Adaptive mutations in the nuclear export protein of human-derived H5N1 strains facilitate a polymerase activity-enhancing conformation.
نویسندگان
چکیده
The nuclear export protein (NEP) (NS2) of the highly pathogenic human-derived H5N1 strain A/Thailand/1(KAN-1)/2004 with the adaptive mutation M16I greatly enhances the polymerase activity in human cells in a concentration-dependent manner. While low NEP levels enhance the polymerase activity, high levels are inhibitory. To gain insights into the underlying mechanism, we analyzed the effect of NEP deletion mutants on polymerase activity after reconstitution in human cells. This revealed that the polymerase-enhancing function of NEP resides in the C-terminal moiety and that removal of the last three amino acids completely abrogates this activity. Moreover, compared to full-length NEP, the C-terminal moiety alone exhibited significantly higher activity and seemed to be deregulated, since even the highest concentration did not result in an inhibition of polymerase activity. To determine transient interactions between the N- and C-terminal domains in cis, we fused both ends of NEP to a split click beetle luciferase and performed fragment complementation assays. With decreasing temperature, increased luciferase activity was observed, suggesting that intramolecular binding between the C- and N-terminal domains is preferentially stabilized at low temperatures. This stabilizing effect was significantly reduced with the adaptive mutation M16I or a combination of adaptive mutations (M16I, Y41C, and E75G), which further increased polymerase activity also at 34°C. We therefore propose a model in which the N-terminal moiety of NEP exerts an inhibitory function by back-folding to the C-terminal domain. In this model, adaptive mutations in NEP decrease binding between the C- and N-terminal domains, thereby allowing the protein to "open up" and become active already at a low temperature.
منابع مشابه
Adaptive mutations in the nuclear export protein of human-derived H5N1 strains
にの The nuclear export protein NEP (NS2) of the highly pathogenic human-derived H5N1 strain には A/Thailand/1(KAN-1)/2004 with the adaptive mutation M16I greatly enhances the polymerase にば activity in human cells in a concentration-dependent manner. While low NEP levels enhance にぱ the polymerase activity, high levels act inhibitory. To gain insights into the underlying にひ mechanism, we analyzed th...
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ورودعنوان ژورنال:
- Journal of virology
دوره 88 1 شماره
صفحات -
تاریخ انتشار 2014