TBEV causes epidemics of acute encephalitis in forested regions of Europe and Asia. The disease takes a characteristic biphasic course: first an unspecific flu-like phase of approximately 5 days followed by a 7-day period of apparent recovery and a second, specific phase involving often severe neurological symptoms (35
). Encephalitis is supposed to be caused by CD8+
T-cell-mediated immunopathology along with overshooting inflammatory responses and direct damage by the virus (22
). However, only about 20 to 30% of infections enter the second phase and result in full-blown disease (35
For the TBEV-related Langat virus as well as for other encephalitic flaviviruses, it is known that the pretreatment of cells with type I IFNs can hamper virus multiplication (3
). It was thus expected that TBEV disturbs the activation of the IFN system, i.e., IFN induction, in order to efficiently spread in the host. Our results suggest that TBEV does not entirely block the transcriptional induction of the IFN-β promoter but slows it down considerably. The delayed onset of IFN transcription results in a late synthesis of biologically active IFNs. No IFN was measured in supernatants of TBEV-infected cells even at up to 24 h p.i. Measurable IFN production by infected cells occurred between 24 h p.i. and 48 h p.i., when large amounts of infectious TBEV particles had already been produced. Thus, TBEV appears to employ a “runaway” strategy to escape the antiviral effects of the IFN system. This is in line with previously reported findings for WNV, where it was shown that the onset of the IFN response occurred at a late stage of infection (15
). Moreover, for influenza A virus, it was previously demonstrated in vivo
that accelerated virus multiplication is a viral strategy to outcompete the IFN response (18
Our attempts to identify a specific TBEV protein responsible for the delay in IFN induction were unsuccessful. In a similar manner, for WNV, it was shown previously that infected cells can normally activate IRF-3 in response to Sendai virus superinfection, arguing against the presence of an active mechanism to inhibit cytoplasmic IFN induction by this flavivirus as well (15
). It must be added, however, that WNV is able to inhibit IFN induction by ectopic dsRNA, most likely because the viral NS1 gene inhibits Toll-like receptor 3, an IFN-activating PRR that recognizes dsRNA in the extracellular phase (64
). Also, the NS2A protein of KUNV, which is a variant of WNV, was shown previously to directly suppress IFN induction (36
). For TBEV, however, neither NS1 nor NS2A or NS2B had any obvious inhibitory effect on IFN induction. Although we cannot exclude the existence of an IFN antagonist that has escaped our attention, our finding that no viral dsRNA can be immunodetected outside internal membranes favors the model that TBEV relies on hiding its dsRNA in membrane compartments. For intracellular PRRs, the access to this important pathogen marker is hence restricted. It is important that most, if not all, viruses with a positive-strand RNA genome like TBEV rearrange cytoplasmic membranes for the formation of virus factories (1
). Moreover, these viruses are known to produce significant amounts of dsRNA during their life cycle (32
). For some of the viruses, it was shown previously that viral dsRNA is indeed sequestered inside virus-induced membranous structures (32
). Nonetheless, many of them express specific factors inhibiting IFN induction (57
). Possible explanations for this discrepancy are that these viruses are vulnerable to PRR detection in the early phase of infection, when membrane reorganization is not yet completed, or that vesicles may rupture. TBEV also appears to expose certain amounts of biologically active dsRNA late in infection. In this context, it should be noted that one molecule of dsRNA is enough to trigger an IFN response under certain conditions (46
) and that, at least for severe acute respiratory syndrome (SARS) coronavirus, dsRNA outside viral membrane compartments has been detected (32
). SARS coronavirus expresses a set of IFN induction antagonists (69
), which together completely block the activation of IRF-3 in infected cells (66
). TBEV, which partially activates IRF-3, may outrun IFN induction by fast replication. It is feasible that the speed of replication also plays a role in IFN escape for other viruses besides TBEV, WNV, and influenza A virus (11
). Quantitative time course measurements of IFN induction (transcription and IFN release) and virus multiplication, as we have performed for TBEV, could help gain a better understanding of the kinetic aspect of IFN escape by these viruses.
Using different strains of TBEV, we observed that the weak IFN induction by TBEV was directly proportional to the level of accumulated virus RNA. This finding is in agreement with data from a previous study of WNV that showed that IFN induction is dependent on genome replication (6
). Moreover, IFN induction by TBEV is strongly dependent on IRF-3 and on IPS-1. Moreover, TBEV activates the phosphorylation of eIF2α, another signaling pathway triggered by viral dsRNA. It is thus conceivable that, at least late in infection, some viral molecules activate cytoplasmic PRRs. Whether the dsRNA that we have detected in large amounts in infected cells is the only viral molecule triggering IFN induction or whether 5′-triphosphorylated RNA, e.g., the viral antigenome, also plays a role remains to be solved. Also, the contribution of individual cytoplasmic PRRs and Toll-like receptors to IFN induction by TBEV will be the subject of future studies.
TBEV efficiently rearranges internal cytoplasmic membranes to host the viral replication factories. ER membrane rearrangements and the formation of replication factories were first described for KUNV (78
). Small vesicle packets (VPs) inside the ER-derived compartments were reported previously to contain the viral proteins NS1, NS2A, NS3, and NS4A and dsRNA (41
). The ultrastructure of VPs was also studied for DENV, and the VP was identified as the site for RNA replication. These vesicles contained a small pore toward the cytoplasm where metabolites and RNA could be exchanged with the cytoplasm (75
). For TBEV (this study) and KUNV (44
), it was shown that the disruption of the membrane compartments with BFA at 12 h p.i. did not interfere with virus multiplication. However, the treatment rendered KUNV sensitive to the antiviral action of the IFN-induced protein MxA (25
), suggesting a certain exposure. For TBEV, we observed that BFA did not alter the inaccessibility of dsRNA to cellular recognition and that the level of viral IFN induction is not elevated (data not shown). This finding indicates that the membrane compartments induced by TBEV are robustly protecting the viral replication complexes.
We found that at the 24-h time point of infection, levels of IFN transcripts were comparable between TBEV and the inducer virus clone 13, but levels of released IFN were different. Only a burst experiment measuring the IFN released de novo between 24 h and 48 h p.i. revealed that cells are able to secrete similar amounts of IFN in both cases. Time course analyses showed that TBEV-infected cells lagged behind the inducer control because final IFN-β mRNA levels were reached with a delay of at least 8 h. If we had measured only mRNAs and viral titers in supernatants at 24 h p.i., the wrong impression of a TBEV-imposed block of IFN secretion would have been produced. This demonstrates that in cases of such observations, rigorous quantitative and kinetic analyses are mandatory before conclusions can be drawn. In general, our quantitative time course analyses of virus multiplication, IFN-β mRNA levels, and IFN protein synthesis indicate that IFN-β mRNAs have to be synthesized over an extended period of time until detectable levels of IFN are released by infected cells. We are not aware that these dependencies have been previously investigated to this detail.
In summary, we propose a kinetic model of TBEV IFN escape. The virus rearranges internal cell membranes to provide a compartment for its dsRNA, which is inaccessible for PRRs. This delays the onset of IFN induction sufficiently to give progeny particle production a head start. Cells start to secrete IFN only later than 24 h after infection. At this time point, however, secondary infections of surrounding cells have already occurred. The well-documented ability of TBEV to block JAK/STAT signaling (3
) makes sure that late-point IFN is unable to develop its antiviral activity. This combination of an IFN induction delay and an IFN signaling block may allow the virus to enter the central nervous system before an efficient antiviral response is launched.