Previous studies have shown that both members of the filovirus family, MARV and EBOV, impair cellular responses to IFNs 
. While ZEBOV blocks the nuclear accumulation of tyrosine-phosphorylated STAT1 
, the present study demonstrates that MARV has evolved a different mechanism to counteract IFN signaling. We show that MARV inhibits the IFNα-induced tyrosine phosphorylation of not only STAT1 and STAT2 but also of the upstream kinases Jak1 and Tyk2. This inhibition prevents the IFN-induced nuclear accumulation of STAT1 and STAT2. Further, MARV infection inhibits the IFNγ-induced tyrosine phosphorylation of STAT1. The inhibition extends even beyond the IFNα/β and IFNγ signaling pathways to another Jak1 dependent signaling pathway, the IL-6 pathway, where the phosphorylation of STAT1 and STAT3 was inhibited. Significantly, the study also identifies a single MARV protein, the matrix protein VP40, sufficient to mediate these inhibitory effects, whereas ZEBOV-induced inhibition of IFN signaling is mediated by VP24 
. Emphasizing the specificity of the inhibitory function for MARV VP40, neither ZEBOV infection nor ZEBOV VP40 expression impairs Jak or STAT phosphorylation. Moreover, MARV VP24, including VP24s corresponding to the Musoke strain and the Angola strain, which caused an outbreak with a very high fatality rate 
, did not detectably inhibit IFNα/β-induced gene expression ( and data not shown). Musoke MARV VP24 was also unable to inhibit IFNα/β-, IFNγ- or IL-6-induced phosphorylation of Jaks or STATs.
The striking differences in the strategies employed by filoviruses to block IFN signaling may have been driven by the different evolutionary paths taken by Marburg and Ebola viruses. Bayesian analysis of genome sequence differences indicates that Ebola and Marburg viruses diverged from a common ancestor several thousands of years ago (S.T. Nichol, personal communication). Evolution in and adaptation to different host species might account for different immune evasion mechanisms. So far, there is only limited information available about the natural host spectrum of filoviruses. Various species of African fruit bats were found to be seropositive or RT-PCR-positive for EBOV 
, however, as yet Ebola viruses have not been isolated from bats. In contrast, Towner and colleagues reported the successful isolation of MARV from the Egyptian fruit bat Rousettus aegyptiacus 
. Since this bat species is also discussed as a potential reservoir for EBOV 
, it remains unclear if Marburg and Ebola viruses differ in their host tropism. Recently, the Asian EBOV species Reston ebolavirus
(REBOV), which is thought to be non-pathogenic for humans, was isolated from pigs 
. Phylogenetic analyses suggested that the REBOV clade has evolved separately from the African Ebola viruses 
. Interestingly, REBOV VP24 was also shown to interfere with the nuclear translocation of STAT1 
, indicating that the ability of VP24 to counteract IFN signaling was evolved among Ebola viruses prior to the separation of the African and Asian species. Notably, VP24 contributes to the host specificity of ZEBOV 
. Whether VP40 plays a similar role in MARV host tropism has yet to be determined; however, it is intriguing that a mouse-adapted MARV acquired amino acid changes in VP40 
The effects of MARV infection and MARV VP40 expression on IFNα/β, IFNγ and IL-6 signaling mirror the impact of Jak1 knock-out on these pathways. In cells lacking Jak1, no STAT or Jak phosphorylation was observed upon IFNα/β or IFNγ treatment 
. Similarly, the absence of Jak1 profoundly affects the IL-6 pathway as elimination of Jak1 was sufficient to fully abrogate any detectable phospho-STAT1 and greatly reduce phospho-STAT3 following IL-6 addition 
. Interestingly, MARV infection and individual expression of MARV VP40 closely mirror this phenotype, where following IL-6 addition, phospho-STAT1 was undetectable but residual phospho-STAT3 was present (). Further studies will reveal to what extent the observed residual STAT3 phosphorylation may mediate IL-6 signaling.
Our data are consistent with a model in which MARV VP40 targets Jak1 function, either directly or indirectly, although the possibility remains that MARV VP40 can also impair signaling of other Jak family kinases. A possible indirect mechanism of the observed inhibition could be a modulating effect of MARV VP40 on PTPs targeting Jak kinases. Recently, it has been reported that transgenic mice with reduced expression of the PTP CD45 were protected against lethal EBOV infection 
. Interestingly, CD45 acts as a negative regulator of Jak1 in cells of hematopoietic origin 
. However, our data suggest that PTPs are not involved in MARV-mediated inhibition of Jak1 signaling in cells of non-hematopoietic origin. Therefore, it is of interest to further extend those studies and to analyze Jak/STAT signaling in human hematopoietic cells in the context of MARV and EBOV infection.
The observed inhibitory effects of MARV VP40 on both IFNα/β-induced gene expression and the antiviral effects of IFNβ may explain the capacity of MARV to prevent cellular responses to exogenously-added IFNα 
. In this respect, MARV VP40 appears to serve the same purpose as the EBOV VP24 proteins which also counteract IFNα/β signaling. It is likely that counteracting IFNα/β signaling has a significant impact on viral pathogenesis in vivo
, because, despite the presence of viral VP35 proteins that suppress IFNα/β production 
, filovirus replication in vivo
results in significant IFNα production 
. The presence of IFNα/β signaling inhibitors likely also contributes to the relative insensitivity of filoviruses to IFNα/β as an antiviral therapy 
. IFNγ also has antiviral properties 
, however, suppression of IFNγ signaling may also modulate adaptive immune responses to infection. For example, human cytomegalovirus down-regulates Jak1 expression in a proteasome-dependent manner, and although a specific viral gene product that mediates this effect has not been defined, this function prevents the IFNγ-induced upregulation of MHC class II on infected cells 
. Another viral protein that interacts with Jak1 and blocks the type I IFN signaling pathway is the measles virus V protein, but the consequence of this function for adaptive immunity has not been defined 
. The possible impact of MARV infection and MARV VP40 expression on other cytokine signaling pathways involving Jak1 remains to be defined. Given the prominent role of Jak1 in numerous pathways, the impact of MARV VP40 on cytokine signaling may be quite broad.
Filovirus VP40 proteins are matrix proteins sufficient to drive budding of virus-like particles, and they are thought to be the driving force for the budding of infectious virus 
. The finding that MARV VP40 also serves as an inhibitor of IFN signaling is surprising and novel. Another example of a negative-strand RNA virus matrix protein that inhibits IFN responses is the vesicular stomatitis virus (VSV) matrix protein (M). VSV M inhibits innate immune responses, including IFNβ production, by a mechanism different from MARV VP40, inhibiting host cell transcription as well as nucleo-cytoplasmic transport of cellular mRNAs 
Host factors that interact with filovirus VP40 proteins have been described 
. The most fully characterized interactions occur via the VP40 late domain which facilitates budding and release of virus particles. ZEBOV VP40 possesses two late domains, a PTAP motif and an overlapping PXXP motif 
. These mediate interaction with Tsg101, Nedd4, and Rsp5 
. MARV VP40 possesses a single PPPY motif that allows interaction with Tsg101 
. To address the potential role of these well-characterized motifs in MARV VP40 inhibition of Jak/STAT signaling, a 16-PPPY-19 to 16-AAAA-19 mutant MARV VP40 was generated. As previously described, this mutation severely impaired MARV VP40 budding () 
. Yet this mutation had no detectable impact on MARV VP40 inhibition of IFNα/β signaling (). Therefore, the late domain is dispensable for the IFN signaling function of VP40, and the budding and signaling functions of MARV VP40 appear to be separable. Of note, IFN-induced cellular inhibitors of filovirus VP40 budding have recently been described. These include the IFN stimulated ISG15 and tetherin 
. ISG15 is an IFN-induced protein which inhibits budding of EBOV VP40. ISG15 inhibits the ubiquitin ligase Nedd4, which interacts with EBOV VP40 through the PPXY motif to promote VP40 ubiquitination and budding 
. Tetherin is constitutively-expressed in some cell types but is also IFN-inducible. Its expression can prevent release of VLPs produced following expression of EBOV or MARV VP40 
. Co-expression of EBOV GP has been shown capable of counteracting this antiviral function 
. Whether MARV GP can also inhibit tetherin has not yet been addressed; however, because MARV VP40 can inhibit IFN signaling, it appears to have a built-in capacity to resist IFN-induced mechanisms that target viral budding.
This study has identified an important difference in the biology of MARV and EBOV, defined a novel function for the MARV VP40 matrix protein and suggests that MARV may inhibit multiple Jak1-dependent cytokine signaling pathways. Future studies will determine whether the different means by which EBOV and MARV counteract cell signaling pathways result in significant differences in the pathologenesis of these viruses. Determining the molecular mechanisms by which MARV VP40 blocks signaling may facilitate development of new anti-MARV therapies.