Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen that causes COVID-19, has severely impacted health and socioeconomic conditions around the world. Supporting and enabling research to help us better understand the host-pathogen response and evaluate new therapies continues to be of the utmost importance in the continued fight against COVID-19.

The structure of the SARS-CoV-2 virion consists of four proteins: the nucleocapsid, the membrane, the envelope, and the spike. SARS-CoV-2 enters the host cell through the binding of the spike protein to ACE2 receptors on the surface of the host cell. A major avenue of COVID-19 research continues to be the evaluation of new drugs and antibodies that disrupt the ability of the SARS-CoV-2 spike protein to interact with the host cell.

Current guidelines recommend that SARS-CoV-2 virus be handled in a biosafety level 3 (BSL-3) or higher facility, which severely limits the number of researchers that can work on new therapies or treatments for COVID-19. To circumvent the use of highly infectious SARS-CoV-2 particles, scientists have utilized recombinant lentivirus particles that have been pseudotyped with the SARS-CoV-2 spike protein. Pseudotying creates lentiviral particles that mimic the infectious behavior of SARS-CoV-2 virus variants without the pathological effects of the live virus. Pseudotyping makes SARS-CoV-2 research safer and more cost-effective, and previous studies have shown that experiments performed with pseudotyped virus yield similar, if not the same, results as those performed with the more dangerous, live virus (Johnson et al. 2020).

SARS-CoV-2 pseudovirus has been an indispensable research tool throughout the COVID-19 pandemic. However, every time a new SARS-CoV-2 variant with new spike protein mutations emerges, a new pseudovirus encoding the mutated spike protein must be produced to evaluate existing and new therapies. Additionally, pseudovirus production can be time-consuming and require extensive optimization. Lenti-X SARS-CoV-2 Packaging Single Shots (Universal) takes the guesswork out of pseudovirus production while allowing the flexibility to use any SARS-CoV-2 spike variant.

4th-generation packaging technology: increased viral titer and safety

The process of pseudovirus production involves transfecting packaging cells, such as Lenti-X 293T cells, with a suite of packaging plasmids that express essential lentiviral structure proteins (Figure 1). The lentiviral pseudovirus is then used in desired assays.

Lenti-X SARS-CoV-2 Packaging Single Shots (Universal) is a complete, preoptimized system that utilizes Takara Bio’s fourth-generation lentiviral packaging system (Figure 1). In this system, ultra-high expression of essential viral components are driven by Tet-Off and Tat activators. Additionally, the lentiviral gag-pro and pol genes are separated onto different packaging plasmids, which decreases the likelihood of recombination events leading to a replication-competent virus.

Flexibility to produce any spike variant

Lenti-X SARS-CoV-2 Packaging Single Shots includes the pEmpty Vector for streamlined spike variant cloning and expression. Once the desired spike variant is cloned into the pEmpty Vector, it can be used directly in the Single Shots reaction. In addition, the kit includes B.1.351, B.1.1.7, and D614G SARS-CoV-2 spike protein variant plasmids to use directly or as template for the creation of additional SARS-CoV-2 spike variant mutants. Watch the tutorial below to learn how to skip the PCR and directly use double-stranded DNA (dsDNA) fragments to seamlessly clone any desired SARS-CoV-2 spike variant into the pEmpty Vector with In-Fusion Snap Assembly:

Designed for ease of use

Optimizing transfection reagent and plasmid ratios for the transfection reaction is a time-consuming step in the production of pseudovirus particles. Lenti-X SARS-CoV-2 Packaging Single Shots (Universal) includes lyophilized, single tubes of Xfect Transfection Reagent premixed with an optimal formulation of Lenti-X lentiviral packaging plasmids (Figure 1). To produce pseudovirus, the pEmpty vector cloned with the desired SARS-CoV-2 spike variant mutant sequence is mixed with a reporter vector of choice. The kit includes options for ZsGreen1 or luciferase reporters. The mixture is then simply added to a tube of lyophilized single shots, and applied to packaging cells—no optimization is required (Figure 2).

Efficient transduction of ACE2-positive cells

Since binding of the SARS-CoV-2 spike protein to the ACE2 receptor enables virus entry into the host cell, we wanted to determine if pseudovirus produced with Lenti-X SARS-CoV-2 Packaging Single Shots (Universal) could transduce ACE2-expressing cells. We used Lenti-X SARS-CoV-2 Packaging Single Shots (Universal) to clone the B.1.1.7 SARS-CoV-2 spike variant gene sequence into the pEmpty Vector provided with the kit. The spike variant protein plasmid was then mixed with either a ZsGreen1 or luciferase reporter vector in the Lenti-X SARS-CoV-2 Packaging Single Shots (Universal) workflow. Pseudovirus was then collected, concentrated using Lenti-X Concentrator (Cat. # 631231), and used to transduce an ACE2-expressing HEK293T cells (Cat. # 631289). Greater than 90% of ACE2-expressing cells were transduced with ZsGreen1-expressing SARS-CoV-2 pseudovirus (Figure 3, Panel A). Additionally, luciferase-expressing pseudovirus selectively transduced ACE2-expressing cells over those that did not express ACE2 (Figure 3, Panel B). These data demonstrate that Lenti-X SARS-CoV-2 Packaging Single Shots effectively produce SARS-CoV-2 pseudotyped lentivirus that efficiently transduce cells expressing the ACE2 receptor.

SARS-CoV-2 pseudovirus has been and still is an indispensable research tool in the fight against COVID-19. Lenti-X SARS-CoV-2 Packaging Single Shots (Universal) allows for safe, fast, and flexible pseudovirus production that results in high-quality, high-titer pseudovirus for the evaluation of COVID-19 therapies.

Johnson, M. C. et al. Optimized pseudotyping conditions for the SARS-COV-2 spike glycoprotein. J. Virol. 94, (2020).