An understanding of the determinants of virulence in different host species is fundamental to our understanding of the zoonotic nature of filoviruses. Rousettus aegyptiacus
bats have been implicated as reservoirs of Marburg viruses (37
). In the laboratory, examining the adaptation of these viruses to rodents may provide clues to viral and host factors that determine the outcome of these infections. Experimental infections with filovirus isolates that cause severe disease in humans do not typically result in disease in rodents. Experiments in mice previously demonstrated a role for the host IFN-α/β responses in the resistance of this species to lethal infection by nonadapted filoviruses. For example, intraperitoneal infection of adult wild-type mice did not result in lethal infection with nonadapted Marburg viruses or EBOVs. In contrast, IFN receptor knockout mice succumbed to infection with nonadapted Marburg viruses or EBOVs. Administration to animals of anti-IFN-α/β antibodies also potentiates filovirus disease (4
). Further, adaptation of Marburg viruses or EBOVs overcomes their inability to cause disease in IFN-competent animals (5
). These observations implicate the host IFN-α/β response in host restriction of filoviruses, at least in mice. However, the molecular basis by which adaptation allows disease to occur in IFN-competent animals remains incompletely defined.
The present study demonstrates that both the nonadapted MARV VP40 and the VP40 of nonadapted RAVV, a marburgvirus from a different clade than MARV, inhibit IFN-α/β signaling and prevent signaling in Jak1-overexpressing human cells. These observations are consistent with the prior observation that MARV infection or expression of MARV VP40 inhibits Jak1-dependent signaling in primate cells (40
). This results in a loss of cellular responsiveness to IFN-α/β or IFN-γ, mimicking the phenotype of Jak1-knockout cells. Thus, the MARV-infected or VP40-expressing cells exhibited a general loss of tyrosine phosphorylation of Jak tyrosine kinases and STAT proteins after the addition of IFN-α/β or IFN-γ to cells. These cells also exhibited resistance to the antiviral effects of IFN-α/β. When Jak family tyrosine kinases are overexpressed in Huh7 cells, the overexpressed kinase becomes tyrosine phosphorylated, as do cellular STAT proteins. In support of the view that Jak1 is a critical and specific target of VP40, expression of either RAVV or MARV VP40 inhibits the tyrosine phosphorylation of Jak1 and STAT proteins in Jak1-overexpressing human cells (40
Notably, neither MARV nor RAVV VP40 could efficiently inhibit IFN-α/β signaling in the mouse cells tested (). Although a modest reduction in STAT1 or STAT2 phosphorylation could be seen in VP40-expressing mouse cells, relative to empty vector-transfected control cells, this inhibition was not sufficient to counteract the antiviral effects of IFN (). Strikingly, however, adaptation of RAVV to mice resulted in a VP40 fully capable of inhibiting IFN signaling and blocking the antiviral effects of IFN-β in mouse cells ( and 4). Moreover, maRAVV VP40 was able to inhibit the phosphorylation of either mouse or human Jak1 and STAT1 in human and mouse cells, while the nonadapted VP40s inhibited the phosphorylation of human Jak1 and mouse Jak1 in human cells but not in mouse cells (). These observations are consistent with a model where VP40 inhibition of Jak1 requires an additional host factor(s) to carry out its inhibitory function.
Given that the mouse-adapted RAVV exhibits increased virulence in mice, relative to the parental strain from which it was derived, the enhanced IFN antagonist function is consistent with a role for VP40 IFN antagonist function in mouse pathogenesis. Whether the mouse-adaptive changes in VP40 are sufficient for enhanced virulence in the absence of other changes remains to be determined. It is intriguing that the mouse-adapted RAVV also accumulated several amino acid changes in its VP35 protein. In EBOV, VP35 has been demonstrated to inhibit the production of IFN-α/β by antagonizing the RIG-I signaling pathway (6
). Whether these VP35 changes influence its function in mouse cells is therefore of interest.
Of the 7 amino acid changes that accumulated in RAVV VP40 during mouse adaptation, 2 residues (V57 and T165 in the nonadapted RAVV VP40) proved to be critical for IFN antagonist function in mouse cells. This was evidenced by the gain of IFN antagonist function in mouse cells when only these two residues were mutated to alanine in the context of an otherwise nonadapted RAVV VP40. Both changes appear to be required for full activity, although very modest increases in inhibitory function were seen with individual changes at these residues (e.g., ). Also, the individual mutation of either residue back to V57 or T165 in the context of the mouse-adapted VP40 was sufficient to abrogate function in mouse cells. When the V57A/T165A mutations were examined in the context of the MARV VP40, this also conferred the ability to inhibit IFN signaling in mouse cells, further emphasizing the importance of these residues. A working hypothesis would be that these residues allow VP40 to interact with an unidentified factor in mouse cells that mediates inhibition of Jak1. How the very conservative valine to alanine change at residue 57 dramatically affects function remains to be determined. It is noteworthy that VP40s with an alanine at this position reproducibly exhibit slightly retarded migration on SDS-PAGE, suggesting a possible structural change or posttranslational modification of the viral protein.
Expression of VP40 alone is able to drive the formation of VLPs that bud from the cell membrane. Filoviral VP40s have well-characterized late domains important for the budding process: ZEBOV VP40 possesses two late domains, a PTAP motif and an overlapping PPXY motif, which mediate interaction with Tsg101, Nedd4, and Rsp5. MARV VP40 possesses a single 13
motif that allows interaction with Tsg101, although it has been proposed that NP can also recruit Tsg101 to sites of MARV VP40 budding (8
). Our previous study suggested that the two functions of MARV VP40—IFN signaling antagonism and VLP formation—are independent (40
). During the adaptation of RAVV, one of the seven amino acids that were changed was the tyrosine at position 16 (43
). This particular amino acid has been shown to be involved in interaction with Tsg101 (39
). However, in mouse cells, change of this amino acid to a histidine only reduced the ability of the protein to bud by 2-fold (). None of the other mutations that are involved in IFN antagonism in mouse cells altered the protein's ability to bud. Therefore, our data suggest that the budding function of RAVV VP40 did not need to adapt to the mouse.
The present study identifies a specific molecular function that changed during RAVV adaptation to mice. The host factor(s) that interact with VP40 to mediate its IFN antagonist function should shed light upon its mechanism of action, and the availability of VP40s that do or do not function in mouse cells provides a useful system that may allow the identification of these critical host factors. Additional studies will be required to define the contribution of these changes to virulence in mice and determine the contribution to host adaptation of other changes in VP40.