The lentivirus, from Latin lenti which means slow, they are viruses that require a long time, between months and years, from the initial infection to the onset of the disease. These viruses belong to the genus Lentivirus and retroviruses (family Retroviridae), which have an RNA genome that is transcribed into DNA by reverse transcriptase (TR).
In nature, lentiviruses are present in primates, ungulates, and felines. For example, in primates there are two phylogenetically related lineages: simian immunodeficiency viruses (SIV) and human immunodeficiency viruses (HIV). Both are the causative agents of acquired immunodeficiency syndrome (AIDS).
Lentivectors, obtained from lentiviruses, have been widely used for basic research in biology, functional genomics, and gene therapy..
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The life cycle of all retroviruses begins with the body's binding to a specific receptor on the cell surface, followed by internalization of the virus through endocytosis..
The cycle continues with the shedding of the virus coat and the formation of a viral nucleus protein complex (VNC), which consists of the viral genome associated with viral and cellular proteins. The composition of the complex changes over time and is related to the conversion, by TR, of the genome of the invader into a DNA double helix.
The integration of the virus genome with that of the cell will depend on the ability of the viral genome to penetrate the host nucleus. VNC reorganization plays an important role in importation into the nucleus, although important cellular proteins also play a role, such as transportin-SR2 / TNPO3, importin-alpha3, and importin7.
Viral proteins, such as integrase, and host cell transcription factors, such as LEDCF, are key in the integration of the viral genome.
It uses the host cell machinery to transcribe and translate viral proteins and to assemble virions, releasing them into extracellular space..
The retrovirus genome has three open reading frames (MLA) for the different viral elements. For example, capsidia and matrix (gene gag), enzymes (gene pol), and envelope (gene env).
The construction of a viral vector consists of the elimination of some genes of the wild virus, such as those related to virulence. In this way, a viral vector can infect eukaryotic cells, retro-transcribe, integrate into the genome of the host eukaryotic cell, and express the transgene (inserted therapeutic gene) without causing disease..
One method of lentivector construction is transient transfection. It is based on the use of viral minigenomes (called constructs) that carry only the genes of interest. Transient transfection consists of the independent delivery of constructs.
Some retrovectors have only main elements for the assembly of viral particles, called non-functional retrovectors. They are used to transfect packaging cells.
Vectors with a transgene expression cassette are capable of infecting, transforming cells (transduction) and expressing the transgene..
The use of separate constructs is intended to avoid recombination events that could restore the wild type phenotype..
Lentivector technology is widely used in basic biology and translational studies for stable transgene overexpression, site-directed gene editing, persistent gene silencing, stem cell modification, transgenic animal generation, and stem cell induction.
Lentivectors are easy to handle and produce systems. They are irreversibly and securely integrated into the host genome. They infect cells that are or are not dividing.
They show tropism towards certain tissues, facilitating therapy. They do not express viral proteins, therefore they have low immunogenicity. They can send complex genetic elements.
In basic research, HIV-based lentivectors have been used as RNA interference (RNAi) delivery systems to eliminate the function of a specific gene, thus allowing the interaction with other different genes to be studied..
In the early 1990s, the first lentivectors were built from HVI-1, which is closely related to chimpanzee SIV. LVH-1 is responsible for AIDS worldwide.
The first generation of lentivectors has a significant part of the HIV genome. Includes genes gal Y pol, and several additional viral proteins. This generation was created using two constructs. One of them, which expresses Env, supplies the packaging functions. Another expresses all MLAs, with the exception of Env.
The transfer vector consists of an expression cassette marked by two types of long repeats (LTRs) and genes necessary for packaging and reverse transcription..
The second generation of packaging vectors lacks most accessory genes and retains Tat and Rev. These genes were removed in the third generation and provided by a fourth construct..
Third generation transfer vectors are composed of two packaging constructs. One encodes gal Y pol. Other encodes rev. A third construct encodes the envelope, which is derived from VSV-G. The one encoding the gene of interest contains inactivated LTR lentiviral sequences to prevent recombination.
In the latter case, the transcriptional regulatory elements increase the performance of the transfer genes..
The HIV-2 virus is closely related to the gray magabey SIV (SIVYE), and is responsible for AIDS in West Africa. First and second generation vectors have been obtained from this virus..
Similar to HVI-1, from SIVYE, Three generation vectors have been constructed from EIAV (equine infectious anemia virus), FIV (feline immunodeficiency virus) and BIV (bovine immunodeficiency virus (BIV). Vectors based on EIAV have been developed for use clinical.
First and third generation vectors have been constructed from the caprine arthritis-encephalitis virus (CAEV). While first-generation vectors have been constructed from the SIV of the African green monkey.
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