The Lentivirus - A Multifunctional Research Tool

Lentiviruses are used as viral vectors to transfer a specific genetic sequence into target cells. They play an important role in research as well as in gene therapy. But what are lentiviruses, how do these viral research tools work, and what are the potential applications? We will go into these questions in more detail in the following blog article.

These topics await you:

1) What are Lentiviruses?

2) Lentiviruses in Gene Therapy - an Application Example

3) Lentiviruses as Vectors

4) Top 10 best-selling Lentivirus Products

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What are Lentiviruses?

Lentus is Latin for "slow" and gives the name to the genus of lentiviruses. They belong to the family of retroviruses, many of them causing slow-progressing degenerative diseases in their host, which is how they got their name. Lentiviruses are enveloped single-stranded RNA viruses and are 80-100 nm in diameter [1]. They remain in their host for life and show a pronounced host specificity. For example, bovine immunodeficiency virus (BIV) exclusively infects cattle, while feline immunodeficiency virus (FIV) only infects domestic and large cats. Probably the best-known representative within the retrovirus family is the human immunodeficiency virus (HIV) with the two types HIV-1 and HIV-2, which can cause the disease AIDS in humans.

Lentiviruses belong to the complex retroviruses. They are different from other retroviruses in that their genome contains additional regulatory genes known as accessory genes. One example is the nef- (negative factor) gene. This gene encodes an intracellular protein which is able to downregulate the CD4 receptor so that it is no longer presented at the cell surface [2]. This results in a weakening of the host's immune system and thus facilitates the spread of infection. A low number of CD4-positive cells indicates a far advanced immune deficiency [3].

The replication cycle of lentiviruses is similar to that of other retroviruses (see Fig. 1). In order to integrate the viral genome into the host cell genome, the viral RNA must first be transcribed into DNA using a virus-specific reverse transcriptase. In contrast to e.g. gammaretroviruses, lentiviruses can overcome the nuclear envelope of the infected cell "on their own" and do not need to take advantage of the division of this cell to integrate their genetic information into that of the host [2]. This property makes them so interesting as viral vectors in research and therapeutic applications. Thus, when lentiviruses are used as vectors, they can transfect cells at any point in their cell cycle. Thus, in practical applications, it is possible to transfect quiescent or dividing cells, immortalized or primary cell lines [5].

Fig. 1: Replication cycle of HIV. Binding to the host cell represents the first step of infection. It involves, among other components, the gp120 envelope protein of the HI-virus. The virus enters the cell through clathrin-mediated endocytosis, where it releases its viral RNA. By the virus's own reverse transcriptase the RNA is transcribed into cDNA, which is then integrated into the host genome. With the use of the protein biosynthesis machinery of the host cell, new virus particles are produced and new viruses are assembled. The final step is the release of the new viruses. 

Lentiviruses in Gene Therapy - an Application Example

Wound healing in diabetic patients is known to be disturbed and remains a challenge in the treatment of diabetic wounds. In 2005, a publication by Lee et al. was published showing that PDGF-B gene therapy using lentiviruses leads to improved wound healing [4]. PDGF is the abbreviation for "platelet-derived growth factor." It is a growth factor that has a mitotic-stimulating effect on cells. Platelets produce PDGF in the event of injury to initiate wound healing at the damaged site. PDGF is also involved in angiogenesis and collagen organization in connective tissue, leading to the formation of new and functional tissue. To promote these mechanisms in diabetic wounds, and thus their healing, PDGF cDNA was inserted into lentiviral vectors along with a GFP reporter protein tag. First, dermal fibroblasts were transfected with these vectors to detect PDGF mRNA production. Subsequently, the vectors were tested for the treatment of diabetic wounds in mice. After 21 days, re-epithelialization was the same compared to untreated wounds as the control group, but the group receiving PDGF gene therapy showed significant improvement in neoangiogenesis as well as much thicker, more cohesively aligned collagen fibers. From this, the authors concluded that this successful lentivirus-mediated gene therapy holds promise for clinical application.

Lentiviruses as Vectors

Lentiviruses are a multifunctional tool in research and development. They can be used to generate targeted gene sequences and stable transfected cell lines. They have a high infection rate (>90%), provide high levels of downstream protein expression and can be used both in vitro and in vivo. Another major advantage that lentiviruses inherently bring is their aforementioned ability to cross the nuclear envelope of their host cell.

Lentivirus infection systems are often offered as so-called pseudoviruses. A pseudovirus is a modified lentivirus in which the original envelope protein is exchanged. For example, the human immunodeficiency virus (HIV) envelope protein gp120 (see Fig. 1), which is required for binding to CD4-positive T helper cells, can be replaced with VSV G (vesicular stomatitis virus G protein). VSV G binds to the human LDL receptor, allowing a variety of cell types to be transfected - rather than just one type of cell. An important new development in SARS-CoV-2 research is the availability of spike pseudoviruses. Here, the envelope proteins are replaced with functional spike proteins that can infect ACE2-positive cells. Spike-mediated infection activates, for example, the expression of a reporter gene to allow quantitative determination of the spike/ACE2 interaction. For instance, the effect of therapeutic antibodies or other substances can be analyzed. Such reporter systems (e.g. eGFP or luciferase) are also available for other pseudoviruses and allow easy analysis of transfection and production of stable cell lines.

In general, there are different vector systems to fall back on. A distinction is made between integrating and non-integrating lentiviral vectors. Integrating vectors are stably integrated into the host genome and passed on to daughter cells, whereas non-integrating vectors remain episomal (outside the genome). Depending on the application, one or the other system may be advantageous. Lentiviruses can also be used for gene knock-out (ko) or gene knock-in (ki). A CRISPR/Cas9 lentivirus system is used for this purpose. To turn genes on or off using the CRISPR/Cas9 method, the gene for the DNA-cutting enzyme Cas9 must first be introduced into the target cells. To do this, the cells are transfected with a Cas9 lentivirus vector. Cas9-positive cells can then be identified, for example, via puromycin selection. These cells are now suitable for ko- or ki-experiments [5]. Due to their high transfection rate and their ability to infect very many cell types through modifications, lentiviruses are particularly well adapted for this application.

Are you interested in lentiviruses? We have the suitable products. Our supplier BPS Bioscience has developed a large portfolio of lentivirus infection systems.

Top 10 best-selling Lentivirus Products

To see all Lentivirus products from BPS Bioscience, click here:     All Lentivirus Products 

For even more information on lentiviruses and the matching products, you can find a webinar and a brochure from BPS Bioscience on our homepage.

To the Lentivirus Webinar

To the Lentivirus Brochure

Sources: 

[1] https://flexikon.doccheck.com/de/Lentivirinae

[2] Molecular biology and pathogenesis of animal lentivirus infections, Clements and Zink, Clinical Microbiology Reviews, Vol. 9, No. 1

[3] https://www.fz-juelich.de/SharedDocs/Pressemitteilungen/UK/DE/2017/2017-08-03-autophagie.html?nn=535780

[4] Lentiviral Transfection with the PDGF-B Gene Improves Diabetic Wound Healing
Lee et al., Plastic and Reconstructive Surgery: August 2005 - Volume 116 - Issue 2 - p 532-538

[5] Lentiviren Webinar BPS Biocscience https://www.biomol.com/resources/webinars/

Fig. 1: Jmarchn Own work, CC BY SA 3.0, https://commons.wikimedia.org/w/index.php?curid=5818847