In this study, we demonstrate that reovirus represses the IFN-α/β signaling pathway and that this repression is virus strain specific. Repression is mediated by the M1 gene product (μ2) causing nuclear accumulation of IRF9. Virus strain-specific differences in this repressor function are associated with virus strain-specific differences in induction of myocarditis, a disease modulated by the IFN response (44
). Together, these data provide the first report of an association between viral subversion of the IFN response and nuclear accumulation of IRF9 and link strain-specific differences in IFN antagonism to viral pathogenesis.
The inhibitory effect of T1L infection on IFN signaling cosegregated with the T1L M1 gene (Fig. and ; Table ), and ectopic expression of the T1L M1 gene inhibited IFN-β-induced reporter gene expression (Fig. ). Reovirus strain-specific differences in induction of and sensitivity to IFN-α/β are associated with the M1, S2, and L2 genes (62
), suggesting that repression of IFN signaling either provides the underlying mechanism for these two properties or involves interactions with similar host factors. Reovirus induction of IFN-β is substantially reduced in cells derived from mice lacking the IFN-α/β receptor (B. Sherry, unpublished observations), indicating that most of the IFN-β induced by reovirus infection results from the positive amplification loop, where IFN-mediated induction of IRF7 induces additional IFN-β (56
). Therefore, repression of IFN signaling would reduce reovirus induction of IFN, providing a mechanism for poor induction of IFN by T1L (62
). Repression of IFN signaling also would provide resistance to the antiviral effects of IFN, providing a mechanism for the relative resistance of T1L to IFN (27
). Thus, T1L repression of IFN signaling provides a mechanism for strain-specific differences in both reovirus induction of and sensitivity to IFN-α/β.
Studies with pathogenic viruses have demonstrated that the type I IFN response is essential in protection against viral disease. The virulence of many viruses is enhanced in mice lacking the IFN-α/β receptor (14
), and absence of the IFN-α/β response can result in broader tissue tropism and lethality following infection (18
). Inhibition of the host IFN response is a critical determinant of viral virulence, as evidenced by the resultant attenuation when viral IFN repressor proteins are mutated (11
). Indeed, targeting these IFN repressors provides the conceptual basis for a new generation of vaccine candidates (69
). Virus strain-specific differences in antagonism of the IFN response can be determinants of strain-specific differences in disease, but results are mixed. For example, while virus strain-specific differences in the NS1 protein of H1N1 influenza virus result in differences in inhibition of the IFN response (31
), there is no evidence that these NS1 protein differences result in differences in influenza virus pathogenicity (46
). However, studies using the NS1 gene from H5N1 influenza virus strains link NS1 effects on the IFN response with pathogenesis (59
). Finally, a study using both H1N1 and H5N1 strains found that NS1 protein differences are associated with differences in pathogenicity but not through differences in modulation of the IFN response (26
). Identification of an association between reovirus repression of the IFN response and reovirus-induced myocarditis here (Table [EW26 in particular]) provides strong evidence that viral IFN antagonism serves as a virulence determinant in the heart.
Infection with T1L or reassortant or recombinant viruses containing the T1L M1 gene results in accumulation of IRF9 in the nucleus, an effect not previously described for any virus. In addition, nuclear accumulation of IRF9 occurs in both murine and human cell lines infected with T1L, indicating that this effect is not species specific. Paramyxovirus inhibitors of IFN are either species specific (35
) or operant across a broad host range (20
). The events underlying T1L M1-mediated nuclear accumulation of IRF9 are not yet understood. The M1- encoded μ2 protein (57
) is an RNA-binding protein (6
) present in low copy number in the virion but expressed abundantly in infected cells, where it associates with microtubules (8
) and contributes to formation of viral factories (47
). Interestingly, despite the exclusively cytoplasmic replication strategy of reovirus, and in contrast to most reovirus proteins, μ2 also distributes to the nucleus and contains a predicted NLS (7
). The function of μ2 in the nucleus is unknown, but it may alter IRF9 structure or function there. In sum, none of the known properties of μ2 suggest a specific mechanism for modulation of IRF9 localization.
Nuclear-cytoplasmic shuttling of IRF9 has been well characterized (1
). IRF9 contains an NLS but lacks a nuclear export signal, while STAT2 lacks a functional NLS but contains a nuclear export signal. They form a heterodimeric cytoplasmic complex in unstimulated cells, resulting in IRF9-mediated interaction with importins for nuclear localization. However, STAT2-mediated interactions with CRM1 in the nucleus result in nuclear export, and this dominant function determines a primarily cytoplasmic localization for both STAT2 and its partner IRF9. Upon IFN stimulation, STAT phosphorylation, and formation of ISGF3, IRF9 is again transported to the nucleus. Nuclear dephosphorylation of STAT2 results in association with CRM1, and STAT2 then escorts IRF9 back to the cytoplasm. Accordingly, we envision four possible models for μ2-mediated nuclear localization of IRF9 and repression of IFN signaling. First, nuclear accumulation of IRF9 in T1L-infected cells could reflect an upregulation of IRF9 expression, and abundant free nuclear IRF9 could compete with ISGF3 to bind but not induce expression from ISREs, thereby functioning as a dominant-negative inhibitor of IFN signaling. However, total cellular IRF9 was not increased in T1L-infected cells (Fig. ), and qRT-PCR experiments demonstrate that T1L does not induce IRF9 mRNA between 4 and 24 h postinfection (data not shown), together indicating that T1L does not upregulate IRF9 expression. Moreover, while IRF9 does bind ISREs without inducing expression (35
), IRF9 overexpression can compensate for IRF9 deficiencies with no evidence of dominant-negative function (37
). Second, T1L μ2 could enhance importin function to increase IRF9 nuclear translocation, and once there, abundant free IRF9 could act as a dominant-negative inhibitor of IFN signaling. Viral modulation of importins or nuclear-cytoplasmic trafficking is well precedented, although in each case, the virus acts to impede rather than enhance normal cell function (16
). However, this model lacks precedent for IRF9 dominant-negative function. Third, T1L μ2 could inhibit CRM1 function, thereby blocking STAT2-mediated IRF9 export. However, STAT2 nuclear localization was not increased by T1L infection (Fig. ), eliminating this possibility. Finally, in a fourth model, which we favor, T1L μ2 might prevent IRF9 from binding to STAT2, thereby affecting both ISGF3 function and IRF9 export. This process could occur through μ2 association with IRF9 in the cytoplasm, consistent with T1L induction of IRF9 nuclear accumulation even in the absence of IFN stimulation (Fig. ), or in the nucleus, consistent with μ2 nuclear localization. Alternatively, μ2 could mediate structural modifications of IRF9. Our future studies will determine whether μ2 affects the interaction of IRF9 with ISRE promoters and other components of the ISG transcriptional apparatus to attenuate ISG expression.
Previous studies of viral mechanisms for antagonizing the IFN response involving IRF9 have documented a decrease in IRF9 levels (37
) or sequestration of IRF9 to prevent its translocation to the nucleus upon IFN stimulation (2
) but not nuclear accumulation of IRF9 as seen here. The nonstructural protein 1 of rotavirus, another member of the Reoviridae
family, modulates the IFN response by recognizing a common element of IRF proteins to mediate degradation of IRF3, IRF5, and IRF7 (3
). While our results suggest that modulation of IRFs may be a common mechanism by which Reoviridae
members subvert the IFN response, reovirus does not express a homolog of rotavirus nonstructural protein 1, and reovirus has not been shown to inhibit the activity of other IRF proteins.
Other viruses use multiple independent mechanisms to inhibit the IFN response. For example, the influenza virus NS1 protein (31
), paramyxovirus V proteins (21
), and herpes simplex virus type 1 (29
) each block both induction of IFN and IFN signaling. The reovirus S4 gene encodes a dsRNA-binding protein, σ3, which is capable of preventing activation of the antiviral ISG PKR (57
). While dsRNA is not thought to be exposed in the cytoplasm during reovirus replication and σ3 has not been implicated genetically or biochemically in modulating the IFN response during reovirus infection, a role for σ3 in countering the host response remains possible. The results reported here provide the first direct evidence for a reovirus protein modulating the IFN response during infection. If σ3 also inhibits the IFN pathway by sequestering dsRNA, then reovirus, encoding only 11 proteins, has evolved at least two independent mechanisms for subverting the antiviral actions of IFN.