Infection with C mutant HPIV1s [F170S and P(C−)], but not WT HPIV1, effectively stimulates IFN-β production in epithelial A549 cells (64
). In addition, F170S, but not WT, HPIV1 trigger IRF3 dimerization and nuclear translocation, indicating that the wild-type C proteins block the activation of IRF3 (64
). In the present study, the mechanisms by which the C proteins prevent IFN-β induction were explored in more detail. IFN-β production is optimally induced by the assembly of an enhanceosome comprised of IRF3, NF-κB, and AP-1 at the IFN-β promoter. Here, we found that the F170S and P(C−) mutants were unable to inhibit IRF3 and NF-κB activation, whereas WT HPIV1 inhibited their activation, suggesting an interaction of the HPIV1 C proteins with the RIG-I/MDA5 pathway upstream of IRF3 and NF-κB activation. Numerous examples of viral proteins that actively inhibit this pathway are known (10
). For instance, the influenza A virus NS1 protein inhibits RIG-I activation by binding to TRIM25 and the hepatitis C NS3/4A protease cleaves and inactivates MAVS (17
). In this context, we hypothesized that the HPIV1 C proteins acted to inhibit IFN-β production by binding to or otherwise interfering with one or more components of the RIG-I/MDA5 pathway. However, four observations led us to reject this hypothesis: (i) none of the known members of the RIG-I/MDA5 pathway that we examined could be immunoprecipitated with the C proteins; (ii) there was no significant decrease in the abundance or shift in the gel mobility of any of these host proteins indicative of C protein-mediated modification or degradation; (iii) the yeast two-hybrid assay and immunoprecipitation in conjunction with mass spectrometry failed to detect interactions; and (iv) supplying the C proteins in trans
failed to block IFN-β induction by RSV and poly(I:C), known RIG-I and MDA5 stimuli, respectively. This inability of the HPIV1 C proteins to prevent heterologous MDA5- and RIG-I-mediated IFN-β induction suggested that they do not antagonize the components of the IFN production pathway at or downstream of RIG-I and MDA5 but rather prevent the activation of these pathways.
Knockout of TBK1 and IKK
completely ablated the IFN-β response to F170S and P(C−) HPIV1 infection, confirming that the RIG-I/MDA5 pathway was necessary for IFN-β induction by these two viruses. Surprisingly, and in contrast to earlier suggestions that paramyxovirus sensing depended mainly on RIG-I (23
), we found that IFN-β induction in murine cells relied more strongly on MDA5 than on RIG-I. However, these results are consistent with more recent studies using SeV that indicated that both RIG-I and MDA5 could contribute to IFN-β production in vitro
and resistance in vivo
). However, these findings cannot be readily extrapolated to HPIV1, because SeV, in contrast to HPIV1, also expresses a V protein, a known inhibitor of MDA5 (2
). Since MDA5 primarily recognizes long dsRNA molecules (or, more specifically, webs of dsRNA and single-stranded RNA [ssRNA] [51
]), its involvement in IFN-β expression during infection with F170S and P(C−) HPIV1 suggested that accumulation of dsRNA might occur, and this was indeed found to be the case. Although earlier studies had reported dsRNA accumulation after infection with positive-strand RNA viruses, double-stranded RNA viruses, and DNA viruses but not with negative-strand RNA viruses (36
), more recent studies also detected dsRNA in cells infected with a C deletion mutant of SeV (59
). Our findings of dsRNA accumulation in cells infected with C protein mutants but not in WT HPIV1 suggest that the HPIV1 C proteins are indeed required to prevent the accumulation of dsRNA that would otherwise trigger a potent host innate antiviral response.
The appearance of dsRNA was temporally associated with phosphorylation of PKR and eIF2α and decreased accumulation of the N, P, and C proteins in F170S-infected cells. PKR knockdown studies confirmed that PKR is a major contributor to the loss of the N, P, and CF170S
proteins. In addition to its role in inhibiting translation during viral infection, PKR has also been implicated in NF-κB activation, IRF1 activation, and IFN induction by poly(I:C) via MAVS, effects that are independent of its kinase activity and that provide another mechanism of innate antiviral host defense (3
). Our data indicate that PKR contributes significantly to IRF3 phosphorylation and induction of IFN-β following infection with HPIV1 C protein mutants that generated dsRNA. These finding are consistent with a previous report on a C-protein-deficient measles virus mutant that required PKR for maximal IFN-β induction (43
). Thus, both MDA5 and PKR contribute to the generation of IFN-β during infection with C mutant HPIV1s, and both are activated by dsRNA.
Quantitation of viral RNA levels showed that accumulation of genomic and antigenomic RNA, but also viral mRNA, was more pronounced in P(C−)- and F170S-infected A549 cells than in WT HPIV1-infected cells. Concomitant with the increase in viral RNA species, N and P protein levels decreased, which led us to speculate that this imbalance could give rise to unencapsidated RNAs that could anneal with each other or with excess viral mRNA to form dsRNA. However, we have not yet confirmed the identity of the dsRNA species detected in A549 cells. The current model of genome replication by nonsegmented negative-sense RNA viruses involves N protein encapsidation of the template. In the absence of concurrent encapsidation, naked replication products are made that terminate prematurely (65
). These RNAs could stimulate the innate immune system.
For SeV, the C proteins have previously been implicated in inhibiting viral RNA synthesis and regulating the balance between genome and antigenome levels (14
). Interestingly, one study reported that infection of primate LLC-MK2 and Vero cells with a SeV C deletion strain led to a predominance of antigenomic RNA compared to genomic RNA and to an increase in viral protein synthesis (29
). Our results differ from these in that the accumulation of viral proteins was decreased rather than increased, and deletion of the HPIV1 C proteins did not alter the ratio of viral genomic RNA to antigenomic RNA. Our results provide an alternative explanation for the observation that a recombinant SeV strain expressing HPIV1 C in place of SeV C failed to block IRF3 activation in murine cells (11
). This originally was interpreted to indicate that the respirovirus C proteins, like the rubulavirus V protein, are determinants of host range (11
). However, it may be that the HPIV1 C proteins do not function efficiently in the context of the heterologous SeV N, P, and L proteins to prevent the accumulation of dsRNA and subsequent IRF3 activation. We (unpublished data) and others (22
) have observed an association between the HPIV1 C proteins and proteins of the homologous replication complex, and this association might be virus specific.
This study has a number of limitations that need to be considered when interpreting our results. First of all, the experimental models used (A549, MEF, and 239 cells) have their own limitations. For example, although the A549 cell line is an accepted model for human respiratory epithelial cells, it is an immortalized cell line, and as such its signaling pathways might be altered. To address this concern, we have previously used primary human airway epithelial cells to confirm that the F170S mutant readily induced type I IFN induction in this organized epithelium. However, the P(C−) mutant was too restricted in replication in this organized epithelium to permit a valid comparison to WT HPIV1 (6
). Another limitation is the uncertainty as to whether PKR- and IFN-β-induced inhibition of protein synthesis can fully account for the reduced accumulation of viral proteins during infection with the HPIV1 mutants. For example, in cells infected with the F170S mutant, the levels of N and P protein were reduced compared to those in WT-infected cells even at 24 h, a time point before the activation of PKR was detected. Also, while the rapid accumulation of genomic and antigenomic RNA with the P(C−) mutant could be imagined to outpace the synthesis of N and P protein and thus initiate dsRNA formation, this proposed mechanism seems less convincing for the F170S mutant with its more gradual dsRNA accumulation. Thus, there may be additional host inhibitory effects on viral protein accumulation that are activated in C mutant-infected cells or, alternatively, the C proteins may have a positive effect on viral protein synthesis. Further studies to explore these alternative mechanisms of regulating viral protein synthesis are clearly needed.
To our surprise, we were unable to detect a physical interaction between wild-type C proteins and any of the examined members of the RIG-I/MDA5 pathway, either by co-IP or by yeast two-hybrid assay or mass spectroscopy. Rather, the C proteins seem to regulate viral RNA synthesis to prevent the production of dsRNA that would otherwise activate MDA5 and PKR which, in turn, would activate IRF3 and NF-κB, result in the induction of IFN-β, and inhibit protein synthesis. We propose that the following series of events occur during infection with P(C−) HPIV1. dsRNA is generated early, within the first 24 h of infection, due to the combined rapid increase of mRNA, genomic RNA, and antigenomic RNA. The dsRNA activates PKR and also induces IFN-β via the RIG-I/MDA5 pathway. PKR activation and IFN-β induction decrease viral protein synthesis, thereby maintaining low levels of N protein. IFN-β might also inhibit protein synthesis by the induction of inhibitors such as IFIT1 and IFIT2 (61
). In addition, apoptosis is activated early in P(C−)-infected cells, but the mechanism underlying this induction remains undefined (6
). The combined effects of PKR activation, IFN-β induction, and the initiation of apoptosis are likely responsible for the decline in viral macromolecular synthesis that starts between 24 and 48 h after infection with P(C−) HPIV1. The events occurring during infection with the F170S mutant are delayed but could be similar to those in P(C−) HPIV1-infected cells. The kinetics of dsRNA accumulation and decreased expression of mutant C proteins described here suggest that a race between the evasion of host antiviral responses and the elimination of viral antagonists of dsRNA accumulation is key in determining the outcome of HPIV1 infections.