Genomic HIV RNA induces innate immune responses through RIG-I-dependent sensing of secondary-structured RNA.
ABSTRACT Innate immune responses have recently been appreciated to play an important role in the pathogenesis of HIV infection. Whereas inadequate innate immune sensing of HIV during acute infection may contribute to failure to control and eradicate infection, persistent inflammatory responses later during infection contribute in driving chronic immune activation and development of immunodeficiency. However, knowledge on specific HIV PAMPs and cellular PRRs responsible for inducing innate immune responses remains sparse.
Here we demonstrate a major role for RIG-I and the adaptor protein MAVS in induction of innate immune responses to HIV genomic RNA. We found that secondary structured HIV-derived RNAs induced a response similar to genomic RNA. In primary human peripheral blood mononuclear cells and primary human macrophages, HIV RNA induced expression of IFN-stimulated genes, whereas only low levels of type I IFN and tumor necrosis factor α were produced. Furthermore, secondary structured HIV-derived RNA activated pathways to NF-κB, MAP kinases, and IRF3 and co-localized with peroxisomes, suggesting a role for this organelle in RIG-I-mediated innate immune sensing of HIV RNA.
These results establish RIG-I as an innate immune sensor of cytosolic HIV genomic RNA with secondary structure, thereby expanding current knowledge on HIV molecules capable of stimulating the innate immune system.
- SourceAvailable from: PubMed Central[show abstract] [hide abstract]
ABSTRACT: The mechanisms underlying CD4(+) T cell depletion in human immunodeficiency virus (HIV) infection are not well understood. Comparative studies of lymphoid tissues, where the vast majority of T cells reside, and peripheral blood can potentially illuminate the pathogenesis of HIV-associated disease. Here, we studied the effect of HIV infection on the activation and depletion of defined subsets of CD4(+) and CD8(+) T cells in the blood, gastrointestinal (GI) tract, and lymph node (LN). We also measured HIV-specific T cell frequencies in LNs and blood, and LN collagen deposition to define architectural changes associated with chronic inflammation. The major findings to emerge are the following: the GI tract has the most substantial CD4(+) T cell depletion at all stages of HIV disease; this depletion occurs preferentially within CCR5(+) CD4(+) T cells; HIV-associated immune activation results in abnormal accumulation of effector-type T cells within LNs; HIV-specific T cells in LNs do not account for all effector T cells; and T cell activation in LNs is associated with abnormal collagen deposition. Taken together, these findings define the nature and extent of CD4(+) T cell depletion in lymphoid tissue and point to mechanisms of profound depletion of specific T cell subsets related to elimination of CCR5(+) CD4(+) T cell targets and disruption of T cell homeostasis that accompanies chronic immune activation.Journal of Experimental Medicine 10/2004; 200(6):749-59. · 13.21 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: To monitor changes in the numbers of CD8 lymphocytes expressing the activated CD38++ phenotype in peripheral blood samples from patients with primary HIV infection (PHI) treated with highly active antiretroviral therapy (HAART). Zidovudine, lamivudine, abacavir and amprenavir were initiated during PHI as part of the Quest study. Absolute numbers of CD8+/CD38++ T cells were determined using three-colour flow cytometry, and plasma viral load (VL) was measured using the Roche Amplicor method. The median, pre-therapy CD8+/CD38++ T cell count was 461/mm(3)(interquartile range 216, 974) in 131 patients compared with normal control values of less than 20 cells/mm(3). Levels fell markedly in parallel with VL within the first 2 weeks of HAART initiation, to a median of 47 cells/mm(3) at 28 weeks (median 436 cell decline; P < 0.001). At that time, 80% of patients had a VL less than 50 copies/ml, and 16.3% of all patients had less than 20 CD8+/CD38++ T cells/mm(3). A continued decrease in CD8+/CD38++ T cell count occurred in 67.2% of patients whose VL was maintained below 50 copies/ml (median change from first to last value -18 cells/mm(3); P < 0.001). After the initiation of HAART in PHI, CD8+/CD38++ lymphocytes declined rapidly in parallel with VL, and allowed for a normalization of CD8+/CD38++ T cell numbers in a subset of patients at week 28. Cell numbers continued to decline in patients who maintained VL below 50 copies/ml, indicating that the CD8+/CD38++ T cell count may represent a marker of residual viral replication when VL falls below detectable levels after HAART intervention.AIDS 04/2002; 16(4):589-96. · 6.41 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Recognition of virus presence via RIG-I (retinoic acid inducible gene I) and/or MDA5 (melanoma differentiation-associated protein 5) initiates a signaling cascade that culminates in transcription of innate response genes such as those encoding the alpha/beta interferon (IFN-alpha/beta) cytokines. It is generally assumed that MDA5 is activated by long molecules of double-stranded RNA (dsRNA) produced by annealing of complementary RNAs generated during viral infection. Here, we used an antibody to dsRNA to show that the presence of immunoreactivity in virus-infected cells does indeed correlate with the ability of RNA extracted from these cells to activate MDA5. Furthermore, RNA from cells infected with encephalomyocarditis virus or with vaccinia virus and precipitated with the anti-dsRNA antibody can bind to MDA5 and induce MDA5-dependent IFN-alpha/beta production upon transfection into indicator cells. However, a prominent band of dsRNA apparent in cells infected with either virus does not stimulate IFN-alpha/beta production. Instead, stimulatory activity resides in higher-order structured RNA that contains single-stranded RNA and dsRNA. These results suggest that MDA5 activation requires an RNA web rather than simply long molecules of dsRNA.Journal of Virology 09/2009; 83(20):10761-9. · 5.08 Impact Factor
Genomic HIV RNA Induces Innate Immune Responses
through RIG-I-Dependent Sensing of Secondary-
Randi K. Berg1, Jesper Melchjorsen1, Johanna Rintahaka2, Elisabeth Diget1, Stine Søby1, Kristy A.
Horan3, Robert J. Gorelick4, Sampsa Matikainen2, Carsten S. Larsen1, Lars Ostergaard1, Søren R.
Paludan3, Trine H. Mogensen1*
1Department of Infectious Diseases, Aarhus University Hospital - Skejby, Aarhus, Denmark, 2Unit of Excellence for Immunotoxicology, Finnish Institute of Occupational
Health, Helsinki, Finland, 3Department of Biomedicine, University of Aarhus, Aarhus, Denmark, 4AIDS and Cancer Virus Program, SAIC-Frederick, Inc., National Cancer
Institute-Frederick, Frederick, Maryland, United States of America
Background: Innate immune responses have recently been appreciated to play an important role in the pathogenesis of
HIV infection. Whereas inadequate innate immune sensing of HIV during acute infection may contribute to failure to control
and eradicate infection, persistent inflammatory responses later during infection contribute in driving chronic immune
activation and development of immunodeficiency. However, knowledge on specific HIV PAMPs and cellular PRRs
responsible for inducing innate immune responses remains sparse.
Methods/Principal Findings: Here we demonstrate a major role for RIG-I and the adaptor protein MAVS in induction of
innate immune responses to HIV genomic RNA. We found that secondary structured HIV-derived RNAs induced a response
similar to genomic RNA. In primary human peripheral blood mononuclear cells and primary human macrophages, HIV RNA
induced expression of IFN-stimulated genes, whereas only low levels of type I IFN and tumor necrosis factor a were
produced. Furthermore, secondary structured HIV-derived RNA activated pathways to NF-kB, MAP kinases, and IRF3 and co-
localized with peroxisomes, suggesting a role for this organelle in RIG-I-mediated innate immune sensing of HIV RNA.
Conclusions/Significance: These results establish RIG-I as an innate immune sensor of cytosolic HIV genomic RNA with
secondary structure, thereby expanding current knowledge on HIV molecules capable of stimulating the innate immune
Citation: Berg RK, Melchjorsen J, Rintahaka J, Diget E, Søby S, et al. (2012) Genomic HIV RNA Induces Innate Immune Responses through RIG-I-Dependent
Sensing of Secondary-Structured RNA. PLoS ONE 7(1): e29291. doi:10.1371/journal.pone.0029291
Editor: Peter Sommer, Institut Pasteur Korea, Republic of Korea
Received June 28, 2011; Accepted November 24, 2011; Published January 3, 2012
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported by The Danish Medical Research Council (THM), Kong Christian IX and Dronning Louises Jubilæumslegat (THM), Fonden til
Lægevidenskabens Fremme (THM), and Scandinavian Society of Antimicrobial Chemotherapy (JM). KAH is recipient of a Marie Curie Incoming International
Fellowship and RKB was supported by a PhD Stipend from the Faculty of Health Science, Aarhus University. This project has been funded in whole or in part with
federal funds from the National Cancer Institute, National Institutes of Health, under contract HHSN261200800001E with SAIC-Frederick, Inc. (RJG). The content of
this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial
products, or organizations imply endorsement by the U.S. Government. Neither SAIC-Frederick, Inc., nor the funders had any role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have the following competing interest: One of the authors (RJG) has an affiliation to SAIC-Frederick, Inc., which is a U.S.
Government contractor, thus funding comes from the U.S. Government. There are no patents, products in development or marketed products to declare. This
does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors.
* E-mail: firstname.lastname@example.org
HIV is a retrovirus that targets mononuclear cells of the
immune system and establish lifelong infection with progressive
immunodeficiency, susceptibility to opportunistic infections, and
the development of AIDS if left untreated . The natural history
of HIV infection is characterized by an acute infection with high
levels of viraemia and irreversible damage to the immune system,
in particular the gut associated lymphoid tissue. This is followed by
a chronic phase with persistent immune activation and depletion
of CD4 T cells, ultimately resulting in progressive immune
exhaustion and profound immunodeficiency [2–5]. Based on the
observation that initiation of highly active antiretroviral therapy
leads to a rapid decline in immune activation, which is correlated
with a significant reduction in HIV viraemia, a direct contribution
of HIV particles to immune activation has been proposed [6,7].
Since one of the fundamental characteristics of HIV pathogen-
esis is the failure of the immune system to initially recognize and
control viral infection, early events taking place during the very
first hours and days of infection are likely to be of central
importance. The innate immune system constitutes the first line of
defence against invading pathogens and is also a prerequisite for
the subsequent activation and maturation of adaptive immune
responses [8,9]. PRRs have been assigned a central role in innate
immune responses due to their ability to recognize and respond to
evolutionarily conserved structures on pathogens termed PAMPs
PLoS ONE | www.plosone.org1January 2012 | Volume 7 | Issue 1 | e29291
. Activation of PRRs induces a proinflammatory and
antimicrobial response by triggering signal transduction pathways
involving NF-kB and the MAPK pathway, as well as IFN
regulatory factors (IRF)s ultimately resulting in the synthesis of
cytokines, chemokines, and antiviral type I IFN [11,12].
PRRs are divided into several families, including TLRs, retinoid
acid inducible gene (RIG)-like receptors (RLR)s, nucleotide-
binding and oligomerization domain-like receptors, and most
recently emerging families of DNA receptors [11,12]. In the
context of viral infection, the receptors for nucleic acids are
particularly important . Among the TLRs, the endosomally
located TLRs sense foreign nucleic acids with TLR3 recognizing
dsRNA and TLR7/8 sensing ssRNA, whereas TLR9 is activated
by unmethylated DNA [11,14–17]. In the cytosolic compartment,
the RLRs RIG-I and melanoma-differentiation-associated gene 5
are RNA helicases that play a pivotal role in sensing of cytoplasmic
RNA . Studies have suggested differential roles of these
receptors with RIG-I being responsible for recognizing 59tripho-
sphorylated RNA and short dsRNAs (e.g. stem-loop structures
[19–21], whereas melanoma-differentiation-associated gene 5
responds to long dsRNA and higher order RNA structures
[21,22]. Finally, cytosolic DNA receptors, which sense most types
of dsDNA, have been identified [23–27].
During the replication cycle of HIV, the HIV genome
consisting of two identical copies of positive strand ssRNA
together with the viral capsid is introduced into the cytoplasm of
the cell. During the process of reverse transcription, RNA:DNA
hybrids are generated, followed by cDNA and finally dsDNA,
which is transported into the nucleus and integrated into the host
cell DNA genome. Later during the HIV replication cycle, viral
genomic ssRNA and mRNA, the latter of which encodes structural
proteins, are synthesized and present in the cytoplasm . Thus,
several of the above mentioned PRRs may theoretically be
involved in recognizing various HIV nucleic acid structures and
trigger innate immune responses. The fact that HIV replication
takes place in the cytosolic compartment allows for HIV PAMPs
either present in the viral genome or synthesized during the viral
replication cycle to be be recognized by cytosolic PRRs.
One of the first links between HIV and PRRs was obtained
from a study demonstrating that U-rich ssRNA derived from HIV
is recognized by TLR7/8 and stimulates DCs and macrophages to
secrete IFN-a and proinflammatory cytokines . In agreement
with this, MyD88-dependent activation of plasmacytoid DCs
(pDC)s and monocytes by U-rich ssRNA from the HIV long
terminal repeat (ssRNA40) has been demonstrated . The
demonstration that endocytosis of HIV particles and the presence
of viral nucleic acids in the endocytic compartment is required for
pDC activation and IFN-a secretion further supports a role for
endosomal TLRs, including TLRs 3, 7/8, and 9 . Addition-
ally, histopathological studies in mice have revealed that sustained
TLR7/8-triggering results in lymphopenia, abolished antibody
production, and alterations in lymphoid micro-architecture
resembling HIV-mediated pathology . TLR7 has also been
attributed an important role in a recent study by Lepelley et al., in
which evidence was presented suggesting innate sensing of HIV-
infected lymphocytes by both endosomal TLR7-mediated- and
cytoplasmic pathways, the latter of which was dependent on
incoming viral material and IRF3 . Although the specific
cytosolic viral sensor was not identified, the work adds to a number
of other reports providing strong support for PRRs other than
TLRs in HIV recognition [32,33]. Recently, Hiscott and colleages
demonstrated that purified genomic RNA from HIV is detected by
the cytosolic RNA-sensor RIG-I and induces a type I IFN
response . However, evasion strategies in human monocyte-
derived macrophages seem to evade such responses during HIV
infection . Regarding sensing of HIV DNA, Stetson et al. have
demonstrated that retroviral cDNA activates a type I IFN response
through an unidentified DNA receptor . More recently,
Lieberman and associates presented evidence that the cytosolic
exonuclease TREX1 inhibits the innate immune response to HIV
by degrading ssDNA derived from integrated provirus . It was
demonstrated that in the absence of TREX1, a type I IFN
response is induced that inhibits HIV replication and spreading,
although the specific DNA sensor responsible remains to be
identified . Finally, a cell-intrinsic sensor for HIV has been
identified which mediates an antiviral immune response in DCs
dependent on the interaction with newly synthesized HIV capsid
and cellular cyclophilin A .
Here, we have investigated the innate immune response
induced by HIV genomic RNA in PBMCs. We report that HIV
genomic RNA and HIV-derived secondary structured RNA
localize to peroxisomes to induce innate immune responses with
low induction of type I IFN and higher induction of IFN-
stimulated genes (ISG)s by a mechanism dependent on RIG-I and
its down-stream adaptor protein MAVS.
Genomic HIV RNA induces innate immune responses in
Previous studies have identified a U-rich ssRNA sequence
derived from the HIV long terminal repeat (ssRNA40) with a
capacity to activate pDCs and monocytes through TLR7 and 8
[16,29]. However, the immuno-stimulatory potential of the entire
HIV genome has never been assessed in primary human cells. We
therefore transfected purified virion-derived HIV genomic RNA
into human PBMCs. As shown in Fig. 1A, HIV genomic RNA
induced expression of the chemokine CXCL10 in a dose-
dependent manner in PBMCs. Although the induction of
CXCL10 was weaker than the one observed using ssRNA40,
which is on a thio-ester backbone, we observed a robust induction,
when the cells we treated with 3 mg/ml of RNA. In addition,
introduction of RNA isolated from HIV virions into the cytoplasm
of cells led to modest induction of IFN-b (Fig. 1B). Finally, HIV
genomic RNA affected IFN-a, TNF-a, and IL-6 levels to only
marginal extent (Fig. 1 C–E).
RNA oligos derived from the HIV genome induce innate
immune responses in PBMCs
A structural model of the HIV genome has recently been
reported, suggesting a high degree of secondary structure . To
examine if the secondary structured regions of the HIV RNA
genome may contribute to its immuno-stimulatory activity, we
designed HIV-derived RNA oligos with varying degrees of
secondary structure based on biophysical predictions in RNAfold
(Fig. 2A). When comparing the ability of these RNA oligos to
induce CXCL10 and IFN-b expression in PBMCs we found that
the RNA oligos with secondary structure (Oligo 2/Tar and oligo
3) induced significantly higher amounts of CXCL10 and IFN-b
mRNA than oligo 1, 4, and 5, which were predicted not to possess
extensive secondary structure (Fig. 2B–D). The same observation
was done when CXCL10 expression was examined at the protein
level (Fig. 2E).
In order to characterize the innate response induced by
secondary structured HIV RNA, we performed stimulation of
primary human monocyte-derived macrophages with Tar. By RT-
qPCR analysis we found that Tar potently induced expression of
the ISGs CXCL10 and RIG-I within 6 hours of stimulation
Innate Immune Activation by HIV Genomic RNA
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(Fig. 2H–I), and also stimulated expression of IL-1b mRNA to
largely the same extent as the dsRNA mimic Polyinosinic:polycy-
tidylic acid (poly(I:C)), which was used as a positive control (Fig. 2J).
Although detectable, more modest levels of type I (IFN-b) and type
III IFN (IFN-l1) were induced by HIV Tar compared to Poly(I:C)
(Fig. 2F–G), and the levels of TNF-a mRNA were only affected
marginally by the HIV-derived RNA (Fig. 2K).
HIV RNA activates the NF-kB, p38, and IRF signaling
To characterize signaling triggered by HIV-derived RNA, we
next transfected human PBMCs with HIV Tar and oligo 4 and
harvested whole-cell extracts. As shown in Fig. 3A and B, Tar
induced phosphorylation of IkBa and p38 corresponding to
activation of the NF-kB and MAPK pathways, respectively.
Importantly, Tar RNA activated the NF-kB pathway to a
significantly higher extent than oligo 4 did (Fig. 3A).
With the apparent preference for induction of ISGs by HIV
RNA, we were particularly interested in examing the ability of
HIV Tar to activate transcription factors of the IRF family.
Nuclear extracts were isolated from cells treated for 0, 1, 3, and
6 h and the levels of binding of IRF-1, 3, and 7 to the ISRE
consensus sequence was determined by TransAM. As shown in
Fig. 3 C–E, stimulation with HIV Tar led to a modest activation of
DNA binding by IRF-1, a more robust activation of IRF-3, and no
detectable activation of IRF-7.
HIV Tar localizes to peroxisomes but not to mitochondria
Several publications within the last few years have underscored
the importance of the cytosolic context and involvement of
different organelles in the final outcome of cellular stimulation
with different PAMPs [39–41]. Most recently, signaling mediated
by RLRs from peroxisomes has been reported to result in an
innate response qualitatively different from RLR signaling from
mitochondria, with activation of IRF-1 and 3 and induction of a
subset of ISGs . Therefore, we were interested in identifying
subcellular localization of the HIV RNA after transfection into
PBMCs. To address this issue, we transfected cells with FAM-
labelled HIV Tar and stained with mitotracker (mitochondria) and
catalase (peroxisomes) to achieve organelle-specific staining. The
cells were fixed and examined by confocal microscopy (Fig. 4). In
agreement with previous publications, we found that synthetic
oligos transfected into cells exhibited distinct punctate locations
[27,42]. This is in contrast to the more even distribution of the
RNA observed after transfection of siRNAs, which are generally
shorter . We observed that the HIV-derived RNA oligo
localized to distinct areas in the cell, and did not display any co-
localization with mitochondria (Fig. 4A). By contrast, in a large
percentage of the cells we found remarkable co-localization
between HIV Tar RNA and the peroxisome marker catalase
(Fig. 4B). This was seen at both 1 and 3 h post RNA transfection
(Fig. 4 and data not shown). Thus, HIV-derived secondary
structured RNA co-localizes with peroxisomes but not mitochon-
dria after transfection into PBMCs.
HIV RNA induces innate responses dependent on RIG-I
To gain insight into the molecular mechanisms of HIV RNA
sensing, we incubated PBMCs with bafilomycin A1 (which inhibits
endosomal acidification and hence inhibits signaling by the
endosomal TLRs – TLR3, 7, 8, and 9) prior to stimulation with
HIV genomic RNA and HIV RNA oligos. Cell culture
supernatants were harvested for measurement of CXCL10
(Fig. 5A). As expected, bafilomycin abrogated CXCL10 produc-
tion in response to the TLR7/8 agonist ssRNA40. Likewise, we
observed a strong inhibition of the response induced by the non-
structured oligo 1. However, in the case of the HIV RNA oligos
with higher order secondary RNA structure, bafilomycin only
Figure 1. Genomic HIV RNA induces innate immune responses dominated by ISGs. PBMCs were stimulated for 16 h with HIV genomic RNA
in increasing doses (from 0.3 to 3.0 mg/ml). Positive and negative controls included ssRNA40 (2 mg/ml) and ssRNA41 (2 mg/ml), respectively.
Supernatants were harvested for measurement of (A) CXCL10, (B) IFN-b, (C) IFN-a, (D) TNF-a, and (E) IL-6. Data are shown as means of triplicates +/2
st.dev. Similar results were obtained in two or three independent experiments. Mock, Lipofectamine 2000 alone.
Innate Immune Activation by HIV Genomic RNA
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Innate Immune Activation by HIV Genomic RNA
PLoS ONE | www.plosone.org4January 2012 | Volume 7 | Issue 1 | e29291
exerted a minor inhibitory effect on CXCL10 production.
Importantly, genomic RNA displayed the same insensitivity to
bafilomycin A1 as the secondary structured oligos by inducing
CXCL10 in a manner not inhibited by bafilomycin A1. Thus, the
HIV RNA tested induced innate immune responses dependent
and independent of endosomal TLRs with the secondary
structured RNA and genomic RNA stimulating CXCL10
expression largely independently of these PRRs.
Given the observed TLR-independency of the response
triggered by genomic HIV RNA and the secondary structured
oligos, we were interested in examining the possible contribution
from alternative PRRs in sensing of HIV RNA and induction of
an ISG response. Since we were particularly interested in the role
of the RLR pathway, we focused on MAVS, an adaptor molecule
essential for RLR signaling . We generated mouse bone-
marrow-derived macrophages (BMM)s from C57BL/6 wildtype
and MAVS2/2 mice and stimulated the cells with HIV Tar and
viral genomic RNA. Importantly, both stimuli induced expression
of CXCL10 in the wild-type BMMs, but this response was strongly
inhibited in cells deficient in MAVS, and hence RLR signaling
(Fig. 5B). Measurement of CXCL10 expression after stimulations
with IFN-a Sendai virus, ssRNA40, and R848 showed that the
MAVS-deficient cells did have the capacity to induce CXCL10
and were specifically defect in the response to a well-characterised
RIG-I activating virus (Fig. 5B). In order to examine if RIG-I
could be responsible for the observed MAVS dependent activation
of innate immune responses, we used Huh7 cells and the derived
cell line Huh7.5 with defect RIG-I function . As seen in
Fig. 5C, transfection with HIV Tar led to induction of CXCL10 in
the parental Huh7 cells, which was not observed in the RIG-I
mutant cell line Huh7.5. Reconstitution of the cells with RIG-I
restored the response to HIV Tar RNA (Fig. 5D). In summary,
these data demonstrate that secondary-structured HIV RNA is
recognized by RIG-I, which induces innate immune response
Recently, innate immune responses have been appreciated to
play an important role in both control and pathogenesis of HIV
infection [13,29,36,45]. However, knowledge on specific HIV
PAMPs and cellular PRRs responsible for inducing innate
immune responses has remained relatively sparse. Here we have
identified the RIG-I/MAVS pathway as a sensor system of HIV
genomic RNA in PBMCs. In contrast, only a minor contribution
from endosomal TLRs was revealed. We investigated the
importance of secondary structures in the HIV genome and
found that the response to secondary structured but not
unstructured RNA oligos derived from the HIV-1 genome
mirrored the response to genomic RNA. Finally, we observed
co-localization between HIV Tar and peroxisomes suggesting a
possible involvement of this organelle as a signaling platform to
NF-kB and IRFs in activation of innate immune responses
directed against HIV.
Since most previous studies have been conducted mainly in cells
that are not natural hosts for HIV-1 infection and have been based
on short synthetic RNA oligos (often on the unnatural phosphor-
othioate backbone) derived from the HIV genome [16,29], our
study is unique in examining virion-derived HIV genomic RNA in
PBMCs. Given that the HIV genome is highly structured ,
Figure 3. HIV RNA activates the NF-kB, p38, and IRF signaling pathways. PBMCs were stimulated with HIV Tar (oligo 2, 2 mg/ml) and in panel
A also with oligo 4 (2 mg/ml). (A–B) Whole-cell lysates were isolated 2 h post treatment, and (C–E) nuclear extracts were isolated at the indicated time
points (IFN-c, 10 ng/ml, 6 h; SeV, MOI 1, 6 h; R848, 500 ng/ml, 6 h). (A–B) P-IkBa and P-p38 were measured by Luminex technology and (C–E) DNA
binding of IRF-1, 3, and 7 to an ISRE consensus sequence was measured by TransAM. Data are shown as means of duplicates or triplicates +/2 st.dev.
Similar results were obtained in two or three independent experiments. RU, relative units. Mock, Lipofectamine 2000 alone. *, p,0.05.
Figure 2. HIV RNA oligos derived from the HIV genome induce an innate immune response in human PBMCs and primary
macrophages. (A) Schematic illustration of the size, position and secondary structure of the HIV-derived RNA oligos used in this study. (B-E) PBMCs
were stimulated with different RNA oligos derived from the HIV genome (2 mg/ml) or with ssRNA40 (2 mg/ml). Total RNA and supernatants were
harvested after 6 and 20 h, respectively, and CXCL10 (mRNA and protein) and IFN-b (mRNA) levels were measured. Data are shown as means of
triplicates +/2 st.dev. Similar results were obtained in three independent experiments. (F–K) Primary macrophages were differentiated from human
PBMCs and stimulated for either 2 or 6 h with HIV Tar (oligo 2, 2 mg/ml). Transfected poly(I:C) (t-pIC) was included as a positive control (2 mg/ml).
Total RNA was isolated at the indicated time points and analyzed by qPCR for the presence of IFN-b (E), IFN-l1 (F), CXCL10 (G), RIG-I (H), IL-1b (I), and
TNF-a (J). Data are presented as mean values from analysis of 2 independent donors +/2 st.dev. Mock, Lipofectamine 2000 alone. RU, relative units.
Innate Immune Activation by HIV Genomic RNA
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such secondary structures may influence, how HIV RNA is
recognized in vivo by cellular PRRs. In the present study we found
that genomic HIV RNA induced the ISG CXCL10 and also to a
lesser extent IFN-b in human PBMCs. When we compared the
early response to different HIV oligos with varying degrees of
secondary structure, the more structured oligos induced higher
responses and displayed a PRR requirement resembling that of
genomic RNA. Specifically, we found that both genomic RNA
and synthetic HIV Tar induced CXCL10 expression in a RIG-I/
MAVS-dependent manner, whereas the non-structured RNA
induced a pathway dependent on endosomal acidification, most
likely TLR7. The recognition of HIV RNA by RIG-I is in
agreement with a recent report published by Hiscott and associates
. These authors reported a stronger IFN-response to
monomeric HIV RNA than to dimeric HIV RNA , but the
degree of secondary structure and possible implications of such
differences were not addressed. Given that full-length genomic
HIV RNA, is endowed with secondary modifications, including
capping and a poly-A-tail, which normally prevent mRNA from
being recognized by the innate immune system, it remains
unresolved how the HIV genome is recognized as foreign. One
possibility is that genomic HIV RNA may be organized with a
higher degree of secondary structures than most mRNA, the latter
of which may be associated with host factors that reduce secondary
structures and transport mRNA directly to the translational
complex in the endoplasmic reticulum.
During recent years, growing evidence suggests that subcellular
localization of signaling platforms and association with cellular
organelles play important roles in the timing and shaping of
inflammatory responses. For instance, mitochondria have been
demonstrated to play an essential part in the response to both
RNA and DNA through the involvement of the adaptor MAVS
linked to mitochondria and essential in signaling complexes
downstream of RLRs [25,39]. This allows for integration of signals
of viral infection or cellular stress, which may need coordination of
both pro-inflammatory and pro-apoptotic responses. Likewise, the
adaptor stimulator of interferon genes (also called STING) has
been demonstrated to be associated with membranes of both the
endoplasmic reticulum, the Golgi apparatus, and perinuclear
vesicles [40,46]. Therefore, we were interested in visualizing the
cellular fate of the transfected HIV oligos, and in particular their
possible co-localization with cellular organelles in the cytosol. By
confocal microscopy and colorization of relevant cellular organ-
elles, we observed that HIV Tar oligos tended to assemble in
specific areas, which also stained for markers for peroxisomes but
not mitochondria. This finding suggests that the Tar oligo used
may interact with specific molecules, including adaptor proteins or
receptors, linked to peroxisomal structures. Indeed, a recent report
by Kagan and associates have implicated peroxisomes in immune
defences by demonstrating localization of MAVS to peroxisomal
membranes, where it is essential for inducing a rapid IFN-
independent response, including ISGs, and dependent on the
transcription factors IRF-1 and 3 . The authors suggested that
peroxisomal MAVS may induce a rapid antiviral defence
programme serving to provide short term protection, until
mitochondrial MAVS is able to induce an IFN-dependent
signaling pathway with delayed kinetics aiming at amplifying
and stabilizing the antiviral response . In the present study, we
found that HIV RNA co-localized with peroxisomes and activated
IRF-1 and 3, as well as NF-kB and p38. By contrast, co-
localization between RNA and mitochondria was not observed,
even at later time points (data not shown). The involvement of
signaling from the peroxisome platform in response to HIV RNA
was further supported by the gene expression pattern dominated
by ISGs with lower induction of type I and III IFN. Thus, during
HIV infection, genomic RNA present in the cytosolic compart-
ment soon after viral entry may induce a rapid antiviral response
via peroxisomal MAVS, whereas other viral PAMPs present later
during the replication cycle may induce a more sustained IFN-
dependent response involving mitochondrial MAVS.
Previous studies using the synthetic HIV RNA oligo ssRNA40
have demonstrated MyD88-dependent signaling mediated by
TLR7 in pDCs resulting in production of IFN-a . Several
other studies have also provided evidence for the involvement of
additional endosomal TLRs, including TLR3 and TLR9 in
response to retroviruses [47,48]. However, the confinement to the
endosomal compartment of these receptors requires either
endocytic uptake of viral material, including HIV RNA, from
the extracellular environment, or alternatively transport of these
structures from the cytosol into the endolysosomal compartment
Figure 4. HIV Tar co-localizes with peroxisomes but not with mitochondria. PBMCs stimulated with 1.2 mg/ml FAM-labeled HIV-Tar RNA
(green) for 180 min were labeled for mitochondria (red) or peroxisomes (red). Nuclei were stained with DAPI (blue). Co-localization is shown in yellow,
scale bar 20 mm. Representative images are shown in A, while panel B shows a quantification of percentage RNA-organelle co-localization as obtained
by counting more than 100 cells per group. Similar results were obtained in two independent experiments.
Innate Immune Activation by HIV Genomic RNA
PLoS ONE | www.plosone.org6 January 2012 | Volume 7 | Issue 1 | e29291
by autophagy . Using bafilomycin A1, an inhibitor of
endolysosomal acidification and TLR signaling, we observed no
significant inhibition of the innate response to genomic RNA or
secondary structured RNA oligos. Together with the strong
reduction of CXCL10 synthesis observed in cells deficient in RLR-
triggered signaling through MAVS, this corroborates the finding
that HIV genomic RNA is sensed primarily by cytosolic RIG-I
rather than endocytic TLRs in PBMCs. This is in agreement with
Figure 5. The innate immune response induced by HIV genomic RNA or RNA oligos is dependent on RIG-I and MAVS. (A) PBMCs were
treated with bafilomycin A1 (0.5 mM) as indicated 15 min prior to stimulation with genomic HIV RNA, RNA oligos, or ssRNA40 (all 2 mg/ml). IFN-a was
included as a positive control (10 ng/ml). Supernatants were harvested 18 h post stimulation for measurement of CXCL10. (B) BMMs from C57BL/6
wildtype and MAVS2/2 mice were stimulated with genomic HIV RNA (2 mg/ml), Tar (2 mg/ml), Sendai virus (MOI 1), IFN-a (10 ng/ml), ssRNA40 (2 mg/
ml), or R848 (2 mg/ml). Supernatants were harvested after 16 h and CXCL10 was measured by ELISA. UT, untreated cells. Data are shown as means of
triplicates +/2 st.dev. (C) Huh7, Huh7.5 (RIG-I mutant), Huh7.5 EV (empty vector), and Huh7.5 RIG-I cells were transfected with Tar RNA (2 mg/ml), or
subjected to mock transfection with Lipofectamine 2000. Total RNA was harvested 6 h later and CXCL10 mRNA levels were analysed by qPCR. Data
are shown as means of triplicates +/2 st.dev. Similar results were obtained in two or three independent experiments. Mock, Lipofectamine 2000
alone. RU, relative units *, p,0.05.
Innate Immune Activation by HIV Genomic RNA
PLoS ONE | www.plosone.org7 January 2012 | Volume 7 | Issue 1 | e29291
a study by Solis et al. in which genomic HIV RNA was reported to
activate RIG-I and elicit a type I IFN response . However, the
authors went on to show that de novo HIV replication in
amounts of IFN. This could be attributed at least in part to an
HIV protease-dependent targeting of RIG-I to lysosomes resulting
in inhibition of IRF-3 phosphorylation and decreased expression
of IFN and ISGs . Reduced levels of IRF3 have also been
demonstrated in T cells in the presence of productive HIV
replication. Interestingly this observation could be extrapolated to
HIV-infected patients, in whom reduced IRF3 levels in CD4+ T
cells were found only in acute HIV infection but not in long-term
non-progressors . Evidence for the involvement of both TLR-
and cytosolic HIV-sensing depending on the cell type studied has
been reported by Lepelley et al. In a study where HIV-infected
cells were co-cultured with target cells (PBMCs or pDCs), it was
demonstrated that virus-infected cells in contrast to cell-free virions
elicited a strong IFN and ISG response . The authors
suggested that incoming viral material may be taken up by target
cells via endocytosis, thereby allowing direct delivery of the viral
RNA genome to endosomal TLRs. Indeed, recognition was
reported to occur largely through TLR7 in pDCs and pDC-like
cells rather than through cytosolic sensors , although evidence
was also provided for the participation of cytosolic IRF3 activation
in the absence of TLR7. However, the cytosolic HIV sensor
involved was not definitively identified, but possible candidates are
RIG-I or DNA receptors. Taken together, these studies together
with our work illustrate that important cell type differences exist in
sensing of HIV PAMPs and suggest that the PRR(s) involved may
be dependent on the mechanism, by which the virus and viral
replication products are presented to the cell, as well as the evasion
strategies employed by the virus.
The establishment of RIG-I in recognition of the HIV RNA
genome in the present work is novel and contributes, together with
the report by Solis et al. reaching similar conclusions , to
extending the current knowledge on innate immune sensing of HIV
PAMPs by host PRRs. This finding may also have important
implications for therapeutic aspects of HIV infection, both in
vaccine design and in future development of novel antiretroviral
treatment strategies. From a clinical perspective,it has recentlybeen
reported that chloroquine therapy during chronic HIV infection
reduces immune activation, suggesting a role of endosomal TLR
signaling induced by HIV . In a similar manner, RIG-I may
represent a novel target of antiretroviral treatment. Depending on
whether RIG-I turns out to play beneficial roles for the host or the
virus, targeting this PRR with either agonists or antagonists could
have impact on host restriction of viral replication, or alternatively
inhibit chronic immune activation, hence affecting the immuno-
pathogenesis and preventing the progression to immunodeficiency
during chronic HIV infection.
There is now evidence that HIV-1 has the potential to stimulate
the innate immune system through its RNA, DNA, and capsid
[16,34,36,37]. However this virus seems to be capable of efficiently
evading these sensing systems. It will be interesting to learn if RIG-
I is also operative during actual HIV infection in natural host cells
and to determine to what extent this PRR system contributes to
activation of early defence mechanisms against HIV.
Materials and Methods
The work does not contain experiments using living animals.
Experiments involving cells from mice sacrificed prior to any
experimental procedures do not require permission according to
Danish law (The Animal Protection Law). The work does contain
human studies. Approval was received from the local ethical
committee (Committee for Research Ethics for Mid-Jutland
County, permission number M-20110108) and informed written
consent from all participating subjects was obtained.
The primary human cell populations used were PBMCs, and
monocyte-derived macrophages. PBMCs were isolated using
Ficoll-Plaque purchased from GE Healthcare. Briefly, the blood
was placed under a layer of Ficoll-plaque and centrifuged at 6006
g for 30 min. Cells from the interphase layer was harvested,
washed twice in phosphate-buffered saline, and resuspended in
RPMI-1640 medium containing 10% heat-inactivated FCS, and
antibiotics (penicillin and streptomycin). For experiments, cells
were seeded in 96-, 24- or 6-well plates at a density of 26105,
16106, or 46106cells per well, respectively, and left for at least
6 hours before further treatment.
The human hepatocarcinoma cell line Huh7, and the derived
cell lines Huh7.5, Huh7.5 EV (empty vector) and Huh7.5-RIG-I
(kindly donated by Ralf Bartenschlager, Heidelberg, Germany)
were maintained in DMEM supplemented with 10% FCS. For the
latter 2 cell lines, the medium was supplemented with 250 mg/ml
of G418 (Roche). For experiments, 76105cells were seeded per
well in 6-well plates and left 3–4 h prior to further treatment.
For generation of monocyte-derived macrophages, PBMCs
were isolated from blood (leukocyte-rich buffy coats). Mononu-
clear cells were allowed to adhere onto plastic six-well plates
(Falcon Multiwell; BD Biosciences) or plastic 24-well plates for 1 h
at 37uC in RPMI 1640 medium supplemented with penicillin
(0.6 mg/ml), streptomycin (60 mg/ml), glutamine (2 mM) and
HEPES (20 mM). After monocyte binding, non-adherent cells
were removed and the wells were washed twice with PBS, pH 7.4.
Adherent cells were then grown for 7–8 days in Macrophage-SFM
medium (Life Technologies) supplemented with antibiotics and
granulocyte–macrophage colony-stimulating factor (GM-CSF;
Nordic Biosite) at 10 ng/ml. GM-CSF-containing medium was
removed from the cells 1 day before further stimulation . The
isolated cells were determined to be macrophages by their typical
morphology and cell-surface CD14 expression . Cells from
individual blood donors were grown separately, but after
stimulation experiments, they were pooled.
Mouse BMMs were obtained as follows: femur and tibia were
surgically removed from C57BL/6 wildtype and MAVS-deficient
mice (kindly provided by Professor Z.J. Chen, Southwestern Medical
Center, Dallas Texas), freed of muscles and tendons, and briefly
suspended in 70% ethanol. Ends were cut, the marrow was flushed
with 10% RPMI 1640, and the cell suspension was filtered over a 70-
mm cell strainer (BD Falcon) and centrifuged for 5 min at 1330 rpm.
After 2 washes, cells were resuspended at 26105cells/ml in RPMI
1640 with 10% FCS and GM-CSF (10 ng/ml), seeded in
bacteriological petri dishes, and incubated at 37uC with 5% CO2
and media changed after 3 and 5 days. On day 7, adherent cells were
harvested from the dishes with medium containing GM-CSF (10 ng/
ml). The cells were centrifuged, washed, and resuspended in RPMI
1640, 10% FCS, and GM-CSF (20 ng/ml), and examined by flow
cytometryforexpression of CD11b and CD11c(datanot shown).For
in vitro experiments, the cells were used at a concentration of 1.06106
cells per well in 96-well plates in 100 ml medium.
HIV genomic RNA and RNA oligos
HIV genomic RNA was isolated as previously described
[38,53]. Briefly, the virus was treated with DNase I and RNase
Cocktain (RNase T1 and RNase A, Ambion) to remove extra-
Innate Immune Activation by HIV Genomic RNA
PLoS ONE | www.plosone.org8January 2012 | Volume 7 | Issue 1 | e29291
virion nucleic acids, and then subjected to subtilisin digestion. The
lysates were phenol:chloroform:isoamyl alcohol-extracted and
ethanol-preciptated. The RNA was re-suspended in water and
used for experiments. The synthetic RNA oligos used were
ssRNA40 (InvivoGen), 59-GCCCGUCUGU UGUGUGACUC-
39; Oligo 1, 59-AGGAAGAAGC GGAGACAGCG ACGAA-
GAGCU CAUCAGAACA GUCAGACUCA -39; Oligo 2/Tar,
59-UCUCUGGUUA GACCAGAUCU GAGCCUGGGA GCU-
CUCUGGC UAACUAGGGA-39; Oligo 3, 59- GUCAACAUAA
UUGGAAGAAA UAUGUUGACU CAGAUUGGUU GCA-
CUUUAAA UUUUCCAAUU -39; Oligo 4, 59-GAGTAGTAGA
ATCTATGAAT AAAGAATTAA AGAAAATTAT AGGA-
CAGGTA–39; Oligo 5, 59-CACGGACAAT GCTAAAACCA
TAATAGTACA GCTGAACACA TCTGTAGAAA-39 (all DNA
Technology) (Fig. 2A). ssRNA40 is on phosphorothioate back-
bone, whereas oligos 1–5 were synthesized with 39 and 59 OH-
ends and on phosphodiester backbone. Secondary structures of the
RNA oligos used were predicted using the RNAfold Webserver
RNA oligos or genomic HIV RNA was transfected into cells
using Lipofectamine 2000 (Invitrogen). RNA and Lipofectamine
2000 complexes were prepared in OptiMEM in the ratio of 10 ml
lipofectamine per 4 mg RNA. After 20 min of incubation, the
RNA mixture was added to cells resulting in a final concentration
of RNA between 1 and 3 mg/ml.
Reagents for cell stimulation, infections and transfections
Control stimuli were added directly to the media and included
R848 (TLR7/8 agonist, 5 mg/ml), ssRNA40 (TLR7 agonist,
2 mg/ml), ssRNA41 (negative TLR7 control, 2 mg/ml) (all from
InvivoGen), IFN-a (10 ng/ml, PBL InterferonSource), IFN-c
(100 ng/ml, R&D Systems), Sendai virus (MOI 1). Inhibition of
endosomal acidification, and thereby TLR3, 7/8, and 9 signaling
was achieved by pre-incubating the cells with bafilomycin A1
(0.5 mM, Sigma Aldrich) 15 min prior to stimulation. Sendai virus
was used at multiplicity of infection (MOI) of 1.
RNA isolation and RT
Macrophages were washed once with PBS and lysed, and total
cellularRNA was recovered using an RNA purification kit (Macherey-
Nagel NucleoSpin RNA II or Qiagen Midi kit) as described by the
manufacturer. Total RNA from PBMCs was recovered by Trizol
(Invitrogen) as described by the manufacturer. Purified RNA was
dissolved in water and stored at 280uC until analysis. cDNA synthesis
was performed with 1 to 2 mg RNA by using a Moloney murine
leukemia virus reverse transcriptase kit (Invitrogen) according to the
manufacturer’s instructions with an oligo(dT)18 primer (DNA
Technology, Aarhus, Denmark)  or as described previously .
Quantitative real-time PCR
cDNA obtained from macrophages was quantified by TaqMan
real-time PCR using primers and probes from Applied Biosystems
for IFN-b, IFN-l1, CXCL10, RIG-I, TNF-a, and IL-1b, as
previously described . cDNA from PBMCs was amplified by
realtime PCR using primers for GAPDH, IFN-b, and CXCL10
(DNA Technology), as previously described .
Detection of Phosphoproteins
For detection of the phosphorylation status of IkBa and p38
Luminex technology was used. Briefly, the filter plate was washed
with assay buffer, and 50 ml of freshly vortexed antibody-
conjugated beads were added to each well. The plate was washed
with assay buffer, and samples of whole-cell lysates were added.
After a brief shake (30 s at 1100 rpm), the plate was incubated at
4uC overnight in the dark with light shaking (300 rpm). After one
wash step, 25 ml of the detection antibody was added to each well,
and the plate was shaken and incubated as above. Subsequently,
the plate was washed and incubated for 30 min with 50 ml of a
streptavidin-PE solution with shaking (30 s at 1100 rpm, 10 min at
300 rpm). Finally, the plate was washed, 125 ml of assay buffer was
added to each well, and the plate was shaken for 10 s at 1100 rpm
and read immediately on a Bio-Plex reader.
IRF DNA-binding activity
ELISA-based measurement of DNA-binding activity of nuclear
IRF family members was performed using TransAM, following the
protocol of the manufacturer (Active Motif). Briefly, 5 mg of nuclear
extract, isolated with the Nuclear Extract Kit (Active Motif), was
used per sample in duplets in a 96-well plate pre-coated with
consensus oligonucleotides for the IFN-stimulated response element.
After washing to remove nonspecific binding, antibodies specific for
IRF-1, -3, and -7 were added. After antibody binding, the plate was
washed again before adding a HRP-conjugated secondary antibody.
The peroxidase substrate was added, and colorimetric change was
measured at an optical density of 450 (OD450).
Measurement of cytokines
Human and murine CXCL10 were detected by Cytoset (Invitro-
gen) and Duoset (R&D Sytems), respectively. IFN-b was measured by
an ELISA kit from PBL InterferonSource. Analysis was performed as
described by the manufacturers. The result was visualized by the
tetramethylbenzidine system (R&D Systems or KemEnTec) and
IL-6, and TNF-a were measured by Luminex technology, using a
custom-made three-plex kit, purchased from Bio-Rad (Hercules, CA),
following the instructions of the manufacturer. Detection limits for the
cytokine analysisassays:CXCL10,17 pg/ml;IFN-b,2 U/ml;IFN-a,
25 pg/ml; TNF-a, 13 pg/ml; IL-6, 20 pg/ml.
Human PBMCs were plated at 16106cells/ml overnight in
RPMI containing 10% FCS onto 12 mm coverslips. Cells were
transfected, with 1.2 mg/ml FAM-labelled HIV-Tar RNA at a
RNA:Lipofectamine 2000 (Invitrogen) ratio of 1:2.5 (mass:volume)
for 180 min. Cells were subsequently fixed with methanol at
220uC for 5 min and stained with anti-Catalase (Abcam) to label
peroxisomes or with DAPI to label nuclear DNA. To label
mitochondria 200 nM Mitotracker Far Red (Invitrogen) was
added to cells 15 min prior to fixation. Images were gathered
using Zeiss LSM 710 confocal microscope, 6361.4 oil objective.
Image processing was performed by MacBiophotonics Image J.
The data are presented as means 6 standard deviations
(st.dev.). The statistical significance of differences between
observations was estimated with the Wilcoxon rank sum test. P
values of ,0.05 were considered statistically significant.
The technical assistance of Erik Hagen, Lene Svinth, and Kirsten S.
Petersen is greatly appreciated. We thank professor Z.J. Chen for
generously providing bones from MAVS2/2 mice and professor Ilkka
Julkunen, Helsinki, for Sendai virus. Huh7 and derivative cell lines were
kindly donated by Ralf Bartenschlager, Heidelberg, Germany.
Innate Immune Activation by HIV Genomic RNA
PLoS ONE | www.plosone.org 9January 2012 | Volume 7 | Issue 1 | e29291
Conceived and designed the experiments: RKB JM SRP THM. Performed
the experiments: RKB JM JR ED SS KAH. Analyzed the data: RKB JM
SRP THM. Contributed reagents/materials/analysis tools: RJG CSL LO
SM. Wrote the paper: RKB THM.
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