The type I interferon (IFN) system plays a critical role in the control of acute viral infections. IFNs are produced In response to virus infection and induce the synthesis of about 300 proteins whose combined antiviral activities generate a so called antiviral state. One of these effector proteins the human MxA protein, has been shown to efficiently block the replication of avian influenza A viruses. Influenza A viruses cause a severe respiratory disease in humans and are responsible for periodic wide-spread epidemics, or pandemics. Many viruses including influenza viruses have developed various strategies to counteract the antiviral activity of the IFN system. We recently made two intriguing observations: Firstly, we found that several isolates of human influenza A virus are largely resistant to the antiviral activity of human MxA. Secondly we have identified the protein encoded by influenza A virus that blocks the IFN-mediated signalling cascade and hence the expression of IFN-induced effector proteins such as MxA.
Molecular mechanism of antiviral activity of the interferon-induced human MxA protein
Christian Wisskirchen, Milos Tatarski, Thomas Ludersdorfer and Jovan Pavlovic
The interferon-α/ß inducible Mx proteins belong to the functionally diverse dynamin family of large GTPases. Characteristic features are a highly conserved GTP-binding region, an intrinsic GTPase activity and their capacity to form oligomers. The human MxA protein exerts antiviral activity by inhibiting the replication of certain RNA viruses in a GTP-dependent fashion. We have recently demonstrated that backfolding of the C-terminal end of human MxA protein onto a more proximal part of the molecule is a prerequisite for oligomerization. The backfolding of MxA is stabilized by an amphipathic helix LZ1. Prevention of backfolding by mutation of LZ1 at the amino acid position 612 (L612K) leads to the loss of GTPase activity and capacity to oligomerize, but the antiviral activity is retained. We show that similarly to dynamin, the intrinsic GTPase activity of Mx proteins is mediated by a GTPase effector domain (GED). Based on these results we propose a model where Mx proteins are synthesized in response to interferon-α/ß to form a pre-activated oligomeric structure. In the presence of viral components, MxA may then convert in a GTP-dependent manner to a monomeric, activated form. Once this step is completed MxA no longer requires GTP for its antiviral activity.
The aim of this project is to identify the viral target molecule of MxA and to elucidate the molecular basis of the resistance of human influenza A viruses to the antiviral activity of MxA. Preliminary evidence suggests that the observed resistance of human influenza virus to the activity of human MxA is mediated by components of the viral polymerase complex. To elucidate the molecular mechanism of resistance of human influenza A viruses to MxA, we take advantage of the plasmid-based minimal transcription /replication system for human and avian influenza viruses. We also plan to employ this reverse genetics approach to identify the viral target molecule of MxA. In addition we have introduced mutations into potentially important domains of Mx proteins and want to test the resulting Mx variants for their functional activity.
Negative regulation of cytokines signalling by the influenza matrix protein (M1)
Thomas Ludersdorfer, Bettina T. Oberle, Ursula Broder, Jovan Pavlovic
Complex organisms have evolved sophisticated mechanisms to prevent and control infection by various pathogens or viruses. Among these mechanisms the IFN system, which is part of the innate immune system, represents the first step of defense against viral infection in vertebrates.
The influenza A virus is counteracting the IFN system by inhibiting the type I IFN production through the non-structural viral protein 1 (NS1). However, ∆NS1, the NS1 deletion mutant of influenza virus, still propagates in IFN competent host cells, and actively interferes with IFN type I-mediated signaling, suggesting a second mechanism by which the virus is able to circumvent the cellular defense mechanisms. However, no conclusive results have been obtained.
The goal of this project is to elucidate the molecular mechanism of the inhibition of the IFN-mediated signalling by the influenza virus protein M1. First experiments demonstrated that the M1-mediated block of IFN-dependent signalling is due to the interaction of M1 with the IFN receptor complex, in particular the scaffold protein RACK1. To address the IFN-anatgonistic function of the viral protein we plan to use classical biochemisty such as -immunoprecipitation experiments as well as mutational analysis of the influenza virus M1 protein and reverse genetics.
Inhibition of Semliki Forest virus by the interferon system
Stefan Deuber, Thomas Ludersdorfer, Christian Wisskirchen and Jovan Pavlovic
We are investigating two closely related Semliki forest viruses (SFV) with different sensitivities to IFN. The virulent V45 strain is lethal for wild-type (wt) mice whereas infection of mice with the avirulent V42 strain remains asymptomatic. Upon IFN treatment in vitro, MEFs (mouse embryo fibroblasts) derived from wt mice are completely protected from the avirulent V42 strain while IFN-stimulated cells are killed by the virulent V45 strain. We were interested in defining the viral sequence determining the IFN sensitivity of SFV. We have sequenced both virus strains and constructed recombinant cDNA clones derived from both strains. Several differences were detected in the coding region of the non-structural and structural proteins but also in the non-translated region (NTR). Unexpectedly, we discovered differences in the 5' NTR compared with the known published SFV 5' NTR. The avirulent V42 strain contains an additional sequence of 20 nucleotides (nts) at the extreme 5' end, which are lacking in the IFN-resistant virus. These additional nts are predicted to form a hairpin structure preceding the highly structured 5' NTR of SFV. Generation of chimeric viruses containing swapped segments revealed that the additional 20 nucleotides are sufficient to confer IFN sensitivity and a reduced cytopathic effect in insect cells. Therefore, we termed this sequence Interferon Sensitivity Determining Element (ISDE). The Goal of the ongoing study will be now to identify the IFN-induced effector protein responsible for the inhibition of the IFN-sensitive SFV V42 strain.