HIV
A Biotechnology Tool
HIV - a Retrovirus
The HIV Genome and Function
gag
• core nucleocapsid proteins
p55, p40
• p24 (capsid, or "core"
antigen)
• p17 (matrix)
• p7 (nucleocapsid);
pol
• the enzyme proteins p66
and p51 (reverse
transcriptase)
• p11 (protease)
• p32 (integrase).
env
• The Envelope Glycoproteins:
– outer envelope glycoprotein
gp120
– transmembrane glycoprotein
gp41
• derived from glycoprotein
precursor gp160
Regulatory Genes
• Lentiviruses differ from other classes
of retroviruses
– presence of regulatory genes in their
genome
– are additional to the structural genes gag,
pro-pol and env
– subdivided into two groups :
• the essential genes tat and rev
• the accessory or auxiliary genes vif, vpr, vpu
and nef.
The Essential Genes
• Tat:
– promotes annealing of tRNA primer to the viral
RNA genome
– suppresses RT activity at later stages in the viral
life cycle.
• Rev:
– regulates the expression of HIV proteins by
controlling the export rate of mRNAs
Rev
• The HIV mRNAs are produced from the primary
transcript by three different splicings:
– Unspliced
– singly spliced
– doubly spliced
• Rev is a doubly spliced product
– Inhibts second splicing step
– => TS and TL of un-, singly spliced Products
How Rev works
The Accessory Genes
• Vpr:
– activates HIV at low concentrations
– cell cycle arrest
– apoptosis in dividing cells
• Vif:
– associated with the infectious activity of the virus
– may also be involved in viral replication,
• Vpu: is required for the efficient assembly and release of
new HIV viruses
• Nef: interacts with host cell signal transduction proteins
– for long term survival of infected T cells
– for destruction of non-infected T cells by inducing apoptosis
Replication
Developement of a selfinactivating Lentivirus Vector
Based on the paper of:
HIROYUKI MIYOSHI, ULRIKE BLO¨MER,† MASAYO
TAKAHASHI,‡
FRED H. GAGE, AND INDER M. VERMA*
Laboratory of Genetics, The Salk Institute for Biological
Studies,
La Jolla, California 92037
Why HIV?
• retrovirus vectors derived from
oncoretroviruses (like murine leukemia virus
(MLV)) require proliferation of the target cells
for integration
– Cannot be used for gene transfer into nondividing
cells such as hepatocytes, myoblasts, neurons,
and hematopoietic stem cells
• Lentiviruses such as human
immunodeficiency virus type 1 (HIV-1) can
infect nondividing cells
HIV - Pseudotyped
• HIV vectors were pseudotyped with the
vesicular stomatitis virus G glycoprotein
(VSV-G)
– Are able to infect a variety of tissues
Risks & Precautions
• HIV-1 is the etiologic agent of AIDS
• Possible generation of replicationcompetent virus during the production of
vectors
– => three-plasmid expression system :
• packaging,
• envelope
• vector constructs
Elimination of all accessory
genes
• Elimination of all accessory genes (vif,
vpr, vpu, and nef) from a packaging
construct is possible without losing the
ability to transduce nondividing cells
Risks & Precautions
• possibility of insertional activation of
cellular oncogenes by random
integration of the vector provirus into the
host genome
– => construction of a self-inactivating (SIN)
vector in which the viral enhancer and
promoter sequences have been deleted.
LTR Modification
• The transcriptional inactivation of the
long terminal repeat (LTR) in the SIN
provirus
– prevents the mobilization by replicationcompetent virus
– enable the regulated expression of genes
from internal promoters by eliminating any
cis-acting effects of the LTR.
Another Modification
• Replacement of the U3 region of the 5’ LTR
with the cytomegalovirus (CMV) promoter
– Tat-independent transcription with no decreases in
viral titer
– Hybrid 5’LTR reduces possibilty of recombination
(and thus the possible generation of replication
competent virus)
Immune Response
• No cellular immune response could be
detected at the site of injection
• second injection of the HIV vector into
the animals is possible
– lack of any potent humoral immune
response to the vector
Germline Transmission and
Tissue-Specific Expression of
Transgenes Delivered by
Lentiviral Vectors
Carlos Lois,* Elizabeth J. Hong,* Shirley
Pease,
Eric J. Brown, David Baltimore†
Science (VOL 295/1 FEBRUARY 2002)
TG Animals
• The ability to introduce and express
exogenous genes of interest in animals
has become an indispensable tool to
modern biologists
Pronuclear Injection
• Transgenic mice are
currently generated by
pronuclear injection
• this technique is
–
–
–
–
relatively inefficient
Technically demanding
costly
impractical in most other
animal species.
Retroviruses
• Another approach to transgenesis:
• retroviruses as gene delivery vehicles
– Can stably integrate into the genome of
cells.
• Moloney murine leukemia virus
(MoMLV):
– impractical for creating transgenic animals
• silencing of the provirus during development:
• results in low to undetectable levels of
transgene expression
Lentiviruses
• Lentiviruses are a class of retroviruses:
– that cause chronic illnesses in the host
organisms they infect. Among retroviruses,
lentiviruses
– are able to infect both dividing and
nondividing cells
– lentiviruses might be immune to
developmental silencing ( in contrast to
oncoretroviruses)
• => development as gene delivery vehicles
Lentiviral Backbone
The Vector FUGW
• 5‘LTR -CMV Enhancer/
Promotor
The Vector FUGW
• To increase the titer of the virus:
– the human immunodeficiency virus–1
(HIV-1) flap element
• inserted between the 5’ long terminal
repeat (LTR) and the human ubiquitin-C
internal promoter
The Vector FUGW
• internal promoter driving the GFP
reporter gene
– human ubiquitin-C promoter:
• expression across different cell types
The Vector FUGW
• Enhanced GFP (EGFP)
The Vector FUGW
• Increase in Transcription:
– the woodchuck hepatitis virus
posttranscriptional regulatory
element (WRE) downstream of GFP
Two Infection Methods
• A: Injection into the perivitelline space of
single-cell mouse embryos
• B: Removal of the zona pellucidae and
incubation of denuded embryos with
lentiviral suspension
•
Injection into perivitelline
space
Injection of virus into the perivitelline space of
single-cell mouse embryos
Injection into perivitelline
space
• 72 hours in culture
– GFP expression was apparent in the
blastula- or morula-stage embryos
developing from the infected zygotes
• Embryos were implanted into
pseudopregnant females and were carried
to term
Results of Perivitelline Injection
• 82% of founder animals carried at least one
copy of the integrated transgene
• 76% showed GFP fluorescence paws, tails,
and face
• All GFP-positive animals carried an
integrated provirus
• all animals with two or more copies of the
provirus expressed the transgene at levels
detectable by direct viewing of GFP
fluorescence.
Expression of GFP
Expression of GFP
Expression of GFP
Discussion
• The delivery of the virus by injection into the
perivitelline space yielded transgenics with
high efficiency;
– however, the number of integrated proviruses in
the genome varied, ranging from 0 to more than
20
• source of this variability
– difficulty in controlling the volume of virus
delivered into the perivitelline space during the
injection.
Incubation of denuded
embryos
• Removal of the zona pellucidae
Incubation of denuded
embryos
• Incubation of denuded embryos for 3
days to the morula or blastocyst stage
• Implantation into the uterus of timed
pseudopregnant females
– Denuded embryos
• delayed in their development in vitro
• the rate of implantation was lower than that of
virusinjected embryos implanted with intact
zona pellucidae
Features of Incubation Method
• there is still some nonlinearity and
irreproducibility
• But:
– this method of virus delivery allows for
better control of the number of proviral
integrations per genome
– requires no specialized equipment and
may be easier for many laboratories that
wish to use this technique
The F1 Generation of TG Mice
• Founders carrying transgene(s)
transmitted most of them to a fraction of
their progeny
• Furthermore, ubiquitous GFP
expression in transgenic F1 progeny
– provirus is not inactivated through one
round of gametogenesis and development
GFP Expression in skeletal muscles
• FMHGW vector:
– histone 2B–GFP (H2BGFP) fusion gene
• The H2B-GFP concentrates the fluorescence
in the nuclei, making the signal more intense
– Myogenin promtor
• activity specific to skeletal muscle
GFP Expression in skeletal
muscles
• 11.5 Embryos:
• showed GFP fluorescence in
– the paraxial and cephalic somites
– limb buds
– Extraocular muscles in the pattern
expected for the myogenin protein muscle
Tissue-specific GFP Expression
• H2B-GFP is expressed in the
somites and emerging musceles
of the limb buds, eye and jaw
(A)
•(B) Higher magnification view of
A showing the boundaries
between somites
Focus on nuclei
• Immunofluorescence of frozen tissue
sections (GFP Antiody):
• Expression was limited to the nuclei of cells in
the
• skeletal muscle lineage
• Negative were: cells of the
–
–
–
–
–
–
Skin
cartilage
neural tube
heart
lung
intestines
Focus on the nuclei
• Image (C) corresponds to a coronal
section in the lumbar region
showing the localization of GFP
immunofluorescence to the somites
and its exclusion from flanking skin
and cartilage.
•Image in (D) is a frontal section
through the cervical region showing
the localization of GFP
immunofluorescence to the somites
on either side of the neural tube (nt).
GFP Expression in Thymus
• GFP driven by the T lymphocyte–
specific proximal lck
• injection to mouse embryos
• => mice expressing GFP exclusively in
the thymus
Lentiviral Vector System in other
Mammals
• FUGW lentivirus delivered to single-cell
rat embryos by perivitelline injection
• Showed expression throughout all
tissues
=> Vectorsystem works also in other
mammals
Possibilities
• overcomes many of the limitations of
pronuclear injection:
– more efficient
– less invasive to the embryos
– more cost-effective
– technically less demanding
– Incubation with denuded embryos obviates
the need for micromanipulation and may
be easier
Possibilities
• it has the potential to be extended to
other animal species.
• VSVG protein mediates viral entry:
– finds receptors on the cells of all
vertebrates, including primates
– Also in birds, a class of animals for which
no satisfactory method exists for creating
transgenics
Limitations
• Use may be limited by
– sequences that can decrease viral titers
• splicing or polyadenylation signals inthe transgene
• insertion of transgenes larger than 10 kilobases between
the LTRs.
• In cases where multiple proviral insertions are
necessary for high levels of expression:
– the establishment of pure breeding transgenic
lines might be complicated by the independent
segregation of the proviruses.
Questions and Comments
Greg@sglab.org
http://www.sglab.org
->News
->Seminars