Overexpressing Virus
Due to the recent description of the pandemic caused by the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the world is facing a major public health crisis. This disease (COVID-19) causes panic worldwide because of its severity, rapid spread, affecting the ability of both lungs and the chance of missed diagnosis. With rich experience in the field of gene therapy, Creative Biolabs has actively developed recombinant overexpression virus based on the targets which play important roles in the infection of SARS-CoV-2, so as to assist the treatment of COVID-19.
Figure 1 COVID-19 virus.1
Creative Biolabs' Products
Viral gene overexpression is a process that leads to the subsequent acquisition of a large number of target gene products, which can be used for research or production and the biological function of target gene products. Based on the important role of S protein and its receptor ACE-2 in the pathogenesis of SARS-CoV-2, we developed the recombinant overexpression virus as a tool for the study of SARS-CoV-2 infection, which will contribute to the development of COVID-19 drugs and vaccines. At present, we have developed the following four kinds of recombinant overexpression viruses based on lentivirus (LV) and adeno-associated virus (AAV):
LV: LVs are versatile tools because they can transform undifferentiated cells. These viruses can make gene expression stable for a long time and integrate into the host genome permanently.
- LV-CMV-SARS-CoV2-S
- LV-CMV-ACE2
AAV: AAVs are widely used for gene transfer in vivo because of their mild immune function.
- AAV-CMV-ACE2
- AAV-CMV-SARS-CoV2-S
SARS-CoV-2 Infection Introduction
SARS-CoV-2 is a positive-stranded enveloped RNA virus of the Coronaviridae family. After entering the host, usually through atomized virus particles or contact with contaminated surfaces, the virus needs to undergo its biological cycle. Studies on SARS-CoV-2 infected patients have shown that the respiratory tract is the main site of the virus infection.
Two of the participating factors play key roles:
Spike (S) Glycoprotein of SARS-CoV-2
The SARS-CoV-2 S glycoprotein plays an important role in the combination with host cells during viral infection. According to reports, SARS-CoV-2 can infect human respiratory epithelial cells through interaction with human angiotensin converting enzyme 2 (ACE-2) receptors. S protein is a large type I transmembrane protein, containing two subunits S1 and S2. S1 mainly contains a receptor binding domain (RBD), which is responsible for identifying cell surface receptors. S2 contains the basic element fusion required for the membrane.
ACE-2
ACE-2 is an 805 amino acid transmembrane protein, an important member of the renin-angiotensin system, and plays a key role in blood pressure regulation. The common expression of ACE-2 on the surface of the gastrointestinal lumen, most commonly in intestinal epithelial cells, maybe the secondary site of SARS-CoV-2 intestinal infection. Studies have shown that SARS-CoV-2 down-regulates ACE-2 through its S protein, causing severe lung damage. This is the main reason why coronaviruses often cause respiratory diseases.
Key Viral and Host Targets in SARS-CoV-2 Infection
| Category | Target | Function in SARS-CoV-2 Infection |
|---|---|---|
| Viral Proteins | Spike (S) Protein | The main viral protein responsible for binding to host cell receptors. |
| Nucleocapsid (N) Protein | Encapsidates the viral RNA genome, protecting it from degradation. | |
| Membrane (M) Protein | A structural protein that is crucial for the formation of viral envelope and the assembly of new viral particles. | |
| Host Proteins | Angiotensin-Converting Enzyme 2 (ACE2) | The main receptor on the surface of human cells that binds to virus S protein. |
| Transmembrane Serine Protease 2 (TMPRSS2) | A host cell protease that can cleave and activate the viral S protein, which is a necessary step for the virus to fuse with the cell membrane and enter the cell. | |
| Cathepsin L | A type of endoplasmic reticulum protease that can also cleave and activate S protein. |
Application of Overexpressed Virus in SARS-CoV-2 Research
Overexpression of the virus has fundamentally changed the research and fight against SARS-CoV-2, providing a multifunctional platform for a wide range of applications.
- Drug screening and development: Overexpression of viruses can build powerful high-throughput screening platforms. This rapid cell-based approach significantly accelerates the drug development process. Once the lead drug is found, overexpression virus models can be used for functional validation in more physiologically relevant systems.
- Vaccine and treatment development: The most prominent application of overexpressing viruses is gene vaccines. Adenovirus vector is an overexpression virus that has been successfully used in various approved COVID-19 vaccines. In addition to vaccines, this technology also brings hope for gene therapy.
Comparison of Viral Vectors
| Feature | Adeno-Associated Virus (AAV) | Lentivirus (LV) | Adenovirus (AdV) |
|---|---|---|---|
| Genome Type | Single-stranded DNA (ssDNA) | Single-stranded RNA (ssRNA) | Double-stranded DNA (dsDNA) |
| Integration | Non-integrating (remains episomal) | Integrating (permanently inserts into host genome) | Non-integrating (remains episomal) |
| Expression | Long-term, stable (episomal) | Stable, permanent | High, but transient |
| Target Cells | Both dividing and non-dividing cells | Both dividing and non-dividing cells | Mostly non-dividing cells |
| Immunogenicity | Low | Low | High (strong host immune response) |
| Cargo Capacity | Small (~4.7 kb) | Medium (~8 kb) | Large (~7.5 kb - 30 kb) |
| Main Use Cases | In vivo gene therapy, animal models, gene function studies (especially for long-term expression without genome integration). | Stable cell line generation, difficult-to-transfect cell types, long-term in vitro studies. | Vaccine development, transient high-level expression, in vivo studies where strong immune response is desired. |
| Relevance to SARS-CoV-2 | Ideal for delivering ACE2 as a therapeutic to the lung. | Excellent for creating stable cell lines expressing S or N protein for drug screening. | Widely used in successful COVID-19 vaccines (e.g., AstraZeneca, J&J). |
Reasons to Choose Creative Biolabs
- Unparalleled expertise: Our team is composed of doctoral scientists with years of experience in virology, molecular biology, and genetic engineering. We not only provide virus production, but also offer consultation to design the best experimental methods for you.
- Customization and flexibility: Our services are fully customizable to meet your project needs. We can clone your specific virus serotype, promoter, and even difficult genes.
- Strict quality control: Each overexpression virus we produce undergoes multiple quality assurance steps, including titer determination, sterility testing, and gene expression validation, to ensure that the products we provide to you have high purity, high titer, and guaranteed performance.
- Cutting edge technology platform: We use two of the most popular and powerful viral vector systems: lentivirus (LV) and adeno-associated virus (AAV), both of which can stably integrate into the host genome for long-term expression.
- Comprehensive product portfolio: Our catalog includes various ready-made vectors, such as LV-CMV-SARS-CoV2-S for studying S protein function and AAV-CMV-ACE2 for exploring host receptor action. This enables you to use validated ready-made products to initiate your research.
Frequently Asked Questions
Q: What is the difference between overexpression viruses and pseudo viruses?
A: Overexpression viruses (eg. our AAV or LV) are viruses that we use to overexpress certain genes (eg. S protein) in the target cells. Pseudoviruses are non-replicating viruses, which are used to carry reporter genes (eg. luciferase) and envelope proteins from other viruses (eg. SARS-CoV-2 S protein).
Q: How long does it usually take to complete a customized overexpression virus project?
A: The timeline may vary according to the complexity of the request. For a more accurate quote, please feel free to contact our technical support.
Q: Are these viruses safe to use in animals?
A: They are replication-incompetent and have had their pathogenic genes removed, making them safe for use in appropriate laboratory settings and animal models. We provide a full certificate of analysis (CoA) with each shipment.
Q: How do I choose between lentivirus (LV) and adeno-associated virus (AAV) for my project?
A: The choice of LV and AAV depends on the different research needs. Slow viruses are more suitable for experiments requiring stable and long-term gene expression, as they can be integrated into the genome of the host cell. Adeno associated viruses are mainly used for transient expression and are the first choice for in vivo studies for its low immunogenicity and safety. Adeno associated viruses also offer various serotypes for cell type specific targeting.
Q: What quality control (QC) steps do you take to ensure the product quality?
A: Our QC process is strict and thorough. We perform titer determination by qPCR and/or infectious titer determination method, perform sterile test to ensure there is no bacterial/fungal contamination, perform validation to test for gene expression by Western blot or fluorescence microscopy to verify that the target protein is being correctly expressed, and perform endotoxin testing on all AAV products to be used in vivo.
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Viral overexpression has been demonstrated to be a critical technology for fighting severe acute respiratory syndrome coronavirus type 2. From discovering molecular pathogenesis to speeding up drug and vaccine development, the technology has propelled virology and host immunology to new levels. Manipulating and expressing critical viruses and host proteins will be key to overcoming future public health threats. Please contact us at any time if the product you need is not listed above. If the product you need is not on the list above, please feel free to contact us. We can customize the overexpressing virus according to your needs.
Reference
- Khanifar A, Najafi A, Hemmati J, et al. Investigation of COVID-19 virus mutagenicity and the effect of the NSP13, NSP14, and NSP16 on the-1 ribosomal frameshifting. Frontiers in Virology, 2024, 4: 1405680. https://doi.org/10.3389/fviro.2024.1405680 (Distributed under Open Access license CC BY 4.0, without modification.)
