A virus is a small parasite that cannot
reproduce by itself. Once it infects a susceptible cell, however, a virus can direct
the cell machinery to produce more viruses. Most viruses have either RNA or DNA as
their genetic material. The nucleic acid may be single- or double-stranded. The
entire infectious virus particle, called a virion, consists of the nucleic acid and an outer shell of protein. The
simplest viruses contain only enough RNA or DNA to encode four proteins. The most
complex can encode 100 – 200 proteins.
The study of plant viruses inspired some
of the first experiments in molecular biology. In 1935, Wendell Stanley purified and
partly crystallized tobacco mosaic virus (TMV); other plant viruses were
crystallized soon thereafter. Pure proteins had been crystallized only a short time
before Stanley’s work, and it was considered very surprising at the time
that a replicating organism could be crystallized.
A wealth of subsequent research with bacterial viruses and animal viruses has
provided detailed understanding of viral structure, and virus-infected cells have
proved extremely useful as model systems for the study of basic aspects of cell
biology. In many cases, DNA viruses utilize cellular enzymes for synthesis of their
DNA genomes and mRNAs; all viruses utilize normal cellular ribosomes, tRNAs, and
translation factors for synthesis of their proteins. Most viruses comman-deer the
cellular machinery for macromolecular synthesis during the late phase of infection,
directing it to synthesize large amounts of a small number of viral mRNAs and
proteins instead of the thousands of normal cellular macromolecules. For instance,
animal cells infected by influenza or vesicular stomatitis virus synthesize only one
or two types of glycoproteins, which are encoded by viral genes, whereas uninfected
cells produce hundreds of glycoproteins. Such virus-infected cells have been used
extensively in studies on synthesis of cell-surface glycoproteins. Similarly, much
information about the mechanism of DNA replication has come from studies with
bacterial cells and animal cells infected with simple DNA viruses, since these
viruses depend almost entirely on cellular proteins to replicate their DNA. Viruses
also often express proteins that modify host-cell processes so as to maximize viral
replication. For example, the roles of certain cellular factors in initiation of
protein synthesis were revealed because viral proteins interrupt their action.
Finally, when certain genes carried by cancer-causing viruses integrate into
chromosomes of a normal animal cell, the normal cell can be converted to a cancer
cell.
Since many viruses can infect a large number of different cell types, genetically
modified viruses often are used to carry foreign DNA into a cell. This approach
provides the basis for a growing list of experimental gene therapy treatments.
Because of the extensive use of viruses in cell biology research and their potential
as therapeutic agents, we describe the basic aspects of viral structure and function
in this section.
Viral Capsids Are Regular Arrays of One or a Few Types of Protein
The nucleic acid of a virion is enclosed within a protein coat, or capsid, composed of multiple copies
of one protein or a few different proteins, each of which is encoded by a single
viral gene. Because of this structure, a virus is able to encode all the
information for making a relatively large capsid in a small number of genes.
This efficient use of genetic information is important, since only a limited
amount of RNA or DNA, and therefore a limited number of genes, can fit into a
virion capsid. A capsid plus the enclosed nucleic acid is called a nucleocapsid.
Figure 6-11
.
Two basic geometric shapes of viruses
(a) In some viruses, the protein subunits form helical arrays around
an RNA or DNA molecule (red), which runs in a helical groove within
the enclosing protein tube. The electron micrograph to the right is
of tobacco mosaic virus (TMV), illustrating the rodlike shape of
this type of virus. (b, c) In other viruses, the capsid proteins
associate to form polyhedrons with icosahedral (20-sided) symmetry.
In the simplest and smallest of these quasi-spherical viruses (b),
three identical capsid protein subunits form each triangular face
(red) of the icosahedron. The subunits meet in fivefold symmetry at
each vertex. In some larger viruses of this type, each triangular
face is composed of four subunits (c). The contact between subunits
not at the vertices is quasi-equivalent: the subunits on the
vertices maintain fivefold symmetry, but those making up the
surfaces in between exhibit sixfold symmetry. Although the actual
shape of the protein subunits in these viruses is not a flat
triangle as illustrated, the overall effect when the subunits are
assembled is of a roughly spherical structure with triangular faces.
The electron micrograph beneath (b) and (c) is of an adenovirus.
Viral proteins that attach to host-cell receptors project from the
vertices. [After S. E. Luria et al., 1978, General
Virology, 3d ed., Wiley, pp.
39 – 40. Photograph of TMV courtesy
of R. C. Valentine; photograph of adenovirus courtesy of Robley C.
Williams, University of California.]
Nature has found two basic ways of arranging the multiple
capsid protein subunits
and the viral
genome into a
nucleocapsid. The simpler structure is a
protein
helix with the RNA or DNA protected within. Tobacco mosaic
virus (TMV) is a
classic example of the helical
nucleocapsid. In TMV the
protein subunits form
broken disklike structures, like lock washers, which form the helical shell of a
long rodlike
virus when stacked together ().
The other major structural class of
viruses, called
icosahedral
or
quasi-spherical viruses, is based on the icosahedron, a
solid object built of 20 identical faces, each of which is an equilateral
triangle. In the simplest type of icosahedral
virion each of the 20 triangular
faces is constructed of three identical
capsid protein subunits, making a total
of 60 subunits per
capsid. At each of the 12 vertices, five subunits make
contact symmetrically ().
Thus all
protein subunits are in
equivalent contact with one
another. Tobacco satellite necrosis
virus has such a simple icosahedral
structure. However, most quasi-spherical
viruses are larger, requiring the
assembly of more than three subunits per face of the icosahedron. These
proteins
form shells whose subunits are in
quasi-equivalent contact.
Here, the
proteins at the icosahedral vertices remain arranged in a fivefold
symmetry, but additional subunits cover the surfaces between in a pattern of
sixfold symmetry ().
Figure 6-12
.
Structure of picornaviruses
These icosahedral viruses include poliovirus and the rhinoviruses,
which cause the common cold. (a) The picornavirus capsid is composed
of four proteins (VP1, VP2, VP3, and VP4; VP4 is located in the
interior). This model of a picornavirus, based on x-ray
crystallographic analyses, shows that the vertices with fivefold
symmetry contain five VP1 molecules (blue); the surfaces with
sixfold symmetry contain three VP2 molecules and three VP3 molecules
(red and yellow). (b) Many picornaviruses have an indentation, or
“canyon,” encircling each vertex of the
icosahedron. In rhinoviruses, this canyon interacts with ICAM-1, a
cell-adhesion molecule on the surface of respiratory epithelial
cells, allowing the virus to bind to these cells in the first step
of infection. Neutralizing antibodies also bind to the canyon,
thereby preventing ICAM-1 from entering the canyon. Only a portion
of the ICAM-1 and antibody molecules are depicted. [Part (a) from J.
M. Hogle et al., 1987, Sci. Am.
256(3):42; courtesy of James M. Hogle. Part (b) adapted
from T. J. Smith et al., 1996, Nature
383:350.]
The atomic structures of a number of icosahedral
viruses have been determined by
x-ray crystallography ().
The first three such
viruses to be
analyzed — tomato bushy stunt
virus, poliovirus,
and rhinovirus (the common cold
virus) — exhibit
a remarkably similar design, in terms of the rules of icosahedral symmetry as
well as in the details of their surface
proteins. In each
virus, at atomic
resolution, clefts (“canyons”) are observed encircling each
of the vertices of the icosahedral structure. Interaction of these clefts with
cell-surface
receptors attaches the
virus to a host cell, the first step in
viral infection ().
Neutralizing antibodies specific for a particular
virus also interact with these
clefts, thereby inhibiting attachment of the
virus to the host cell.
Figure 6-13
.
Electron micrograph of a negatively stained influenza virus
virion
The virion is surrounded by a phospholipid bilayer; the large spikes
protruding outward from the membrane are composed of trimers of
hemagglutinin protein and tetramers of neuraminidase protein. Inside
is the nucleocapsid. [Courtesy of A. Helenius and J. White.]
In some
viruses, the symmetrically arranged
nucleocapsid is covered by an
external
membrane, or
envelope, which consists mainly of a
phospholipid bilayer but also contains one or two types of
virus-encoded
glycoproteins (). The
phospholipids in the viral envelope are similar to those in the
plasma membrane
of an infected host cell. The viral envelope is, in fact, derived by budding
from that
membrane, but contains mainly viral
glycoproteins.
The components of simple viruses such as TMV, which consists of a single RNA
molecule and one protein species, undergo self-assembly if they are mixed in
solution. More complex viruses containing a dozen or more protein species do not
spontaneously assemble in vitro. The multiple components of such viruses
assemble within infected cells in stages, first into subviral particles and then
into completed virions. The genomes of these complex viruses encode proteins
that assist in the assembly of the virion, but the assembly proteins are not
themselves components of the completed virion.
Most Viral Host Ranges Are Narrow
The fact that the host range — the group of cell
types that a virus can infect — is generally
restricted serves as a basis for classifying viruses. A virus that infects only
bacteria is called a bacteriophage,
or simply a phage. Viruses that
infect animal or plant cells are referred to generally as animal
viruses or plant viruses. A few viruses can grow
in both plants and the insects that feed on them. The highly mobile insects
serve as vectors for transferring such viruses between susceptible plant hosts.
An example is potato yellow dwarf virus, which can grow in leafhoppers (insects
that feed on potato plant leaves) as well as in potato plants. Wide host ranges
are characteristic of some strictly animal viruses, such as vesicular stomatitis
virus, which grows in insects and in many different types of mammalian cells.
Most animal viruses, however, do not cross phyla, and some (e.g., poliovirus)
infect only closely related species such as primates. The host-cell range of
some animal viruses is further restricted to a limited number of cell types
because only these cells have appropriate surface receptors to which the virions
can attach.
Viruses Can Be Cloned and Counted in Plaque Assays
Figure 6-14
.
Plaque assay for determining number of infectious particles in a
viral suspension
(a) Each lesion, or plaque, which develops where a single virion
initially infected a single cell, constitutes a pure viral clone.
(b) Plate illuminated from behind shows plaques formed by
λ bacteriophage plated on E. coli. (c)
Plate showing plaques produced by poliovirus plated on HeLa cells.
[Part (b) courtesy of Barbara Morris; part (c) from S. E. Luria et
al., 1978, General Virology, 3d ed., Wiley, p.
26.]
The number of infectious viral particles in a sample can be quantified by a
plaque assay. This assay is performed
by culturing a dilute sample of viral particles on a plate covered with host
cells and then counting the number of local lesions, called
plaques, that develop (). A plaque develops on the plate wherever a single
virion initially infects a single cell. The
virus replicates in this initial
host cell and then lyses the cell, releasing many progeny
virions that infect
the neighboring cells on the plate. After a few such cycles of infection, enough
cells are lysed to produce a visible plaque in the layer of remaining uninfected
cells.
Since all the progeny virions in a plaque are derived from a single parental
virus, they constitute a virus clone. This type of plaque assay is in standard
use for bacterial and animal viruses. Plant viruses can be assayed similarly by
counting local lesions on plant leaves inoculated with viruses. Analysis of
viral mutants, which are commonly isolated by plaque assays, has contributed
extensively to current understanding of molecular cellular processes. The plaque
assay also is critical in isolating λ bacteriophage clones carrying
segments of cellular DNA, as discussed in Chapter 7.
Viral Growth Cycles Are Classified as Lytic or Lysogenic
Figure 6-15
.
Electron micrograph of a T4 bacteriophage adsorbed onto an E.
coli cell
Once viral surface proteins interact with receptors on the host cell,
the viral DNA is injected into the cell. [From A. Levine, 1991,
Viruses, Scientific American Library, p.
20.]
The surface of
viruses includes many copies of one type of
protein that binds, or
adsorbs, specifically to multiple copies of a
receptor protein on a host cell.
This interaction determines the host range of a
virus and begins the infection
process (). Then, in one of
various ways, the viral DNA or RNA crosses the
plasma membrane into the
cytoplasm. The entering genetic material may still be accompanied by inner viral
proteins, although in the case of many bacteriophages, all
capsid proteins
remain outside an infected cell. The
genome of most DNA-containing
viruses that
infect eukaryotic cells is transported (with some associated
proteins) into the
cell
nucleus, where the cellular DNA is, of course, also found. Once inside the
cell, the viral DNA interacts with the host’s machinery for
transcribing DNA into mRNA. The viral mRNA that is produced then is translated
into viral
proteins by host-cell
ribosomes, tRNA, and
translation factors.
Most viral protein products fall into one of three categories: special enzymes
needed for viral replication; inhibitory factors that stop host-cell DNA, RNA,
and protein synthesis; and structural proteins used in the construction of new
virions. These last proteins generally are made in much larger amounts than the
other two types. After the synthesis of hundreds to thousands of new virions has
been completed, most infected bacterial cells and some infected plant and animal
cells rupture, or lyse, releasing all the virions at once. In many plant and
animal viral infections, however, no discrete lytic event occurs; rather, the
dead host cell releases the virions as it gradually disintegrates.
Figure 6-16
.
The steps in the lytic replication cycle of a nonenveloped virus
are illustrated for E. coli bacteriophage T4, which has a
double-stranded DNA genome
During adsorption (step 1), viral coat proteins (at the
tip of the tail in T4) interact with specific receptor proteins on
the exterior of the host cell. The viral genome is then injected
into the host. Next, host-cell enzymes transcribe viral
“early” genes into mRNAs and subsequently
translate these into viral “early” proteins
(step 2), which replicate the viral DNA and induce
expression of viral “late” proteins by host-cell
enzymes (step 3). The viral late proteins include
capsid and assembly proteins and enzymes that degrade the host-cell
DNA, supplying nucleotides for synthesis of viral DNA. Progeny
virions are assembled in the cell (step 4) and released
(step 5) when the cell is lysed by viral proteins.
Newly liberated viruses initiate another cycle of infection in other
host cells.
These events — adsorption, penetration,
replication, and release — describe the
lytic cycle of viral replication. The
outcome is the production of a new round of viral particles and death of the
cell. illustrates the lytic
cycle for T4 bacteriophage. Adsorption and release of enveloped animal
viruses
are somewhat more complicated processes. In this case, the
virions
“bud” from the host cell, thereby acquiring their outer
phospholipid envelope, which contains mostly viral
glycoproteins.
Figure 6-17
.
The steps in the lytic replication cycle of an enveloped virus
are illustrated for rabies virus, which has a single-stranded RNA
genome
The structural components of this virus are depicted at the
top. Note that the nucleocapsid of this virus
is helical rather than icosahedral. After a virion adsorbs to a
specific host membrane protein (step 1), the cell
engulfs it in an endosome (step 2). A protein in the
endosome membrane pumps protons from the cytosol into the endosome
interior. The resulting decrease in endosomal pH induces a
conformational change in the viral glycoprotein, leading to fusion
of the viral envelope with the endosomal lipid bilayer membrane and
release of the nucleocapsid into the cytosol (steps 3
and 4). Viral RNA polymerase uses ribonucleoside
triphosphates in the cytosol to replicate the viral RNA genome (step
5) and synthesize viral mRNAs (step
6). One of the viral mRNAs encodes the viral
transmembrane glycoprotein (blue), which is inserted into the lumen
of the endoplasmic reticulum (ER) as it is synthesized on ER-bound
ribosomes (step 7). Carbohydrate is added to the large
folded domain inside the ER lumen and is modified as the membrane
and the associated glycoprotein pass through the Golgi apparatus
(step 8). Vesicles with mature glycoprotein fuse with
the plasma membrane, depositing viral glycoprotein on the cell
surface with the large folded domain outside the cell, the
transmembrane α helix spanning the plasma membrane, and
the small cytoplasmic domain within the cell (step 9).
Meanwhile, other viral mRNAs are translated on host-cell ribosomes
into nucleocapsid protein, matrix protein, and viral RNA polymerase
(step 10). These proteins are assembled with replicated
viral genomic RNA (dark red) into progeny nucleocapsids (step
11), which then associate with the viral
transmembrane glycoprotein in the plasma membrane (step
12). As additional copies of the matrix protein on
a single nucleocapsid associate with the cytoplasmic domain of
additional copies of the viral transmembrane glycoprotein, the
plasma membrane is folded around the nucleocapsid, forming a
“bud” that eventually is released (step
13).
Figure 6-18
.
Transmission electron micrograph of measles virus budding from
the surface of an infected cell
[From A. Levine, 1991, Viruses, Scientific American
Library, p. 22.]
We illustrate the
lytic cycle of enveloped
viruses with the rabies
virus, whose
nucleocapsid consists of a single-stranded RNA
genome surrounded by multiple
copies of
nucleocapsid protein (,
upper left). Within the
nucleocapsid of rabies
virions are viral
enzymes for synthesizing viral mRNA and replicating the viral
genome. The envelope around the
nucleocapsid is a
phospholipid bilayer
containing multiple copies of a viral transmembrane
glycoprotein. This
receptor-binding, or “attachment,”
protein has a large
external folded
domain on the outside of the viral envelope, an
α-helical transmembrane
domain that spans the viral envelope, and a
short internal
domain. The internal
domain interacts with the viral matrix
protein, which functions as a bridge between the transmembrane
glycoprotein and
nucleocapsid protein.
outlines the events involved in adsorption of a rabies
virion, assembly of
progeny
nucleocapsids, and release of progeny
virions by budding from the
host-cell
plasma membrane. Budding
virions are clearly visible in electron
micrographs, as illustrated by .
Figure 6-19
.
λ bacteriophage undergoes either lytic replication or
lysogeny following infection of E. coli
The linear double-stranded DNA is converted to a circular form
immediately after infection.
(Left) If the
nutritional state of the host cell is favorable, most infected cells
undergo lytic replication, similar to lytic replication of cells by
bacteriophage T4 (see ).
(Right) If the nutritional state
of the host cell cannot support production of large numbers of
progeny
phages,
lysogeny is established. In this case, viral
genes
required for the
lytic cycle are repressed, and host-cell
enzymes
synthesize viral
proteins that integrate the viral DNA into a
specific sequence in the host-cell
chromosome where no host-cell
genes are disrupted. The prophage DNA then is replicated along with
the host-cell
chromosome as the lysogenized cell (called a
lysogen) grows and divides. Repression of the
viral
genes required for lytic replication is maintained in progeny
cells. At infrequent intervals, the prophage in a lysogen is
induced, or activated, leading to
expression of viral
proteins that
precisely remove the prophage DNA from the host-cell
chromosome and
to derepression of the
genes required for the
lytic cycle. As a
result, a normal cycle of lytic replication ensues.
In some cases, after a bacteriophage DNA molecule enters a bacterial cell, it
becomes integrated into the host-cell
chromosome, where it remains
quiescent and
is replicated as part of the cell’s DNA from one generation to the
next. This association is called
lysogeny, and the integrated
phage DNA is referred to as a
prophage (). Under certain conditions, the prophage DNA is activated,
leading to its excision from the host-cell
chromosome and entrance into the
lytic cycle. Bacterial
viruses of this type are called
temperate
phages. The
genomes of a number of animal
viruses also can
integrate into the host-cell
genome. Probably the most important are the
retroviruses, described briefly later in this chapter.
A few phages and animal viruses can infect a cell and cause new virion production
without killing the cell or becoming integrated.
Four Types of Bacterial Viruses Are Widely Used in Biochemical and Genetic
Research
Bacterial viruses have played a crucial role in the development of molecular cell
biology. Thousands of different bacteriophages have been isolated; many of these
are particularly well suited for studies of specific biochemical or genetic
events. Here, we briefly describe four types of bacteriophages, all of which
infect E. coli, that have been especially useful in molecular
biology research.
DNA Phages of the T Series
The T
phages of E. coli are large lytic
phages that contain a single molecule
of double-stranded DNA. This molecule is about
2 × 10
5 base pairs long in
T2, T4, and T6
viruses and about
4 × 10
4 base pairs long in
T1, T3, T5, and T7
viruses. T-
phage virions consist of a helical
protein
“tail” attached to an icosahedral
“head” filled with the viral DNA. After the tip of a
T-
phage tail adsorbs to
receptors on the surface of an E. coli cell, the DNA
in the head enters the cell through the tail (see ). The
phage DNA then directs a program of
events that produces approximately 100 new
phage particles in about 20
minutes, at which time the infected cell lyses and releases the new
phages.
The initial discovery of the role of
messenger RNA in
protein synthesis was
based on studies of E. coli cells infected with bacteriophage T2. By 20
minutes after infection, infected cells synthesize T2
proteins only. The
finding that the RNA synthesized at this time had the same
base composition
as T2 DNA (not E. coli DNA) implied that mRNA copies of T2 DNA were
synthesized and used to direct cellular
ribosomes to synthesize T2
proteins.
Temperate Phages
Bacteriophage λ, which infects E. coli, typifies the temperate
phages. This
phage has one of the most studied
genomes and is used
extensively in
DNA cloning (
Chapter
7). On entering an E. coli cell, the double-stranded λ
DNA assumes a circular form, which can enter either the
lytic cycle (as T
phages do) or the
lysogenic cycle (see ). In the latter case,
proteins expressed from the
viral DNA bind a specific sequence on the circular viral DNA to a similar
specific sequence on the circular bacterial DNA. The viral
proteins then
break both circular molecules of DNA and rejoin the broken ends, so that the
viral DNA becomes inserted into the host DNA. The carefully controlled
action of viral
genes maintains λ DNA as part of the host
chromosome by repressing the lytic functions of the
phage. Under appropriate
stimulation, the λ prophage is activated and undergoes lytic
replication.
Small DNA Phages
The genome of some bacteriophages encodes only
10 – 12 proteins, roughly
5 – 10 percent of the number encoded by T
phages. These small DNA phages are typified by the ΦΧ174
and the filamentous M13 phages. These were the first organisms in which the
entire DNA sequence of a genome was determined, permitting extensive
understanding of the viral life cycle. The viruses in this group are so
simple that they do not encode most of the proteins required for replication
of their DNA but depend on cellular proteins for this purpose. For this
reason, they have been particularly useful in identifying and analyzing the
cellular proteins involved in DNA replication (Chapter 12).
RNA Phages
Some E. coli bacteriophages contain a genome composed of RNA instead of DNA.
Because they are easy to grow in large amounts and because their RNA genomes
also serve as their mRNA, these phages are a ready source of a pure species
of mRNA. In one of the earliest demonstrations that cell-free protein
synthesis can be mediated by mRNA, RNA from these phages was shown to direct
the synthesis of viral coat protein when added to an extract of E. coli
cells containing all the other components needed for protein synthesis.
Also, the first long mRNA molecule to be sequenced was the genome of an RNA
phage. These viruses, among the smallest known, encode only four proteins:
an RNA polymerase for replication of the viral RNA, two capsid proteins, and
an enzyme that dissolves the bacterial cell wall and allows release of the
intracellular virus particles into the medium.
Animal Viruses Are Classified by Genome Type and mRNA Synthesis
Pathway
Animal viruses come in a variety of shapes, sizes, and genetic strategies. In
this book, we are concerned with viruses that exhibit at least one of two
features: they utilize important cellular pathways to form their molecules,
thereby closely mimicking a normal cellular function, or they can integrate
their genomes into those of normal cells.
The names of many viruses are based on
the names of the diseases they cause or of the animals or plants they infect.
Common examples include poliovirus, which causes poliomyelitis; tobacco mosaic
virus, which causes a mottling disease of tobacco leaves; and human
immunodeficiency virus (HIV), which causes acquired immunodeficiency syndrome
(AIDS). However, many different kinds of viruses often produce the same symptoms
or the same apparent disease states; for example, several dozen different
viruses can cause the red eyes, runny nose, and sneezing referred to as the
common cold. Clearly, any attempt to classify viruses on the basis of the
symptoms they produce or their hosts obscures many important differences in
their structures and life cycles.
What are central to the life cycle of a virus are the types of
nucleic acids formed during its replication and the pathway by which mRNA is
produced. The relation between the viral mRNA and the nucleic acid of the
infectious particle is the basis of a simple means of classifying viruses. In
this system, a viral mRNA is designated as a plus strand and
its complementary sequence, which cannot function as an mRNA, is a minus
strand. A strand of DNA complementary to a viral mRNA is also a
minus strand. Production of a plus strand of mRNA requires that a minus strand
of RNA or DNA be used as a template. Using this system, six classes of animal
viruses are recognized. Bacteriophages and plant viruses also can be classified
in this way, but the system has been used most widely in animal virology because
representatives of all six classes have been identified.
Figure 6-20
.
Classification of animal viruses based on the composition of
their genomes and pathway of mRNA formation
DNA is shown in blue; RNA, in red. The viral mRNA is designated as a
plus strand, which is synthesized from a minus strand of DNA or RNA.
Class VI
viruses (
retroviruses) have two identical plus strands of
genomic RNA, although the reason for this is unclear. See
Table 6-3 for examples of
viruses in each class.
Table 6-3
Animal Viruses Commonly Used in Molecular Biology
| CLASS I (DNA) | | | | | |
| Adenoviruses (class Ia) | Vertebrates | 36 | No | Replicate in host-cell nucleus; use host enzymes
for viral mRNA synthesis | mRNA synthesis and regulation: DNA replication;
cell transformation; gene therapy (as vectors) |
| Herpesviruses (class Ia) | Vertebrates | 150 | Yes | Replicate in host-cell nucleus; use host enzymes
for viral mRNA synthesis | mRNA synthesis and regulation: DNA replication;
cell transformation; gene therapy (as vectors) |
| SV40 (class Ia) | Primates | 5.5 | No | Replicate in host-cell nucleus; use host enzymes for viral mRNA
synthesis | mRNA synthesis and regulation: DNA replication; cell
transformation; gene therapy (as vectors) |
| Vaccinia virus (class Ib) | Vertebrates | 200 | Yes | Replicates in host-cell cytoplasm using viral
enzymes | Genome structure; mRNA synthesis by viral
enzymes |
| CLASS II (DNA) | | | | | |
| Parvoviruses | Vertebrates | 5 | No | Have linear ssDNA genome | DNA replication; gene therapy (as vectors) |
| CLASS III (RNA) | | | | | |
| Reoviruses | Vertebrates | 1.2 – 4.00‡ | No | Have a genome of 10 dsRNA segments; use viral
enzymes to replicate | mRNA snythesis by viral enzymes; mRNA
translation |
| CLASS IV (RNA) | | | | | |
| Poliovirus (class IVa) | Primates | 7 | No | Synthesizes a single mRNA, which is translated into
a polyprotein that is cleaved to yield functional proteins | Viral RNA replication; interruption of host mRNA
translation; polyprotein cleavage |
| Sindbis virus (class IVb) | Vertebrates, insects | 10 | Yes | Synthesizes at least two mRNAs, each of which is
translated into a polyprotein that is cleaved to yield
functional proteins | Membrane formation; glycoprotein biosynthesis and
intracellular transport |
| CLASS V (RNA) | | | | | |
| Vesicular stomatitis virus (class Va) | Vertebrates, | 12 | Yes | Has a virus-specific RNA polymerase that produces
several mRNAs from its nonsegmented genome | Membrane formation; glycoprotein biosynthesis and
intracellular transport |
| Influenza virus (class Vb) | Mammals, birds | 1.0 – 3.3‡ | Yes | Has a genome of 8 ssRNA segments; uses a
virus-specific RNA polymerase to produce mRNAs | Membrane formation; glycoprotein biosynthesis and
intracellular transport; disease prevention |
| CLASS VI (RNA) | | | | | |
| Retroviruses | Vertebrates, insects, yeasts | 5 – 8 | Yes | Copy RNA genome into DNA with viral reverse
transcriptase; integrates viral DNA into host genome | Cell transformation; function of oncogenes; AIDS;
gene therapy (as vectors) |
Figure 6-21
.
Structures of viruses determined by cryo-electron microscopy and
image analysis
Cowpea mosaic virus (CPMV) is a plant RNA virus, poliovirus (polio) a
human RNA virus, nudaureila capensis β virus (NβV)
an insect RNA virus, simian virus 40 (SV40) a monkey DNA virus, and
adenovirus (adeno) a human DNA virus. During infection, adenovirus
binds first to cell-surface receptors through the tips of the fibers
(green), and then interacts with integrins through mobile portions
(red) of the penton base (yellow). All viruses are shown at the same
magnification. [See P. L. Stewart et al., 1997, EMBO
J.
16:1189; CPMV, poliovirus, NβV, and SV40
courtesy of T. S. Baker; adenovirus courtesy of P. L. Stewart.]
The composition of the viral
genome and its relationship to the viral mRNA are
illustrated in for each of
the six classes of
virus.
Table 6-3
summarizes important properties of common animal
viruses in each class and the
research areas in which they have been widely used. Structural models of several
virions are shown in .
DNA Viruses (Classes I and II)
The genomes of both class I and class II viruses consist of DNA. Various
types of DNA viruses are commonly used in studies on DNA replication, genome
structure, mRNA production, and oncogenic cell transformation.
Class I viruses contain a single molecule of double-stranded
DNA (dsDNA). In the case of the most common type of class I animal virus,
viral DNA enters the cell nucleus, where cellular enzymes transcribe the DNA
and process the resulting RNA into viral mRNA. Examples of these viruses
include the following:
-
Adenoviruses, which cause infections in the upper
respiratory tract and gastrointestinal tract in many animals
-
SV40 (simian virus 40), a monkey virus that was
accidentally discovered in kidney cell cultures from wild monkeys
used in the production of poliovirus vaccines
-
Herpesviruses, which cause various inflammatory
skin diseases (e.g., chickenpox) and latent infections that recur
after long intervals (e.g., cold sores and shingles)
-
Human papillomaviruses (HPVs), which cause warts
and other insignificant skin lesions and occasionally cause
malignant transformation of cervical cells
Some types of HPV are passed
through sexual contact. In some infected women, the HPV genome integrates
into the chromosome of a cervical epithelial cell. This rare integration
event initiates an intensively studied process that can lead to development
of cervical carcinoma, one of the most common types of human cancers.
Routine Pap smears performed for early detection of cervical carcinoma are
done to identify cells in the early stages of the transformation process
initiated by HPV integration.
The second type of class I virus, collectively referred to as
poxviruses, replicates in the host-cell cytoplasm.
Typical of class Ib viruses are variola, which causes smallpox, and
vaccinia, an attenuated (weakened) poxvirus used in vaccinations to induce
immunity to smallpox. These very large, brick-shaped viruses
(0.1 × 0.1 × 0.2
μm) carry their own enzymes for synthesizing viral mRNA and DNA in
the cytoplasm.
Class II viruses, called parvoviruses (from
Latin parvo, “poor”), are simple
viruses that contain one molecule of single-stranded DNA (ssDNA). Some
parvoviruses encapsidate (enclose) both plus and minus strands of DNA, but
in separate virions; others encapsidate only the minus strand. In both
cases, the ssDNA is copied inside the cell into dsDNA, which is then itself
copied into mRNA.
RNA Viruses (Classes III – VI)
All the animal viruses belonging to classes
III – VI have RNA genomes. A wide range of
animals, from insects to human beings, are infected by viruses in each of
these classes. These viruses have been particularly useful in studies on
mRNA synthesis and translation (class III); glycoprotein synthesis, membrane
formation, and intracellular transport (classes IV and V); and cell
transformation and oncogenes (class VI).
Class III viruses contain double-stranded genomic RNA
(dsRNA). The minus RNA strand acts as a template for the synthesis of plus
strands of mRNA. The virions of all class III viruses known to date have
genomes containing 10 – 12 separate
double-stranded RNA molecules, each of which encodes one or two
polypeptides. Consequently, these viruses are said to have
“segmented” genomes. In these viruses, the virion itself
contains a complete set of enzymes that can utilize the minus strand of the
genomic RNA as a template for synthesis of mRNA in the test tube as well as
in the cell cytoplasm after infection. A number of important studies have
used class III viruses as a source of pure mRNA.
Class IV viruses contain a single plus strand of genomic
RNA, which is identical with the viral mRNA. Since the genomic RNA encodes
proteins, it is infectious by itself. During replication of class IV
viruses, the genomic RNA is copied into a minus strand, which then acts as a
template for synthesis of more plus strands, or mRNA. Two types of class IV
viruses are known. In class IVa viruses, typified by poliovirus, viral
proteins are first synthesized, from a single mRNA species, as a long
polypeptide chain, or polyprotein, which is then cleaved to
yield the various functional proteins. Class IVb viruses synthesize at least
two species of mRNA in a host cell. One of these mRNAs is the same length as
the virion’s genomic RNA; the other corresponds to the
3′ third of the genomic RNA. Both mRNAs are translated into
polyproteins. Included in class IVb are a large number of rare insect-borne
viruses including Sindbis virus and those causing yellow fever and viral
encephalitis in human beings. These viruses once were called
arboviruses (arthropod-borne viruses), but now are
called togaviruses (from Latin toga,
cover) because the virions are surrounded by a lipid envelope.
Class V viruses contain a single negative strand of genomic
RNA, whose sequence is complementary to that of the viral mRNA. The genomic
RNA in the virion acts as a template for synthesis of mRNA but does not
itself encode proteins. Two types of class V viruses can be distinguished.
The genome in class Va viruses, which include the viruses causing measles
and mumps, is a single molecule of RNA. A virus-specific RNA polymerase
present in the virion catalyzes synthesis of several mRNAs, each encoding a
single protein, from the genomic template strand. Class Vb viruses, typified
by influenza virus, have segmented genomes; each segment acts as a template
for the synthesis of a different mRNA species. In most cases, each mRNA
produced by a class Vb virus encodes a single protein; however, some mRNAs
can be read in two different frames to yield two distinct proteins. As with
class Va viruses, a class Vb virion contains a virus-specific polymerase
that catalyzes synthesis of the viral mRNA. Thus the genomic RNA (a minus
strand) in both types of class V viruses is not infectious in the absence of
the virus-specific polymerase. The influenza RNA polymerase initiates
synthesis of each mRNA by a unique mechanism. In the host-cell nucleus, the
polymerase cuts off 12 – 15 nucleotides from
the 5′ end of a cellular mRNA or mRNA precursor; this
oligonucleotide acts as a “primer” that is elongated by
the polymerase to form viral (+) mRNAs, using the genomic
(−) RNA as a template.
Class VI viruses are enveloped
viruses whose
genome consists
of two identical plus strands of RNA. These
viruses are also known as
retroviruses because their RNA
genome directs the formation
of a DNA molecule. The DNA molecule ultimately acts as the
template for
synthesis of viral mRNA (). Initially, a viral
enzyme called
reverse transcriptase copies the viral RNA
genome
into a single minus strand of DNA; the same
enzyme then catalyzes synthesis
of a
complementary plus strand. (This complex reaction is detailed in
Chapter 9.) The resulting dsDNA
is integrated into the chromosomal DNA of the infected cell. Finally, the
integrated proviral DNA is transcribed by the cell’s own machinery
into (+) RNA, which either is translated into viral
proteins or is
packaged within
virion coat
proteins to form progeny
virions, which are
released by budding from the host-cell
membrane. Because most
retroviruses
do not kill their host cells, infected cells can replicate, producing
daughter cells with integrated proviral DNA. These daughter cells continue
to transcribe the proviral DNA and bud progeny
virions.
Some retroviruses contain
cancer-causing genes (called oncogenes). Cells infected by such
retroviruses are oncogenically transformed into tumor cells. Studies of
oncogenic retroviruses (mostly viruses of birds and mice) have revealed a
great deal about the processes that lead to oncogenic transformation. Among
the known human retroviruses are human T-cell lymphotrophic virus (HTLV),
which causes a form of leukemia, and human immunodeficiency virus (HIV),
which causes acquired immune deficiency syndrome (AIDS). Both of these
viruses can infect only specific cell types, primarily certain cells of the
immune system and, in the case of HIV, some central nervous system neurons
and glial cells. Only these cells have cell-surface receptors that interact
with viral proteins, accounting for the host-cell specificity of these
viruses.
Viral Vectors Can Be Used to Introduce Specific Genes into Cells
Knowledge about mechanisms of viral replication has allowed virologists to modify
viruses for various purposes. For instance, the ability of virions to introduce
their contents into the cytoplasm and nuclei of infected cells has been adapted
for use in DNA cloning and offers possibilities in the treatment of certain
diseases. The introduction of new genes into cells by packaging them into virion
particles is called viral gene transduction, and the virions
used for this purpose are called viral vectors.
By use of recombinant DNA techniques
described in Chapter 7, it is a
relatively straightforward process to construct human adenovirus
recombinants in which potentially therapeutic genes replace
the viral genes required for the lytic cycle of infection. Because adenovirus
has a very broad host range for different types of human cells, these vectors
can introduce the engineered gene into the cells of tissues where they are
applied. If the transduced gene encodes the normal form of a protein that is
missing or defective in a particular disease, then such gene
therapy may successfully treat the disease. One type of adenovirus,
for example, efficiently infects cells lining the air passages in the lungs,
causing a type of common cold. Researchers have replaced some of the
disease-causing genes in this adenovirus with the CFTR gene,
which is defective in individuals with cystic fibrosis. This recombinant
adenovirus currently is being used to introduce a normal CFTR
gene into the airway-lining cells of cystic fibrosis patients. Unfortunately,
with most of the adenovirus vectors currently available, the transduced gene
usually is expressed only for a limited period of 2 to 3 weeks. This
significantly limits their usefulness in gene therapy.
Viral vectors have also been developed from viruses that integrate their genomes
into host-cell chromosomes. Such vectors have the advantage that progeny of the
initially infected cell also contain and express the transduced gene because it
is replicated and segregated to daughter cells along with the rest of the
chromosome into which it is integrated. Retroviral vectors, which can
efficiently integrate transduced genes at approximately random positions in
host-cell chromosomes are now widely used experimentally to generate cultured
cells expressing specific, desired proteins. However, technical limitations in
producing the large numbers of retroviral vectors required to infect a
significant fraction of cells in the tissues of a human or vertebrate currently
limit their use as gene therapy vectors. Another concern with retroviral vectors
is that their random integration might disturb the normal expression of cellular
genes encoding proteins regulating cellular replication. This type of cellular
gene deregulation occurs naturally following infection with certain
retroviruses, such as avian leukosis virus and murine leukemia viruses, leading
to development of leukemia in birds and mice, respectively.
Adeno-associated virus (AAV) is a
“satellite” parvovirus that replicates only in cells that
are co-infected with adenovirus or herpes simplex virus. When AAV infects human
cells in the absence of these “helper” viruses, its ssDNA
genome is copied into dsDNA by host-cell DNA polymerase and then is integrated
into a single region on chromosome 19, where it does not have any known
deleterious effects. Research is under way to adapt the AAV integration
mechanism that operates in the absence of helper virus to the development of a
safe and effective integrating viral vector.
SUMMARY
-
Viruses are intracellular parasites that
replicate only after infecting specific host cells. Viral infection
begins when proteins on the surface of a virion bind to specific
receptor proteins on the surface of host cells. The specificity of this
interaction determines the host range of a virus.
-
Aside from being the causative agents of
many diseases, viruses are important tools in cell biology research,
particularly in studies on macromolecular synthesis (see Table 6-3). -
Viruses can be counted and cloned by the
plaque assay (see ).
All the virions in a single plaque compose a clone derived from the
single parental virion that infected the first cell at the center of the
plaque. -
Individual viral particles (virions)
generally contain either an RNA or a DNA genome, surrounded by multiple
copies of one or a small number of coat proteins, forming the
nucleocapsid. The nucleocapsid of many animal viruses is surrounded by a
phospholipid bilayer, or envelope.
-
During lytic replication, host-cell
ribosomes and enzymes are used to express viral proteins, which then
replicate the viral genome and package it into viral coats. The multiple
progeny virions produced within a single infected cell eventually are
released, following cell lysis or gradual disintegration of the cell
(see ). Progeny
nucleocapsids of enveloped viruses are released by budding of the
host-cell membrane in which viral membrane proteins have been deposited
(see ). -
Some bacterial viruses (bacteriophages) may
undergo lysogeny following infection of host cells. In this case, the
viral genome is integrated into host-cell chromosomes, forming a
prophage that is replicated along with the host genome. When suitably
activated, a prophage enters the lytic cycle (see ). -
All retroviruses and some other animal
viruses can integrate their genomes into host-cell chromosomes (see
). In some cases,
this leads to abnormal cell replication and the eventual development of
cancers. -
Recombinant viruses can be used as vectors
to carry (transduce) selected genes into cells. In this approach, viral
genes required for the lytic cycle are replaced by other genes. The use
of viral vectors for gene therapy is still in its infancy, but has great
potential for treatment of various diseases.
ǀ
