Our studies reveal a specific depletion of IRF-3 in HIV-1-infected cells that results in a loss of PRR signaling of innate antiviral defenses. The decline in IRF-3 levels occurred with the accumulation of viral proteins and was dependent on HIV-1 replication initiation. That HIV-1 mediates the targeted depletion of IRF-3 is supported by our observations that neither IRF-7 levels nor IRF-9-dependent signaling was impacted by HIV-1. In addition to the depletion of IRF-3 during infection within the CD4+ cell lines and primary cells, we observed IRF-3 dysregulation by HIV-1 in HEK293 cells expressing transfected HIV-1 provirus DNA. Thus, IRF-3 antagonism by HIV-1 is not restricted to a specific cell type. Our data show that IRF-3 depletion is a general property shared by R5- and X4-tropic viruses and is a corresponding early event of acute HIV-1 infection. Our studies indicate that, when activated, IRF-3 directs an intracellular innate antiviral response that can potently suppress HIV infection. Thus, reduction of IRF-3 levels provides a strategy for HIV-1 to evade the host innate immune response and to promote host cell permissiveness for infection.
Our results confirm previous work suggesting that HIV-1 targets IRF-3 for protein depletion, in which the authors concluded that HIV accessory proteins mediated early postentry depletion of IRF-3 by stimulating its ubiquitination and targeting to the proteosome (32
). This conclusion was in part based on an observation that IRF-3 depletion by HIV-1 was insensitive to preinfection treatment of cells with AZT and was blocked by treatment of cells with proteosome inhibitors (32
). Conversely, we found that similar treatment of cells with AZT actually prevented IRF-3 depletion concomitant with the ablation of provirus production, whereas provirus expression was necessary and sufficient for IRF-3 depletion and occurred independently of HIV-1 protease function. We also found that proteosome inhibitor treatment of cells could partially restore IRF-3 levels during HIV-1 infection (B. P. Doehle and M. Gale, Jr., unpublished results). While these differences between studies might be attributed to variation among the HIV strains and experimental systems used by each group, we note that proteosome inhibitors are deleterious to HIV-1 infection and that viral replication is suppressed in treated cells (32
). Thus, we conclude that HIV-1 replication and provirus expression are required for IRF-3 suppression wherein one or more viral proteins, including possibly virion-associated proteins, direct the rapid destabilization and degradation of IRF-3. We found that the active, nuclear isoform of IRF-3 did not accumulate within HIV-1-infected cells even prior to the large reduction of IRF-3 levels early in acute infection. These observations may implicate a direct or indirect viral protein interaction with IRF-3 that interferes with its activation and may serve to catalyze protein degradation. Expression of the HIV-1 Vpr protein had a modest effect to attenuate virus-induced IFN-β promoter expression (Fig. ), which we attribute to an associated reduction in the level of coexpressed IRF-3 (Fig. ), further supporting a possible role for Vpr in IRF-3 regulation (32
). However, Vif did not associate with reduced IFN-β promoter signaling or IRF-3 levels in our coexpression experiments. Thus, Vpr could have a dominant role among these viral accessory proteins in directing the suppression of IRF-3, or Vif may require additional viral factors to further influence IRF-3 levels, possibly explaining the differences between our results and the previous report (32
). These possibilities are being explored.
HIV-1 infection is typically initiated at mucosal sites of transmission, where innate antiviral defenses of resident mucosal CD4+
T cells and macrophages provide the first level of immune protection against infection (18
). Importantly, we found that IRF-3 is rapidly depleted within vaginal mucosal CD4+
T cells upon HIV-1 infection in an ex vivo tissue model. Our studies clearly demonstrate that PBMCs and T-cell lines can mediate RLR signaling in response to specific ligand treatment or SenV infection of cells. Disruption of RLR signaling compromises IFN and ISG expression to enhance susceptibility to RNA virus infection (27
). In contrast, TLR3 and TLR4 signal through TRIF/TRAM-dependent pathways, conferring cellular responsiveness to the dsRNA ligand and bacterial lipopolysaccharide, respectively (33
). The disruption of signaling by these PRRs has been shown to attenuate both innate and adaptive antimicrobial immunity (33
). Thus, the depletion of IRF-3 in mucosal cells of infection overall serves to compromise the integrity of mucosal antiviral defenses by limiting IFN production and secretion from infected resident macrophages and abrogating PRR signaling of the antiviral state, including signaling by RIG-I, MDA5, TLR3, and TLR4, in infected mucosal T cells. The HIV-1-mediated depletion of IRF-3 within a short time frame during initial acute infection therefore allows for unchallenged virus amplification and dissemination from the mucosa to peripheral sites of infection. We found that IRF-3 levels were not uniformly reduced in CD4+
T cells across all HIV-1-infected patients examined in our study, with differential levels observed among patients undergoing acute infection and normal IRF-3 levels observed among LTNP. While further studies of larger patient cohorts are required in order to understand these differences, it is possible that viral polymorphisms and/or host factor distinctions could impart the differential regulation of IRF-3 and innate defenses to impact the virologic features of HIV-1 infection. These differences in IRF-3 levels may also be due to the impact of viral load and could be subject to regulation by antiretroviral therapy, possibly leading to a rebound in IRF-3 levels. Such possibilities are being investigated.
In addition to decreased IRF-3 levels in CD4+ cells, our data reveal overall low levels of IRF-7 in CD4+ cell populations in general. These observations implicate IRF-3 as a central transcription factor of PRR signaling in CD4+ T cells and further affirm the importance of IRF-3-dependent innate response pathways in pathogen sensing and control. IRF-7 in T cells appears to have a less prominent role in infection than IRF-7 in dendritic cells, which have high constitutive IRF-7 levels. This difference may have played a role in the evolution of HIV-1 countermeasures against T-cell innate immune programs. Indeed, our data suggest that a strong evolutionary pressure to block these host defenses exists, as we observed that IRF-3 activation can severely limit HIV-1 infection.
HIV-1 is not unique in targeting IRF-3 as a countermeasure to host innate defenses, as other viruses have been shown to antagonize IRF-3 function through disruption of upstream signaling programs (27
) and degradation of IRF-3 during infection (3
). While HIV-1 targeting of IRF-3 is particularly effective in disrupting several host PRR pathways, we found that IRF-9-dependent IFN signaling and PRR signaling of NF-κB remain intact in HIV-1-infected cells. These observations underscore the specificity of IRF-3 regulation by HIV-1 and likely reflect the requirement for NF-κB function in viral mRNA transcription from the HIV-1 provirus (1
). In particular, RLR signaling and the actions of TLR3 and TLR4 drive the activation of IRF-3 and NF-κB concomitantly through adaptor protein signaling bifurcation (33
). By specifically targeting IRF-3, HIV-1 therefore ensures the attenuation of IRF-3-dependent immunity while preserving pathways of NF-κB activation that might assist virus replication (1
). IRF-3 is central to the stimulation of IFN production in many cell types, and a decrease in IRF-3 levels functionally disrupts IFN production and the ISG response normally triggered by a variety of microbial pathogens through processes of PRR signaling (27
). Overall, IRF-3 depletion in T cells and macrophages could provide a framework for opportunistic pathogens to invade mucosal sites of HIV-1 infection. A similar effect on secondary infection has recently been demonstrated with a chronic lymphocytic choriomeningitis virus infection model (45
). Our studies of the SenV paramyxovirus model provide direct evidence that HIV-1 depletion of IRF-3 may support polymicrobial opportunistic infection.
Innate immune suppression through IRF-3 depletion could be expected to overall slow the kinetics of the immune response against HIV to support a systemic chronic infection. In support of this idea, studies of herpes simplex virus infection have demonstrated that immune suppression in the local infection environment can delay pDC infiltration to the mucosal site of acute infection and associates with increased viral production (28
). IRF-3 depletion may have an analogous effect in HIV-1 infection, increasing peak viral load and therefore contributing to the viral set point and disease progression to AIDS. TLR7 and TLR9 recognition of HIV-1 has previously been demonstrated with pDCs and leads to IFN production in an IRF-7-dependent fashion (5
). This recognition is important for the induction of the adaptive immune response that initially controls acute viremia in HIV-1 infection (22
). Recent work with simian immunodeficiency virus-infected nonhuman primates has suggested that chronic pDC activation through TLR7 and TLR9 may contribute to AIDS pathogenesis (29
). We propose that a higher HIV set point caused by IRF-3 depletion in infected cells could support a larger pool of chronically infected cells and hence provide an increased number of TLR ligands to trigger and sustain chronic immune activation.
Our results demonstrate that active IRF-3 directs a robust and lethal antiviral hit to HIV-1 that can suppress de novo virus production. This inhibition of HIV-1 in the face of activated IRF-3 unveils an additional avenue for both therapeutics and vaccine design to control HIV-1 replication and dissemination. IRF-3 activation or the rescue of IRF-3 levels may benefit infected patients by inducing antiviral defenses that suppress HIV-1 infection and replication while serving to enhance overall immunity to infection.