We initiated these studies to determine the mechanism through which the HSV-1 vhs protein blocks dendritic cell activation upon infection. Our key findings are as follows: (i) vhs did not degrade certain proinflammatory gene mRNAs under experimental conditions where HSV-1-infected cells were treated with TLR agonists; (ii) DC activation by wild-type HSV-1 and the vhs− mutant virus occurs by an RLR/MAVS-independent mechanism; (iii) while the vhs effect on DC cytokine responses is independent of the type I IFN receptor, the IFN receptor-dependent ISG response remains intact during wild-type HSV-1 infection; and most importantly, (iv) a novel activity of vhs was identified that targets an immediate activation of NF-κB during productive HSV infection.
HSV-1 and NF-κB share an interesting relationship. Activation of NF-κB has been shown to occur in two distinct phases (2
). UV-inactivated virions have been shown to activate the NF-κB signaling pathway at early times postinfection (2
). Mechanistically, this has been proposed to occur via either HSV glycoprotein D (gD) interacting at the cell surface with the receptor for HSV-1, the herpesvirus entry mediator (HVEM), or via tegument protein UL
37-mediated TRAF6 activation (28
). Importantly for the work presented in this study, the viral glycoproteins have been shown to induce cytokine secretion and costimulatory marker upregulation in DCs (46
). However, this report also showed that a gD mutant that fails to bind to HVEM and nectin-1 still activated the DCs. Interestingly, the gD mutant did not activate the DCs to the same degree as wild-type gD (46
). One possible explanation for this phenotype is that in DCs a synergistic activation involving both the gD-HVEM/nectin-1 interaction and UL
37 may be required for complete NF-κB activation and, ultimately, DC activation. Alternatively, there may be cell type specificity in what determines activation of NF-κB in DCs whereby UL
37 alone mediates activation, not the gD-HVEM interaction. A second wave of NF-κB activation occurs around the time of the onset of virus replication, and this has been reported to play a role in cell survival during HSV-1 infection (16
). Interestingly, one report describes how HSV-1 hijacks this pathway for its own benefit, directing NF-κB subunits to the immediate-early viral promoter of ICP0 to enhance its transcription (2
). Since HSV-1 relies on activated NF-κB for efficient virus replication and to interfere with apoptosis, it is also placed in a potentially problematic situation where an immune response will also be initiated as a consequence. How the virus reconciles this may be critical for efficient host pathogenesis—a sufficient amount to limit apoptosis and aid in viral gene expression yet not so much as to generate large toxic quantities of proinflammatory cytokines (IL-6 and TNF-α). Our work suggests that the vhs protein contained in the virion particle helps to maintain an appropriate balance of cellular levels of activated NF-κB in the early phase of infection.
Important insights into how HSV-1 regulates transcription factor activation in DCs were obtained by comparing infections with live and UV-inactivated wild-type and vhs−
mutant viruses (). Our data demonstrate (i) that the biphasic induction of NF-κB occurs in DCs and (ii) that vhs inhibits the early replication-independent induction (A). Biphasic induction of NF-κB refers to the fact that while NF-κB activation is detected with wild-type HSV-1 only between 3 and 5 h p.i. (16
), there is also an earlier activation, which can be slightly detected under certain conditions (2
). It is this immediate activation of NF-κB that vhs prevents. These results highlight the value in our study of using the vhs−
mutant virus as a tool to better understand the early molecular pathways involved in DC cytokine production: a clear view of the dual nature of NF-κB activation (replication-dependent versus -independent triggering) is observed that otherwise would not be simply by analyzing wild-type HSV treatments via Western blotting.
An important question that remains is why vhs blocks the replication-independent activation of NF-κB but not the replication-dependent triggering. vhs may be important in the early hours (between 0 and 5 h p.i.) of the infection for limiting the host response to HSV-1 (blocking NF-κB activation and thus preventing a robust induction of proinflammatory cytokines). Later in the infection, when NF-κB plays an opposite role in limiting the host response (through its antiapoptotic function [16
]), vhs blocking the response would seem counterproductive. The fact that we observed more activated NF-κB during infection with the vhs−
mutant virus in DCs fits well with previous observations that there was less background apoptosis during infection with vhs−
mutants in other cell types (3
). The data in clearly show equivalent NF-κB activation, as measured by the accumulation of phospho-IκB-α, between live wild-type and vhs−
mutant viruses at 5 h p.i. The virion form of vhs may become inactivated (in its capacity to block NF-κB signaling) early during the course of infection (sometime between 3 and 5 h p.i.). This inactivation phenomenon has been described for its nuclease function (54
). It is also a possibility that by 5 h into the infection, the stimulation of the DCs by replicating virus is so strong that vhs can no longer effectively block NF-κB signaling. Moreover, by this time, de novo
synthesis of other viral proteins with immune antagonistic functions will also be under way.
Alternatively, as different signaling pathways presumably contribute to the two different waves of NF-κB activation, vhs may specifically target one but not the other. This could potentially explain our results when we coinfected HSV with RNA viruses or cotreated with TLR agonists ( and ). At least one report describes different roles for some of the signaling components involved in NF-κB and IRF activation when comparing TLR to RLR activation of cells (24
). Recently, the HSV virulence factor γ(1)34.5 was described to interfere with NF-κB activation by impairing the function of the upstream IκB kinase (IKK) when DCs are triggered by TLR agonists (20
). Experiments to identify putative binding partners for vhs and to address precisely where in the NF-κB signaling pathway vhs may be acting are required to fully decipher the mechanism underlying this new functionality for vhs.
The presence of phosphorylated IRF3 in DCs infected with live virus (but not UV-inactivated virus) during our early time course experiments was unexpected (B). Reports of other nonimmune cell types tested show that IRF3 functionality as a transcriptional regulator is impaired following wild-type HSV infection by the immediate-early protein ICP0 (33
). Specifically, in fibroblasts, infection with wild-type HSV resulted in no detection of phospho-IRF3 (39
). We observed the opposite in DCs infected with HSV-1; the wild type showed accumulation of the phosphoprotein, whereas UV-inactivated viruses did not. We speculate that the differential activation of IRF3 in different cell types (e.g., DCs compared to fibroblasts) following HSV infection may be due to the relationship between the pathways of virus detection in these different cell types (TLR versus non-TLR signaling pathways) and ICP0. Alternatively, the functionality, localization, and behavior of ICP0 might be different or compromised in DCs relative to other cell types, which may impact its ability to interfere with IRF3 activation.
vhs, which has homology to several other nonviral endonucleases, was initially believed to indiscriminately degrade all mRNA molecules in infected cells in a global fashion (13
). However, multiple reports point to the possibility that there may be some selectivity as to which mRNA substrates vhs degrades (7
). Furthermore, some cell types (neurons and dendritic cells) appear to show some resistance to vhs-mediated RNA degradation (7
). When we stimulated wild-type HSV-infected DCs with either LPS or CpG, we failed to detect any significant differences in the mRNA expression of certain proinflammatory genes. If vhs had an affinity for these mRNA molecules (with respect to nuclease-driven degradation), the nature of what drives their expression would presumably be irrelevant, and there would be at least some measurable decrease in their cellular expression levels. However, to definitively examine the contribution of the nuclease function of vhs to the block to DC activation, experiments comparing wild-type HSV-1 to point mutants that have inactivated the nuclease motif of vhs are required. In contrast to TLR costimulation, coinfecting DCs with wild-type HSV and NDV showed an impairment in accumulation of these same proinflammatory cytokine mRNAs. It is tempting to speculate that NDV-specific factors might act to modulate vhs activity. Alternatively, these results also raise the intriguing possibility that HSV might actually affect 5′-triphosphate-containing RNA (5′-ppp-RNA) or double-stranded RNA (dsRNA), targets of RIG-I and MDA5, respectively. Such an effect might explain the mechanism by which HSV blunts cytokine expression during NDV/SeV coinfection with HSV. This could be an alternative explanation for (or a factor contributing to) the results (D) when we observed a decrease in levels of phospho-IκB-α during wild-type HSV-1 and SeV coinfection.
In DCs, NDV is recognized by members of the RLR family of cytosolic proteins, and maturation requires type I IFN signaling. Based on the repression of immune gene transcription during NDV coinfection (), we evaluated both of those signaling pathways in the response to HSV-1. Analysis of cytokine and type I IFN mRNA expression following infection of DCs prepared from MAVS KO mice with wild-type HSV revealed that the RLR/MAVS-dependent signaling pathways are not essential for their production by DCs in vitro
(A). Moreover, vhs−
virus infection resulted in higher cytokine release than wild-type HSV infection for both control and MAVS KO DCs (B). Recently, the characterization of MAVS-independent, RIG-I-dependent immune responses to RNA viruses has been reported (43
). However, these authors show that the induction of type I IFN requires MAVS, and this signaling pathway driving NF-κB activation was observed during RNA virus infection and not triggered by synthetic DNA (43
). Presently, it is unknown whether this pathway contributes to the NF-κB response in DCs following infection with a DNA virus, such as HSV-1. The DNA-sensing pathway mediated by IFI16 and STING and involved in HSV detection in various immune cell types is the likely receptor recognizing replication-competent HSV here in the 3- to 5-h p.i. window (19
). Importantly, the data from show that the RNA polymerase III-dependent RIG-I signaling pathway is not necessary for the DC response to HSV (1
We chose to evaluate the role of the type I IFN signaling pathway in DC infections by comparing wild-type HSV to the vhs mutant virus for several reasons. Our hypothesis was that by interfering with key components of this signaling cascade (STAT1 phosphorylation [41
] and ISG expression [10
]), the vhs protein was exerting its block on the activation of DCs during HSV-1 infection. The vhs mutant stimulated DCs with or without type I IFN signaling equally, indicating that unimpeded IFN signaling was not responsible for the enhanced activation of the DCs achieved with the vhs mutant virus (C). Suppression of the IFN signaling pathway alone could not fully explain how vhs blocks DC activation during HSV infection. Our data showing that vhs can block the activation of NF-κB () clarify how the vhs−
mutant virus can activate the DCs even when the cells lack components of the type I IFN signaling pathway (F). We generated a model describing the insights into the modulation of DC activation by the vhs protein during HSV-1 infection gained from this study ().
Fig. 5. Model describing the modulation of HSV-1-induced DC maturation by the vhs protein. HSV-1 infection of DCs begins with binding of the virus to the necessary receptors expressed on the surface of the cell. The binding of HSV-1 gD to the HVEM receptor has (more ...)
In summary, the work presented here reveals a novel function for the vhs protein of HSV-1 contained in the virion as an early inhibitor of the NF-κB signaling pathway in dendritic cells. In addition, the power of using a potently stimulatory virus in understanding the complex interplay between HSV and various immune signaling pathways is also highlighted throughout the study. These findings contribute to our understanding of immune antagonism by HSV while potentially having implications for future DC-based vaccine design and gene therapies.