HIV-1 mediates global disruption of innate antiviral signaling and immune defenses within infected

37 Pages·2009·2.46 MB·English

JVI Accepts, published online ahead of print on 12 August 2009
J. Virol. doi:10.1128/JVI.00849-09
Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

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2 HIV-1 mediates global disruption of innate antiviral signaling and immune
3 defenses within infected cells
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5 Brian P. Doehle1, Florian Hladik2,3,5, John P. McNevin2, M. Juliana McElrath2,3,4, D
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6 and Michael Gale Jr.1 n
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8 1University of Washington School of Medicine, Department of Immunology f
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9 Seattle, WA 98195; 2Vaccine and Infectious Disease Institute, Fred Hutchinson Cancer Research

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10 Center, Seattle, Washington 98109; Departments of 3Medicine,4 Laboratory Medicine, and tp
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11 5Obstetrics and Gynecology, University of Washington School of Medicine, Seattle, Washington jvi.
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12 98105.
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14 Address correspondence to: Dr. Michael Gale Jr., University of Washington School of
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15 Medicine, Department of Immunology, BOX 357650, Seattle, WA 98195. Phone: [phone] il
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16 Fax: [phone] Email: [email protected] 2
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17 Running Title: HIV-1 antagonism of IRF-3
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19 Manuscript information: 31 text pages, 6 figures, Abstract 220 words, Text 5874 words, 45 e
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20 References
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1 Abstract:
2 Interferon regulatory factor (IRF)-3 is essential for innate intracellular immune defenses
3 that limit virus replication but these defenses fail to suppress HIV infection, which can ultimately
4 associate with opportunistic co-infections and the progression to AIDS. Here, we examined
5 antiviral defenses in CD4+ cells during virus infection and co-infection, revealing that HIV-1
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6 directs a global disruption of innate immune signaling and supports a co-infection model through o
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7 suppression of IRF-3. T cells responded to paramyxovirus infection to activate IRF-3 and lo
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8 interferon–stimulated gene expression but they failed to mount a response against HIV-1. The e
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9 lack of response associated with a marked depletion of IRF-3 but not IRF-7 in HIV-1-infected o
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10 cells that supported robust viral replication, whereas ectopic expression of active IRF-3 h
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11 suppressed HIV-1 infection. IRF-3 depletion was dependent on a productive HIV-1 replication //
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12 cycle, and caused the specific disruption of Toll-like receptor and RIG-I-like receptor innate s
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13 immune signaling that rendered cells permissive to secondary virus infection. IRF-3 levels were o
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14 reduced in vivo within CD4+ T cells from acutely HIV-1 infected patients but not from long- o
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15 term non-progressors. Our results indicate that viral suppression of IRF-3 promotes HIV-1 p
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16 infection by disrupting IRF-3-dependent signaling pathways and innate antiviral defenses of the 2
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17 host cell. IRF-3 may direct an innate antiviral response that regulates HIV-1 replication and viral 1
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18 set point while governing susceptibility to opportunistic virus co-infections. y

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1 Introduction:
2 Immune evasion and dysregulation of the immune response to infection are major
3 features that support HIV-1 infection and pathogenesis. Acute exposure to HIV-1 through direct
4 mucosal contact initiates infection in resident CD4+ cells, in which cell-intrinsic innate antiviral
5 defenses impose the first level of immunity and restriction against infection (18,23). Innate
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6 immune host factors induced by type I interferons (IFN), including members of the o
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7 apolipoprotein B mRNA editing catalytic enzyme (APOBEC) and TRIM families, and products lo
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8 of certain interferon stimulated genes (ISGs) such as ISG15 and ISG20, have been defined as e
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9 HIV restriction factors because their effector actions can limit HIV infection (9,10,33,34). o
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10 However, innate antiviral defenses are overall largely ineffective at suppressing acute HIV-1 h
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11 infection in vivo, and the virus most often progresses to a chronic infection after acute exposure. //
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12 This inability to control HIV-1 infection has in part been attributed to properties of the virus that s
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13 inhibit specific host defense factors but the overall impact of HIV-1 on global intracellular innate o
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14 immune programs has not been defined (9,34). o
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15 Innate antiviral immune defenses are triggered during virus infection through the p
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16 recognition of viral products by host cell pathogen recognition receptors (PRRs). RIG-I-like 2
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17 receptors (RLRs) and the Toll-like receptors (TLRs) are PRR families that recognize microbial 1
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18 ligands known as pathogen associated molecular patterns (PAMP) to initiate intracellular y

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19 signaling cascades in the infected cell that induce IFN expression and production to direct a e
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20 cellular antiviral state mediated by ISGs. ISG products, including IFN-induced proinflammatory
21 cytokines, have antiviral and/or immunomodulatory functions that serve to suppress virus
22 replication and enhance adaptive immunity, thus mediating a response that controls the viral “set
23 point” and limits virus dissemination to peripheral sites (27,35). A central feature of
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1 PRR signaling involves the activation of interferon regulatory factors (IRFs) and NF-κβ. Among
2 the IRF gene family IRF-3, IRF-7, and IRF-9 play critical roles in inducing IFN and ISG
3 expression. Whereas IRF-3 is widely expressed and highly abundant in most tissues, including T
4 cells and macrophages, IRF-7 expression is more restricted. While IRF-7 constitutively
5 expressed in plasmacytoid dendritic cells (pDCs) and certain hematopoetic cells, it is typically
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6 induced by IFN in most tissues where it serves to amplify the innate response (27). IRF-9 is o
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7 widely expressed at a low level and is induced by IFN to play a pivotal role in mediating IFN lo
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8 signaling of ISG expression through its interactions with the signal transducer and activator of e
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9 transcription (STAT)-1 and STAT-2 (27). In this respect IRF-7 and IRF-9 lie downstream of o
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10 IRF-3 in a variety of cell types. RLRs signal innate defenses through the activation IRF-3, and t
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11 signaling bifurcates to trigger the additional activation of NF-κβ, thereby directing the /jv
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12 expression of both IRF-3 and NF-κβ-target genes 7. Moreover, TLR3 and TLR4 signal innate s
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13 defenses and IFN production through the TRIF or TRAM adaptor proteins that activate IRF-3 r
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14 and also converge on the NF-κβ activation pathway (31). Thus, processes that regulate signaling n

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15 outcome of the RLRs, TLR3 or TLR4 globally impact innate immune gene expression. r
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16 Many pathogenic viruses direct strategies to antagonize innate defenses and IRF ,
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17 activation in order to support viral replication (27), and control of IRF-3 has been defined as a 9

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18 major determinant of evasion from innate antiviral defenses. Recent observations suggest that
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19 HIV-1 may antagonize IRF-3 (32), but its effects on mucosal infection and PRR pathway s
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20 function, and how it impacts IRF-3 within CD4+ T cells from HIV-1 infected patients has not
21 been defined. Moreover, various studies have implicated HIV-1 or the related SIV in the
22 regulation of IFN defenses but the mechanisms underlying such regulation are not known
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1 (20,29,40,44).
2 In the present study we examined IRFs and PRR signaling during HIV infection. We
3 observed the suppression of RLR signaling and TRIF/TRAM-dependent TLR signaling in HIV-
4 1-infected cells attributed to viral suppression of IRF-3 and that supported acute infection in
5 mucosal tissue. Our studies demonstrate that IRF-3 can direct a robust innate antiviral response
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6 that controls HIV-1 replication but that HIV-1 suppression of IRF-3 abrogates these defenses o
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7 lending support of chronic infection and increasing the potential for opportunistic virus co- lo
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8 infection. These data validate observations of innate immune regulation by HIV and extend to e
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9 implicate immune regulation at the virus/IRF-3 interface as a critical feature that contributes to o
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10 the direction of HIV-1 infection outcome through immune evasion and support of opportunistic h
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11 co-infections. //
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13 Materials and Methods o
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14 Cell culture and transfections. All cells were grown under standard conditions. SupT1, o
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16 and antibiotics. PBMCs were additionally activated with hIL2 (Roche) and PHA (Sigma) prior 2
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17 to use. HEK293, 293T, 293-TLR3, HeLa CD4+, and Tzm-bl cells were cultured in DMEM 1
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18 supplemented with 10% FBS, L-glutamine, and antibiotics. The 293-TLR3 cells (Invivogen) y

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19 were additionally supplemented with 10 µg/ml blasticidin to maintain expression of TLR3. e
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20 PBMCs were obtained from random screened blood packs from the American Red Cross (ARC
21 Portland) and purified using standard procedures on Ficol gradients except those used in Figure 6
22 (see below). Transfection of adherent cells was performed using the calcium phosphate method.
23 SupT1 cells were transfected using Fugene6 transfection reagent (Roche) according to the
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1 manufacture’s suggested protocol. RNA transfections were preformed using TransMessenger
2 (QIAGEN) according to the manufacture’s instructions.
3 HIV study population and cell isolation. We evaluated person with newly diagnosed
4 HIV-1 infection (acute infection), with long-term non-progressive HIV disease (LTNP), and with
5 no evidence of HIV-1 infection (control). Subjects with acute infection, enrolled based on
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6 previously defined criteria and followed longitudinally at the University of Washington Primary o
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7 Infection Clinic (6), were selected based upon the availability of cryopreserved PBMCs from lo
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8 leukapheresis performed during primary infection. Primary infection was documented by signs e
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9 and symptoms consistent with mucosa, obtained following an acute retroviral syndrome o
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10 (6,38,39). LTNP persons (defined as HIV-infected >10 years, viral load <10,000 copies/ml, and h
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11 sustained CD4+ counts ≥500 cells/uL in the absence of antiretroviral therapy (ART)) as well as //
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12 seronegative subjects, were enrolled and followed at the Seattle HIV Vaccine Trials Unit. The s
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13 appropriate IRB approved the studies, and volunteers provided written consent. o
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14 PBMCs were isolated and cryopreserved as described previously (7). Cryopreserved o
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15 PBMC were thawed and rested overnight at 37°C/5% CO2 prior to isolation of CD4+ and CD4- p
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16 T cell subsets. CD4+ and CD4- T cell subsets were isolated using the RoboSep Automated Cell 2
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17 Separator and CD4 T cell enrichment kits according to the manufacturer’s protocol (STEMCELL 1
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18 Technologies, Vancouver, BC). Purity of CD4+ T cell fractions were assessed by flow y

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19 cytometry with the average purity of CD4+ T cell fractions >95% (data not shown). Isolation of e
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20 T cells from healthy vaginal mucosa obtained from patients undergoing vaginal repair surgeries,
21 was carried our as described in detail previously (16,17), and followed our Institutional Review
22 Boards (IRB) approved protocol with written subject consent.
23 Cell treatments. CHX (Sigma) and IFN-β (Toray) were used in the indicated
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1 experiments. CHX was used at 75ug/ml, and IFN-β was used at 50U/ml. pI:C (Sigma) was used
2 at the indicated concentrations.
3 Viral stocks and infection. HIV-1 was propagated using standard procedures through
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4 CEM-SS cells grown in RPMI supplemented with 10% FBS and antibiotics. HIV-1 strains
5 pNL4-3, JR-CSF, and BA-L were obtained as proviral clones and transfected into 293T cells as
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6 described previously to generate infectious virus (16,17). All HIV-1 strains were titered on Tzm- o
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7 bl cells to determine concentration of infectious virus, unless otherwise noted. Sendai virus lo
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8 (SenV) strain Cantell was obtained from Charles River Laboratory. e
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9 Immunoblot analysis and immunofluorescent imaging. SDS-PAGE, immunoblot o
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10 analysis, and immunofluorescence imaging were performed using standard procedures as h
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11 described previously (26). The following antibodies were used in the study: Rabbit (Rb) αISG15 /jv
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12 (A. Haas), Rb αISG56 (G. Sen), Mouse (m) αp24, Goat (Gt) αB-actin (Santa Cruz), Rb total s
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13 αIRF-3 (a gift from Michael David), Rb IRF-3-p (Cell Signaling), Rb IRF-7 (Santa Cruz), Gt r
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14 αSenV (Charles River Laboratory). For immunoblot applications the appropriate HRP- n

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15 conjugated secondary antibody was used (Jackson Immunoresearch Laboratories) followed by r
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16 treatment of the membrane with ECL-plus reagent (Roche) and imaging on X-ray film. ,
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17 Densitometry was performed using ImageJ (NIH) on unsaturated blots. For immunofluorescence 9

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18 imaging, appropriate Alexa Flour secondary antibodies (Invitrogen) used along with DAPI
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19 during secondary staining for each slide. All images were photographed on a Nikon TE2000-E s
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20 microscope and processed with Nikon EIS-Elements software.
21 qPCR. Quantitative RT-PCR (qPCR) using SYBR Green technology has been described
22 previously (37). IRF-3, IFN-β, and GAPDH specific primers are commercially available
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1 (SuperArray). GAPDH was used for normalization in all cases. RNA was extracted using the
2 RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions.
3 Dual luciferase assays. Dual Luciferase assays (Promega) were performed according to
4 the manufacturer’s specifications. The IFN-β, ISG56, ISRE, PRDII, and NF-κβ promoter
5 plasmids have all be described previously (11,12). Transfections were preformed at the ratio
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6 3.75: 1: 0.25 of provirus/viral construct: promoter luciferase: internal control (Renilla) for all o
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7 experiments. lo
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8 Statistical analysis. Differences between groups were analyzed for statistical e
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9 significance by the Student's t-test. o
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10 The following reagents were obtained through the AIDS Research and Reference t
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11 Reagent Program, Division of AIDS, NIAID, NIH: Tzm-bl cells from Dr. John C. Kappes, Dr. //jv
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12 Xiaoyun Wu and Tranzyme Inc, monoclonal antibody to HIV-1 p24 (AG3.0) from Dr. Jonathan s
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13 Allan, pNL4-3 from Dr. Malcolm Martin, CEM-SS from Dr. Peter L. Nara, pcDNA-HVif from r
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14 Dr. Stephan Bour and Dr. Klaus Strebel, pEGFP-Vpr from Dr. Warner C. Greene, darunavir o
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15 from Tibotec, Inc., and indinavir sulfate. p
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16 ,

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17 Results: 1
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19 To determine how HIV-1 impacts the host cell innate antiviral response we examined e
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20 PRR signaling and ISG expression during acute virus infection in vitro. Infection of CD4+
21 SupT1 T cells with Sendai virus (SenV), a model paramyxovirus, triggered the expression of
22 IRF-3 target genes, including ISG15 and ISG56 (13). Expression of both ISG15 and ISG56 in
23 SenV-infected cells occurred concomitant with the activation of IRF-3, the latter observed as a
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1 transient reduction of IRF-3 coupled with the appearance of the slower migrating IRF-3 species
2 representing the phosphorylated, active IRF-3 isoform (Fig 1A) (2,19). In contrast, ISG
3 expression was not induced in SupT1 cells infected with HIV-1 even under conditions of high
4 multiplicity of infection (MOI). A MOI of 10 was used to ensure we would not miss any low
5 level ISG induction. The lack of ISG expression in HIV-1 infected cells associated with a
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6 marked depletion of IRF-3 levels coincident with the appearance and accumulation of viral o
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7 proteins (Fig 1A). Similar results were obtained when Jurkat, H9, CEM174 and CEM-SS cells lo
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8 were infected with HIV-1 (data not shown). We performed the same experiments using cultures e
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9 of purified PBMCs as targets of HIV-1 infection. As shown in Fig. 1A, SenV infection of o
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10 PBMCs induced a robust expression of ISG15 and ISG56 in association with the transient h
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11 activation of IRF-3 but ISGs were not expressed in HIV-1-infected PBMCs. We observed a //
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12 relative reduction of IRF-3 protein levels in PBMC cultures that occurred in association with s
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13 viral protein accumulation (Fig. 1A right panels). The IRF-3 decrease in PBMC was less o
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14 extensive and took place with delayed kinetics compared to HIV-1-infected SupT1 cells, o
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15 probably corresponding to difference in infection frequency and viral replication kinetics p
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16 between cultures. These results confirm that HIV-I infection associates with a reduction of IRF- 2
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17 3 protein in infected cells, and link the reduction in IRF-3 with a lack of ISG expression during 1
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19 To further assess the impact of HIV-1 on IRF-3 levels we examined IRF-3 in CD4+ e
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20 HeLa cells infected with HIV-1. Cells were immunostained with antibodies specific to IRF-3 or
21 HIV-1 p24 gag protein, and were visualized by fluorescence microscopy at various time points
22 post-infection. As shown in Fig. 1B, IRF-3 was highly abundant in the cytoplasm of non-
23 infected control CD4+ HeLa cells but within 24 hours post-infection the intracellular levels of
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1 IRF-3 were reduced concomitant with HIV-1 protein expression, and by 72 hours post-infection
2 IRF-3 levels were completely ablated. We also examined IRF-3 levels in THP1 cells, a human
3 monocyte/macrophage like cell line, as macrophages are an important reservoir of HIV-1 in vivo
4 (42). As seen in Figure 1C, the THP1 cells exhibited high abundance of IRF-3 but levels were
5 markedly reduced as HIV-1 infection progressed over a 48 hour period. We found similar results
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6 using the U937 monocyte cell line (data not shown). Importantly, these experiments were o
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7 performed with HIV-1JR-CSF, a R5-tropic HIV-1 strain, in contrast to the X4-tropic virus (HIV- lo
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8 1 ) used in Figure 1A and B. Collectively these results demonstrate that HIV-1 infection e
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9 associates and a rapid depletion of IRF-3 within CD4+ cells in a cell-type unrestricted manner, o
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11 //
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12 HIV-1 promotes the specific decay of IRF-3 protein levels independently of mRNA s
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13 expression. o
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14 To determine if IRF-3 ablation by HIV-1 is specific or part of a host metabolic shutdown o
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15 of innate antiviral pathways, we examined IRF-3 and IRF-7 levels in HIV-1 infected cultures. p
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16 Within HIV-1-infected SupT1 cells IRF-3 levels started to decrease 24 hours post infection, with 2
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17 less than 20% remaining by 48 hours, whereas IRF-7 levels remained relatively unchanged over 1
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18 the infection time course compared to mock-infected cells (Fig 2A). While IRF-3 protein levels y

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19 were specifically suppressed during HIV-1 infection, we found that IRF-3 mRNA levels e
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20 remained constant and relatively unchanged compared to mock-infected control cells (Fig 2B).
21 Thus, the observed decrease in IRF-3 protein levels was not due to reduced IRF-3 mRNA
22 expression.
23 We therefore assessed the stability of IRF-3 and IRF-7 protein during HIV-1
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