Download The viral manipulation of the host cellular and immune environments

The viral manipulation of the host cellular and immune
environments to enhance propagation and survival: a focus
on RNA viruses
Surendran Mahalingam,* Jayesh Meanger,† Paul S. Foster,* and Brett A. Lidbury‡
*Division of Molecular Biosciences, The John Curtin School of Medical Research, The Australian National
University, Canberra; †Macfarlane Burnet Institute for Medical Research and Public Health, Fairfield, Victoria,
Australia; and ‡Gadi Research Centre, Division of Science and Design, University of Canberra, Australia
Abstract: Virus infection presents a significant
challenge to host survival. The capacity of the virus
to replicate and persist in the host is dependent on
the status of the host antiviral defense mechanisms.
The study of antiviral immunity has revealed effective antiviral host immune responses and enhanced
our knowledge of the diversity of viral immunomodulatory strategies that undermine these defences. This review describes the diverse approaches that are used by RNA viruses to trick or
evade immune detection and response systems.
Some of these approaches include the specific targeting of the major histocompatibility complexrestricted antigen presentation pathways, apoptosis, disruption of cytokine function and signaling,
exploitation of the chemokine system, and interference with humoral immune responses. A detailed insight into interactions of viruses with the
immune system may provide direction in the development of new vaccine strategies and novel antiviral compounds. J. Leukoc. Biol. 72: 429 – 439;
2002.
Key Words: transcription factors 䡠 apoptosis 䡠 immune modulation
䡠 cytokines 䡠 chemokines 䡠 antibody 䡠 HIV 䡠 antigen processing
䡠 antigen presentation 䡠 immune evasion
INTRODUCTION
Viruses serve as parasites and genetic elements in their hosts
and drive the evolutionary process [1]. Not only do they have
considerable plasticity, enabling them to evolve in new directions, but their genetic and metabolic interactions with cells
uniquely position them to mediate subtle, cumulative evolutionary changes in their hosts as well [1]. The past decade has
seen an explosion of interest in mechanisms of immune evasion
and host manipulation by viruses. The intense focus stems from
a desire to gain a fundamental understanding of the complexities of virus-host interactions, mechanisms of viral pathogenesis, as well as a reflection through viral evasion mechanisms
of key antiviral immune pathways and cellular functions. Understanding how viruses manipulate cells may also provide
some important insights into new approaches to rational drug
design and vaccines. As a measure of the activity of this field,
many outstanding reviews have already been written on this
subject [2– 4]. These past reviews summarize a vast array of
strategies that viruses use in their quest to avoid immune
detection and effect. The number of strategies uncovered has
allowed for a classification of viral immune avoidance mechanisms into groupings, such as “viral inhibitors of antigen
presentation,” “viral inhibitors of humoral immunity,” “viral
interference of interferon,” “modulators of cytokine and chemokine activity,” and “inhibitors of apoptosis” [3–5] (see below). It is clear from such classifications that viruses have
“learned” to target all arms of the immune response as well as
normal cellular processes, such as apoptosis, during their long
co-evolutionary host relationships. As such strategies are used
by viruses, we also accept that the immune response has
evolved more effective tools with which to repel invading
pathogens; hence, we often use an “arms race” metaphor to
describe virus-host interactions.
Much of the focus has been on large DNA viruses, which are
thought to have “stolen genes from the host that were subsequently modified for the benefit of the virus” [4, 6] in addition
to possibly developing some nonhost homologous genes, which,
through the co-evolutionary relationship, have also been beneficial to the virus, subsequently selected for and exploited.
The case for the smaller genome RNA viruses is emerging, but
provides fewer examples of immune evasion techniques. The
fundamental molecular biology of RNA viruses restricts their
capacity to build large genomes with low fidelity RNA polymerases [4], so therefore leaves little if any genomic capacity to
develop individual evasion genes. This molecular scenario
implies that small genome RNA viruses have needed to be in
some ways more ingenious in surviving the rigors of the mammalian immune response. A theme of this review will be to look
more closely at “tricky” RNA virus evasion strategies and
explore how it has been possible for them to survive long term.
In this context, we will particularly consider the impact of host
Correspondence: Surendran Mahalingam, Ph.D., Division of Molecular Biosciences, John Curtin School of Medical Research, Australian National University, Canberra, ACT 0200 Australia. E-mail: Surendran.Mahalingam
@anu.edu.au
Received January 31, 2002; revised April 24, 2002; accepted April 25,
2002.
Journal of Leukocyte Biology Volume 72, September 2002 429
immune proteins manipulation by viruses and how this allows
a virus to transform a host cellular environment to meet its
needs, often at the expense of the host’s requirements.
VIRAL GENETIC “BUDGETS” AND THE “COST
TO THE INFECTED HOST”
We have recently proposed a theory on the “genetic budget of
viruses and the cost to the infected host” [7]. This theory
proposes that large genome DNA viruses, as alluded to in the
introduction, have developed an “acquisition” strategy for survival in the hostile host environment, while small genome RNA
viruses have survived via “erroneous replication” strategies.
The central thrust of the theory posits that the acquisition
strategy is less likely to be detrimental to the infected host, as
the close genetic relationship has allowed the virus to very
precisely target host pathways and functions. This has resulted
in a much-reduced impact on the infected host, on whom the
virus ultimately depends for survival. Conversely, erroneous
replication strategies used by RNA viruses are not specifically
tailored to host responses, leading to many random virus mutations that are more likely to result in inappropriate or overzealous host responses and have a detrimental effect on the
host while a relationship equilibrium is negotiated.
In the context of this review, we will consider the following
human disease-causing RNA viruses: measles virus (MV;
paramyxoviridae), influenza (orthomyxoviridae), respiratory
syncytial virus (RSV; paramyxoviridae), ebola virus (EV; filoviridae), Ross River virus (RRV; alphaviridae), hepatitis C
virus (HCV; flaviviridae), and HIV (lentiviridae). These examples comprise four negative-strand RNA viruses (virus with a
single-stranded RNA genome of the opposite polarity as
mRNA), two positive-strand viruses (virus with a singlestranded RNA genome of the same polarity as mRNA), and one
retrovirus (virus with two copies of single-stranded RNA genome of the same polarity as mRNA), respectively. With the
possible exception of MV, the RNA virus examples mentioned
above represent significant challenges to the formulation of
long-term and effective vaccines. What special characteristics
of RNA virus immune evasion need to be better understood
before effective vaccines can be developed?
STRATEGIES OF RNA VIRUS IMMUNE
EVASION
Antiviral defense mechanisms are numerous and range from
relatively primitive, constitutively expressed, nonspecific defenses to sophisticated mechanisms that are specifically induced in response to viral antigens [8]. Described below are
several strategies that RNA viruses have evolved to counteract
the various compartments of these defense mechanisms (Table 1).
Interference with antigen presentation
T cells recognize antigens in association with host major histocompatibility complex (MHC) molecules on antigen-present-
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ing cells. The MHC class I-restricted CD8⫹ cytotoxic T lymphocytes (CTLs) recognize antigenic peptides synthesized
within target cells. The role of CD8⫹, MHC class I-restricted
CTLs is critical in the recovery from primary virus infection
[9]. On the other hand, class II MHC-restricted CD4⫹ T helper
(Th) cells recognize peptides derived exogenously. CD4⫹ T
cells are activated during virus infections and can therefore
influence antibody production, CTL, and macrophage activity
as well as production of antiviral cytokines [10]. Expression of
these cell surface molecules is important to initiate and sustain
an effective immune response. It is not surprising that the HIV
has evolved strategies to down-regulate the surface expression
of class I, class II, and CD4 molecules [11]. Down-regulation of
CD4 expression prevents activation of infected Th cells via the
MHC class II pathway and thus helps virus evade immune
detection.
Inhibition of cytokine action
Cytokines are the messenger molecules that play an important
role in inflammation, cellular activation, proliferation, and
differentiation [12]. Their effects involve a wide range of mechanisms including alteration of the expression of MHC molecules, adhesion molecules, and costimulatory molecules and
direct activation or deactivation of immune cells [8]. Cytokines
such as interferons (IFNs), tumor necrosis factor (TNF), and
interleukin-12 (IL-12) are frequently targeted by viruses to
divert their potent antiviral effects. In this context, we will
briefly describe the IFN system. The IFN response represents
an early host defense mechanism against viral infections (inhibitory against a number of DNA and RNA viruses) and is
known to be an important component of innate immunity [13].
The antiviral activity of IFNs, the property that led to their
discovery almost 40 years ago, is mediated by a number of
intracellular, antiviral pathways that are activated by IFNs
(Fig. 1) [14]. The binding of IFN to its receptor results in the
phosphorylation of transcription factor complexes [signal transducer and activator of transcription (STAT) complexes], which
translocate to the nucleus and bind to the transcription coactivator elements on order to stimulate the downstream, antiviral
genes. Examples of these antiviral genes are IFN-␤, RNAdependent protein kinase (PKR), 2⬘ 5⬘ A synthetase, nitric
oxide (NO), and secondary transcription factors [e.g., IFNregulatory factor 1 (IRF-1), IRF-3, and IRF-7]. The latter
factors are also important for the transcription of many antiviral
genes. These IFN-inducible proteins mediate antiviral effects
by interfering with the regulation of viral and cellular macromolecular synthesis and degradation. Given the efficiency by
which the IFN system can inhibit replication of a multitude of
viruses, it is perhaps not surprising that some viruses have
evolved mechanisms to evade this host defense. Several RNA
viruses are known to inhibit the IFN system by different
mechanisms including targeting the IFN-inducible protein
PKR and 2⬘ 5⬘ A synthetase as well as suppression of primary
(STAT complexes) and secondary (IRFs) transcription factor
activation.
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TABLE 1.
Summary of Strategies Employed by RNA Viruses to Avoid Immune Detection and/or Clearance by the Infected Host
Virus
Human Immunodeficiency Virus
Measles Virus
Influenza Virus
Ebola Virus
Ross River Virus (ADE Infection)
Respiratory Syncytial Virus
Hepatitis C Virus
Immune/antiviral evasion mechanism (viral gene/protein involved)
1) Inhibition of humoral immunity:
a) 2 soluble complement (gp120–41)
b) The incorporation of host complement regulatory proteins such as CD59 into the HIV
envelope to inhibit complement activation
2) Interference of interferon:
a) 2 PKR activity (Tar RNA & Tat)
b) 2 2⬘ 5⬘ A synthetase/RNase L (Tar RNA and Tat)
3) Cytokines & cytokine receptors:
Chemokine similarity; attraction of monocytes (Tat)
4) Interference of MHC functions:
a) Endocytosis of surface class I (Nef)
b) Class I destabilization (Vpu)
c) Class II processing interference (Nef)
1) Cytokine activity modulation & inhibition:
a) Blockade of macrophage IL-12 induction via CD46 binding (HA)
2) Humoral immunity disruption:
a) Fc␥RII ligation (NP)
3) Interference of interferon:
a) Failure of viral RNA to activate PKR/NF-␬B in neurons (unknown)
b) 2 IFN post-PHA stimulation of PBLs (unknown)
1) Interference of interferon:
a) 2 PKR activity (NS1)
b) 2 NF-␬B (NS1)
c) 2 IRF-3 (NS1)
d) 2 PKR via p581PK induction
1) Interference of interferon:
a) Antagonism of type I interferon (VP35)
2) Functions associated with viral glycoprotein (GP):
a) 2 ␤1-integrin
b) 2 CR3 ⫹ Fc-␥-RIIIB linkage
c) 2 Mitogen-stimulated lymphocyte proliferation
1) Disruption of inflammatory antiviral response:
a) Ablation of TNF & NOS2 expression (unknown)
b) 2 NF-␬B & STAT complexes (unknown)
1) Cytokines & cytokine receptors:
a) Chemokine mimicry (G glycoprotein)
1) Interference of interferon:
a) Inhibit PKR activity (NS5A and E2)
b) Enhance IL-8 production, which suppresses type I IFN (NS5A)
2) Inhibition of cell-mediated immunity
a) Down-regulation of CTL function (core protein)
Abbreviations: 2, Antagonism/suppression; gp/GP, glycoprotein; PKR, dsRNA-dependent protein kinase; TAR, transacting-response element; MHC, major
histocompatibility complex; IL, interleukin; HA, haemagglutinin; Fc␥R, Fc ␥ receptor; NP, nucleoprotein; NF-␬B, nuclear factor-␬ B; IRF, interferon-regulatory
factor; TNF, tumour necrosis factor; NOS, nitric oxide synthase; PHA, phytohemagglutinin; PBL, peripheral blood lymphocytes; CR3, complement receptor type
3; ADE, antibody-dependent enhancement.
Modulation of chemokine activity
Leukocyte trafficking to sites of viral infection is an important
component of the early host inflammatory response, and chemokines are key effector molecules that orchestrate this process [15, 16]. They are produced in response to exogenous
stimuli such as viruses and bacterial lipopolysaccharide (LPS)
and endogenous stimuli such as IL-1, TNF, and IFNs [17]. The
chemokine superfamily mediates development and recruitment
of immune cells to sites of insult by signaling through a family
of G protein-coupled receptors. Given that the virus relies on a
cell to replicate, reproduce, and survive, it makes sense that
RNA viruses, like many DNA viruses, would need to modulate
chemokine action to encourage migration of suitable cells to
the site of infection. There is no doubt that chemokine and
chemokine receptors are critical for defense against viruses; however, it is also clear that viruses such as HIV and RSV have
evolved to accommodate the workings of the host chemokine
system.
Modulation of apoptosis
Programmed cell death, or apoptosis, is a natural cellular
response to injury or virus infection. Following viral infections,
T cells and natural killer (NK) cells are triggered to secrete
cytotoxic cytokines such as TNF and lymphotoxin [18]. In
addition, contact between these immune cells and virally infected cells results in the release of perforin and granzyme
proteins or delivery of FasL to Fas on the target cell [18].
Apoptosis before virus replication has been completed would
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Fig. 1. The IFN-␥ and IFN-␣ JAK-STAT signaling cascades. IFN-␥ stimulates the induction of immediate early genes (IEGs) through a signaling pathway that
employs Jak-1, Jak-2, Stat-1 and Stat binding elements. Activated Stat1 homodimer translocates to the nucleus where it binds the gamma-activation site (GAS)
and activates transcription of a subset of genes that includes the PKR, 2⬘ 5 A synthetase, IRF-1, and Stat1. Newly generated IRF-1 bind to an IFN-response
stimulation (IRS) site and activate (in concert with other factors) transcription of genes as inducible nitric oxide synthase (NOS2) and IFN-␤. In contrast, IFN-␣
stimulates the induction of immediate early genes through a pathway that employs Jak-1, Tyk-2, Stat-2, IRF-9/p48, and the interferon-stimulated response element
(ISRE). Phosphorylated Stat1/Stat2-heterodimer in concert with IRF-9 (p48) forms the interferon-stimulated gene factor 3 (ISGF3) complex that binds to the
element ISRE and increases transcription of a subset of genes that includes the PKR, IRF-1, IRF-7, 2⬘ 5⬘ A synthetase and Stat1.
be a disastrous outcome for the virus; consequently, viruses
such as HIV have evolved means to defuse this pathway to
create a suitable environment for their replication.
Manipulation of humoral immunity
Antibodies are important in preventing reinfection with many
viruses. Antibody-mediated mechanisms that are thought to
control virus infections include the neutralization of virus
particles and the cytolysis of antibody-coated, infected cells
[19]. The killing of virus-infected cells can also be mediated by
the binding of complement to antibody on virus-infected cells.
The importance of complement in virus infection is also reflected by the ability of some viruses to block the complement
pathway. The humoral immune response relies on the ability to
effectively process and eliminate immune complexes, a process
in which complement and Fc receptors play key roles. We
discuss some examples of viruses that manipulate this response.
NEGATIVE-STRAND RNA VIRUSES
Measles virus (MV)
MV is a highly contagious agent that is responsible for many
childhood deaths, particularly in the developing world (⬎1
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million deaths per annum in children in the Third World) and
is transmitted via respiratory/oral secretions. After initial infection, virus can disseminate to other areas of the body. Of
particular concern are neurological infections that may lead to
subacute sclerosing panencephalitis some years after the primary infection. Despite the generation of a vigorous immune
response against MV, immunity to other pathogens is depressed. This generalized immunosuppression allows the establishment of opportunistic infections and results in many
complications associated with measles [20].
Recent findings have revealed several mechanisms on MVmediated immunosuppression. For instance, it has been demonstrated in neuronal tissue that MV-RNA fails to activate
double-stranded, RNA-activated PKR. PKR is believed to be a
key component in the control of protein synthesis in virusinfected cells. Induction of PKR by IFNs leads to phosphorylation of eukaryotic initiation factor 2␣ (eIF2␣), which inhibits
protein synthesis and protects cells from virus infection [14].
The inability to activate this antiviral protein leads to virusmediated disruption to transcription factor nuclear factor
(NF)-␬B binding, subsequent blockage to the IFN-␤ response,
and ultimately a lack of MHC class I expression [21]. The
authors suggested that this mechanism allowed the virus to
hide and persist in neuronal tissue by escaping the attention of
CTLs. As neuronal cells apparently lack alternative activation
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pathways for IFN-␤, this could explain why long-term disease
might manifest in the brain. Furthermore, the MV-mediated
disruption to type I IFN induction has been found not only to
be restricted to neuronal cells, but also in phytohemagglutinstimulated peripheral blood lymphocytes [22]. At the time of
writing this review, the viral product responsible for type I IFN
interference was not known, although it has been speculated
based on evidence from studies on the close MV relative
Sendai virus that the nonstructural C protein is a likely candidate [22].
The MV repertoire also includes the blockage of IL-12
induction in macrophages via MV hemagglutinin (HA) binding
to the cellular complement receptor CD46 [3, 4, 23, 24]. This
may result in the suppression of several facets of the immune
component such as IFN-␥ secretion by immune cells, development of Th1 responses, enhancement of lytic activity in NK
cells, and CTL [23–25].
Furthermore, work by Ravanel and colleagues [26] have
shown that MV nucleoprotein (NP) can bind to the surface of B
cells. It was demonstrated that the murine and human Fc-␥
receptor II (Fc␥RII) are receptors for MV-NP and that the
binding of NP inhibits immunoglobulin synthesis by activated
B cells.
Influenza virus
Influenza virus remains a significant cause of morbidity and
mortality worldwide, particularly in the elderly and immunosuppressed individuals. Up to 20% of the population can
become ill during a single epidemic, with 50,000 deaths per
year occurring in the United States alone [27].
The fragmented influenza genome allows genetic recombination within and between species (humans, pigs, poultry),
leading to the problems of “antigenic drift” and “antigenic
shift.” The difference between antigenic drift and antigenic
shift is as follows: antigenic drift refers to point mutation in
major epitopes of HA that are recognized by immune cells and
prevents highly efficient immune clearance of virus; antigenic
shift is the reassortment of genes between influenza viruses that
infect different species of host that result in major changes in
the viral HA, which prevents existing antibodies from clearing
the virus rapidly. Problems of antigenic drift manifest, for
at-risk groups, as a yearly requirement to be vaccinated. Longterm immunity does not significantly develop against influenza
via wild-infection or vaccination. The problems associated with
antigenic shift can be catastrophic as changes to viral antigenic
properties are so pronounced that large proportions of the
population may have no immunity at all to the new strain,
which could lead to serious pandemics.
As a leading infectious disease concern, influenza has traditionally been at the forefront of virus pathogenesis research.
Beyond the already appreciated problems of antigenic shift and
drift, recent studies have shown that the sole nonstructural
protein of influenza A virus, NS1, is a key virulence factor for
its ability to inhibit type I IFN (IFN-␣/␤) responses in the
infected host (Fig. 2) [28, 29]. This ability of NS1 to block
IFN-␣/␤ activation has been found to be associated with the
perturbation of PKR activation [30]. It is known that transactivation of the IFN-␤ promoter depends on NF-␬B and several
other transcription factors. Further investigation subsequently
found that the activation of IRF-3 and NF-␬B was also inhibited by NS1 [31, 32]. This evidence points to viral proteins
performing dual or multiple functions; in addition to its polymerase activity, NS1 has been shown to be capable of perturbing type I IFN expression via compromising transcriptional
Fig. 2. RNA viruses subversion of the IFN system. The figure shows various strategies that RNA viruses use to antagonize the IFN system. Dotted arrow represents
suppression or inhibition [29].
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activation pathways in infected cells. Consistent with these
observations, it was also demonstrated that infection of tissue
culture cells with deleted NS1 virus (delNS1), but not with
wild-type influenza A virus, induced high levels of mRNA
synthesis from IFN-␣/␤ genes, including IFN-␤ [30]. Interestingly, cells infected with delNS1 virus showed high levels of
NF-␬B activation compared with those infected with wild-type
virus [32].
Another approach used by influenza virus to inhibit PKRmediated phosphorylation of eIF2␣ is through the activation of
a host PKR inhibitory protein, P58IPK [33, 34]. In normal
conditions, P58IPK is bound to I-P58IPK in an inactive complex.
However, this complex is disrupted in cells infected with
influenza virus resulting in the release of P58IPK, which then
interacts with PKR and inhibits its kinase activity.
Respiratory syncytial virus (RSV)
RSV is the principal etiological agent of bronchiolitis and
pneumonia in infants and young children worldwide, causing
an estimated 4500 deaths and 91,000 hospitalizations annually
in the United States. RSV is also responsible for an estimated
3.3 million cases of respiratory tract diseases in the elderly
annually in the United States. Thus, there is an urgent need for
a safe and effective RSV vaccine. Protective immunity against
RSV is provided by virus-neutralizing antibodies against the
surface fusion and attachment (G) proteins.
More recently, Tripp and colleagues [35] have made an
exciting discovery on chemokine mimicry by RSV. They re-
ported that the G glycoprotein (GP) of RSV has structural
similarities to a CX3C chemokine Fractalkine and binds to
cells in a manner similar to Fractalkine through the chemokine
receptor CX3CR1. Interestingly, this interaction appears to
have two important functions in RSV infection [35]. First, the
interaction of the CX3C motif on the G GP with CX3CR1 on
cells is capable of inducing migration of leukocytes and thus
modulating the immune response (Fig. 3a). Second, G GP
binding via CX3CR1 appears to facilitate infection. In this
regard, it is likely that G GP of RSV competes with Fractalkine
for binding to CX3CR1 on cells and evades Fractalkine-mediated immune responses, which result in delayed virus clearance.
In the context of IFN antagonistic effects, like influenza,
bovine RSV NS1 and NS2 proteins have been shown to cooperatively antagonize an ␣/␤ IFN-induced antiviral response
[36]. Although not known, it is possible that the NS1 and NS2
proteins of human RSV may be mediating similar processes
(Klaus Conzelmann, personal communication).
Ebola virus (EV)
EV, a member of the Filoviridae, burst from obscurity with
spectacular outbreaks of severe, haemorrhagic fever. It was
first associated with an outbreak of 318 cases and a casefatality rate of 90% in Zaire; it caused 150 deaths among 250
cases in Sudan. Explanations for its immense virulence and
detrimental impact on the host are slowly emerging, with viral
genes and proteins observed to alter host responses. The prop-
Fig. 3. Strategies used by viruses to subvert the host chemokine system. (a) RSV: Virus-encoded, chemokine-like protein that can compete with host chemokines
for binding to host chemokine receptor. This process can result in the delay in viral clearance as well as enhancement of viral infectivity. (b) HIV: Virus-encoded,
chemokine-like protein (Tat) by HIV that can promote chemotaxis of monocytes/macrophages to enhance infection.
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erty of type 1 IFN antagonism described above for influenza
has also been identified for EV and has been attributed to the
viral VP35 protein [37], suggesting again the roles for proteins
encoded by small, genome-size RNA viruses in cell interactions and immune evasion. In addition to VP35, the EV GP has
been recognized as a key determinant of immune evasion
capacity. Immune evasion, cell-altering activities recognized
thus far are down-regulation of ␤1 integrin [38], significant
reductions in complement receptor type 3/Fc␥RIIIB linkage in
neutrophils [39], and suppression of mitogen-stimulated lymphocyte proliferation [40]. Furthermore, the mucin domain of
EV GP has been proposed as the mediator of viral pathogenicity, with studies showing enhanced cytotoxicity and vascular permeability in endothelial cell cultures and blood vessel
explants [41]. Recently, it was determined that this virus
envelope GP binds to the human folate receptor as a mediator
of entry [42]. With such an array of activities attributable to
individual viral proteins such as GP, vaccination strategies
focused on viral determinants will be very challenging for EV,
particularly with an inactivated virus capable of eliciting reactions that are potentially damaging to the host [40].
Fig. 4. ADE of RRV infection in vitro. Suppression of NF-␬B complex in
LPS-stimulated macrophages infected with RRV in the presence of anti-RRV
antibody. NMS, normal mouse serum.
POSITIVE-STRAND RNA VIRUSES
Ross River virus (RRV)
RRV is an indigenous Australian alphavirus and the agent
responsible for the greatest incidence of arboviral disease in
Australia. Disease resulting from infection is not fatal but
involves a syndrome of symptoms, which include arthritis/
arthralgia, myalgia, lethargy, and/or rash. These symptoms are
often episodic but can be responsible for persistent debilitation
for over 12 months after primary infection [43].
Macrophage and monocyte infiltrates have been associated
with human disease [43, 44], and F4/80⫹ cells have been
recently identified as the cellular agent of severe muscle damage in RRV-infected mice [45]. Furthermore, RRV grows in
human and murine macrophages after infection via a “natural”
cellular receptor or through FcRs involving “antibody-dependent enhancement” (ADE) mechanisms of infection [45, 46]. In
studies using LPS-stimulated murine macrophage cultures
(RAW 264.7), RRV was found to specifically ablate at the
RNA and protein level the expression of the antivirals TNF and
inducible NO synthase (NOS2) post-ADE infection [47]. Similar to IFN evasion mechanisms described earlier for measles
and influenza infections, the ablation of TNF and NOS2 production by RRV was found to be associated with the perturbation of NF-␬B (Fig. 4) and STAT 1 complexes. These
observations explained why RRV could grow to high titers in
macrophages despite LPS stimulation and may provide insights
into ADE associated with other human, disease-causing viruses. In this regard, ADE of dengue virus infections has long
been implicated in the pathogenesis of dengue hemorrhagic
fever [48]. Interestingly, a recent study by Yang and colleagues
[49] showed suppression of IFN-␥ production in the ADE of
heterotypic dengue infections. However, others have reported
an increase in IFN-␥ production in dengue infections [50]. The
reasons for these differences are not clear but may be related
to experimental conditions and cells used in these studies.
The RRV gene/protein responsible for the ablation of antiviral factors post-ADE infection is still unknown. There has
been traditional interest in the structural protein E2 as important to RRV virulence and antibody evasion [51, 52], but based
on the observations of NF-␬B and IRF disruption for influenza,
the role of nonstructural viral genes/proteins will also need to
be closely considered in future studies on antiviral evasion
by RRV.
Hepatitis C virus (HCV)
HCV is an emerging virus of great medical importance and
almost always causes chronic infections. The high incidence of
HCV persistence after infection suggests that this virus has
evolved mechanisms in order to evade the host response. Little
is known about the mechanisms that allow HCV to achieve
lifelong persistence in infected individuals because the lack of
an effective in vitro culture system has impaired virologic
studies. However, recent discoveries may explain the long-term
persistence of HCV in the host. One hypothesis to explain this
phenomenon is that HCV escapes immune recognition through
its intrinsic hypermutability. Here, altered peptide ligands with
antagonistic activity can be an effective mechanism to shut off
antiviral CTL responses to HCV [53]. Furthermore, as observed
in HIV infection (see below), evasion from CD4⫹ T cell responses may be particularly effective during HCV infection, as
strong CD4⫹ T cell responses have been associated with an
improved disease outcome [54 –56]. It has also been shown that
HCV core protein can interact with cellular RNA helicases and
potentiate TNF-mediated triggering of NF-␬B activity, and may
block proapoptotic signals in HCV-infected cells [57, 58]. It is
believed that signaling through the TNF receptor may be partly
responsible for the chronic state of HCV infection, as the core
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435
protein alone when administered to mice results in general
immunosuppression [59]. HCV may also suppress immune
response(s), leading to dampening of cellular immunity. This
observation is supported by recent studies demonstrating that
vaccinia virus (VV) expressing HCV structural protein can
suppress host immune responses to VV by down-regulating
viral-specific CTL responses and cytokine production. Using a
series of VV recombinants expressing various C-terminally
truncated polyproteins, this immunosuppressive effect was
mapped to the core protein [59].
One of the nonstructural proteins of HCV, NS5A, has been
shown to bind and inhibit PKR [60], while another study
showed that the HCV envelope protein E2 contains a sequence
identical with phosphorylation sites of PKR and eIF2␣ [61]. E2
inhibited the kinase activity of PKR and blocked its inhibitory
effect on protein synthesis and cell growth. Furthermore, the
expression of NS5A in human cells can induce IL-8 expression, and this effect correlated with the inhibition of antiviral
effects of IFN-␣ via reduced 2⬘ 5⬘ A synthetase activity [62,
63]. Optimal activity of 2⬘ 5⬘ A synthetase is important for the
activation of latent RNase (RNase L), which induces the degradation of RNAs followed by inhibition of protein synthesis.
RETROVIRUS
Human immunodeficiency virus (HIV)
HIV is the viral agent spread by contact with infected blood or
semen that causes AIDS. Although rates of infection have
stabilized in many western countries, this virus is poised to
inflict an enormous disease impact on many African and some
Asian communities. Therefore, HIV/AIDS remains a primary
worldwide health concern.
HIV induces a strong antiviral response, while simultaneously and progressively disrupting the immune system. The
question remains as to how HIV manages to persist in the face
of such a strong antiviral response. One of the answers lies in
the ability of HIV to mutate key epitopes, which are recognized
by the immune response (“antigenic variation”). The range of
immune evasion and host-altering mechanisms used by HIV
have been the subject of immense scientific interest, as clues
are sought into basic questions of pathogenesis and the virus’s
resistance to the formulation of effective vaccine and therapeutic approaches. As summarized in Table 1, HIV has the
most extensive repertoire of immune-evasion tactics thus far
identified, covering all aspects of the host response to infection,
from early type I IFN activity to the disruption of MHC function. Corresponding knowledge of the viral gene products responsible for the impact on host responses is also quite extensive, and the HIV genes Tat, Nef, Env, and Vpu feature
prominently, thus further enhancing the earlier comments on
the amazing, multifunctional capacities of viral RNA genomes.
As a retrovirus, there is no guarantee that HIV will be a
reliable guide to immune evasion potentials across the broad
range of RNA virus families, but what HIV does emphasize is
the enormous extent to which apparently simple viruses have
been able to combat the sophisticated mammalian immune
system.
There are several mechanisms that HIV uses to modulate
immune responses. For instance, the HIV-1 Nef, Env, and Vpu
proteins are engaged in down-regulating the expression of the
surface CD4 molecule [64, 65]. Because Nef is an early gene
product, it acts more rapidly. By contrast, Env and Vpu are late
viral proteins that modulate CD4 expression along its biosynthetic pathway. Thus, the combined actions of Nef, Env, and
Vpu almost completely eliminate CD4 from the surface of
HIV-1-infected cells [11, 66]. Down-regulation of CD4 may
also prevent activation of infected Th cells via the MHC class
II antigen-presentation pathway and thus help the virus evade
immune detection. In addition, Nef protein is also capable of
down-regulating human leukocyte antigen (HLA) class I molecules, which can result in impaired CTL recognition in vitro
(Fig. 5) [67]. Such events expose an infected cell to lysis by
NK cells. However, this does not appear to be the case, as
HIV-1 Nef leads to the down-regulation of HLA-A and HLA-B,
but not HLA-C and HLA-E [68]; therefore, infected cells are
protected from NK-mediated destruction via HLA-C and
HLA-E expression. The elements on HIV Nef that are involved
Fig. 5. Nef-mediated MHC-I down-regulation. (a)
In the absence of Nef, prominent expression of
MHC class I-presenting viral peptides results in an
efficient lysis of infected cells by CTLs. (b) In the
presence of Nef, lack of MHC-I expression results
in CTLs unable to recognize infected cells and
therefore is protected from lysis.
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in the selective down-regulation of HLA molecules are different from the ones that are involved in the Nef-dependent CD4
down-regulation, suggesting a dichotomous effect of Nef on
these two cell molecules [69, 70].
The Tat protein of HIV, which is expressed early in the viral
life cycle, also influences a variety of immune regulatory
processes through diverse mechanisms. The HIV-1 Tat protein
is a potent chemoattractant for monocytes [71]. It was shown
that Tat displays conserved amino acids corresponding to
critical sequences of the chemokines. This viral protein serves
to recruit monocytes/macrophages toward HIV-producing cells
and facilitates activation and infection (Fig. 3b). The reported
down-regulation of HLA class I and class II molecules by Tat
remains controversial, with some investigators reporting no
effect and others observing an effect [72–74]. Tat may also
have direct effects on the development of B cell lymphomas
and display a profound impact on the replication of viruses
such as Kaposi’s sarcoma herpesvirus, human papillomavirus,
and human papovavirus [70]. These viruses themselves have
immune-modulating mechanisms and thereby contribute to
HIV-associated diseases.
HIV-infected cells contain a number of molecules capable of
modulating the activity of PKR and 2⬘ 5⬘ A synthetase. The
HIV-1 transactivation response (Tar) RNA binding protein was
shown to be a potent inhibitor of double stranded RNA-mediated activation of PKR [75]. On the other hand, Tar RNA has
been reported to bind and activate 2⬘ 5⬘ A synthetase in vitro
[76]. However, this activation by Tar was inhibited by Tat
protein [77].
In the mid 1990s, virus interaction with the chemokine
system took center stage after the discovery that HIV exploits
chemokine receptors as coreceptors for entry into CD4⫹ cells
[78]. Structural proteins of HIV (gp120), by virtue of its interaction with the cellular receptors for viral entry, may influence
the activity of cells expressing CD4, CCR5, and CXCR4.
Beside induction of apoptosis in human endothelial cells and
CD4⫹ T cells, the binding of gp120 to chemokine receptors
CCR5 and CXCR4 may have functional consequences such as
dysregulated lymphocyte homing or neurodegenerative effects
[79]. Another study found that recombinant gp120/gp41 complex (gp160) from macrophage-tropic HIV-1 induces a signal
through CCR5 on CD4⫹ T cells and that this envelope-mediated signal transduction induces chemotaxis of T cells [80].
This chemotactic response may contribute to the pathogenesis
of HIV in vivo by chemoattracting activated CD4⫹ cells to sites
of viral replication. HIV-mediated signaling through CCR5
may also enhance viral replication in vivo by increasing the
activation state of target cells. Alternatively, envelope-mediated CCR5 signal transduction may influence viral-associated
cytopathicity or apoptosis. It is clear that these strategies point
out the potential for viral gene products to alter multiple steps
in the host response(s) to infection with HIV.
THE PROSPECT OF EFFECTIVE VACCINES
AGAINST DECEPTIVE RNA VIRUSES
It is clear that traditional vaccine strategies that rely on generating antibody and/or CTL responses will not be sufficient to
combat elusive RNA viruses (examples of which have been
highlighted by this review) as a result of the evolved capacity
of these viruses to counter sophisticated immune responses
[81]. In fact, it could be hypothesized that the application of
ineffective vaccine-mediated immune responses may hasten
the evolution of additional immune-evasion activities. The
fundamental study of RNA virus immune-evasion tactics may
eventually expose viral weaknesses that can be exploited by
vaccines, but beyond such obvious conclusions, innovative and
lateral strategies must be identified. Such innovative solutions
will not eventuate without fuller appreciation of the nuances of
virus-host interaction. Future vaccines may not be only designed to stimulate T-B lymphocytes, but may be targeted at
the actual infected cell or tissue to activate a natural, innate
antiviral response before infection is established. Also, stimulating or suppressing particular receptors could aid the host
response or diminish the ability of viruses to penetrate cells.
What special characteristics of RNA virus immune evasion
have we learned that can be applied in the design of effective
new generation vaccines? As mentioned earlier in this review,
the influenza A virus NS1 protein exhibits IFN antagonist
activity, allowing influenza virus to replicate in IFN-competent
systems. Talon and colleagues [82] have recently proposed an
alternative, rational approach to the design of live virus vaccines by alteration of viral IFN antagonists. They reported that
deletion of virally encoded IFN antagonists or mutagenesis of
these proteins to reduce activity can be used as a general
strategy to construct live viral vaccines that are optimally
attenuated and immunogenic. Indeed, these viruses show significant growth attenuation in immunologically mature, embryonated chicken eggs and in mice. Furthermore, they demonstrated that immunization of mice with NS1-altered flu viruses
provides protective immunity in mice against the replication
and/or pathogenicity of wild-type influenza virus.
CONCLUDING REMARKS
Research studies conducted in the past several years have
clearly demonstrated how RNA viruses have evolved diverse
mechanisms to evade the host immune response. In summary,
HIV has the broadest array of immune evasion techniques so
far identified, targeting humoral and cell-mediated (via MHC
perturbation) immunity, type I IFN activity and cytokine/chemokine responses to infection. In the context of the earlier
discussed theory on the viral “genetic budget,” it must be
considered that a virus-like HIV, which uses the faulty enzyme
reverse transcriptase in routine replication, has additional molecular opportunities to develop via “erroneous replication”
strategies an expanded immune-evasion arsenal. It is likely
that other RNA viruses will have additional immune-evasion
strategies identified in due course, but whether nonretroviruses
ultimately have the same range of mechanisms to repel the
host’s immune response will be decided only by continuing
investigations. Considering that the error rate introduced by
RNA polymerases is sufficiently high, however, it is highly
possible that other RNA viruses have the ability to develop
multiple immune-evasion strategies. Such a question will be
ultimately answered only once the full multifunctionality of
Mahalingam et al. Modulation of the host immune responses by RNA viruses
437
viral RNA genomes is completely realized. With such additional insight, traditional vaccine strategies will be abandoned
for many RNA viruses, challenging scientists to devise new
approaches that circumvent or in some way neutralize the
impact of viral evasion factors.
23.
24.
ACKNOWLEDGMENTS
25.
S. M. is a recipient of the Australian National Health and
Medical Research Council Peter Doherty Fellowship. We
thank Mr. Geoff Sjollema for excellent technical assistance.
26.
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