-CoV-2 more widely available for research was based on similar techniques applied to studying human immunodeficiency virus (HIV-1), the virus that causes acquired immunodeficiency syndrome (AIDS). (Virologists love their acronyms!) Specifically, a replication-defective SARS-CoV-2 that could be studied in the common Biosafety Level 2 laboratories versus the more limited Biosafety Level 3 facilities was engineered as follows:
- Create a synthetic DNA version of the RNA virus containing only the desired genetic elements.
- Remove the coding sequences for structural proteins responsible for generating a virus that can infect more cells.
- Replace the structural proteins in the DNA version of the viral genome with reporter genes.
- Control viral genome expression using an inducible promoter.
- Create separate constructs for the membrane and envelope structural proteins that can be added to cultured cells for packaging the replication-defective virus.
Generating a Synthetic SARS-CoV-2 Genome
Creating a synthetic DNA version of an RNA virus seems simple enough because SARS-CoV-2 had been sequenced and published shortly after its identification. By synthesizing the viral genome, there is no need to work with an infectious virus at any point when creating this version of SARS-CoV-2. Instead, the synthesized DNA fragments just need to be ligated together to create a full-length version of the virus. However, this is no easy task. The SARS-CoV-2 genome is 30,000 bases long and the longest DNA fragment that can be generated is 2,000 bases. Furthermore, this synthetic fragment needs to be grown in bacteria and harvested to generate enough DNA for use. In some cases, the synthesized fragments can be toxic to the bacteria. Thus, it takes time and effort to create a full-length synthetic DNA version of the SARS-CoV-2 RNA genome that correctly codes for the elements desired, not the wild-type virus.
Another advantage to building the SARS-CoV-2 from the ground up is that the codons used for synthesizing the needed viral proteins can be optimized for mammalian cells. The group from McArdle took the time to implement this step when working with the SARS-CoV-2 sequence.
When you can synthesize the DNA sequence from scratch, removing the structural elements responsible for infectiousness and replacing them with other coding sequences is easy. For the 30kb DNA version of SARS-CoV-2, these structural elements are the envelope and membrane proteins. Without the membrane and envelope proteins that make up most of the viral particle outer layer, the virus is incapable of infecting other cells. Rather than leaving this space in the DNA derivative genome empty, the group from McArdle chose to replace the structural coding regions with two reporter genes: NanoLuc® luciferase and green fluorescent protein. Both reporter genes offer options to detect this modified SARS-CoV-2 both inside cells and in the cultured cell medium while providing a method to monitor production of the virus. Promega helped support the Sugden lab by providing a GloMax® Discover System for measuring the fluorescence and luminescence of the replication-defective SARS-CoV-2 DNA construct.
Repressing Expression of Viral Proteins
Another level of control over viral expression would be to add a promoter for the viral genome that needs to be induced to start transcription and translation. The research team selected the KRAB domain of human Kox1 fused to the Tet repressor or Tet-KRAB. This protein fusion is constitutively expressed in cells, binding to tet elements in the DNA, silencing any expression downstream. However, once doxycycline is added, Tet-KRAB no longer binds and the promoter becomes active. The Tet-KRAB system was implemented for the replication-defective SARS-CoV-2 DNA construct. Thus, when this construct is transfected into cultured cells, the viral proteins are not expressed until the inducer, doxycycline, is added.
Separately Expressing Structural Proteins
Similar to other viral packaging systems, the Sudgen lab developed a technique to provide the structural proteins separately to recreate an infectious viral particle, but one limited to a single round of infection. That is, the synthetic viral genome lacks the membrane and envelope proteins so even if the virus infects and replicates inside a cell, the defective genome cannot assemble an infectious viral particle, halting subsequent infections of surrounding cultured cells. The human 293 cell line was used as the basis for producing the membrane protein by integrating the DNA encoding the protein under control of the inducible Tet-KRAB element into the cell line genome. Like the replication-defective SARS-CoV-2, the membrane protein can only be produced when doxycycline is added. Envelope protein is produced using a different technique: RNA transfection. By introducing the short-lived RNA and the replication-defective SARS-CoV-2 construct into 293 cells with integrated membrane protein, an infectious SARS-CoV-2 can be produced, albeit one limited to a single round of infection. However, by combining all the elements together, this work would need first to be performed in the more restrictive Biosafety Level 3 facility because it can infect humans. This would prove the engineered system works and is safe to use in commonly available Biosafety Level 2 labs.
All these components—a synthetic DNA construct of SARS-CoV-2 that contains an inducible promoter and two reporter genes but lacks the envelope and membrane proteins, a 293 human cell line expressing the membrane protein, also under control of an inducible promoter, and an RNA encoding the envelope protein for transfection into cells—come together to produce a replication defective, safer SARS-CoV-2 that can be used by researchers to study the virus in commonly available Biosafety Level 2 laboratory facilities found on university research campuses. Work is still ongoing to complete this SARS-CoV-2 system. By engineering this SARS-CoV-2 component system, the scientists from McArdle plan to share this version with other researchers around the world for the benefit of everyone doing research on this novel coronavirus.
Looking for some basic background information on SARS-CoV-2? This Promega Connections blog talks about some of the basics.
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