LENTIVIRUS

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As a group, lentiviruses have a number of common features that are instructive with respect
to HIV. In terms of their biology and genetic composition, key properties of lentiviruses are:
1. Lentiviruses can infect non-dividing cells. Unlike oncoretroviruses, lentiviruses can
efficiently infect terminally-differentiated, non-dividing cells such as macrophages. Tissue
resident macrophages, such as brain macrophages (or microglia) may be important for
development of specific aspects of virally-induced disease.
2. Lentivirus genomes encode regulatory proteins. In addition to structural (gag, env) and
enzymatic (pol) proteins, which are found in oncoretroviruses, lentiviruses also encode
regulatory proteins. Most notable among these are Tat-like and Rev-like proteins which,
respectively, regulate viral transcription and viral RNA transport.

In terms of the pathogenesis of lentivirus infections, some key properties are as follows:
1. Lentiviruses persist lifelong. This is a function both of their ability to integrate into the
host chromosome and of their ability to evade host immunity. This ability to evade host
immunity may be related both to the high mutation rates of these viruses, and to their ability
to infect immune cells (macrophages, and in the case of HIV, T-cells).
2. Lentiviruses have high mutation rates. Lentiviruses replicate, mutate and undergo
selection by host immune responses.
3. Infection proceeds through at least three stages.
(A) Initial (acute) lentivirus infection is associated with rapid viral replication and
dissemination, which is often accompanied by a transient period of disease.
(B) This is followed by a latent period, during which the virus is brought under immune
control and no disease occurs.
(C) High levels of viral replication then resume at some later time, resulting in disease.

Lentiviral Vectors

Lentiviral vectors can deliver large cDNAs to a variety of dividing and non dividing cells,
including terminally differentiated mammalian cells e.g. neurons, lymphocytes and
macrophages. Lentiviral vectors have proven to be effective in transducing brain, liver,
muscle, and retina in vivo without toxicity or immune responses. This feature makes
lentiviruses a very attractive tool for gene delivery and possibly further use for human gene
therapy. The safety of the lentiviral vectors has been improved with the generation of self
inactivating vectors and a minimal packaging system.

HIV-BASED VECTORS

Most of the lentiviral vectors presently in use for gene delivery and gene therapy approaches
are HIV-derived vectors. The cis- and trans-acting factors of lentiviruses can be separated
while preserving their functions. The lentiviral packaging systems provide in trans the viral
proteins that are required for the assembly of viral particles in the packaging cells. The vector
constructs contain the viral cis elements, packaging sequences, the Rev response element
(RRE), and the transgene.
In addition to the polypurine tract (PPT), located close to the U3 region of the LTR,
lentiviruses contain a central copy of the PPT (cPPT) at which synthesis of the downstream
plus strand is initiated. Inclusion of the upstream cPPT element enhances nuclear import of
the HIV-1derived vector genome and significantly improves their transduction efficiency in
vivo. Another important improvement of the lentiviral vectors is the inclusion of cis-acting
transcriptional regulatory elements, such as the WPRE, which enhances transgene expression
in the target cells. Furthermore the development of regulatable lentiviral vector allows for
tetracycline-dependent induction of transgene expression (see Kafri Lab, below).

VEKTOR HIV BERBASIS

Sebagian besar vektor lentiviral saat digunakan untuk pengiriman gen dan
pendekatan terapi gen adalah vektor HIV diturunkan. Faktor cis dan trans-acting
lentivirus dapat dipisahkan sambil menjaga fungsi mereka. Sistem kemasan
lentiviral menyediakan dalam trans protein virus yang diperlukan untuk
perakitan partikel virus dalam sel kemasan. Konstruksi vektor mengandung
unsur-unsur cis virus, urutan kemasan, elemen respon Rev (RRE), dan transgen.
Selain saluran polypurine (PPT), yang terletak dekat dengan wilayah U3 dari LTR,
lentivirus berisi salinan sentral dari PPT (cPPT) di mana sintesis ditambah untai
hilir dimulai. Pencantuman unsur cPPT hulu meningkatkan impor nuklir dari
genom vektor HIV-1derived dan secara signifikan meningkatkan efisiensi
transduksi mereka in vivo. peningkatan penting dari vektor lentiviral adalah
dimasukkannya unsur pengaturan transkripsi cis-acting, seperti WPRE, yang
meningkatkan ekspresi transgen di sel target. Selanjutnya pengembangan vektor
lentiviral regulatable memungkinkan untuk induksi tetrasiklin tergantung
ekspresi transgen
Lentiviral vectors.
(A) Schematic representation of the wild-type HIV provirus.
(B) The latest generation of SIN-lentiviral vector constructs incorporates a central polypurine
tract (cPPT) to enhance nuclear translocation of the vector in the target cell. In addition, a
WPRE is included. Black triangle, SIN mutation; RRE, Rev response element.
(C) Third generation, tat-free, nonoverlapping split genome packaging system. One plasmid
codes for gag and pol, whereas rev is expressed in trans from another plasmid.

vektor lentiviral.
(A) Skema representasi dari tipe liar provirus HIV.
(B) Generasi terbaru dari SIN-lentiviral konstruksi vektor menggabungkan
polypurine saluran pusat (cPPT) untuk meningkatkan translokasi nuklir dari
vektor dalam sel target. Selain itu, WPRE disertakan. Hitam segitiga, SIN mutasi;
RRE, respon elemen Rev.
(C) generasi ketiga, tat-bebas, nonoverlapping perpecahan sistem genom
kemasan. Satu kode plasmid untuk gag dan pol, sedangkan rev dinyatakan
dalam trans dari plasmid lain.

LENTIVIRAL PACKAGING SYSTEMS

Replacement of the HIV envelope glycoprotein with VSV-G enables the design of lentiviral
vectors useful for gene delivery and possibly gene therapy. The major advantage provided by
VSV-G pseudotyped lentivirusesapart from broadening the host-range of the vectorsis that
the viral particles can be easily concentrated (a 1000-fold) by ultracentrifugation of the cell
culture medium harvested from the packaging cells. To minimize the amount of HIV coding
regions present in the packaging system, a second generation of the packaging system was
develolped with extensive deletions of the viral genome that contain only the gag, pol, tat,
and rev genes of HIV-1and lacking all the accessory genes, since accessory genes (vpr, vpu,
vif, and nef) are not required for efficient production of viral particles. Third generation of
packaging constructs lack the HIV-1 tat gene (a strong transcriptional activator of the HIV-1
LTR promoter essential for viral replication) and the residual HIV genome is divided into two
expression constructs. Furthermore replacement of the 50 LTR promoter/enhancer regions
with strong constitutive promoters, such as CMV or RSV, creates vectors that are efficiently
transcribed in the packaging cells in the absence of Tat. The remaining HIV genome was split
into two separate plasmids: one carrying the HIV RRE and gag, pol, whose expression are,
therefore, dependent on the presence of Rev, which is encoded on a separate expression
plasmid. Lentiviral vectors are produced by transient transfection into 293T cells of
packaging and vector plasmids, followed by concentration of the virus particles by
ultracentrifugation.