The nature of human immune responses to CHIKV and the strategies used by the virus to withstand these are poorly characterized phenomena. In light of this, we undertook an investigation of the innate antiviral reactions to CHIKV by human fibroblasts, a known in vivo
target of the virus. Our goal was to acquire information on some of the basic responses, processes, and molecules involved in immune activation by CHIKV in host cells that are both nontransformed (see reference 4
) and relevant to the virus's replication and pathogenesis. We focused on one of the most rapid and consequential innate immune responses to alphaviral infection, namely, the induction of type I IFN and the expression of ISGs through the activation of IRF3. Since CHIKV is highly susceptible to the actions of IFN-α/β, information regarding the molecular and biochemical bases of IFN induction by the virus and its ability to replicate in the face of this induction is likely to be of great utility to the design of antiviral therapies.
Our data show MOI-dependent upregulation of IFN-β, ISG56, and Viperin mRNA during CHIKV infection. We further observed Ser398 phosphorylation and nuclear accumulation of IRF3 during infection that occurs after the appearance of dsRNA. While shown for other alphaviruses in nonhuman cells (11
), activation of IRF3 during CHIKV infection of human cells has until this point not been described. Importantly, we also show that CHIKV-triggered IFN-β/ISG mRNA accumulation is directly dependent on IRF3 and does not require JAK/STAT activity since (i) Transcription of these genes does not occur following siRNA-directed depletion or NPro-mediated degradation of IRF3, (ii) these genes are induced despite the fact that CHIKV does not stimulate IFN-α/β secretion in these cells (Fig. ), and (iii) infection does not induce mRNA accumulation of the IFN-dependent ISG Mx1. Interestingly, however, while IFN-β/ISG expression is evident at an MOI of 0.1 (Fig. ) and as early as 6 h postinfection (Fig. ), IRF3 Ser398 phosphorylation is only weakly detected at this MOI and is not substantial until after 8 h postinfection (Fig. ). It is possible that the amount of Ser398-phosphorylated IRF3 protein is below the detection limit of this assay yet is still functionally active at this MOI and time point. It is also possible that innate responses to CHIKV at early times postinfection or following low MOI exposure result in the phosphorylation of other serine or threonine residues that result in activation of the protein (e.g., see reference 57
). We are currently attempting to distinguish between these alternatives. To our knowledge, this represents the first demonstration of the direct requirement of IRF3 for alphavirus-mediated induction of IFN-β and ISGs. It is worth noting that IRF3 is not required for such transcriptional induction by all viruses, however. Prescott et al. showed that ISG56 and Mx1 were transcriptionally induced in HUH-7 cells infected with Sin Nombre virus (a Hantavirus) following siRNA-mediated knockdown of IRF3 (65
). Daffis et al. recently showed type I IFN secretion in mice lacking both IRF3 and IRF7 (DKO) after infection with West Nile virus (WNV) (17
). Interestingly, these authors also examined virus-triggered IFN-β transcription in macrophages harvested from these mice and saw no difference between WT and DKO macrophages infected with WNV, encephalomyocarditis virus, or CHIKV strain 142 (17
). While this result may appear to contrast with data presented here, the disparity could be related to differences in cell type or viral strain.
We also show that CHIKV-mediated phosphorylation of IRF3 and subsequent activation of IRF3-dependent transcription requires the adaptor protein IPS-1. As shown in Fig. , CHIKV infection of HFs involves cytoplasmic accumulation of dsRNA, a strong stimulator of IRF3-dependent gene expression. Cytoplasmic dsRNA is detected by two known IRF3-terminal PRRs, MDA5 and RIG-I, that both signal via IPS-1. Our results are thus consistent with activation of IRF3 through detection of dsRNA by MDA5 and/or RIG-I. However, while we are currently examining the potential roles of these proteins for CHIKV-triggered IRF3 activation, we have thus far been unable to determine the essentiality of either molecule. Our results in human cells agree with recent data from Schilte et al. in which an essential role was found for IPS-1 in IFN-β induction by murine cells following CHIKV infection (76
). Consistent with this, IFN-α/β secretion was diminished relative to WT cells in MDA5−/−
MEFs infected with SINV (11
). However, Schilte et al. observed a reduction in CHIKV-triggered IFN-β transcription in RIG-I−/−
murine embryonic fibroblasts (MEFs) relative to WT cells exceeding that seen in MDA5−/−
). In another recent study, IFN-stimulatory total RNA was harvested from cells infected with the alphavirus SFV and transfected into MEFs, but no difference in IFN-α/β secretion was observed between WT, RIG-I−/−
, or MDA5−/−
). Thus, great observational disparities exist regarding Alphavirus
-triggered IFN responses that may be due to biological differences between viral species or strains or perhaps is related to the species or genetic background of host cells (see reference 76
). However, the role of an IPS-1-dependent signaling pathway in CHIKV-induced IFN-β synthesis appears clear even across host species.
In contrast to these previous reports, however, transcriptional induction of IFN-β and ISGs by CHIKV was not reflected in the synthesis of corresponding proteins. Thus, unlike the RNA virus SeV and the Alphavirus
SINV, infection of our target cells with CHIKV (even at high MOI) led to no appreciable IFN-β, ISG56, or Viperin protein, and yet viral protein synthesis was obvious. While these findings represent, to our knowledge, the first examination of ISG protein induction by CHIKV, the results we obtained for IFN-β secretion seemingly contrast with those published in a previous study (76
). The authors of that study show that CHIKV-triggered IFN-β secretion from human lung (MRC-5) and foreskin fibroblasts that is MOI dependent. Although barely detectable IFN secretion from CHIKV-infected relative to untreated HFs was observed in our case, this was in no way MOI dependent. Our data are in agreement, however, with a study by Burke et al. in which the authors detect no type I IFN secretion from MEFs infected with CHIKV for 8, 12, 24, or 32 h (11
). It is possible that the disparity between the two studies is related to the strains of CHIKV used. While Burke et al. (11
) and the present study used the CHIKV-LR strain (2
), Schilte et al. (76
) used CHIKV-21 strain. Whether this is responsible for the differences observed between these studies will require further exploration.
We next examined whether the absence of IFN-β secretion and ISG protein synthesis in response to CHIKV infection could be due to a virus-associated, widespread block in cellular translation. To address this, we examined polypeptide-incorporated puromycin using SDS-PAGE in CHIKV-infected cells. Infection of HFs led to the diminishment of puromycin in cellular protein by 8 h postinfection and puromycin was undetectable by 16 h postinfection. Based on this, we conclude that a global shutoff of cellular gene translation may contribute to the lack of IFN-β and ISG protein synthesis during CHIKV infection. Determining whether cellular translational shutoff is essential to the lack of these proteins will require experimental approaches involving inhibition of this response. We nevertheless hypothesize that this represents a strategy of immune evasion in which virus infection leads to prevention of the synthesis of cellular proteins, especially those that are actually or potentially detrimental to virus replication (e.g., ISGs). Although translational shutoff has not been previously investigated for CHIKV, it has been described for other alphaviruses such as SINV (28
) and SFV (53
). In SFV this is at least partially due to virus-triggered phosphorylation of eIF2α (53
). Moreover, the nsP2 proteins of SINV (32
) and SFV (7
) have been implicated in both the suppression of host transcription and IFN evasion, although the molecular bases for these have not been characterized. Interestingly, we also observed translational shutoff in SINV-infected HFs (data not shown) and, as such, this phenomenon is unlikely to provide the sole explanation for the absence of CHIKV-induced IFN/ISG protein. The causal relationship between antiviral gene translation and the Alphavirus
-associated shutoff of protein synthesis clearly requires more thorough examination. Intriguing work by Frolov and coworkers has shown that little to no IFN is secreted from NIH 3T3 mouse cells infected with wild-type SINV (strain TE12) yet when a mutant is used that fails to induce translational shutoff strong IFN secretion is induced (28
). This result is consistent with our observations for and inferences about CHIKV replication in human cells. However, in contrast to those findings we detected high levels of secreted IFN-α/β from SINV-infected cells (Fig. ). It is also important to note that we observed translational shutoff in both MEFs and human fibroblasts after infection with SINV and in MEFs after CHIKV infection (data not shown). As such, differences in host cell species are not likely to account completely for the observed SINV and CHIKV differences in IFNα/β secretion. This may, however, be related to differences in the virus strain (TE12 versus Ar339), and our results do agree with other studies examining SINV-triggered IFN secretion (29
). The bases of these disparities will require more careful examination.
Phosphorylation of eIF2α on Ser51 leads to shutoff of mRNA translation by reducing levels of ternary complex eIF2-GTP-Met-
. While never examined for CHIKV, phosphorylation of eIF2α Ser51 has been demonstrated for SINV (85
) and SFV (53
). eIF2α phosphorylation can occur via four upstream kinases that react to cytoplasmic dsRNA (PKR), heme deficiency, heat shock and oxidative stress (heme-regulated inhibitor [HRI]), amino acid or serum deprivation or UV irradiation (general control nonderepressible-2 [GCN2]), or unfolded proteins (PKR-like endoplasmic reticulum kinase [PERK]). Since replication of alphaviruses (including CHIKV; Fig. ) involves the synthesis of dsRNA, a role for PKR in eIF2α phosphorylation has been hypothesized. Indeed, Gorchakov et al. have shown SINV-induced eIF2α phosphorylation in PKR-positive NIH 3T3 MEFs but not PKR−/−
). These authors also describe increased eIF2α phosphorylation in NIH 3T3 cells infected with SINV replicons that express WT PKR and a lack of phosphorylation in cells infected with replicons expressing a PKR dominant-negative mutant (35
). Ventoso et al. have also described dependence of eIF2α phosphorylation on PKR during SINV infection (85
). However, in contrast to these data, Berlanga et al. have shown diminished SINV-associated eIF2α phosphorylation in GCN2−/−
MEFs relative to WT cells (5
). Curiously, however, these authors observed no change in eIF2α phosphorylation in PKR−/−
MEFs relative to WT cells following SINV infection (5
). Our finding that CHIKV-induced eIF2α phosphorylation is greatly diminished following shRNA-mediated knockdown of PKR is in agreement with the data of Gorchakov et al. and Ventoso et al., and yet a potential role for GCN2 remains to be examined.
Since CHIKV infection leads to both PKR-mediated eIF2α phosphorylation and shutoff of cellular protein synthesis, we next sought to determine whether the two events were causally linked. However, a CHIKV-associated block to cellular protein translation was still seen following siRNA-mediated knockdown of PKR expression (Fig. ). In addition, knockdown of PKR failed to reestablish synthesis of ISG56, Viperin, or IFN-β protein following CHIKV infection (Fig. ). Based on these results, we conclude that the translational shutoff observed does not result from CHIKV-induced, PKR-dependent phosphorylation of eIF2α. Although a specific role for PKR in CHIKV infection has not previously been examined, the molecule has been investigated with respect to SINV replication. In this case translational inhibition was shown to occur following SINV infection as efficiently in PKR−/−
MEFs, as in wild-type NIH 3T3 cells (35
). In addition, viable SINV mutants have been constructed that fail to induce a block in cellular translation, suggesting that the virus achieves this effect via an active genomically encoded factor (34
). Much additional work thus remains regarding the molecular basis of Alphavirus
-mediated shutoff of cellular translation. Recently, a related phenomenon was described during in vitro
infection with hepatitis C virus (HCV) (30
). Infection was found to lead to PKR-dependent phosphorylation of eIF2α. Furthermore, this process inhibited cellular (including ISG) protein synthesis, which was restored during PKR knock down. Thus, HCV appears to effectively exploit an innate antiviral response for a proviral objective. It is also possible that this represents a strategy used by many RNA viruses to evade the effects of antiviral gene expression.
We next examined whether CHIKV infection leads to a decrease in RNA transcription, a response described for other alphaviruses and that could contribute to the differences in IFN/ISG translation observed here between SINV and CHIKV. To address this, we used a technique that allows the separation of newly synthesized from preexisting cellular RNA using biotinylation of incorporated 4-thiouridine (22
). Using this approach we found that while infection of HFs with either SINV or CHIKV leads to reduced host cell transcription, CHIKV leads to an overall greater transcriptional reduction at 24 h postinfection (Fig. ). Moreover, while cells infected with SINV appear to be actively synthesizing mRNA as late as 24 h postinfection, cells infected with CHIKV are synthesizing mRNA for housekeeping genes but not IFN-β/ISGs (Fig. ). Based on these observations we conclude that CHIKV and SINV differ in their abilities to inhibit both overall cellular RNA transcription and transcription of IRF3-dependent mRNAs. A role for this more potent and potentially target-specific CHIKV-induced transcriptional block in the lack of IFN-β/ISG protein synthesis observed during infection will require further exploration and is currently being investigated.
Our work aims to describe and characterize basic aspects of innate immune induction and evasion by CHIKV in a clinically relevant cell model. We have shown that the virus strongly induces, via IPS-1, accumulation of IRF3-dependent mRNAs but that it also efficiently prevents synthesis of corresponding proteins, perhaps through blocking global cellular protein synthesis. The extent to which this translational block represents an immune evasion strategy whose purpose is to avoid the antiviral effects of IFN and ISG proteins is unknown but is a current research focus in our laboratory. In addition, we have shown that CHIKV leads to PKR-dependent phosphorylation of eIF2α but that this process is not essential to translational shutoff. It is possible that the action of PKR during CHIKV infection leads to changes in the kinetics of viral or cellular protein synthesis. In addition, CHIKV also induces shutoff of RNA transcription that may specifically target IRF3-dependent genes and the relationship between this effect and the absence of ISG proteins requires further elucidation. The molecular basis of Alphavirus-induced shutoff of cellular transcription and translation remain important areas of inquiry and detailed characterization of these phenomena are likely to have profound implications for the development of anti-Alphavirus therapies.