Innate Immune System - Viruses And Human Disease

to completely eradicate the virus in these areas is due to the

as well as the adaptive system and has been described. Other

difficulty of immunizing infants with a live virus vaccine

components of the innate system include the cytokines, a

while maternal antibodies are still protective. In regions of

complicated set of small proteins that are powerful regula-

Africa, measles is so widely epidemic that many infants con-

tors of the immune system. Many cytokines are critical for

tract the disease as soon as the protection due to maternal

the function of the adaptive immune system, as described

antibodies wears off. Multiple, spaced immunizations for

in part before. Cytokines are also important components of

each infant would be required to successfully immunize the

the innate system. The innate system also includes natural

population of new susceptibles before the epidemic virus

killer cells, cytotoxic lymphocytes that kill cells that do not

gets there, which is difficult to achieve in countries with

express adequate levels of class I MHC molecules. Apoptosis

limited health care facilities. In an effort to get around this

is another innate defense mechanism, because most cells are

problem, very young infants were immunized with higher

programmed to die if they sense that they are infected.

doses of the live virus vaccine in a trial in Senegal, on the

theory that using a larger dose would overcome the effects of

Toll-Like Receptors

maternally derived immunity. This trial was a failure, how-

ever, because for unknown reasons the cumulative mortality

Initiation of an immune response requires the recognition

in infants receiving the high titer vaccine was greater than

of "pathogen-associated molecular patterns" or PAMPs for

that in controls (Fig. 10.14). Thus at present it is very diffi-

short, which are conserved molecular motifs present in or

cult to devise methods that will effectively immunize young

produced by invading pathogens but which are not present

infants in these countries against measles, but programs are

in their hosts. The sensors that recognize PAMPs are called

being developed to multiply immunize infants at different

pattern recognition receptors or PRRs. The best known

ages using national immunization days in order to overcome

PRRs are Toll-like receptors (TLRs) but other PRRs exist,

these problems.

such as the protein called RIG-1 described later.

TLRs are components of ancient pathways of innate

immunity to invading microbes. They recognize a number

INNATE IMMUNE SYSTEM

of conserved structures in microbes and upon activation

lead to the production of antimicrobial peptides or to activa-

The innate immune system is composed of a large number

tion of components of the innate and the adaptive immune

of elements that attempt to control infection by pathogens,

systems. The Toll pathway was first discovered in the fruit

but this system is independent of the identity of the particular

fly, Drosophila melanogaster. Activation of this pathway

pathogen. Complement is a component of the innate system

in Drosophila results in the production of peptides that

200

EZ-HT

150

SW-HT

Standard

100

Age (months)

FIGURE 10.14 Child mortality after high-titer measles vaccination in Senegal. Three groups of children were given

either 105.4 PFU of Edmonston-Zagreb vaccine at 5 months of age (EZ-HT), or 105.4 PFU Schwartz vaccine (SW-HT) at

5 months of age or the standard schedule (placebo at 5 months and 103.7 PFU vaccine at 10 months of age.) Adapted from

Garenne et al. (1991).

render the fly resistant to fungal infection and to infection

intracellular compartments, usually thought to be endosomal

by Gram-positive bacteria. Flies that lack this pathway are

compartments. Examples of TLRs activated by nonself mol-

very sensitive to infection by fungi or Gram-positive bacte-

ecules include TLR4, activated by exposure to lipopolysac-

ria. Resistance of flies to Gram-negative bacteria is medi-

charide (LPS), a component of the cell wall of gram-negative

ated by another pathway called the IMD pathway. Innate

bacteria, as well as by other molecules, including the enve-

defenses against microorganisms are also known to exist in

lope proteins of some viruses; TLR5, activated by exposure

starfish, nematodes, plants, and virtually all other organisms

to bacterial flagellin; and TLR2, activated by components

that have been examined, and many of these defense mecha-

from Gram-negative bacteria, mycoplasma, spirochetes, and

nisms are known to rely on Toll-like proteins.

trypanosomes, among others. TLRs activated by exposure to

Mammalian homologues of Toll have been described

nucleic acids include TLR9, activated by exposure to non-

only recently. There are at least 11 TLRs in humans that can

methylated CpG in DNA, which is rare in mammalian DNA

be grouped into five subfamilies. They are integral mem-

(CpG itself is underrepresented in mammalian DNA and it

brane proteins, some of which are expressed at the cell sur-

is usually methylated) but common in bacteria and in DNA

face and some of which are present in intracellular vesicles.

viruses; TLR3, activated by double-stranded RNA, which

They possess extracytoplasmic leucine-rich domains that are

is produced by most viruses after infection; and TLR7 and

important for recognition of the molecular stimuli and intra-

TLR8, which recognize single-stranded RNA. Overall, then,

cytoplasmic domains that signal, via various intermediates,

a wide variety of molecules produced by invading viruses,

to start production of effector proteins. A partial list of the

bacteria, fungi, and other pathogens is recognized by various

ligands that stimulate various TLRs is shown in Table 10.4.

TLRs and an immune response is generated. Of interest also

Some TLRs recognize nonself molecules as is the case for

is the fact that TLR4 responds to heat shock proteins (HSP60

TLR1, 2, 4, 5, and 6, whereas others recognize nucleic acids

and HSP70), which are produced by the host in response to

in unfamiliar contexts as in the case for TLR3, 7, 8, and 9.

stress, which can include the stress induced by infection.

In general, TLRs in the first class are expressed at the cell

TLRs that are expressed on the cell surface respond to

surface whereas TLRs of the second class are expressed in

extracellular stimuli, whether present there because of cell

TABLE 10.4

Characterization of Toll-Like Receptors

Toll-like receptor

Major cell types

Ligands

Source of ligand

TLRs found predominantly in the cell plasmalemma

TLR1/TLR2 heterodimer

Monocytes, mDCs

Tri-acyl lipopeptides

Bacteria/mycobacteria

GP-1 anchored proteins

Parasites

TLR2/TLR6 heterodimer

Monocytes, mDCs, pDCs

Di-acyl lipopeptides,

Bacteria/mycobacteria,

lipoteichoic acid,

gram-positive bacteria

Zymosan

Fungi

TLR4 (homodimer)

Monocytes, differentiated DCs

LPS

Gram-negative bacteria

Taxol

Plant

HRSV fusion protein

Paramyxoviridae

Heat shock proteins

Mammalian host

Fibrinogen

Mammalian host

Envelope proteins

MMTV (Retroviridae)

TLR 5

Monocytes

Flagellin

Motile bacteria

TLRs found predominantly in intracellular compartments

TLR3

mDCs (also surface of fibroblasts)

dsRNA

Many viruses; Reoviridae

TLR7

???

ssRNA, some siRNAs

Many viruses including: HIV (Retroviridae),

VSV (Rhabdoviridae), Influenza

TLR8

Monocytes, mDCs

ssRNA

Many viruses; Rhabdoviridae

TLR9

pDCs

CpG DNA

Bacteria

CpG DNA

DNA viruses, esp. Herpesviridae

Abbreviations: mDCs, myeloid dendritic cells; pDCs, plasmacytoid dendritic cells; HRSV, human respiratory syncytial virus; MMTV, mouse mammary

tumor virus. Interactions most important for controlling viral infection are shown in red.

Source: Data from Takeda et al. (2003); O'Neill (2005); Iwasaki and Medzhitov (2004); Kawai and Akira (2006).

death that releases these components or because extracellu-

have a net positive charge, and are produced by cleavage

of propeptides. They fall into three subfamilies called α-,

lar microorganisms are present. TLRs in intracellular com-

β-, and θ-defensins. α- and β-defensins are linear polypep-

partments may also be responding to extracellular molecules

tides whereas θ-defensins are cyclic 18mers. Only α- and

that enter the endosomal compartment by endocytosis, or

β-defensins are produced in humans. They are mainly pro-

they may be intracellular molecules that enter the intracel-

lular compartments where TLRs are present during infec-

duced by leukocytes and epithelial cells.

α- and β-defensins have diverged from a common

tion or replication. Many (most?) TLRs function as dimers.

β-defensin ancestral gene that is expressed as far back in

Heterodimers of TLR1 and TLR2 and of TLR6 and TLR2

have been found, and these two heterodimers have different

evolution as snakes. There are multiple genes for them. Six

human α-defensins have been identified, and gene-based

specificities. TLR4 is commonly found as a homodimer.

searches indicate that there are about 30 β-defensin genes

Various TLRs are expressed in most tissues of the body.

Particularly noteworthy is the expression of multiple TLRs

in humans and more than this in mice. These polypeptides

by monocytes, macrophages, dendritic cells, and mast cells,

will kill a number of microbes in vitro, and model studies in

key players in the immune system. Activation of TLRs

mice have shown that they are important in protecting mice

leads to activation of transcription factors such as NFκB

from bacteria that infect the lungs or the gut. Production of

α-defensins is constituitive for the most part, whereas pro-

that results in the production of a number of end products.

duction of β-defensins is usually inducible. Induction can be

In some cases, antimicrobial peptides are produced, includ-

ing in humans various defensins described later. Of equal

through the activation of Toll-like receptors or through the

or greater importance, however, is the expression of proin-

activity of cytokines. Defensins appear to work by permeabi-

flammatory genes such as TNF-α, IL-6, IL-1β, and IL-12 in

lizing the microbial membrane, but they also appear to recruit

response to activation of several TLRs. Of particular impor-

other elements of the immune system to the site of infection.

tance, type I interferons are produced in response to activa-

They may have evolved to fight off bacteria, fungi, and proto-

tion of TLR3, 4, 7, 8, and 9. The inflammatory response that

zoa, but studies have shown that they are also effective against

results from the activation of TLRs is crucial for the activa-

at least some viruses. Their antiviral activity is of two types.

tion of other components of the innate system as well as for

They can directly inactivate some enveloped viruses, probably

the activation of the adaptive immune system. In particular,

in the same way that they inactivate bacteria. They can also

type I IFNs are key players in starting and organizing an

interact with potential target cells to interfere with virus rep-

immune response. TLR-signaling also leads to the matura-

lication by mechanisms that are poorly understood, and these

tion of dendritic cells, key players in the adaptive immune

mechanisms are potentially effective against both enveloped

and nonenveloped viruses. It is noteworthy that both α- and

system. Activation of dendritic cells may be direct, by infec-

β-defensins have been found in breast milk, suggesting that

tion of these cells by viruses, for example, or indirect, as

the result of signaling by other infected cells. There appear

they are important for protecting the infant from infection.

to be multiple kinds of dendritic cells that express different

combinations of TLRs. Upon activation of a dendritic cell, it

Natural Killer Cells

migrates to lymph nodes where it presents antigens to T cells

and stimulates T cells that recognize the antigen presented by

Natural killer (NK) cells are cytolytic cells that kill by an

it to become active. Thus, the maturation of dendritic cells

antigen-independent mechanism. They are important for the

induced by activation of TLRs is critical for the activation of

control of many virus infections, as shown by the fact that

the adaptive immune response. Overall, therefore, TLRs are

ablation of NK cells leads to more serious disease. NK cell

not only important as the first line of defense against invad-

activity increases within the first 23 days after infection by

ing microorganisms via the production of antimicrobial pep-

a virus, stimulated by the presence of interferon and per-

tides or the induction of cytokine expression that slow their

haps by other cytokines, and thereafter the number of cells

growth and in some cases eradicate the infection, but also

declines. One of the functions of these cells is to kill cells

for activation of an adaptive immune response to eliminate

that do not express class I MHC or that express it in only

the invading pathogen and cause the organism to become

low amounts. Such cells, of course, do not present antigen

immune.

to CTLs and therefore escape normal immune surveillance.

NK cells express two sets of receptors on their surface. One

set of receptors interacts with MHC class I molecules on the

Defensins

surface of the target cell. This interaction inhibits killing by

Organisms from plants to mammals produce antimicrobial

the NK cell. The second set of NK receptors interacts with

peptides that are important in defending the organism from

activating molecules on the surface of cells. Interaction with

infection. Mammals produce two classes of such peptides,

activating molecules will stimulate the NK cell to kill the

called cathelicidins and defensins. Defensins are peptides

target cell if the NK cell is not sufficiently inhibited by its

of 18 to 45 amino acids that possess three disulfide bonds,

interaction with class I molecules.

Many different viruses downregulate the production of

cells that are no longer needed, cells that are damaged in

MHC in infected cells in order to escape immune surveil-

some way (such as by exposure to UV light or to free radi-

lance. The elimination of these infected cells by NK cells is

cals), or cells that are dangerous to the host because they are

of considerable importance to the host. It seems clear that

infected or because their cell cycle is deregulated (and they

NK cells evolved to rid the body of cells that are not subject

are therefore potential tumor cells). Cells that are no longer

to normal immune surveillance, which could include tumor

needed include excess cells that are produced during devel-

cells as well as virus-infected cells that no longer express

opment and must be eliminated (a normal occurrence during

adequate levels of MHC. In the one case known of a human

development) and T cells responsible for the control of an

with a deficiency in NK cells, as well as in experimental stud-

infectious agent after the infection has been eliminated (and

ies with mice in which NK cells are depleted, infection by

which may even be dangerous if left around). Thus, apopto-

a number of viruses that downregulate production of MHC

sis serves many roles in an organism and the basic machin-

results in a much more severe disease than that produced in

ery has been conserved throughout evolution. In mammals it

individuals able to mount a normal NK response.

serves as a component of both the adaptive immune response

(ridding the body of unneeded lymphocytes and killing of

infected cells by CTLs) and the innate immune response

Apoptosis

(suicide by infected cells).

Apoptosis or programmed cell death is a defense of last

Several different mechanisms for the induction of apop-

resort by an infected cell. It is a cell suicide pathway in which

tosis exist, as would be expected from the many functions

mitochondria cease to function, chromatin in the nucleus

served by apoptosis. Furthermore, the control of apoptosis is

condenses, nuclear DNA is degraded, and the cell fragments

very complicated, which is necessary to control an event as

into smaller membrane-bound vesicles that are phagocy-

drastic as cell death. Some of the pathways used to induce

tosed by neighboring cells. Apoptosis serves to eliminate

apoptosis are illustrated in Fig. 10.15. One pathway involves

Killling via Receptor

Killing due to External Stimuli

Killing by CTL

UV irradiation

via the Granzyme B Pathway

Unscheduled

Hypoxia

DNA synthesis

E2F

Viral infection

Ligand

FASL

p53

Mdm2

CTL

Receptor

Fas

p53

Perforin Channel

Bax

Granzyme

Cytoplasmic

death domains

FADD

Bcl-2

Death effector

domains

Procaspase

Activation

Procaspase

cleavages

Caspase cascade

Effector caspases

Induction

Apoptosis

Inhibition

FIGURE 10.15 Three pathways that lead to cell death by apoptosis. In each case a signal comes from outside the cell.

This signal may be the binding of a ligand to its receptor (left panel); stress upon the cell caused by viral infection, DNA

damage caused by exposure to UV light, deregulation of the cell cycle, or other stresses (center panel); or from a cytotoxic

T cell (right panel). The signal leads to the activation of a procaspase by proteolytic cleavage. The activated caspase

begins a cascade that leads to apoptosis. Data for this figure came from Ashkenazi and Dixit (1998) and Raff (1998).

death receptors on the cell surface, which initiate apoptotic

anti-apoptotic protein is called Bcl-2, which blocks the action

pathways when their ligands are present and bind to them.

of Bax and interferes with the caspase cascade, among other

One such receptor is the Fas receptor (also known as CD95

activities. Bcl-2 is made by mature neurons, for example,

or Apo-1), which can be stimulated by Fas ligand present

in response to signals received when they form connections

on the surface of CTLs. Fas contains domains known as

with the target cells that they enervate. Immature neurons

death domains in the cytoplasmic region, and trimerization

do not make adequate amounts of Bcl-2 and die when nerve

of Fas induced by ligand binding results in the recruitment of

growth factor is removed. There is a complicated interplay

other cellular proteins by these death domains. This in turn

between Bcl-2 and related proteins and between Bax and

results in the activation, by cleavage, of a protease known as

related proteins that we are only beginning to understand.

caspase-8. A popular model for activation is that two mol-

Mitochondria are also important players in apoptosis.

ecules of caspase-8 must be brought into proximity so that

A decrease in the potential across the mitochondrial mem-

they cleave one another. Caspase-8 then cleaves other cas-

brane makes the cell more sensitive to apoptosis. Decreased

pases, thereby activating them in turn. At least 10 caspases

potential is accompanied by synthesis of reactive oxygen

are known, and they are normally present as inactive pro-

species and the release of cytochrome C into the cytoplasm,

caspases. A cascade of caspase cleavages leads, ultimately,

both of which are proapoptotic. Cytochrome C forms com-

to the activation of effector caspases. The effector caspases

plexes with other cellular proteins that promote the caspase

cause cell death by cleaving many cellular proteins, includ-

cascade. Bax, a proapoptotic protein, may exert its effect by

ing, for example, lamin in the nuclear membrane and a repair

lowering the mitochondrial membrane potential, and Bcl-2

enzyme called poly(ADP-ribose) polymerase.

may prevent this.

Another death receptor that may be present at the cell sur-

The importance of apoptosis for the regulation of the

face is a receptor for TNF (tumor necrosis factor). TNF can

organism is clear. Individuals with deficits in apoptotic

be secreted by CTLs, as well as by other lymphocytes, in

pathways may suffer from many diseases, including devel-

response to inflammatory signals. TNF receptor oligomeri-

opmental abnormalities, neurodegenerative disorders, or

zation also results in cleavage of caspase-8 and the induction

autoimmune diseases. In normal individuals, tumor cells

of the caspase cascade. The sensitivity of cells to the induc-

can only thrive if they avoid apoptosis despite the fact that

tion of apoptosis by these pathways thus depends on the con-

their cell cycle is deregulated, and mutations that suppress

centration of Fas and TNF receptor in the surface, and this

apoptosis are important for the development of tumors.

concentration can be regulated by other events.

Fas and TNF receptors are ubiquitous, present on most

Interferons and Other Cytokines

cells. Many other receptors that can lead to apoptosis are

also known. Some of these are also ubiquitous, others are

The cytokines constitute a large family of proteins, defined

present on only a limited set of cells.

by their structure, that have important regulatory roles in an

The caspase cascade can also be initiated by other mecha-

animal. More than 30 cytokines have been described, and for

nisms. Withdrawal of growth factors, such as occurs during

some of these there is more than one gene. Most have a molecu-

development or occurs when activated T cells are no longer

lar mass of about 30 kDa. Many, but not all, are glycoproteins.

stimulated by interaction with their antigens, can lead to acti-

Some function as monomers but others act as homodimers or

vation of caspases. Events that damage DNA or the protein

homotrimers and at least one acts as a heterodimer. Cytokines

synthetic machinery can lead to apoptosis. At least some of

are inducible agents and constitutive production is normally

these apoptotic signals are delivered through changes in p53

low or absent. On induction, their production is short lived.

concentrations. p53 is a key player in the regulation of the

Cytokines effect their action by binding to receptors on the sur-

cell cycle (Chapter 7), and the apoptotic pathway is induced

face of target cells. These receptors bind cytokines with high

affinity--the dissociation constants range from 10-9 to 10-12

if its concentration is not tightly regulated. These mecha-

nisms involve Bax and related molecules, as well as Bcl-2

M. Binding to the receptor induces a cascade of events in the

and related molecules, as key players in the activation of the

target cell that leads to changes in gene expression within the

caspase cascade.

cell. These changes in gene expression may lead to many dif-

In addition to being able to initiate the caspase cascade

ferent effects. They are important in orchestrating the immune

indirectly by signaling through the Fas receptor or the

response by inducing cell proliferation in cells like T cells

TNF receptor, T cells can also initiate the caspase cascade

and B cells and causing changes in the state of differentiation

directly. They do this by introducing granzymes into the cell

of cells like dendritic cells. Hematopoietic cells are impor-

by way of channels formed by perforin secreted by the T

tant targets of all cytokines, although most cytokines have a

cell. Granzyme B cleaves procaspases downstream of cas-

diverse range of actions. The cytokine system is redundant in

pase-8 to begin the cascade.

that many cytokines evoke a similar spectrum of action, and it

Control of apoptosis is complex. Many cells make pro-

is pleiotrophic in that one cytokine may have many different

teins that render them less susceptible to apoptosis. One such

target cells. This system is also complex because many dif-

ferent cytokines are usually induced at the same time and

hematopoietic system (e.g., maintenance of lymphoid

they may act synergistically or antagonistically to achieve a

organs), whereas the proinflammatory chemokines recruit

result. Cytokines are important players in both the adaptive

immune cells to sites of infection, inflammation, or tissue

and innate immune systems. The importance of cytokines in

damage. The receptors for chemokines are distinct from

the maturation of T cells and B cells has been described. Fig.

those of cytokines. An example of a cytokine receptor is

10.16 presents an overview of cytokine networks that illus-

described later in this chapter for interferon. Chemokine

trates their complexity, and a partial listing of cytokines is

receptors belong to the family of seven-transmembrane-

given in Table 10.5. Because of the complexity of their activi-

domain, G-protein-coupled receptors, and one was illus-

ties, we are only beginning to understand their many func-

trated schematically in Fig. 1.4B as the receptor for HIV.

tions, but as these functions begin to be understood, attempts

In some treatments, chemokines are considered a class of

are being made to use different cytokines therapeutically as

cytokines. Here, because these two classes of molecules dif-

indicated in the table.

fer in structure and mechanism of action, the term cytokine

A second family of proteins that play an important role

refers only to nonchemokine cytokines.

in the regulation of the immune response is the chemokines.

Chemokines are small proteins, 7080 amino acids in size.

Importance of Interferons in the Defense

More than 30 are known in humans. Some serve house-

against Viruses

keeping functions and are produced constitutively; others

are proinflammatory and usually inducible. Chemokines

Among the best known cytokines that function in the

serve to attract leukocytes. The housekeeping chemokines

defense against viruses are the interferons (IFNs). There

are important for the development and homeostasis of the

are two kinds of IFN, called type I and type II. The IFNs

Endothelial cell

MCP-1

IL-1b TNF-a

IL-6

CD8+T cell

CD4+T cell

IFN-a /b

IL-15

IL-18

Macrophage dendritic cell

Th1

Th2

B-cell

IL-12

Cytotoxic

T Lymphocyte

(CTL)

Natural

Killer

IFN-g

cell

LT-a

(NK)

IFN-g

IL-4

IL-5

IL-2

IL-10

LT-a

IgM

IL-13

IFN-g

Cell-mediated response to viral infection

Plasma Cell

IgG

IgA

Developmental

Activation

pathway

IgE

Secretion

Inhibition

Humoral response to viral infection

FIGURE 10.16 Overview of the cytokine networks important for innate and acquired antiviral immune responses.

IFN, interferon; IL, interleukin; Ig, immunoglobulin; TNF, tumor necrosis factor; LT, lymphotoxin; MCP, monocyte

chemotactic protein. Adapted from Griffin (1999) Figure 1 on p. 340.

TABLE 10.5

Some Cytokines and Their Therapeutic Uses

Functional

Name

Therapeutic

Side effects

group

(abbreviation)

Normal biological function

targets

of therapy

Antiviral

Type I interferon

Inhibits viral replication

Chronic hepatitis B,

Fever, malaise, fatigue,

(IFN-α,β)

Cytokines

hepatitis C, herpes

muscle pain; toxic to

zoster, papilloma

kidney, liver, heart,

viruses, rhinovirus,

bone marrow

HIV(?), warts

Type II interferon

Inhibits viral replication,

Lepromatous leprosy,

As above for Type I

(IFN-γ)

upregulates expression of class

leishmaniasis,

interferons

I and class II MHC, enhances

toxoplasmosis

activity of macrophages

Inflammatory

Tumor necrosis factor

Cytotoxic for tumor cells,

Anti-TNF in septic

Shock with marked

Cytokines

(TNF)

induces cytokine secretion

shock

hypotension

by inflammatory cells

Interleukin 1 (IL-1)

Costimulates T-helper cells,

Receptor antagonist in

???

promotes maturation of B cells,

septic shock

enhances activity of NK cells,

attracts macrophages and neutrophils

Interleukin 6 (IL-6)

Promotes differentiation of B cells,

stimulates Ab secretion by plasma cells

Regulators of

Interleukin 2(IL-2)

Induces proliferation of T cells, B cells,

Leprosy, local treatment

Vascular leak syndrome,

lymphocyte

and CTLs, stimulates NK cells

of skin lesions

hypotension, edema,

functions

ascites, renal failure,

hepatic failure, mental

changes, and coma

Interleukin 4 (IL-4)

Stimulates activity of B cells, and

proliferation of activated B cells,

induces class switch to IgG and IgE

Interleukin 5 (IL-5)

Stimulates activity of B cells, and

proliferation of activated B cells,

induces class switch to IgA

Interleukin 7 (IL-7)

Induces differentiation of stem cells,

increases IL-2 in resting cells

Interleukin 9 (IL-9)

Mitogenic activity

Interleukin 10 (IL-10)

Suppresses cytokines in macrophages

Septic shock

Interleukin 12 (IL-12)

Induces differentiation of T cells into CTLs

Interleukin 13 (IL-13)

Regulates inflammatory response in macrophages

Transforming growth

Chemotactically attracts macrophages, limits

Septic shock

Symptoms similar to

factor (TGF-β)

inflammatory response, promotes wound

those for IL-2,

healing

especially shock

and hypotension

Source: Data for this table came from Mims et al. (1993) p. 37.3, and Figure 7.13, and Kuby (1997) pp. 318, 319.

induce cells to become resistant to viral infection, an innate

extremely sensitive to infection by viruses. Viruses grow to

defense against viruses, but also play important roles in the

much higher titer in such animals and, in the more dramatic

adaptive immune response. The importance of IFNs in the

examples, virus infection may be lethal although infection

defense of mammals against viral infection has been shown

by the same virus in animals able to mount a normal IFN

by experiments in which an IFN response is ablated. Early

response may be asymptomatic. Many viruses have evolved

experiments in mice used injection of antibodies against

mechanisms to ablate the activity of IFNs, and in many cases

IFN to block its activity. More stringent ablation of IFN

it has been shown that viral mutants that have lost the ability

activity has been accomplished by using transgenic mice in

to resist IFN are severely crippled, again demonstrating their

which the receptor for either type I or type II IFN has been

importance in controlling viral infection. Interestingly, mice

abolished. In general, mice that lack a type I IFN response are

lacking a type I IFN response are able to handle bacterial

infections reasonably well. Conversely, mice lacking a type

Induction of IFN

II IFN response are extremely sensitive to bacterial infec-

Type I IFNs are induced upon the activation of a

tion but handle viral infections well. One known exception

number of PRRs, as described earlier. Induction may be

is vaccinia virus, which is lethal in mice lacking either IFN

direct (e.g., upon activation of TLR3, 4, 7, 8, 9 or RIG-1)

response. Although the relative importance of the two IFNs

or the result of production of other cytokines (e.g., TNF-α).

in the defense against viruses versus bacteria may differ,

The best studied activating agent that results in type

both are important in the defense against viruses.

I IFN production, and perhaps the most important agent,

is double-stranded RNA (dsRNA), whether infection is

by a DNA- or RNA-containing virus. Two dsRNA sen-

Types of IFNs

sors are known whose activation results in production of

Several characteristics of type I and type II IFNs are shown in

type I IFN, illustrated schematically in Fig. 10.17. One

Table 10.6. The type I IFNs are 165172 amino acids in length

sensor is TLR3 and the second sensor is a protein called

and have been classified into four different subfamilies, called

RIG-1. TLR3 is present in intracellular compartments

α, β, ω, and τ, of which α and β are the best studied. In humans

in many cells but present at the cell surface of fibrob-

there are 14 IFN-α genes and 1 IFN-β gene, none of which

lasts, where it senses external dsRNA. As noted earlier,

contain introns. IFN-β shares 2530% amino acid sequence

it is possible that intracellular TLR3 also senses exter-

identity with any particular IFN-α. In humans, IFN-β is glyco-

nal dsRNA that has been endocytosed or it may sense

sylated whereas IFN-α is not, but in mice both are glycosylated.

replicating RNA that enters the endosomal compart-

Thus, glycosylation is not a fundamental property distinguish-

ment. In any event, upon binding dsRNA, TLR3 sig-

ing α from β IFNs. IFN-α and IFN-β use the same receptors

nals through a protein called TRIF. In contrast, RIG-1,

and therefore evoke the same responses in target cells.

which has an RNA helicase domain that binds dsRNA,

Type II IFN contains only one member, IFN-γ. In humans

is present in the cytosol and therefore detects cytoplas-

there is one IFN-γ gene, which contains three introns. The

mic dsRNA. Upon binding dsRNA, RIG-1 interacts with

receptor used by IFN-γ is distinct from that used by type I

a protein called MAVS (it also has other names),which is

IFNs, but the phosphorylation cascade induced by IFN-γ con-

attached to the mitochondrial membrane by means of a

tains some elements that are shared with the type I cascade.

C-terminal transmembrane anchor. In either case, signal-

Thus the responses to the two IFNs are partially overlapping.

ing proceeds through a number of intermediate steps with

TABLE 10.6

Characteristics of the Interferons

Type I

Type II

INF-α

INF-β

IFN-γ

Characteristics

Alternative name

Leucocyte IFN

Fibroblast IFN

Immune IFN

Location

All cells

T-lymphocytes

Inducing agent

Viral infection or dsRNA

Antigen or mitogen

Number of species

14 (man),

(number of genes)

22 (mouse)

Chromosomal location of gene

9 (man),

12 (man),

4 (mouse)

10 (mouse)

Number of introns

None

Three

Size of IFN protein

165166 aa

166 aa

146 aa, dimerizes

Receptor for both IFN-α and INF-β consists of 2 polypeptides: IFN-α

Receptors

Receptor consists of two proteins: IFN-

R1 and INF-α R2, encoded on chromosome 21 (man) or 16 (mouse)

γR1 encoded on chromosome 6 (man)

or 10 (mouse) and IFN-γR2 encoded on

chromosome 21 (man) or 16 (mouse)

General functions

Anti-viral activity

Macrophage activation

↑ MHC class Ia

↑ MHC class I

↑ MHC class II on macrophages

NK cell activation, some antiviral activity

↓ MHC class II on B cells

↓ IgE, IgG production by B cells

↓ = downregulate; ↑ = upregulate.

Source: Data for this table came from Mims et al. (1993) Figure 12.9 and Fields et al. (1996) Table 3 on p. 378.

ds RNA

TLR3

TRIF

ds RNA

RIG-1 MAVS

TBK1

NFk B

IKKε

TBK1

IkB

Normal

IKKe

Interferon b

IRF3

Phosphorylation

Induction

Homodimerization

Phosphorylation

IRF3

Pathway

Degradation

Phosphorylation

IkB

Homodimerization

NF-kB

IRF3

IFN-b

NF-kB

IRF3

NUCLEUS

Kinase activation

Phosphorylation

Translocation to nucleus

TBK1

Kinases

IKKe

FIGURE 10.17 The induction of interferon β. External double-stranded RNA signals through Toll-like receptor 3

(TLR3) to TRIF, and internal dsRNA signals to MAVS through the retinoic-acid inducible gene I (RIG-I) protein. MAVS

is the mitochondrial antiviral signaling protein. MAVS and TRIF both can activate the kinases TBK1 and IKKε, leading

to the phosphorylation and homodimerization of IRF3, and the phosphorylation and degradation of IκB, and enabling the

translocation of IRF3 and NFκB to the nucleus. In the nucleus activated IRF3 cooperates with NFκB to induce IFN-β

transcription. Adapted from Figure 1 in Freundt and Lenardo (2005).

the final result that transcription factors NFκB and IRF3

cell by any virus. However, some viruses are much more

are activated (Fig. 10.17). These then translocate to the

efficient at the induction of IFN than others, and it has been

nucleus and initiate transcription of a type I IFN gene.

postulated that viruses may differ in the extent of production

Transcription of type I IFNs is regulated at both the tran-

of dsRNA, or perhaps in the production of products that acti-

scriptional and posttranscriptional levels. Regulation at the

vate TLRs other than TLR3.

In contrast to type I IFNs, production of IFN-γ is largely

transcriptional level is complex and incompletely under-

stood. At the posttranscriptional level, IFN mRNAs contain

restricted to T cells and NK cells. Production is induced by

destabilization sequences in the 3′ nontranslated region and

agents that promote T-cell activation. These include exposure

have short half-lives. Thus, shutoff of IFN synthesis occurs

to antigens to which the organism has been presensitized or

fairly quickly once the gene is no longer transcribed.

exposure to IL-12, a cytokine produced by monocytes and

Almost all cell types produce IFN-α or -β, and thus

macrophages after infection by bacteria or protozoa. Other acti-

vators of T cells are also known that induce synthesis of IFN-γ.

induction of these cytokines follows infection of almost any

Many other cytokines are also induced following infec-

membrane protein that contains a cytoplasmic domain. The

tion by viruses. These include TNF-α, TNF-β, IL-1, IL-2,

type I IFN receptor is present in low abundance (around 103

IL-4, IL-5, IL-6, IL-8, and GM-CSF (granulocyte-macro-

receptors per cell) on all major types of cells. It has a very

high affinity for IFN (dissociation constant about 10-10 M).

phage colony-stimulating factor). The spectrum of cytokines

induced is different for different viruses. Some of the mech-

Upon binding of type I or type II IFNs to their cognate

anisms by which many viruses interfere with IFN or other

receptors, tyrosine kinases associated with the cytoplasmic

cytokines are described later.

domains are activated. In the case of type I IFNs, the kinases

activated are called TYK2 and JAK1. Upon activation, they

phosphorylate transcription factors STAT1 and STAT2 which

Interferon Receptors and Signal Transduction

then heterodimerize, recruit IRF-9 (p48), and translocate to

IFNs effect their responses by binding to specific receptors

the nucleus, where they stimulate the transcription of a large

on the cell surface. These receptors are composed of more

number of genes (Fig. 10.18). Most genes that are induced by

than one polypeptide chain, at least one of which is an integral

type I IFN have an upstream element referred to as ISRE, the

INF-g

INF-a

INF-g

INF-a

JAK-2

TYK-2

JAK1

Recruitment

STAT1

STAT2

Phosphorylation

Dimerization

p48

ISRE

GAS

NUCLEUS

Interaction leading to phosphorylation

Phosphorylation

Protein-protein interactions

Migration to nucleus

FIGURE 10.18 Overlapping signal transduction pathways used for gene induction by IFN-α and IFN-γ, which bind

to different cellular receptors. Binding to their respective receptors leads to tyrosine phosphorylation of JAK1 and either

TYK2 or JAK2. These in turn phosphorylate STAT1 and STAT 2 proteins. The phosphorylated STAT proteins dimerize,

migrate to the nucleus, where they form complexes and bind to ISRE (interferon stimulated response element) or GAS

(IFN-gamma activation site), which are present upstream of interferon inducible genes, resulting in transcription of these

genes. Drawn from data in Fields et al. (1996) p. 379, Kalvakolanu (1999), and Nathanson et al. (1996) p. 123.

interferon stimulated response element. Induction of genes

effect is to stimulate the adaptive immune response. Both

controlled by this element is very rapid and happens within

type I and type II IFNs induce increased production of class

minutes after treatment with IFN. Activation is transient, as

I MHC molecules, thus leading to enhanced surveillance

one or more of the proteins induced act in a feedback loop to

by CTLs. Type II IFN also leads to increased production of

repress the continued transcription of these genes. In all, liter-

class II MHC in macrophages, which are important players

ally hundreds of genes are affected by IFN. The production

in the humoral response. The MHC response is augmented

of many genes is stimulated by IFN induction, but, in con-

by the induction of genes in the MHC cluster that encode

trast, many genes are instead repressed upon IFN induction.

components of the proteasome. These cause the proteasome

A few of the genes induced are listed in Table 10.7.

to become more active in producing peptides suitable for

In the case of type II IFN, the tyrosine kinases acti-

presentation by MHC molecules. Production of the TAP

vated are JAK1 and JAK 2. STAT1 is phosphorylated,

transporter system is upregulated, so that increased quanti-

homodimerizes, and translocates to the nucleus. The genes

ties of peptides are transferred across the ER membrane for

that are induced by IFN-γ are under the control of several

binding by MHC molecules. Either type of IFN leads to the

different regulatory elements, one of which is referred to as

activation of monocytes and macrophages, the activation of

GAS, the IFN-gamma activation site. Induction by IFN-γ is

natural killer cells, the activation of CTLs, and the modula-

slower and gene transcription continues for a longer period

tion of the synthesis of Ig by B cells, all important for the

immune response. IFN-γ also inhibits the growth of nonviral

of time after induction. The complex of genes induced by

type I and type II IFNs are overlapping because the tyrosine

intracellular pathogens and induces the increased expression

kinases associated with the type I and type II receptors and

of Fc receptors in monocytes. Fc receptors bind to a con-

the transcription factors that are phosphorylated are overlap-

served domain present in the heavy chain of all antibodies.

ping (Fig. 10.18). In addition, many genes contain cis-acting

This conserved domain is not involved in antibody binding

regulatory elements that respond to both type I and type II

but serves to interact with cells expressing Fc receptors and

IFN-induced transcriptional activators. A partial list of genes

to bind the C1q component of complement. Thus, antibodies

induced by IFN-γ is also given in Table 10.7.

bound to an infected cell or to a pathogen can recruit cells

such as monocytes or recruit complement to kill the patho-

gen or virus infected cell.

Biological Effects of Interferons

Both type I and type II IFNs are also key players in the

Type I or type II IFN induces the expression of many

innate immune defense against viruses. They are pyrogenic,

different genes that have important biological effects. One

inducing fever. High temperatures inhibit the replication of

TABLE 10.7 Genes Induced by Interferons

Induced bya

IFN-α

IFN-β

IFN-γ

Protein

Inducible element

Functions/phenotype

(2′-5′) (An) synthetaseb

(2′-5′) (An) synthesis/induction of antiviral state,

+++

ISRE

esp. antipicornavirus

p68 Kinase (PKR)

+++

ISRE

Protein kinase/induction of antiviral state

Indoleamine 2,3-dioxygenase

+++

Tryptophan degradation

γ56

+++

Trp-tRNA synthetase

GBP/g57

+++

Guanylate binding

MxA

+++

Inhibits replication of influenza and VSV

IRF1/ISGF2

Transcription factor

IRF2

Transcription factor

MHC class I

+++

Upregulation of antigen presentation

MHC class II

Not ISRE nor GAS

Upregulation of antigen presentation

RING 12

+++

Proteosome subunit

RING 4

+++

Putative TAP

β2 microglobulin

+++

MHC light chain

The strength of the induction is indicated by the number of plus signs.

Full induction also requires dsRNA.

Source: Adapted from Nathanson et al. (1996) p. 124.

many viruses or other pathogens. It has been proposed that

require that dsRNA be present for them to be active. Thus,

the major symptoms of influenza infection, which include

not only is dsRNA a primary inducer of IFN synthesis, but

high fever as well as muscle aches and pains, are due to the

these two pathways induced by IFN are also dependent on

induction of IFN rather than to virus replication per se. Both

dsRNA for their activation. This dependence on dsRNA sug-

IFNs also induce what has been called the antiviral state,

gests that it is a common product in viral infection, whether

described next, in which cells are less susceptible to or

the virus is DNA or RNA, but is not normally present in

resistant to infection by viruses.

uninfected cells.

One inhibition pathway involves 2′-5′ oligo(A) syn-

Thus, the induction of IFNs is important for the control of

thetases, 2′-5′ OS. These synthetases, of which several are

viral infection at more than one level. Induction of the anti-

viral state results in lower yield of virus and the stimulation

known, some of which have different subcellular locations,

of immune responses leads to a more effective and faster

polymerize ATP into oligoadenylates that are joined in a

2′-5′ linkage. 2′-5′ Oligo(A) synthetase is induced by IFN but

clearance of the viral infection. Of interest is the fact that

requires dsRNA as a cofactor for activity. 2′-5′ Oligo(A), in

both types of IFNs inhibit cell growth, and treatment with

IFN or with inducers of IFN has been effective in the treat-

turn, is a cofactor for a latent ribonuclease, RNase L, which

ment of at least some types of cancer.

is present in all animal cells, but is inactive in the absence

of 2′-5′ oligo(A). Once activated by its cofactor, which is

bound tightly enough so that the two can be coimmunopre-

The Antiviral State

cipitated, RNase L can cleave single-strand mRNAs. 2′-5′

Expression of the genes induced by interferon results

Oligo(A) is hydrolyzed by a phosphodiesterase present in

in the establishment of the antiviral state in cells, in which

cells, and activation of RNase L is transient.

viruses fail to replicate or replicate to much lower titers.

Thus, RNase L results in a degradation of mRNAs only

The antiviral state is multifaceted and interference with the

in those cells in which dsRNA is present. The picornaviruses

replication of viruses may occur at different stages of their

are particularly sensitive to the action of RNase L, and it has

replication cycle. The effect, therefore, depends on both the

been well established that activation of RNase L is a primary

virus and the host cell. Furthermore, interference can occur

mechanism by which IFN interferes with the replication

at more than one stage of replication for some viruses, and

of these viruses. Other viruses seem to be less affected by

such viruses tend to be more sensitive to the activities of

RNase L, and the possible role of this enzyme in combating

interferon than others.

other virus infections remains to be determined.

Interference with the virus replication cycle may occur

The second pathway leading to the inhibition of trans-

very early, during penetration and uncoating of the virus.

lation of viral mRNAs involves a protein kinase known as

This occurs with SV40 and the retroviruses, for example.

PKR. PKR is present at low levels in most cells, but its con-

The proteins that are responsible for interference at these

centration increases on IFN induction. It requires dsRNA for

early stages of infection are unknown. For some viruses

activity. On binding dsRNA, PKR is autophosphorylated on

the transcription of the infecting viral genome is inhibited,

serine and threonine residues. Once activated, it phosphor-

examples being influenza, vesicular stomatitis virus, and

ylates the translation initiation factor, eIF-2. Phosphorylated

herpes simplex virus. A host protein known as Mx, which

eIF-2 cannot be recycled and protein synthesis is shut down.

is induced by interferon, is responsible, at least in part, for

The importance of PKR for inhibiting viral replication is

interfering with the transcription of influenza virus. In mice

shown by the fact that several viruses encode inhibitors of

that are unable to produce the Mx protein, as is the case for

its activity (see later). In particular, it is believed that reo-

most inbred strains of mice, infection with influenza pro-

viruses, adenoviruses, vaccinia virus, vesicular stomatitis

duces a fatal outcome, demonstrating the importance of this

virus, and influenza virus, among others, are sensitive to the

gene. At a later stage in the infection cycle, interferon treat-

effects of this enzyme.

ment results in reduced translation of many viral mRNAs,

PKR binds dsRNA and thus is another sensor for dsRNA

and the mechanisms by which this occurs are described next.

within the cytoplasm of the cell. Whether it has any activity

Finally, some interferon-induced products interfere with

upon activation by dsRNA other than shutdown of protein

virus assembly. This occurs with the retroviruses, vesicular

synthesis is not clear, but PKR is known to have important

stomatitis virus, and herpes simplex virus, but the proteins

roles in the regulation of cellular processes in addition to its

responsible are unknown.

antiviral activities. For example, it has a role in regulating

several signal transduction cascades. In association with other

factors, PKR can activate NFκB, which is proinflammatory.

Interference with Translation of Viral mRNAs

It also has a role in the activation of certain MAP kinases and

Two distinct pathways induced by IFN result in interfer-

a stress-related protein kinase. It is involved in the regulation

ence with the translation of viral mRNA. These are illus-

of the cell cycle and if PKR is nonfunctional, unregulated cell

trated schematically in Fig. 10.19. Both of these pathways

proliferation and neoplastic transformation occur.

A. Development of the antiviral state

Induction by IFN

Latent RNase L

Latent 2'-5' OS

Latent PKR

dsRNA

ACTIVATION

PKR

2'-5' OS

AUTOPHOSPHORYLATION

RNase L

SYNTHESIS

2'-5'

oligo(A)

EIF2

PHOSPHORYLATION

EIF2

NUCLEUS

UNINFECTED HOST CELL

B. Virus attempts to replicate in a cell in the antiviral state

EIF2

TRANSLATION INITIATION BLOCKED

Uncoating

Viral

mRNA

RNaseL

VIRAL mRNA DEGRADED

NUCLEUS

NO VIRAL REPLICATION

Phosphorylated EIF2

EIF2

Ribosome

dsRNA

Phosphate group

Activated RNase L

FIGURE 10.19 The antiviral state. (A) Development of the antiviral state begins with the action of interferon on an

uninfected cell. The result of the signal transduction cascade shown in Figure 10.18 is the induction of expression of up to

100 genes, of which three are shown: RNase L, the 2′-5′ oligo(A) synthetase (2′-5′ OS), and the dsRNA-dependent protein

kinase (PKR). These proteins are latent until they are activated by viral infection. PKR and 2′-5′ oligo(A) synthetase

are activated by dsRNA that is produced during viral infection. Once activated, PKR autophosphorylates itself, and

then phosphorylates EIF2. The activated synthetase makes trimeric oligonucleotides which in turn activate RNase L.

(B) Phosphorylated EIF2 and activated RNase L are characteristic of the "antiviral state" in which a eukaryotic cell

is refractory to infection by a wide variety of viruses. Phosphorylated EIF2 cannot serve to initiate translation of mRNA

by ribosomes and activated RNase L degrades mRNAs, both viral and cellular, so protein synthesis stops. Without protein

synthesis no virus replication can take place, but the inhibition of protein synthesis is transient and the cell may recover.

Adapted from Nathanson et al. (1996) Figure 6.8 on p. 125.

Degradation of mRNA and shutoff of protein synthesis

investigators and by biotech companies to use these mecha-

are drastic events that represent a defense of last resort. The

nisms to specifically turn off viral genes as a method to con-

dependence of both pathways on dsRNA means that only

trol viruses have followed. It is now clear that almost every

cells that contain dsRNA, presumably only virus-infected

cell can produce such interfering RNAs. These RNAs are

cells, will shut down as a result of these pathways. To sim-

used in the determination of the fate of the cell, in protecting

plify somewhat, one effect of IFN is to prepare the cell for

organisms from transposons and genomic rearrangements,

instant shutdown if it becomes infected, while leaving unin-

in physiological regulation, and in brain morphogenesis.

fected cells essentially alone.

The fact that several viruses have been found to encode

products that interfere with gene silencing (described later

in this chapter) indicates that the gene-silencing pathway is

Therapeutic Uses of Interferon

also important for the control of at least some viruses.

Since the discovery of IFN some 50 years ago, there has

There are two sources of these small RNAs used for gene

been great hope and expectation that IFN therapy would be

silencing. One source is long dsRNA, whether supplied

useful for the treatment of viral infections. In early stud-

externally or synthesized within the cell, which gives rise to

ies, IFN was induced in patients by injection of dsRNA,

RNAs called siRNAs (for short interfering RNAs). The long

but quantities of recombinant IFN-α suitable for injection

dsRNAs can be shortened by RNaseIII but are ultimately

are now available. In general, IFN-α therapy has been dis-

cleaved by the ribonuclease DICER into short dsRNAs that

appointing for treatment of viral infections in humans,

are typically 21 nucleotides long with 2 nucleotide over-

hangs at each 3′ end. These short molecules are then deliv-

although it is useful in a number of diseases (Table 10.5).

Infection with hepatitis B or hepatitis C viruses often results

ered to the RISC (RNA-induced silencing complexes) where

in chronic infection. In at least some of these chronically

they interact with one or more argonaute proteins (Ago1,

infected patients, the virus load can be decreased, or the dis-

Ago2). In the RISC complex the "sense" strand is selectively

ease may go into remission, upon long-term treatment with

removed, and the resulting "guide strand" ssRNA binds to

recombinant IFN-α. However, side effects of IFN treatment

the complementary portion of mRNA from the gene to be

often limit the dose and duration of treatment that can be

silenced. Depending upon the gene, the organism, and the

used. IFN-α has also been useful in treatment of infection

degree of sequence identity between the siRNA and the tar-

with papilloma viruses. More recently, combination therapy

get sequence the silencing may be accomplished by cleavage

in which IFN-α has been combined with other drugs that

of the mRNA or by repression of translation of the mRNA.

interfere with virus replication, such as ribavirin, which

The siRNA may also be used to repress the transcription of

depletes GTP pools in cells, have often proved more suc-

a gene through chromosome modification, but this proc-

cessful than treatment with IFN-α alone, and such methods

ess is not understood at present. Figure 10.20 illustrates the

for treatment of chronic virus infection continue to evolve.

production and use of siRNA to silence genes.

During clinical trials with IFN-α, it was found that this IFN

The second source of interfering RNAs is ssRNA that

is useful for the treatment of at least some cancers, including

is transcribed from the genome of many (all?) organisms,

hairy-cell leukemia and AIDS-related Kaposi's sarcoma. It

including humans. These RNAs form long hairpins of imper-

is assumed that this control is based on the inhibition of cell

fect complementarity, which are processed by a nuclear

growth caused by IFNs. Clinical trials using IFN-α, as well

enzyme called Drosha to form pre-micro RNA (pre-miRNA)

as other cytokines, for treatment of other viral diseases and

of about 70 nts. These are exported to the cytoplasm by the

other neoplasias continue and it is to be expected that further

protein Exportin5 where they are cleaved by DICER into

uses for these agents will be found (Table 10.5).

dsRNAs about 21 nucleotides long, and delivered to RISC.

Here the sense strand is removed and the resulting miRNA

performs the same function as do siRNAs. miRNAs, how-

Gene Silencing

ever, often contain one or more mismatches with their tar-

gets, unlike siRNAs, and thus miRNAs are more likely to

It has been known for many years that plants use RNA

function as translational repressors rather than causing

molecules 21 to 25 nucleotides in length, sometimes referred

mRNA degradation. However, some miRNAs do cause

to as RNAi (i for interference), to silence genes. The discov-

mRNA degradation, and some siRNAs cause the inhibition

ery in 1999 that a similar mechanism exists in animals, in

of translation, so that the functions of these two RNAs are

this case in the worm Caenorhabditis elegans, and that this

overlapping, as are the pathways that lead to their formation

mechanism is used to control a developmental pathway in

(Fig. 10.20). There are approximately 400 miRNA genes in

the worm, created a great deal of excitement and resulted in a

humans, grouped by sequence into 350 families. Many of

Nobel Prize for Andrew Fire and Craig Mello (see Table 1.1).

these are encoded in the 3′ NTRs of mammalian genes and

Since 1999 a great deal of work has elucidated the details

a small fraction of them are tissue specific. Each miRNA

of the mechanisms involved and extended the findings to

can have many targets, since for most genes a 7-nucleotide

many other animals, including mammals. Attempts by many

Pre-miRNA

Long dsRNAs

Drosha

Pre-miRNA

RNaseIII

DICER

Cleavage into 21-nt dsRNAs

with 3 overhangs

siRNA

RISC

ature miRNA

3 m

miRNP

Selective degradation of sense strand

ITS

Ago 1

Ago 2

RISC

DNA methylation

Ago 2

AAA

Histones

CAP

AAAA

CAP

Translational

CAP

AAAA

repression

Transcriptional

mRNA degradation

gene silencing

FIGURE 10.20

Mechanisms of RNA interference. Long dsRNAs are cleaved by RNaseIII and subsequently processed

by DICER into siRNAs, which are short dsRNAs of 2122 nts with 2-nt overhangs on the 3 termini. siRNAs then enter

the RNA-induced silencing complex (RISC) where the sense strand is selectively degraded. miRNAs are encoded as

incompletely self-complementary hairpins in viral and cellular genomes. They are cleaved by Drosha in the nucleus and

the pre-miRNA exported to the cytoplasm by Exportin5. There they are also processed by DICER. siRNAs silence genes

by binding in the context of RISC to mRNAs that are exactly complementary and causing mRNA degradation. miRNAs

often contain some mismatches and generally exert their effects by inhibiting translation. siRNAs can also enter RNA-

induced transcriptional silencing complexes (RITS) which recruit an enzyme to methylate DNA in chromatin, turning it

into inactive heterochromatin. Both RITS and RISC contain argonaute proteins (Agos). The figure is adapted from those

in Novina and Sharp (2004) and Schütz and Sarnow (2006).

match is sufficient for silencing. It is clear that these miRNAs

tions, although expression of the RNA by expression vec-

are important for the development of the organism and for cell

tors in the cell has been used in a few cases. These initial

regulation, but the many functions of these RNAs have as yet

experiments highlight some of the problems with adapt-

to be unraveled.

ing siRNA to therapeutic use. First, many cells do not take

There are ongoing attempts to develop gene silencing

up small RNAs easily or efficiently and expression vec-

as a new antiviral strategy, and numerous trials have been

tors may be required, which pose another set of problems.

made using a variety of viruses. Table 10.8 summarizes

Second, small RNAs have a short half-life in the average

results in both cell culture and in experimental animals

cell. Attempts to extend the half-life have included altering

the siRNAs in a number of ways, such as 2′-O methyla-

for a number of viruses. For many viruses, including HIV,

polio, and foot-and-mouth disease virus, treatment of cells

tion of the bases or covalent attachment of cholesterol at

the 3′ terminus. Third, it is unknown how specific siRNA

with siRNA either previous to or concommitantly with viral

infection reduced viral replication significantly. For the

or miRNAs would be for their intended targets and whether

experiments in cell culture, uptake of siRNA was usually

cellular mRNAs that might share some sequence identity

induced by treatment with lipofectamine or other polyca-

with the gene to be silenced might be downregulated in a

TABLE 10.8 Viruses Suppressed by siRNA

Cell type or

Reduction in viral

Virus

organism

Delivery protocol

Genes targeted

yield

Viral replication in cultured cells

HIV

Astroglioma cells

Lipofectamine

Polymerase, Nef

7080%

HIV

Human primary T cells

Lentiviral vector

CCR5

???

Cultured cell lines

Lipofectamine

Five viral genes and LTR

???

Poliovirus

HeLa Cells

Lipofectamine

Capsid, polymerase

9095%

HRSV

Cultured cell lines

Lipofectamine

???

5′NTR, 3′NTR, VPg, VP4, Pol

FMDV

BHK-21 cell lines

Lipofectamine

9099.9%

Flock house virus

Drosophila cell line

Endogenous cellular siRNA

???

Nearly 100%

Vaccinia

HeLa cells

???

E3L-specific siRNA

97%

Influenza

MDCK, CEF, chicken eggs

Plasmids

NP, PA

???

LCMV

Cultured cells

Recombinant adenovirus vector

L, Z1

Cures chronically infected

cells

Viral replication in whole animals

VSV

C. elegans

Gene products of rde-1, rde-4

???

Influenza

Mice (lung)

Inhale siRNA with polycations

???

99%

HRSV and

Mice (lung)

Inhale siRNA without

Essential viral genes

Protected from simultaneous

parainfluenza

transfection reagents

viral challenge

HSV-2

Mice (vagina)

Oligofectamine

UL27 and UL29

Protected from challenge up

to 3 hours later

Hepatitis B

Mice (intravenous)

siRNA in liposomes

???

Coxsackie B3

Mice (intravenous)

Hydrodynamic injection

???

Reduces virus by 6 logs

Japanese

Mice (intracranial)

Lipid complexed lentiviral

cd-loop domain of envelope

Protects against both JE

encephalitis

vector

protein

and WN encephalitis

Abbreviations: HIV, human immunodeficiency virus; LTR, long terminal repeat; HRSV, human respiratory syncytial virus; FMDV, foot and mouth disease

virus; LCMV, lymphocytic choriomeningitis virus; VSV, vesicular stomatitis virus; HSV-2, herpes simplex virus 2; JE, Japanese encephalitis virus;

WN, West Nile virus.

Source: Data from McManus and Sharp (2002), Lecellier et al. (2005), and Dykxhoorn et al. (2006).

"bystander effect." Obviously, such bystander downregula-

Preliminary experiments in mice have also been performed

tion could potentially be deleterious.

to explore ways of delivering siRNAs to appropriate tissues

More detailed experiments with HIV in cell culture

in sufficient quantities to effect viral silencing (Table 10.8).

have used not only siRNA exogeneously supplied but also

Some success has been achieved for viruses affecting the

lentiviral vectors expressing siRNAs to target a variety of

lungs, for lung tissue appears to be able to take up RNA even

HIV genes, the 5′ LTR, and even the HIV coreceptor CCR5

without transfection reagents such as lipofectamine. Similarly,

(referred to in Table 10.8). What has become apparent from

viruses affecting the genital tract can be suppressed by siRNA

these studies is that HIV mutates rapidly under pressure

inserted into the vagina with oligofectamine. Hepatitis B

from siRNA inhibition and resistant variants arise quickly.

infection has been treated with siRNA introduced intrave-

Even single nucleotide substitutions in the center of the core

nously in liposomes. Other intravenous approaches have so

recognition sequence can make the virus resistant to silenc-

far been less successful, although a Coxsackie B3 infection

ing. Thus, resistant variants can arise that have an unaltered

in mice could be suppressed by a million-fold after siRNA

amino acid sequence because they possess only silent muta-

was introduced by hydrodynamic injection. Hydrodynamic

tions. Similar results with picornaviruses have implied that

injection, however, carries significant risk of cardiac failure

any successful RNAi strategy must involve multiple siRNAs

and is not an option for human treatment. Thus at the current

directed at a variety of targets so as to reduce the probability

time, siRNA appears to be a molecule with much therapeutic

that resistant variants will quickly arise.

potential but no proven efficacy.

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