Engineering a Safer SARS-CoV-2 for Use in the Research Laboratory

This illustration, created at the Centers for Disease Control and Prevention (CDC), reveals ultrastructural morphology exhibited by coronaviruses such as SARS-CoV-2. Photo Credit: Alissa Eckert, MS; Dan Higgins, MAM CDC
SARS-CoV-2 illustration from CDC; Photo Credit: Alissa Eckert, MS; Dan Higgins, MAM
E = envelope; M = membrane

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:

  1. Create a synthetic DNA version of the RNA virus containing only the desired genetic elements.
  2. Remove the coding sequences for structural proteins responsible for generating a virus that can infect more cells.
  3. Replace the structural proteins in the DNA version of the viral genome with reporter genes.
  4. Control viral genome expression using an inducible promoter.
  5. 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.

Conclusion

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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Buckling Down to Scale Up: Providing Support Through the Pandemic

The past year has been a challenge. Amidst the pandemic, we’re thankful for the tireless work of our dedicated employees. With their support, we have continuously stayed engaged and prepared during all stages of the COVID-19 pandemic so that we can serve our customers at the highest levels.

How We Got Here

The persistent work by our teams has made a great impact on the support we can provide for scientists and our community during the pandemic. From scaling up manufacturing to investing in new automation, every effort has helped.

Promega has a long history of manufacturing reagents, assays, and benchtop instruments for both researching and testing viruses. When the pandemic began in 2020, we responded quickly and efficiently to unprecedented demands. In the past year, we experienced an approximately 10-fold increase in demand for finished catalog and custom products for COVID-19 testing. In response to these demands, we increased production lines. One year ago, we ran one shift five days per week. Currently, we run three shifts seven days per week. This change has allowed 50 different Promega products to support SARS-CoV-2 testing globally in hospitals, clinical diagnostic laboratories, and molecular diagnostic manufacturers. Additionally, our clinical diagnostics materials make up about 2/3 of COVID-19 PCR tests on the global market today. Since January 2020, Promega has supplied enough reagents to enable testing an estimated 700 million samples for SARS-CoV-2 worldwide.

Developments and Advances

Promega products are used in viral and vaccine research. This year, our technologies have been leveraged for virtually every step of pandemic response from understanding SARS-CoV-2 to testing to research studies looking at vaccine response.

Promega product: The Lumit™ Dx SARS-CoV-2 Immunoassay

Who Got Us Here

We are extremely grateful for our employees. In the past year, we hired over 100 people and still have positions open today. While welcoming newcomers, this challenging year also reinforced the importance of our collaborative culture. Relationships at Promega have been built over multiple years. The long history of our teams allows us to stay coordinated while prioritizing product distribution to customers across the globe. It also leads to effective communication with colleagues and vendors. Those leading our manufacturing operations team, for example, have an average tenure of 15 years. Their history in collaborating through challenging situations helps them quickly focus where needed most.

Our 600 on-site employees support product manufacturing, quality, and R&D. They do it all while remaining COVID-conscious by social distancing, wearing masks, working split shifts, and restricting movement between buildings. While we continue to practice physical safety precautions, we also prioritize our employees’ mental health and wellness. Promega provides a variety of wellness resources including phone and video mental health sessions, virtual fitness and nutrition classes, and stress and anxiety tools.

What’s to Come

While we acknowledge that the COVID-19 is not over, we are proud of the support we have been able to provide to customers working both on pandemic research and critical research not related to COVID-19. Our policies of long-term planning and investing in the future has allowed us to respond quickly and creatively and learn from the experience.


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Artist conception of coronavirus in the brain. Researchers are investigating the neurotropic effects of SARS-CoV-2

Viruses are both fascinating and terrifying. Stealthy, insidious and often deadly, they turn our own cells against us. Over the past year, we have all had a firsthand view of what a new and unknown virus can do. The SARS-CoV-2 virus has caused a global pandemic, and left scientists and medical professionals scrambling to unravel its mysteries and find ways to stop it.

COVID-19 is considered a respiratory disease, but we know that the SARS-CoV-2 virus can affect other systems in the body including the vascular and central nervous systems. In fact, some of the most noted symptoms of SARS-CoV-2 infection, headache, and the loss of the sense of taste and smell, are neurological— not respiratory— symptoms.

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Intranasal COVID-19 Vaccines: What the Nose Knows

COVID-19 vaccine distribution efforts are underway in several countries. Recently, the Serum Institute of India celebrated the nationwide rollout of its Covishield vaccine, kicking off the country’s largest ever vaccination program. Meanwhile, many other vaccines against the coronavirus that causes COVID-19 are in either preclinical studies or clinical trials. At present, 19 vaccine candidates are in Phase 3 clinical trials, while 8 vaccines have been granted emergency use authorization (EUA) in at least one country.

intranasal covid-19 vaccine coronavirus

In the US, mRNA vaccines from Pfizer/BioNTech and Moderna are in distribution. Adenoviral vector vaccines authorized for distribution include Oxford/AstraZeneca AZD1222 in the UK (Covishield in India) and Gamaleya Sputnik V in Russia. A third type of vaccine consists of inactivated coronavirus particles, such as those developed by Sinopharm and Sinovac in China.

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Ramping Up COVID-19 Testing with the Maxwell® HT Viral TNA Kit

COVID-19 testing with Maxwell HT

John Longshore admits that he was not a big Promega customer before the COVID-19 pandemic. His team uses a wide variety of suppliers to assemble the types of testing protocols needed to serve over 50 hospitals. However, when he began to face supply chain disruptions in early 2020, he needed a supplier he could depend on to support the rapid scale-up of COVID-19 testing, and Promega rose to the occasion.

“When we started working with Promega for bulk isolation reagents, our ask was, ‘Can you supply us with 15,000 isolation reagents per week?’” John says. “The answer was yes, and we have gotten everything we’ve asked for on the dates that it was promised.”

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UWCCC Small Molecule Screening Facility Validates the Lumit™ Dx SARS-CoV-2 Immunoassay for High-Throughput SARS-CoV-2 Antibody Screening

Three researchers from the University of Wisconsin and the Small Molecule Screening Facility (SMSF) at the University of Wisconsin Carbone Cancer Center (UWCCC) have expanded their collaboration in new directions because of COVID-19. Before the pandemic, Gene Ananiev, PhD, Facility Manager of the SMSF, Tim Bugni, PhD, a Professor in the School of Pharmacy, and David Andes, MD, Professor of Medicine and Medical Microbiology and Immunology and Head of the Division of Infection Disease, worked together on antibiotic compound discovery and development, now they have added Covid-19-related projects to that list.

“It was kind of an interesting aside…” said David Andes “To try to see a need, fill a need.”

The need they saw was for tools that are necessary around any pandemic or infectious disease outbreak: Ways to quickly diagnose and manage those who are infected and ways to study the epidemiology of the disease—the distribution pattern and frequency, causes and risk factors for infection within a population. Specifically, the three were interested in an antibody test that could be used not only to understand the proportion of the population that might have already been infected with SARS-CoV-2, but that also could be used to evaluate the response to different vaccine candidates. 

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This illustration, created at the Centers for Disease Control and Prevention (CDC), reveals ultrastructural morphology exhibited by coronaviruses. Photo Credit:  Alissa Eckert, MS; Dan Higgins, MAM CDC It is one is used in several of our top 10 most viewed blogs of 2020
Illustration from CDC; Photo Credit: Alissa Eckert, MS; Dan Higgins, MAM

When you look at our top 10 most viewed blog posts of 2020, there’s no surprise that all relate to COVID-19. We have come a long way since the beginning of the year, thanks to tireless scientists and researchers around the globe. They have led the way in COVID-19 research, treatment, and testing. Let’s take a closer look at this top 10 list:

10. Tips to Maintain Physical Distance in the Lab 

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9. Investigation of Remdesivir as a Possible Treatment for SARS-2-CoV (2019 nCoV) 

Scientists have worked hard to determine possible treatment for COVID-19. This blog post focuses on Remdesivir (RDV or GS-5734), an encouraging treatment used for the first case in the United States. It provides an in-depth look at numerous studies and clinical trials on Remdesivir as treatment for COVID-19. One key finding is that RDV needed to be administered either before or shortly after infection to limit lung damage. 

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The global war against the coronavirus that causes COVID-19 rages on, spearheaded by efforts to develop effective and safe vaccines. At the time of writing, over 100 COVID-19 vaccine clinical trials were listed in the clinicaltrials.gov database. Recent attention has focused on mRNA vaccines developed by Pfizer/BioNTech and Moderna. If licensed, they would become the first mRNA vaccines for human use.

Other vaccine development efforts are relying on more conventional techniques—using an adenoviral vector to deliver a DNA molecule that encodes the SARS-CoV-2 spike (S) protein. Examples of these adenoviral vector vaccines include the vaccines from Oxford University/AstraZeneca (the UK), Cansino Biologics (China), Sputnik V (Russia) and Janssen Pharmaceuticals/Johnson & Johnson (the Netherlands and USA).

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In SARS-CoV-2 infections, the nucleocapsid protein is critical for infection, replication and packaging. The SARS-CoV-2 nucleocapsid protein is not only localized in the cytosol of the host cell but also is translocated into the nucleus. There, it interacts with various cellular proteins that modulate cellular functions, such as the degradation of unneeded or damaged proteins by proteolysis. Researchers have proposed that the protein degradation system plays an important part in coronavirus infection (1).

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