Choices for Measuring Luciferase-Tagged Reporter Pseudotyped Viral Particles in Coronavirus Research

Coronavirus (CoV) researchers are working quickly to understand the entry of SARS-CoV-2 into cells. The Spike or S proteins on the surface of a CoV is trimer. The monomer is composed of an S1 and S2 domain. The division of S1 and S2 happens in the virus producing cell through a furin cleavage site between the two domains. The trimer binds to cell surface proteins. In the case of the SARS-CoV, the receptor is angiotensin converting enzyme 2. (ACE2). The MERS-CoV utilizes the cell-surface dipeptidyl peptidase IV protein. SARS-CoV-2 uses ACE2 as well. Internalized S protein goes though a second cleavage by a host cell protease, near the S1/S2 cleavage site called S2′, which leads to a drastic change in conformation thought to facilitate membrane fusion and entry of the virus into the cell (1).  

CDC / Alissa Eckert, MS; Dan Higgins, MAMS

Rather than work directly with the virus, researchers have chosen to make pseudotyped viral particles. Pseudotyped viral particles contain the envelope proteins of a well-known parent virus (e.g., vesicular stomatitis virus) with the native host cell binding protein (e.g., glycoprotein G) exchanged for the host cell binding protein (S protein) of the virus under investigation. The pseudotyped viral particle typically carries a reporter plasmid, most commonly firefly luciferase (FLuc), with the necessary genetic elements to be packaged in the particle. 

To create the pseudotyped viral particle, plasmids or RNA alone are transfected into cells and the pseudotyped viruses work their way through the endoplasmic reticulum and golgi to bud from the cells into the culture medium. The pseudoviruses are used to study the process of viral entry via the exchanged protein from the virus of interest. Entry is monitored through assay of the reporter. The reporter could be a luciferase or a fluorescent protein.   

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The Surprising Landscape of CDK Inhibitor Selectivity in Live Cells

Cyclin-dependent kinases (CDKs) are promising therapeutic targets in cancer and are currently among the most intensely studied enzymes in drug discovery. The FDA has recently approved three drugs for breast cancer that target members of this kinase subfamily, fueling interest in the entire family. Although broad efforts in drug discovery have produced many CDK inhibitors (CDKIs), few have been characterized in living cells. So just how potent are these compounds in a cellular environment? Are these compounds selective for their intended CDK target, or do they bind many similar kinases in cells? To address these questions, teams at the Structural Genomics Consortium and Promega used the NanoBRET™ Target Engagement technology to uncover surprising patterns of selectivity for touted CDKIs and abandoned clinical leads (1). The results offer exciting opportunities for repurposing some inhibitors as selective chemical probes for lesser-studied CDK family members.

CDKs and CDKIs

nanobret technology for kinase target engagement

Cyclin-dependent kinases (CDKs) regulate a number of key global cellular processes, including cell cycle progression and gene transcription. As the name implies, CDK activity is tightly regulated by interactions with cyclin proteins. In humans, the CDK subfamily consists of 21 members and several are validated drivers of tumorigenesis. For example, CDKs 1, 2, 4 and 6 play a role in cell cycle progression and are validated therapeutic targets in oncology. However, the majority of the remaining CDK family is less studied. For example, some members of the CDK subfamily, such as CDKs 14–18, lack functional annotation and have unclear roles in cell physiology. Others, such as the closely related CDK8/19, are members of multiprotein complexes involved broadly in gene transcription. How these kinases function as members of such large complexes in a cellular context remains unclear, but their activity has been associated with several pathologies, including colorectal cancer. Despite their enormous therapeutic potential, our knowledge of the CDK family members remains incomplete.

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38 Years After First Release, RNasin Protects COVID-19 Tests

A protein first purified and sold by Promega almost four decades ago has emerged as a crucial tool in many COVID-19 testing workflows. RNasin® Ribonuclease Inhibitor was first released in 1982, only four years after the company was started. At that time, the entire Promega catalog fit on a single sheet of 8.5×11” paper, and RNasin was one of the first products to draw widespread attention to Promega. Today, the demand for this foundational product has skyrocketed as it supports labs responding to the COVID-19 pandemic.

What is RNasin® Ribonuclease Inhibitor?

RNA is notoriously vulnerable to contamination by RNases. These enzymes degrade RNA by breaking the phosphodiester bonds forming the backbone of the molecule. To say that RNases are everywhere is barely an exaggeration – almost every known organism produces some form of RNase, and they’re commonly found in all kinds of biological samples. They’re easily introduced into experimental systems, since even human skin secretes a form of RNase. Once they’re present, it’s very hard to get rid of them. Even an autoclave can’t inactivate RNases; the enzymes will refold and retain much of their original activity.

RNasin® Ribonuclease Inhibitor is a protein that has been shown to inhibit many common contaminating RNases, but without disrupting the activity of enzymes like reverse transcriptase that may be essential to an experiment. It works by binding to the RNase enzyme, prevent it from acting on RNA molecules. This is important for ensuring that RNA samples are intact before performing a complex assay.

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Understanding the Structure of SARS-CoV-2 Spike Protein

Glycosylation is the process by which a carbohydrate is covalently attached totarget macromolecules, typically proteins. This modification serves various functions including guiding protein folding (1,2), promoting protein stability (2), and participating signaling functions (3).

ribbon structure of SARS-CoV-2 protein
Ribbon Structure of SARS-CoV-2 Spike Protein

SARS-CoV-2 utilizes an extensively glycosylated spike (S) protein that protrudes from the viral surface to bind to angiotensin-converting enzyme 2 (ACE2) to mediate host-cell entry. Vaccine development has been focused on this protein, which is the focus of the humoral immune response. Understanding the glycan structure of the SARS-CoV-2 virus spike (S) protein will be critical in the development of glycoprotine-based vaccine candidates.

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Working from Home: Finding Productivity in this New Normal

During this time of adjusting to a new normal, one of the most difficult things that I have had to get used to is being productive in my own home. Work from home (WFH) days are embraced by some people and not by others. For me, transitioning from working in an office and school setting, to working at-home and completing online courses, has led me on a search for answers about how to get the most out of my day. After creating a productive at-home work environment for me, I wanted to share some of my findings with you.

Here are some of the tips that I have found useful:

Section out a portion of your home for work only.

When I first started working from home, I moved room to room working wherever I felt most comfortable. I soon found this affected my organization and time management, so I started keeping all my work in one area. Now, as I sit here writing this post, I know where all of my work is, and I also know that when I walk out of this area I can ‘power down’ my mind knowing I no longer have to do work.

Power off your electronics when not working.

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ADCC and Fc Effector Functions: Considerations for COVID-19 Vaccine Development

As we continue navigating the challenges presented by COVID-19, several research areas are crucial for helping us slow the infection rate and ending the pandemic. Advanced testing methods, such as antibody testing, help us understand and predict how the virus will spread, which can inform policy decisions. Effective therapeutics will influence clinical outcomes for individual patients, and several drugs are already being tested or administered. However, an effective vaccine against the SARS-CoV-2 virus is perhaps the most important tool we can use to protect individuals and populations from COVID-19.

Over 90 vaccines against the SARS-CoV-2 virus are currently in development around the world. While there are many different types of vaccines, the overall goal is to create long-lasting protective immunity by stimulating the production of specific antibodies. As these vaccine candidates are further characterized, monitoring ADCC activity can provide important insights into their potential efficacy.

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Celebrating the 100th Cartoon with a Few Words from the Promega Cartoonist

Heading into 2020, we realized that our Cartoon Lab was reaching a milestone: the 100th cartoon! We asked the “official” Promega Cartoonist Ed Himelblau to list his Top Five Cartoons and what inspired them. See what he has chosen in his own words:

This was the first of my cartoons that Promega published and it’s still one of my favorites. The file on my computer is dated February, 1999. I have been an undergraduate in a lab. I’ve mentored undergraduates in lab. Today I have lots of undergraduates working in my plant genetics lab at Cal Poly in San Luis Obispo. For the record, I enjoy having undergraduates in the lab and I never make them dress like robots. In this cartoon, I particularly like the centrifuge and stir plate on the right. I’ve always tried to put something in each cartoon (a tube rack, an enzyme shipping box, a desiccator) that make molecular biologists say, “I know that!”

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Neutralizing Antibodies to SARS-CoV-2 Shown to Lessen Infection in Mice

Here in the US, as around the world, we’re beginning to come out of COVID-19 hiding, whether mandated or voluntary. We are slowly starting to leave the confines of home and “safer at home” orders. Many of us are donning masks and venturing out as needed, still under social distancing considerations.

We’re looking forward to a time when social distancing won’t be necessary, when we can see our relatives and friends, and give them a hug without concern for their safety or ours.

When will that time come? Many believe that it won’t be completely safe until there is an effective vaccine to protect us from SARS-CoV-2.

How does a vaccine protect us? Effective vaccines cause our immune system to produce antibodies that are specific for SARS-CoV-2, so that if we come into contact with the virus, it will be neutralized, preventing infection.

At this time, many questions remain about whether SARS-CoV-2 virus causes production of antibodies. And if antibodies are produced, are they protective?

In some exciting news this week, scientists studying SARS-CoV-2 have shown that neutralizing antibodies to this virus are made in humans. Here’s a look at their work.

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Antibody From Humanized Mice Blocks SARS-CoV-2 Infection in Cells

As the SARS-CoV-2 coronavirus continues to spread throughout the world, the race is on to produce antivirals and vaccines to treat and prevent COVID-19. One potential treatment is the use of human monoclonal antibodies, which are antibodies engineered to target and block specific antigens. A recent study by Wang, C. and colleagues published in Nature Communications showed that human monoclonal antibodies can be used to block SARS-CoV-2 from infecting cells.

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A Specific & Sensitive Matter: The Trouble with COVID-19 Antibody Tests

From the beginning of this pandemic, scientists around the world have been working around the clock in pursuit of answers that can effectively combat the SARS-CoV-2 virus. One of hardest things for people to grapple with, is all the unknowns: When will this end? When can I safely visit my friends and family again? What if I have it or had it and I don’t even know it?  

The increased availability of serological testing has helped ease people’s minds about their personal COVID-19 status. From a distance, serological testing may seem like a huge milestone in the marathon that is this pandemic. Unfortunately, many of these tests provide murky and inconsistent results.

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