Lentiviral Vectors 1st Edition
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Preface
``Felix qui potuit rerum cognoscere causas (Virgil)
(Happy the one who could penetrate the secret causes of things)''
In the 20 years of war against HIV, the cause of one of the most
dreadful health disasters of all times, a few battles have been won:
the virus's major modes of transmission were quickly identi®ed, a
blood test was created, and highly active antiviral therapies were
developed which have changed the face of the disease in wealthy
parts of the world. The ®ght against HIV will not abate short of a
vaccine and of aordable, easy to take and nontoxic therapies.
Yet AIDS research has, in addition, provided insights into many
basic biological and medical questions. One of its unexpected
spin-os has been the development, a few years ago, of a new
system of gene transfer that holds great promises for gene ther-
apy: the lentiviral vectors.
Retroviral vectors had long been considered as formidable
gene delivery tools, owing to their large cloning capacity (close to
10kb), their ability to integrate into the chromosomes of target
cells (a likely requisite for long term expression), and their failure
to transfer virus-derived coding sequence (an immunological
blessing). In spite of these assets, however, the clinical perspec-
tives of retroviral vectors seemed rather narrow because, as de-
rivatives of oncoretroviruses such as MLV, they could not
transfer genes into nondividing cells. A sobering limitation since
most of the potential targets of gene therapy are cells that rarely
if ever proliferate, be they neurons, hepatocytes, myocytes or
hematopoietic stem cells.
The recognition that HIV can infect nonmitotic cells by
hijacking the cell nuclear import machinery and a quite re®ned
mapping of the molecular determinants of this property led to the
development of lentiviral vectors. Following the demonstration
that lentivectors can govern the ecient in vivo delivery, in-
tegration and long-term expression of transgenes into nonmitotic
cells, the last 4 years have witnessed a spectacular eruption of this
system on the scienti®c scene.
VI Preface
Tissues that long appeared irremediably refractory to stable
genetic manipulation can now be reached, and the concrete
proposal for the clinical use of a lentiviral vector seems imminent.
This volume describes these exciting developments. The ®rst
chapter sums up our current understanding of the biology of
lentivirus-mediated gene transfer, an essential starter. We then
move on to describe how this information is utilized to derive
vectors from a variety of primate and nonprimate lentiviruses.
State-of-the-art techniques of lentivector production are dis-
cussed in detail, and the all important question of biosafety is
addressed. Emerging data on vector targeting, whether at the
entry or at the integration level, are also presented. Finally,
special emphasis is given to what are currently the most pro-
mising clinical applications of lentiviral vectors, in particular in
the ®elds of neurological and lympho-hematopoietic disorders,
including AIDS itself.
I hope that this book will encourage nonspecialists to take
advantage of lentiviral vectors. The ability of this delivery system
to transduce cells otherwise refractory to genetic manipulation
could be used broadly, for instance in developmental and stem
cell biology or in the neurosciences. I also hope that the contents
of this volume will stimulate many investigators to embark in
research aimed at pursuing the development of lentivectors, at
dissecting and surmounting the current shortcomings of this tool,
and at exploiting its potential for therapy. Importantly, I hope
that these eorts will contribute to further our comprehension of
HIV virology. This would be a most appropriate payback.
Finally, I wish to thank all the authors who took time from
their many commitments and generously agreed to contribute a
chapter to this collection.
November 2001, Geneva D. TRONO
List of Contents
M. STEVENSON
Molecular Biology of Lentivirus-Mediated
Gene Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
L.E. AILLES and L. NALDINI
HIV-1-Derived Lentiviral Vectors . . . . . . . . . . . . . . . . . 31
D. NEÁGRE, G. DUISIT, P.-E. MANGEOT, P. MOULLIER,
J.-L. DARLIX, and F.-L. COSSET
Lentiviral Vectors Derived from Simian
Immunode®ciency Virus . . . . . . . . . . . . . . . . . . . . . . . . 53
M.A. CURRAN and G.P. NOLAN
Nonprimate Lentiviral Vectors . . . . . . . . . . . . . . . . . . . 75
R. ZUFFEREY
Production of Lentiviral Vectors . . . . . . . . . . . . . . . . . . 107
C. DELENDA, M. AUDIT, and O. DANOS
Biosafety Issues in Lentivector Production . . . . . . . . . . . 123
A. LAROCHELLE, K.-W. PENG, and S.J. RUSSELL
Lentiviral Vector Targeting . . . . . . . . . . . . . . . . . . . . . . 143
F.D. BUSHMAN
Integration Site Selection by Lentiviruses:
Biology and Possible Control . . . . . . . . . . . . . . . . . . . . 165
T. HOPE
Improving the Post-Transcriptional Aspects
of Lentiviral Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . 179
N. DEÂGLON and P. AEBISCHER
Lentiviruses as Vectors for CNS Diseases . . . . . . . . . . . . 191
P. SALMON and D. TRONO
Lentiviral Vectors for the Gene Therapy
of Lympho-Hematological Disorders . . . . . . . . . . . . . . . 211
VIII List of Contents
R.G. AMADO and I.S.Y. CHEN
Lentiviral Vectors for Gene Therapy
of HIV-Induced Disease . . . . . . . . . . . . . . . . . . . . . . . . 229
F. GALIMI and I.M. VERMA
Opportunities for the Use of Lentiviral Vectors
in Human Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . 245
Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
List of Contributors
(Their addresses can be found at the beginning of their respective
chapters.)
AEBISCHER, P. 191 HOPE, T. 179
AILLES, L.E. 31 LAROCHELLE, A. 143
AMADO, R.G. 229 MANGEOT, P.-E. 53
AUDIT, M. 123 MOULLIER, P. 53
BUSHMAN, F.D. 165 NALDINI, L. 31
CHEN, I.S.Y. 229 NEÁGRE, D. 53
COSSET, F.-L. 53 NOLAN, G.P. 75
CURRAN, M.A. 75 PENG, K.-W. 143
DANOS, O. 123 RUSSELL, S.J. 143
DARLIX, J.L. 53 SALMON, P. 211
DEÂGLON, N. 191 STEVENSON, M. 1
DELENDA, C. 123 TRONO, D. 211
DUISIT, G. 53 VERMA, I.M. 245
GALIMI, F. 245 ZUFFEREY, R. 107
Molecular Biology of Lentivirus-Mediated Gene Transfer
M. STEVENSON
1 Overview of the Lentiviral Life Cycle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Viral Replication Cycle: Early Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2 Viral Replication Cycle: Late Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.3 Factors that In¯uence Virion Infectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2 Host-Cell Restrictions to Retrovirus Entry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3 Models of Nuclear Uptake of Primate Lentiviral Genomes . . . . . . . . . . . . . . . . . . . . 14
3.1 The Role of Gag Matrix Proteins in Entry Steps of the Lentiviral Replication Cycle . . . . . . 17
3.2 The Integrase Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.3 The Vpr/Vpx Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3.4 The DNA Flap Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
1 Overview of the Lentiviral Life Cycle
In order to better appreciate issues regarding the design and the utility of lentiviral
vectors, the lentiviral life cycle and, in particular, how the host cell cycle in¯uences
lentiviral replication, will be discussed. Since most of the events in lentiviral rep-
lication have been best characterized for the primate lentiviruses, including human
immunode®ciency virus-1 (HIV-1), HIV-2 and simian immunode®ciency virus
(SIV), the discussion will focus on these viruses. The primate lentiviruses contain
ten open reading frames (Fig. 1). The gag open reading frame directs the synthesis
of structural virion proteins and proteins which direct the encapsidation of genomic
viral RNA. The pol open reading frame encodes the viral enzymes which are
involved in synthesis of viral cDNA and which direct the integration of viral into
cellular DNA. The env open reading frame encodes the structural envelope proteins
which mediate attachment of the virion to the cell surface and fusion of viral with
target cell membranes. Sequences within the long terminal repeat (LTR) regulate
viral gene expression both at the transcriptional and post-transcriptional levels. The
LTR contains cis acting regulatory sequences and sequences which mediate the
Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street,
Worcester, MA 01605, USA
2 M. Stevenson
Fig. 1. Genetic organization of primate and non-primate lentiviruses. The major dierences between the
major primate lentiviral lineages are indicated. The central viral region (the area spanning the vif and Tat
open reading frames) contains a single vpr gene in HIV and SIVcpz/syk/mnd and SIVagm. HIV-2/SIVmac
contain two open reading frames in the central viral region referred to as vpx and vpr. Vpu is encoded by
HIV-1 and the related SIVcpz but absent from the genomes of HIV-2 and other SIV isolates characterized
to date. Vif is common to primate and non-primate lentivirus genomes with the exception of EIAV. For
comparison, the genome of the animal onco-retrovirus, murine leukemia virus (MLV), is shown.
(Adapted from STEVENSON 1999)
binding of trans-acting viral regulatory proteins. Gag, pol, and env open reading
frames are a basic characteristic of retroviral genomes including primate and non-
primate lentiviruses as well as simple animal onco-retroviruses such as murine
leukemia virus (Fig. 1). A number of additional small open reading frames dis-
tinguish the primate and non-primate lentiviruses from simple animal onco-retro-
viruses. The Tat and Rev proteins regulate lentiviral gene expression at the
transcriptional and post-transcriptional levels respectively (CULLEN 1998; JEANG
et al. 1999). Tat protein binds to a cis-acting element (TAR), located within the
LTR, to up-regulate the activity of the promoter. Rev recognizes a cis-acting ele-
ment (RRE) located in the central portion of the viral envelope gene to post-
transcriptionally regulate viral gene expression. The remaining open reading frames
encode what are referred to as the accessory or auxiliary proteins. These terms are
somewhat of a misnomer since they imply that these proteins facilitate, but are not
essential for, viral replication. However, the Vif protein, which is common to all
lentiviruses, with the exception of EIAV, is essential for the replication of the
primate lentiviruses (BORMAN et al. 1995; COURCOUL et al. 1995; HARMACHE et al.
1995; REDDY et al. 1995). Activities of these accessory proteins have been
comprehensively reviewed elsewhere (TRONO 1995; EMERMAN and MALIM 1998).
Molecular Biology of Lentivirus-Mediated Gene Transfer 3
Although, with the exception of Vif, the accessory proteins appear dispensable for
viral replication and pathogenicity in vivo (DESROSIERS et al. 1998) they are likely
to contribute to some unique aspects of primate lentiviral biology. For example,
and as will be discussed later, the Vpr/Vpx proteins may facilitate the entry of the
primate lentiviruses into non-dividing cells by promoting nuclear uptake of the
viral genome. Collectively, these unique proteins contribute to viral ®tness in that
they allow the virus to adapt, or to function within, inhospitable environments. As
such, loss of any of these functions would impair the ability of the virus to compete
with its wild-type counterpart, ultimately leading to loss of the viral variant from
the virus population. This point will become more apparent as the overlapping
functions of some of the viral proteins in promoting viral entry into non-dividing
cells are discussed. What may be considered redundant features of the viral genome
may, in a competitive setting, confer distinct ®tness advantages which allow viruses
bearing these apparent redundancies to predominate. Ultimately, viral evolution
preserves the ®ttest viruses and the fact that primate lentiviruses have several
determinants which may promote nuclear uptake of the viral genome underscores
the essential contribution of these proteins to viral replication and persistence in the
host.
1.1 Viral Replication Cycle: Early Events
In the retroviral life cycle, the ¯ow of genetic information is from RNA to cDNA
and back to RNA (Fig. 2). The viral particle protects genomic viral RNA in transit
from the virus-producing cell to the new target cell (Fig. 3). All lentiviruses, and
retroviruses in general, deposit genomic viral RNA in the cytoplasm of the target
cell after fusion of viral with target cell membranes. In the case of the primate
lentiviruses, fusion domains within the envelope glycoprotein are maintained in a
conformationally closed state. Binding to CD4 promotes the ability of the virus to
bind co-receptor molecules which in turn promotes exposure of fusogenic domains
within envelope that allow viral and target cell membranes to fuse (MICHAEL and
MOORE 1999). While the sequence of events leading up to fusion of the viral with
the host cell membrane is fairly well understood, events involving disassembly of
the viral core (a process referred to as ``uncoating'') are not well understood. The
uncoating process likely involves several sequential steps which result in a reor-
dering of virion proteins such that some of these virion proteins remain associated
with the genomic viral RNA that enters the cell while other proteins distribute in
the cytoplasm or remain associated with the cell membrane at the site of virus entry
(Fig. 3). This reordering of virion proteins leads to the formation of a reverse
transcription complex (also called a pre-integration complex) in which viral cDNA
intermediates are synthesized. The reverse transcription complex contains the viral
enzymes reverse transcriptase, which drives the synthesis of viral cDNA and
integrase, which promotes integration of viral with host cell DNA. In addition,
several other virion proteins remain associated with the viral reverse transcription
complex as it moves from the site of virus entry to the cell nucleus. In the case of
4 M. Stevenson
Fig. 2. Genetic throughput of retroviruses. In the case of primate lentiviruses, reverse transcription is
initiated by interaction of a tRNAlys with the primer binding site. Reverse transcription proceeds to the 50
end of the viral genome resulting in formation of ``minus-strand strong-stop'' cDNA. The viral RNA
portion of the resulting RNA-DNA hybrid is degraded and strong-stop DNA interacts with sequences in
the 30 end of the second RNA template (®rst template switch) which allows continuation of minus-strand
cDNA synthesis. RNase H introduces staggered gaps in the RNA which serve as primers for discon-
tinuous plus-strand synthesis. A second template switch facilitated by base complimentarity at the primer
binding site results in the formation of an ordered intermediate allowing the viral ends to interact prior to
completion of plus-strand synthesis. Linear cDNA molecules comprising full-length, plus- and minus-
strand cDNA translocate to the nucleus where a small percentage undergo recombination and end-end
ligation to form 1- and 2-LTR circles, respectively. The linear form is the immediate precursor to the
integrated provirus. The circular forms appear to be dead-end products of reverse transcription. Thin lines
denote RNA, thick lines denote cDNA. Primer binding sites and polypurine tracks are indicated by circles
and squares, respectively. Although the major subcellular sites for distinct steps in reverse transcription
are indicated, the hatch lines denote uncertainty in the boundaries because cDNA synthesis and nuclear
import are concurrent processes. (Adapted from STEVENSON 1999)
primate lentiviruses, the structural Gag matrix protein (MA) and the Vpr/Vpx
proteins associate with the reverse transcription complex. This is based on studies
demonstrating that genomic viral RNA can be co-immunoprecipitated from
cytoplasmic extracts of acutely infected cells using antibodies to Gag MA and to
Vpr/Vpx proteins (BUKRINSKY et al. 1993; FLETCHER et al. 1996). Whether Gag
Molecular Biology of Lentivirus-Mediated Gene Transfer 5
Fig. 3. The retroviral life cycle. 1, Infectious, fully processed virion; 2, receptor/co-receptor engagement;
3, fusion and uncoating; 4, reverse transcription; 5, nuclear localization; 6, integration and; 7, activation
of basal transcription; 8, transactivation of the LTR and upregulation of viral gene expression; 9,
transport of spliced and unspliced viral RNAs; 10, translation of viral proteins and transport to site of
virus assembly; 11, formation of viral structures; 12, budding of immature virions and processing of
virion proteins. (Adapted from STEVENSON 1999)
MA proteins also co-fractionate with viral reverse transcription complexes (HAN-
SEN and BUSHMAN 1997) is not known. In the case of HIV-1, capsid proteins appear
to enter the cytosol immediately following fusion and uncoating but not in asso-
ciation with high molecular weight reverse transcription complexes. In contrast,
reverse transcription complexes of onco-retroviruses retain capsid molecules from
the virion (BOWERMAN et al. 1989; FASSATI and GOFF 1999). It is unclear whether
these compositional dierences impart functional dierences to the complex. In the
case of primate lentiviruses, the association of Gag MA proteins with the reverse
transcription complex has been suggested to in¯uence the ability of these complexes
to access the cell nucleus. It is unclear whether the association of capsid with reverse
transcription complexes of MLV in¯uences the transport properties of the complex
or whether it provides some protection of viral nucleic acids to host cell nucleases.
6 M. Stevenson
Synthesis of viral cDNA proceeds at approximately one nucleotide per second
in vivo (KLARMANN et al. 1997; PRESTON 1997). However, reverse transcription
complexes of HIV-1 can be detected in the nucleus within ®fteen to thirty minutes
of infection (BUKRINSKAYA et al. 1996). Therefore, it is likely that the synthesis of
viral cDNA and nuclear translocation of viral nucleic acids proceed concurrently
such that completion of viral cDNA synthesis may occur after the complex has
entered the nucleus (BUKRINSKY et al. 1993) (Fig. 2). This does not exclude the
possibility that cellular factors important for completion of reverse transcription
are present in the nucleus. In the case of MLV, integration competent reverse
transcription complexes containing full-length viral cDNA can be detected in the
cytoplasm of acutely infected cells (BROWN et al. 1987). The Vif protein, which is
essential for viral replication in primary cells, has been suggested to in¯uence the
stability of viral nucleic acids within the reverse transcription complex and perhaps
in¯uences the stability of the reverse transcription complex itself (VON SCHWEDLER
et al. 1993; SIMON and MALIM 1996).
Reverse transcription complexes of primate lentiviruses and onco-retroviruses
dier functionally as well as biochemically. As will be discussed later, the major
distinction regards the mechanism by which the complexes are transported into the
host cell nucleus. For onco-retroviruses, nuclear translocation of viral nucleic acids
appears to occur after nuclear envelope breakdown at host cell mitosis while for
primate lentiviruses, nuclear translocation of the reverse transcription complex
appears not to require nuclear envelope breakdown. Upon completion of viral
cDNA synthesis, viral nucleic acids are competent for integration into genomic
cellular DNA. The integration reaction is catalyzed by the viral integrase, which is
derived from the pol open reading frame. This integration event is essential for
completion of the viral replication cycle (STEVENSON et al. 1990). Integration
involves three coordinated steps. Two nucleotides are trimmed from the 30 ends of
plus- and minus-strand cDNA by the integrase enzyme to create a staggered end.
Integrase then cleaves host cell DNA and joins the staggered 30 end of viral cDNA
to the 50 end of host cell DNA. The ®nal ligation of the unjoined ends is directed by
the DNA repair apparatus of the host cell. This reaction occurs in the context of a
nucleoprotein complex, accurately referred to as a pre-integration complex, which
likely comprises, in addition to the viral factors, cellular factors which promote
certain steps of the integration reaction (HANSEN et al. 1998). In vitro integration
reactions can be reconstituted with substrate DNA and recombinant integrase
(BROWN et al. 1989; FUJIWARA and CRAIGIE 1989; ELLISON et al. 1990). However,
concerted integration, in which both ends of viral DNA are modi®ed and coupled
to substrate DNA, requires the addition of pre-integration complexes obtained
from acutely infected cells or using puri®ed virion extracts (FARNET and HASELTINE
1990; GOODARZI et al. 1995). A more comprehensive discussion of cellular co-
factors which promote the integration reaction will be discussed in the chapter by
R. Bushman, this volume. The integrated form of the viral cDNA is referred to as
the provirus, which directs the synthesis of viral transcripts, some of which will be
spliced and translated to produce viral proteins and others of which will remain
unspliced and packaged within progeny virions. Although the provirus serves as a
Molecular Biology of Lentivirus-Mediated Gene Transfer 7
source for viral transcripts and proteins, several studies have suggested that extra-
chromosomal forms of viral cDNA may also serve as a template for the production
of viral proteins. For reasons which are not well understood, some linear cDNA
molecules in the nucleus circularize to form 1-LTR or 2-LTR circles (Fig. 2).
1-LTR circles are the result of homologous recombination between 50 and 30 LTRs
whereas, 2-LTR circles are formed by end-end ligation of the LTRs. These circular
viral genomes are localized speci®cally to the nucleus, most likely because the
enzymes which promote the recombination are located there (BROWN et al. 1987;
BUKRINSKY et al. 1991; ZENNOU et al. 2000). Although the 2-LTR circle was
initially considered to be the immediate precursor to the integrated provirus, it was
subsequently demonstrated that the linear cDNA is the preferred integration pre-
cursor (BROWN et al. 1987). Currently, circular forms of viral cDNA are considered
to be dead-end products of reverse transcription that do not participate in prop-
agation of infectious virus. However, as circles are localized speci®cally in the
nucleus, they have been used as convenient markers with which to examine factors
which regulate the nuclear translocation of the lentiviral genome (BROWN et al.
1987; BUKRINSKY et al. 1992; ZENNOU et al. 2000). Retroviral episomes are func-
tionally distinct from episomal of herpesvirus genomes. For example, Epstein-Barr
virus can replicate from an episomal DNA intermediate. These episomes contain an
origin of replication and are stabilized by transacting viral proteins such that they
can persist in the infected cell (YATES et al. 1984, 1985; REISMAN et al. 1985). In
contrast, retroviral and lentiviral episomes lack such maintenance functions and as
a result, exhibit a short half-life in the infected cell (SHARKEY et al. 2000). Because
of their short half-life, episomes of HIV have been used as a surrogate marker for
recent infection events in patients on highly antiretroviral therapy (FURTADO et al.
1999; SHARKEY et al. 2000). It is currently unclear whether speci®c conditions favor
circularization of viral cDNA. In vitro, 1- and 2-LTR circles constitute about
5%±10% of the total viral cDNA that is synthesized in a spreading viral infection.
However, many of these circular products appear to be a result of super infection,
where infected cells carry multiple copies of the viral genome. Super infection is
unlikely to occur in vivo and infected cells may therefore harbor single copies of the
viral genome. Nevertheless, 1- and 2-LTR circles are clearly detectable in infected
individuals (JURRIAANS et al. 1992; PAUZA et al. 1994; NANDI 1999; PANTHER et al.
1999; SHARKEY et al. 2000). Analysis of the phenotype of HIV-1 mutants with
defects and integrase indicates that formation of circles occurs by default if inte-
gration is inhibited or delayed. Thus, HIV-1 variants containing point mutations
which inhibit viral integration activity produce more circular relative to linear viral
cDNA in acutely infected cells (WISKERCHEN and MUESING 1995). In addition,
infection of cells in the presence of integrase inhibitors leads to accumulation of
circular forms of viral DNA in the nucleus (HAZUDA et al. 2000). There may be
dierences in eciency of integration in dierent cell types, for example, cells in
mitosis versus non-dividing cells. Therefore, there may be a dierential propensity
for circles to form in T cells as opposed to macrophages or dendritic cells. These
caveats are not strictly academic. One of the advantages to using retrovirus vectors
is their ability to stably transduce the cell by inserting into genomic DNA. This is
8 M. Stevenson
also a concern if the insertional event modi®es the regulation of that gene. One
possible adaption to the design of retrovirus vectors may involve inserting episomal
maintenance functions such that viral cDNA can be maintained extra-chromo-
somally as an episome. Some studies have suggested that extra-chromosomal
lentiviral genomes are competent for gene expression (STEVENSON et al. 1990;
ENGELMAN et al. 1994; WISKERCHEN and MUESING 1995). If the retroviral genome
could be maintained episomally, this would allow long-term expression from the
transgene without the hazards that go with insertional mutagenesis by integration.
1.2 Viral Replication Cycle: Late Events
Unspliced viral RNAs that are destined for incorporation into progeny virions as
well as spliced mRNAs for viral protein synthesis are derived from the integrated
viral genome, also known as the provirus (Fig. 3). In the primate lentivirus repli-
cation cycle, gene expression is regulated by Tat and Rev proteins at transcriptional
and post-transcriptional levels respectively (OU and GAYNOR 1995; JEANG 1998).
Spliced messages encoding the Tat and Rev proteins as well as the Nef protein are
produced early after the establishment of the provirus. Rev recognizes a Rev
responsive element (RRE), only present in unspliced and singly spliced transcripts,
to promote nuclear export and translation of RRE containing transcripts which
ultimately drive the synthesis of the structural as well as accessory viral gene
products. The best accepted model on the mechanism of action of the Tat protein is
that it regulates transcription elongation by binding to a stem-loop structure
referred to as TAR (for a review, see LANDAU 1999). A cellular protein (cyclin-T)
has been demonstrated to interact with the transactivation domain of Tat, which
acts to recruit CDK9 and RNA pol II transcription elongation factor which
ultimately drives transcription elongation of TAR containing transcripts (WEI et al.
1998; LANDAU 1999). For a comprehensive review of the actions of the regulatory
proteins, see EMERMAN and MALIM (1998) and FRANKEL and YOUNG (1998).
It is not within the scope of this review to comprehensively discuss the indi-
vidual functions of the structural and enzymatic proteins in the viral life cycle.
There are a number of excellent reviews that cover these topics (FRANKEL and
YOUNG 1998; LANDAU 1999). Instead, the discussion will focus on those viral
functions which in¯uence the infectious nature of the virion since this can be
considered relevant to the design and use of retroviral vectors. The viral Gag
protein products, including the capsid protein (CA), matrix (MA), nucleocapsid
(NC), and p6 proteins, are derived from a polyprotein (Pr55gag) which is processed
by the virus encoded protease. These Gag proteins constitute the bulk of the virion
which protects genomic viral RNA while in transit from the virus-producing cell to
the new target cell. The viral core, which contains genomic viral RNA, is comprised
of the capsid protein while matrix lines the inner phospholipid lea¯et of the host
cell-derived virion membrane (GELDERBLOM 1991; HOGLUND et al. 1992). The NC
protein, in the context of the Gag polyprotein, speci®cally interacts with an ordered
stem-loop structure in the 50 non-translated region of the genome known as the
Molecular Biology of Lentivirus-Mediated Gene Transfer 9
packaging signal or Psi (BERKOWITZ et al. 1993; SAKAGUCHI et al. 1993; DARLIX
et al. 1995). This interaction promotes the recruitment of unspliced genomic-length
viral RNA to the site of virus assembly by the Gag polyprotein such that viral RNA
can be packaged within virions. The C-terminal cleavage product of the Gag
polyprotein (p6) contains a conserved LXXLF motif which mediates the interac-
tion of Gag with the accessory protein Vpr which ultimately leads to the packaging
of Vpr within virions (YU et al. 1988; LU Y et al. 1993; PAXTON et al. 1993; LAV-
ALLEE et al. 1994; WU et al. 1994; KONDO et al. 1995; KONDO and GOTTLINGER
1996).
1.3 Factors that In¯uence Virion Infectivity
Central to the ability of the Gag polyprotein to direct encapsidation of genomic
viral RNA and Vpr/Vpx proteins, as well as the requirement for the MA domain in
promoting incorporation of envelope glycoproteins into the virion (BUGELSKI et al.
1995; COSSON 1996; FREED and MARTIN 1996; GONZA LEZ et al. 1996; LEE
et al. 1997; ONO et al. 1997; OTT et al. 1999; MURAKAMI and FREED 2000a; WYMA
et al. 2000) is that Gag has an unrestricted passage to the site of virus assembly at
the plasma membrane of the host cell. However, several reported functions of the
Gag proteins have the potential to interfere with this normal membrane localiza-
tion. As will be discussed later, a number of studies have demonstrated a role for
the matrix protein in viral infectivity. One of these activities involves promoting the
nuclear uptake of the viral reverse transcription complex (BUKRINSKY et al. 1993;
VON SCHWEDLER et al. 1994) and preservation of the integrity of the reverse tran-
scription complex (BUKRINSKAYA et al. 1998; KIERNAN et al. 1998). One would
predict that untimely nuclear uptake of Gag polyprotein at late stages in the viral
life cycle would misdirect it away from the site of virus assembly. Recent studies
have suggested that HIV has evolved a strategy to ensure that Gag polyproteins
remain cytoplasmic at late stages of the viral life cycle (DUPONT et al. 1999).
Similarly, the Vpr and Vpx proteins of HIV and SIV have been shown to localize to
the nucleus and as such, promote nuclear uptake of viral reverse transcription
complexes (HEINZINGER et al. 1994; YAO et al. 1995; FOUCHIER et al. 1998; JENKINS
et al. 1998; POPOV et al. 1998a,b; VODICKA et al. 1998). In the viral life cycle, Vpr is
directed to the site of virus assembly by Gag. At this point, it is unclear how the
nucelophillic activity of Vpr is suppressed so as to prevent mislocalization of Gag
polyprotein in the nucleus. Similar caveats apply to products of the pol gene. As will
be discussed, integrase, when expressed in the absence of other viral proteins,
localizes to the nucleus (GALLAY et al. 1997; PLUYMERS et al. 1999). It is unclear
whether the nucleophilic activity of integrase is manifest in the context of the Gag-
Pol polyprotein. If it is, a mechanism must exist to suppress the nucleophillic
activity of integrase at late stages in the viral life cycle. Otherwise, Gag-Pol
precursor proteins would be mislocalized.
Several other viral and cellular proteins regulate the infectivity of the virion.
The most dramatic of these is the viral accessory protein Vif, which, with the
10 M. Stevenson
exception of equine infectious anemia virus (EIAV), is contained within the
genomes of all lentiviruses (Fig. 1). Vif is essential for virus replication in primary
T-cells, in macrophages and in certain cell lines (commonly referred to as non-
permissive cell types), yet is dispensable for viral replication in other cell lines
(referred to as permissive cell types) (SODROSKI et al. 1986; FISHER et al. 1987;
STREBEL et al. 1987; GABUZDA et al. 1992a; VON SCHWEDLER et al. 1993). SIV
variants bearing an inactivated Vif gene display a marked replication defect and are
non-pathogenic in vivo (DESROSIERS et al. 1998). Although studies have suggested
that Vif is a virion protein (BORMAN et al. 1995; LIU et al. 1995; CAMAUR and
TRONO 1996; FOUCHIER et al. 1996; KARCZEWSKI and STREBEL 1996; SIMON et al.
1998b), it has more recently been demonstrated, using procedures which better
separate viral particles from contaminating host-cell-derived microvesicles, that Vif
is not speci®cally incorporated into virions (DETTENHOFER and YU 1999). The
major defect in Vif defective viruses is manifest post-entry and appears to be due to
an instability of nascent viral cDNA (SOVA and VOLSKY 1993; COURCOUL et al.
1995; GONCALVES et al. 1996; SIMON AND MALIM 1996). Correction of the defect
can be restored by expression of Vif in transit in the virus producing cell but not in
the infected cell (GABUZDA et al. 1992b; VON SCHWEDLER et al. 1993) suggesting
that Vif in¯uences an essential step in virus production. The best accepted model is
that non-permissive cells contain a negative cellular factor which inhibits a stage
leading to the production of infectious virus particles and that Vif counteracts the
eect of this negative cellular factor. This model is supported by studies with
heterokaryons formed between permissive and non-permissive cells. Vif mutant
viruses produced from these heterokaryons exhibit an infectivity defect suggesting
that Vif counteracts the activity of a negative factor to restore viral replication
capacity (MADANI and KABAT 1998; SIMON et al. 1998a). Vif co-localizes with
structural Gag proteins in vitro and in infected cells (BOUYAC et al. 1997; SIMON
et al. 1997). Since Vif mutant virions have been shown to have defects in core
structure and in reactions of endogenous reverse transcription (HOGLUND et al.
1994; GONCALVES et al. 1996), one possibility is that the negative cellular factor
interferes with appropriate localization, post-translational modi®cation or con-
formation of Gag protein. Studies with viral chimeras are needed to identify which
viral proteins Vif cooperates with to promote viral infectivity.
The infectivity of the virion can further potentially be in¯uenced by intracel-
lular interactions between the viral envelope glycoprotein and cellular receptor/
co-receptor molecules. This could potentially interfere with normal localization of
envelope glycoproteins at the site of virus assembly, thereby reducing incorporation
into virions. Additionally, incorporation of complexes between envelope and
receptor or co-receptor molecules into the virion would reduce the number of
occupancy sites on envelope glycoproteins that are able to bind receptor molecules
on the target cell. Several features of viral replication have been described which
could potentially circumvent these hazards. One mechanism may involve degra-
dation of CD4 molecules so as to reduce their occupancy of receptor binding sites
on nascent viral envelopes. Newly synthesized envelope binds directly to CD4 on
the endoplasmic reticulum and impairs its subsequent translocation to the cell
Molecular Biology of Lentivirus-Mediated Gene Transfer 11
surface (HOXIE et al. 1986; STEVENSON et al. 1987; STEVENSON et al. 1988; JABBAR
and NAYAK 1990). As a result, the amount of CD4 in the cell surface is reduced in
virus infected cells. A similar phenomenon occurs with co-receptor molecules such
as CXCR4 (ENDRES et al. 1996). By reducing the amount of free envelope or of free
receptor molecules, the virus may be able to restrict complexing between those
proteins that could interfere with infectious particle production. The accessory
protein Vpu, binds CD4 in the endoplasmic reticulum to recruit it to the cytosolic
ubiquitin-proteasome pathway, where it is degraded (WILLEY et al. 1994; SCHUBERT
et al. 1998). The recruitment of CD4/Vpu complexes to the proteasome is mediated
through a protein termed beta-TrCP which itself binds to the proteasome targeting
factor Skp1p (MARGOTTIN et al. 1998). The accessory protein, Nef, recruits CD4
from the cell surface to clathrin-coated pits and eventually to degradative lyso-
somes (GUY et al. 1987; GARCIA et al. 1991; AIKEN et al. 1994; GREENBERG et al.
1998; LE GALL et al. 1998; PIGUET et al. 1998). As a result of these CD4 degra-
dation mechanisms, the amount of CD4 that is incorporated into virions and that
can interfere with the infectivity of virions is reduced (LAMA et al. 1999). A similar
mechanism must exist to reduce interference from co-receptor molecules since
virions of HIV-1 appear to exclude incorporation of CXCR4, CCR5, or CCR3
even when cell lines in which viruses are produced express these co-receptors
(LALLOS et al. 1999).
Several cellular proteins have been shown to contribute, to varying degrees,
to the infectivity of the virion. The best characterized of these cellular factors is
cyclophilin A, the cellular ligand of the immunosuppressive drug, cyclosporine A.
Cyclophilin A is packaged into viral particles through an interaction with the viral
capsid protein (FRANKE et al. 1994; THALI et al. 1994). Disruption of the Gag-
cyclophilin interaction, either through treatment of infected cells with cyclosporine
A or by mutations in capsid that inhibit the interaction with cyclophilin A, result in
a marked impairment of viral infectivity (BRAATEN et al. 1996). The best accepted
model is that cyclophilin A participates in the uncoating step of viral entry, perhaps
by promoting disassembly of capsid molecules within the viral core. The trans-
cription factor NF-ATc which is normally involved in regulating expression of the
interleukin-2 gene, appears to in¯uence viral infectivity post-entry by promoting
the reverse transcription step (KINOSHITA et al. 1998). Since there is no evidence
that NF-ATc is virion associated, this factor may interact with the reverse trans-
cription complex post-entry. As NF-ATc is activated in the cell cycle, its expression
in resting T cells may partially account for the refractiveness of resting T cells to
HIV-1 infection in vitro (ZACK et al. 1990).
2 Host-Cell Restrictions to Retrovirus Entry
To support a complete viral replication cycle in vitro, primary CD4+ T cells
must be in cell cycle, i.e. at a stage beyond the G0 stage (also referred to as the
