Figure 1
.
Schematic cross section through a retroviral particle. The viral envelope is
formed by a cell-derived lipid bilayer into which proteins encoded by the
env region of the viral genome are inserted. These
consist of the transmembrane (TM) and the surface (SU) components linked
together by disulfide bonds. Internal nonglycosylated structural proteins
are encoded by the gag region of the viral genome. They are
the matrix (MA) protein, capsid (CA) protein, and nucleocapsid (NC) protein.
The suggested icosahedral structure of the retroviral capsid is not
definitely established (Chapter
2). Major products of the pol-coding region are
reverse transcriptase (RT) and integrase (IN). The protease (PR) is derived
from the pro gene between gag and
pol.
Retroviruses comprise a large and diverse family of enveloped RNA viruses defined by
common taxonomic denominators that include structure, composition, and replicative
properties (
Coffin 1992a,
b,
1996). The virions are 80–100 nm in diameter, and their outer
lipid envelope incorporates and displays the viral glycoproteins (). The shape and location of the internal
protein core are characteristic for various genera of the family. The virion RNA is
7–12 kb in size, and it is linear, single-stranded, nonsegmented, and of
positive polarity. The hallmark of the family is its replicative strategy which
includes as essential steps reverse transcription of the virion RNA into linear
double-stranded DNA and the subsequent integration of this DNA into the genome of
the cell.
Figure 2
.
(A) A simple retroviral genome. The genetic map of an
ALV contains four major coding regions, gag,
pro, pol, and
env. Different reading frames are indicated by vertical
displacement of the coding region. The pro gene is
encoded in the gag reading frame. The terminal
noncoding sequences include two direct repeats (R), a U5
(5′unique), and a U3 (3′unique) sequence.
(B) A complex retroviral genome. The genetic map of
HTLV contains, besides the major coding domains, information for two
regulatory proteins, Tax and Rex, encoded in regions (boxes) joined by
RNA splicing. In this case, gag, pro,
and pol are all in different reading frames.
Table 1
Classification of Retroviruses
| 1. | Avian sarcoma and leukosis viral groupb | Rous sarcoma virus | central, spherical core “C particles” | simple |
| 2. | Mammalian B-type viral group | mouse mammary tumor virus | eccentric, spherical core “B particles” | simple |
| 3. | Murine leukemia-related viral group | Moloney murine leukemia virus | central, spherical core “C particles” | simple |
| 4. | Human T-cell leukemia–bovine leukemia viral | human T-cell leukemia virus | central, spherical core | complex |
| 5. | D-type viral group | Mason-Pfizer monkey virus | cylindrical core “D particles” | simple |
| 6. | Lentiviruses | human immunodeficiency virus | cone-shaped core | complex |
| 7. | Spumaviruses | human foamy virus | central, spherical core | complex |
Retroviruses are broadly divided into two categories—simple and
complex—distinguishable by the organization of their genomes () (
Coffin
1992a;
Murphy et al. 1994). All
retroviruses contain three major coding domains with information for virion
proteins:
gag, which directs the synthesis of internal virion
proteins that form the matrix, the capsid, and the nucleoprotein structures;
pol, which contains the information for the reverse
transcriptase and integrase enzymes; and
env, from which are
derived the surface and transmembrane components of the viral envelope protein. An
additional, smaller, coding domain present in all retroviruses is
pro, which encodes the virion protease. Simple retroviruses
usually carry only this elementary information, whereas complex retroviruses code
for additional regulatory nonvirion proteins derived from multiply spliced messages
(). Retroviruses are further
subdivided into seven groups defined by evolutionary relatedness, each with the
taxonomic rank of
genus (
Table
1). Five of these groups represent retroviruses with oncogenic potential
(formerly referred to as oncoviruses), and the other two groups are the lentiviruses
and the spumaviruses. All oncogenic members except the human T-cell leukemia
virus–bovine leukemia virus (HTLV-BLV) genus are simple retroviruses.
HTLV-BLV and the lentiviruses and spumaviruses are complex.
Oncogenic retroviruses occur in all classes of vertebrates, and many act as natural
carcinogens. Some of the best studied are Rous sarcoma virus (RSV), a highly
pathogenic agent inducing connective tissue tumors in chickens; mouse mammary tumor
and murine leukemia viruses (MMTV and MLV); and more recently HTLV (Table 2). The lentiviruses are also found
ubiquitously, causing disease principally by killing or inducing loss of function of
specific cells and tissues. A representative example is human immunodeficiency virus
(HIV), the causative agent of acquired immunodeficiency syndrome (AIDS). Relatively
less is known about spumaviruses, which cause no known disease, and interest in this
category is relatively recent. The prototypical species is human foamy virus, a
virus that has not been definitely linked to a specific pathology.
Retroviruses as Models and Tools
Figure 3
.
Retroviral replication cycle. The parental virus attaches to a specific
receptor on the surface of a susceptible cell with the SU portion of the
viral Env protein leading to fusion and entry of the core. Reverse
transcription then generates a double-stranded DNA copy of the RNA
genome. The provirus is transported into the nucleus and integrated into
chromosomal DNA. It is then transcribed by cellular RNA polymerase II.
Transcription generates RNA copies with the terminal structures
organized as in the parental genome. These copies become full-length and
spliced messenger RNAs as well as full-length progeny virion RNA. Viral
messages are translated in the cytoplasm. Virion proteins and progeny
RNA assemble at the cell periphery and the plasma membrane, and progeny
virus is released by a process of budding and subsequent maturation into
infectious virus.
The study of retroviruses has had a broad impact on diverse areas of biology and
medicine, notably on molecular genetics, on the study of cellular growth control
and carcinogenesis, and on biotechnology (
Varmus 1988;
Temin 1992).
The effect of retrovirology on our concept of genetic information, its molecular
forms, transmission, and evolution has been nothing short of revolutionary. In
the early years of molecular biology, no exceptions to the unidirectional and
presumed irreversible flow of genetic information from DNA to RNA to protein
were known. This unidirectional flow came to be known as the “Central
Dogma.” It was this Central Dogma that had to be revised when the
replication of retroviruses was understood: The retrovirus growth cycle includes
as an essential step the copying of RNA into DNA by a virus-coded polymerase,
reversing the flow of genetic information, hence the terms
“retroviruses” and “reverse
transcriptase” (). The
reverse transcription of retroviruses is not a singular, odd exception but
rather a paradigm for a process that is shared by viral and nonviral genetic
elements occurring widely in nature. Examples are the Ty elements of yeast and
the
copia and
ulysses elements of
Drosophila (
Boeke and
Corces 1989;
Boeke and Chapman
1991;
Evgen'ev et al. 1992;
Garfinkel 1992). They resemble
retroviruses not only by encoding reverse transcriptase, but also in the
structure of both coding and noncoding regulatory sequences of their DNA forms.
Indeed, these elements can be viewed as degenerate viruses, although they are
more commonly considered to be ancestral to or parallel with Retroviruses in
evolution. Among other viral groups, the hepadnaviruses (hepatitis B virus) and
the plant-pathogenic caulimoviruses (
Shepherd
1989;
Robinson 1990),
although mechanistically and structurally distinct, practice reverse
transcription and are referred to as pararetroviruses. Furthermore, a
significant percentage of the mammalian genome appears to be the product of
reverse transcription, containing sequences whose characteristics point to RNA
as a template precursor. These sequences testify to important evolutionary
processes that have shaped the store of vertebrate genetic information but that
are not fully understood.
The study of retroviral oncogenesis opened up the entire field of cellular growth
control (Bishop 1983; Varmus 1984). It has led to the discovery
of Protooncogenes, cellular genes whose products normally function in the
transduction of signals that regulate cell replication. Current knowledge of the
control of growth and differentiation and of the aberrant growth of cancer has
largely originated from investigations of regulatory genes first identified in
highly oncogenic retroviruses. Studies of these genes have highlighted the
importance of discrete genetic changes in malignant transformation.
Retroviruses have also provided essential tools for biotechnology. Reverse
transcriptases from avian and murine retroviruses are used universally to
generate cDNA copies of RNA which then can be manipulated with relative ease for
cloning and sequencing (Skalka and Goff
1993). Modified retroviral genomes are widely used in transient and
stable expression of cloned genes in vertebrate cells (Miller 1992). They are also promising delivery vehicles
for human gene therapy.
A Unique Genetic Strategy
Figure 4
.
Comparison of the RNA and the DNA forms of the viral genome. Reverse
transcription of the RNA genome generates identical structures referred
to as long terminal repeats (LTRs) found at both ends of the DNA
provirus. Transcription of the provirus between the upstream U3 and
downstream U5 regions generates RNA with the same terminal organization
as in the parental virus. (R) Terminal direct repeat RNA; (U5) unique
regulatory sequences at the 5′end; (U3) unique regulatory
sequences at the 3′end.
The retroviral replication cycle follows the general pattern of enveloped viral
infections, but embedded in it are some highly uncommon features (
Swanstrom and Vogt 1990;
Luciw and Leung 1992;
Coffin 1996). Retroviruses enter the host
cell through the attachment of their surface glycoproteins to specific plasma
membrane receptors, which leads to fusion of virus and cell membranes (). The interaction of virus and cell
surfaces is highly specific; it constitutes the main determinant of viral host
range, defining susceptible animal species and target cells within the host.
After penetration into the cell, the RNA genome, still contained in a core
complex of nonglycosylated proteins and associated with the virion reverse
transcriptase, is transcribed into a double-stranded DNA. Transcription into DNA
involves two jumps of the reverse transcriptase from the 5′terminus to
the 3′terminus of the template molecule. The result of these jumps is
a duplication of sequences located at the 5′and 3′ends of
the virion RNA; these sequences then occur fused in tandem on both ends of the
viral DNA, forming the long terminal repeats (LTRs) (). The LTRs regulate viral gene expression and
therefore replication and pathogenesis.
Reverse transcription takes place in the cytoplasm; the viral DNA is translocated
into the nucleus where the linear copy of the retroviral genome is inserted into
chromosomal DNA with the aid of the virion integrase to form a stable provirus.
Integration does not permute the linear order of the proviral sequences,
LTR-
gag-
pol-
env-LTR
(). The number of possible sites
of integration into the cellular genome is very large and very widely
distributed. With integration, the provirus achieves the status of a cellular
gene and is expressed through the agency of cellular RNA polymerase II and
replicated by cellular enzymes in concert with chromosomal DNA. Control of
proviral transcription remains largely with the noncoding sequences of the viral
LTR. Transcription of the provirus generates spliced and unspliced mRNAs and
full-length progeny RNA genomes. In infection with simple retroviruses, control
of transcription is mediated solely by interaction of cellular factors with the
DNA of the LTR. Complex viruses, in contrast, take a more active role, encoding
trans-activating factors that affect the levels of
transcripts and the relative amounts of the products of the various genes. This
strategy allows these viruses a measure of control of gene expression not seen
with the simple viruses.
Viral messages are translated on cellular ribosomes. The translation products,
together with progeny RNA, are assembled at the cell periphery into viral
particles that are released from the cell by budding of the plasma membrane.
Budding of viruses is followed by proteolytic cleavage of virion polyproteins by
a viral protease and by cellular proteases. Productive Retroviral infection is
not of necessity cytopathic; infected cell cultures often show no visible
effects of viral production. In some congenital infections in animals, virus can
be produced by most cells in most tissues without deleterious effect on the
development and function of the organism (Rubin
et al. 1961, 1962).
In the replication cycle, viral entry, assembly, and release follow common
patterns (pathways) similar to these events in other enveloped RNA viruses. The
unique steps in the retroviral growth cycle are reverse transcription and,
especially, integration. Reverse transcription generates a progenitor proviral
DNA copy from which the entire viral progeny of the cell is derived by
polymerase-II-mediated transcription. Although reverse transcription is much
more error prone than cellular DNA replication, its product, once integrated
into the cellular genome, has the genetic stability of cellular genes. Reverse
transcription and integration make retroviral infection permanent, as integrated
proviruses are only rarely lost from the cellular genome: “A
retrovirus is forever.” Integration of viral DNA into the host genome
is ipso facto mutagenic. This insertional mutagenesis can inactivate or activate
cellular genes; this process is one of the Mechanisms by which retroviruses
induce tumors.
Viral Takeover at the Level of the Gene
Retroviruses have also developed unique mechanisms of pathogenicity involving the
transfer or transcriptional activation of specific cellular genes. These
Mechanisms are based on genetic recombination between virus and cell and between
viral genomes. Retroviral particles contain two copies of their genome linked by
regions near the 5′termini. They are diploid and are the only viruses
so equipped (Billeter et al. 1974; Kung et al. 1975; Beemon et al. 1976). A direct consequence of diploidy is
the formation of heterozygote virions in cells that are infected with two or
more genetically distinct but related retroviruses. Such heterozygotes give
rise, in the next cycle of infection, to stable genetic recombinants that are
formed during the process of reverse transcription of the two parental genomes
from the same viral particle. Rates of recombination between related
retroviruses are high (Coffin 1979; Linial and Blair 1982; Temin 1991).
Figure 5
.
Retroviral transformation and oncogene transduction. From the top right:
Infection of a cell with a retrovirus that contains only viral genes can
occasionally initiate tumor formation by insertion of the provirus next
to a protooncogene. Recombination during subsequent infection can lead
to the incorporation of the cell-derived oncogene. Cells infected with
virus with only the oncogene-containing genome become transformed
nonproducer cells from which transforming virus can be rescued by
superinfection with a replication-competent, nontransforming helper
virus.
Interviral genetic exchange, together with integration into the cellular genome,
formally also a recombination event, probably accounts for the presence of
cellular genes in some retroviruses (). A plausible hypothesis for this acquisition of cellular genetic
material postulates that a provirus integrates upstream of a cellular gene and
leads to the production of chimeric virus-cell transcripts. In the next round of
replication, nonhomologous recombination between virus and cell sequences leads
to the incorporation of the cellular gene into the retroviral genome, so that it
is now transported by the virus from cell to cell and expressed under control of
the viral LTR (
Goldfarb and Weinberg
1981;
Swanstrom et al. 1983;
Raines et al. 1988;
Felder et al. 1991,
1993;
Coffin 1992b;
Swain and Coffin 1992; see
Chapter 4). The usual product of
this transduction process has acquired the host sequence at the cost of one or
more viral genes. Such viruses are therefore generally defective for
replication, requiring the presence of a replication-competent provirus in the
same cell to provide viral proteins for replication (see ). The transduction of cellular genes has been found
only with simple retroviruses and not with complex retroviruses. The reasons for
this difference are not clear, but they may have to do with the mechanism by
which retroviruses acquire cellular sequences or with viral genome organization
that must be tolerant of foreign inserts.
The modified cellular genes carried by retroviruses convey a high degree of
tumorigenicity to the virus. These viral or v-onc genes are
usually mutated growth-regulatory genes. Their cellular progenitors are referred
to as Protooncogenes or c-onc genes (Bishop 1983; Varmus
1984; Cooper 1990).
Overexpression or inappropriate expression, often combined with mutation of an
oncogene that has become part of a viral genome, results in a gain of function
of a positive growth signal. This constitutive gain of function induces and
maintains malignant transformation. Retroviruses with oncogenes in their genomes
are particularly fast-acting carcinogens and in most cases also transform cells
in culture. Retroviruses lacking an oncogene do not transform cultured cells,
but some can induce tumors in animals, a process that is characterized by a long
latent period. Here again, an oncogene is the mediator of neoplastic
transformation, but in this instance, it is the cellular homolog that is
activated through the insertion of a provirus. The viral LTR through its
promoter and enhancer sequences alters expression of the neighboring cellular
oncogene (for reviews, see Lazo and Tsichlis
1990; van Lohuizen and Berns
1990; Kung and Vogt 1991).
Transduction and insertional activation of cellular oncogenes are the two main
mechanisms by which most retroviruses induce tumors. Other mechanisms exist but
are less common. One is exemplified by the highly aggressive leukemias which are
linked to infection with HTLV. This virus neither carries a transduced oncogene
in its genome nor activates a resident oncogene by insertion; it may cause
tumors through one of its regulatory proteins, possibly by changing the
expression levels of cellular genes (Feuer and
Chen 1992). A unique mode of tumor induction is also seen with a form
of the Friend murine leukemia virus. It produces a modified Env protein that
appears to mimic a component of the erythropoietin signaling pathway (Li et al. 1990; D'Andrea et al. 1992; Johnson and Benchimol 1992).
Retroviruses show the entire continuous spectrum of effects on cell replication
and survival from pronounced growth stimulation to cell death. The latter is an
important element in lentiviral infections including infection with HIV.
Although it appears likely that viral Env proteins are involved in some aspects
of retroviral cell killing, as is reinfection of cells leading to accumulation
of large amounts of viral DNA, the full cytopathic mechanisms operating at the
cellular and organismic levels remain to be established.
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