Completion of Hepatitis C Virus Replication Cycle in Heterokaryons Excludes Dominant Restrictions in Human Non-liver and Mouse Liver Cell Lines

Hepatitis C virus (HCV) is hepatotropic and only infects humans and chimpanzees. Consequently, an immunocompetent small animal model is lacking. The restricted tropism of HCV likely reflects specific host factor requirements. We investigated if dominant restriction factors expressed in non-liver or non-human cell lines inhibit HCV propagation thus rendering these cells non-permissive. To this end we explored if HCV completes its replication cycle in heterokaryons between human liver cell lines and non-permissive cell lines from human non-liver or mouse liver origin. Despite functional viral pattern recognition pathways and responsiveness to interferon, virus production was observed in all fused cells and was only ablated when cells were treated with exogenous interferon. These results exclude that constitutive or virus-induced expression of dominant restriction factors prevents propagation of HCV in these cell types, which has important implications for HCV tissue and species tropism. In turn, these data strongly advocate transgenic approaches of crucial human HCV cofactors to establish an immunocompetent small animal model

Analysing molecular virulence determinants of the viral NS1 protein of an avian H5N1 influenza A virus

Zusammenfassung Die Infektionen von Menschen mit hoch-pathogenen aviären Influenza Viren (HPAIV) des Subtyps H5N1 führten bisher zu einer Mortalitätsrate von ca 60 %. Aufgrund des pandemischen Potentials dieser Viren ist die Identifizierung von Virulenzdeterminanten dringend erforderlich, um ein besseres Verständnis über die Pathomechanismen dieser HPAIV zu erhalten. Dieses Wissen ermöglicht es, neue Medikamente zur Behandlung dieser Infektion zu entwickeln. Das Influenza Virus NS1 Protein, dessen primäre Funktionen die Inhibition der Typ I Interferon Antwort und die Limitierung antiviraler Effekte von Interferon induzierten Proteinen wie PKR, OAS/RNase L und RIG-I sind, trägt mit zur Virulenz der Viren in Säugern bei. Ziel dieser Arbeit war es daher, zwei Funktionen des NS1 Proteins eines HPAIV zu analysieren. Zum einen wurde das kürzlich als Virulenzdeterminante postulierte PDZ-Liganden Motiv ESEV des NS1 Proteins auf seine Rolle in der Pathogenität von HPAIV untersucht. Zum anderen wurde analysiert, ob und wie das NS1 Protein eines HPAIV als PKR Inhibitor wirkt und welchen Einfluss diese Interaktion auf die virale Replikation des HPAIV hat. Das NS1 Protein der meisten aviären Influenza Viren enthält ein C-terminales PDZ-Liganden Motiv bestehend aus den Aminosäuren ESEV. Im Gegensatz hierzu enden die meisten saisonalen Influenza Viren auf die Aminosäuresequenz RSKV. Bisherige Studien zeigten, dass das „aviäre“ ESEV Motiv in vitro an verschiedene humane PDZ Domänen bindet und zur Steigerung der Virulenz eines Maus-adaptierten H1N1 Isolates führt. Im Gegensatz hierzu konnte für das „humane“ RSKV Motiv kaum eine Bindung an PDZ Domänen oder ein Einfluss auf die Virulenz der Viren gezeigt werden. In dieser Arbeit wurde ein prototypisches Infleunza Virus des Subtyps H5N1 vom Stamm 1 verwendet, das VN/1203/04 Virus. Das NS1 Protein dieses Virus ist C-terminal verkürzt, so dass das PL Motiv fehlt. Um die Rolle des C-terminalen NS1 Motivs für die Virulenz von H5N1 Viren untersuchen zu können, wurde ein revers- genetisches System etabliert. Dies ermöglichte die Generierung isogener rekombinanter Viren mit einem rekonstituierten C-Terminus, der entweder auf die Aminosäuresequenz ESEV oder RSKV endet. Die Ergebnisse dieser Arbeit zeigen, dass im Vergleich zum WT und RSKV Virus die Replikation der ESEV Variante auf humanen alveolaren Zellen und murinen Fibroblasten leicht attenuiert war, während auf primären Hühnerembryo-Fibroblasten kein Unterschied zu beobachten war. Dennoch verursachten alle drei Viren letale Infektionen in Mäusen und Hühnern, bei denen zum einen nur geringfügige Unterschiede hinsichtlich der viralen Titer in verschiedenen Organen festgestellt werden konnten, und es zum anderen kaum Unterschiede hinsichtlich der letalen Dosis 50 bzw. des Intravenösen Pathogenitätsindexes gab. Diese Ergebnisse lassen daher darauf schließen, dass das C-terminale ESEV Motiv des NS1 Proteins wenig zur Virulenz des VN/1203 Virus in Mäusen und Hühnern beiträgt, jedoch die virale Replikation in humanen Zellen limitiert. Dementsprechend scheint das PL Motiv des NS1 Proteins die virale Pathogenität in einer Stamm- und Wirts-abhängigen Art und Weise zu modulieren. Des Weitern wurde untersucht, ob das NS1 Protein des VN/1203 Virus die Aktivierung der dsRNA abhängigen Protein Kinase R (PKR), die für den Aufbau des antiviralen Status der Wirtszelle wichtig ist, inhibiert. Während einer Infektion erkennt PKR virale Nukleinsäuren, wodurch es zur strukturellen Umlagerung des Proteins und damit verbunden zur Dimeriserung und Autophosphorylierung der Kinase kommt. Aktivierte PKR phosphoryliert die alpha Untereinheit des eukaryotischen Translationsinitiationsfaktors 2 (eIF2α), wodurch die Translation zellulärer und viraler mRNAs verhindert wird. Dies inhibiert die virale Replikation stark. Für die Inhibiton von PKR durch das A/NS1 Protein wurden unterschiedliche Mechanismen postuliert, wie z.B. RNA Sequestrierung (AS R38 und K41) oder direkte NS1-PKR Bindung (AS I123, M124, K126, N127). Daneben konnte für das B/NS1 Protein kürzlich gezeigt werden, dass die Inhibierung der PKR von der Fähigkeit zur Bildung eines physikalischen NS1-PKR Komplexes abhängt. Die Ergebnisse der vorliegenden Arbeit bestätigen, dass das VN/1203 NS1 Protein als PKR Inhibitor wirkt, und zeigen, dass die Inhibierung durch die Aminosäuren R38 und K41 vermittelt wird, welche die Bindung von RNA ermöglichen. Desweiteren sind diese AS für eine effiziente virale Replikation essentiell und werden auch für eine Interaktion von NS1 mit PKR in einer GST- Kopräzipitation benötigt. Dies deutet auf einen ähnlichen Mechanismus der PKR Inhibierung durch die Influenza A und B Viren hin.Summary Highly pathogenic avian influenza viruses (HPAIV) of the subtype H5N1 have caused a high mortality rate of about 60% in humans. Due to the potential pandemic threat by H5N1 viruses, the identification of virulence determinants and their mode of action are fundamental for a better understanding of the pathogenicity mechanisms of these viruses and for the development of new drugs to treat the infections. The influenza virus non-structural NS1 protein, that has a major function in the inhibition of type I IFN and limitation of the antiviral effects of IFN-induced proteins including PKR, OAS/RNase L and RIG-I, is known to contribute to the virulence in mammals. Therefore the goal of this study was to investigate two different functions of the NS1 protein of a HPAIV: On the one hand the influence of a newly predicted virulence determined the PDZ ligand motif was analysed in the background of a HPAIV. On the other hand it was analysed if and how the NS1 protein of HPAIV also functions as PKR inhibitor and which influence this interaction may have on viral replication. The NS1 proteins of most avian influenza viruses contain the C-terminal PDZ ligand (PL) amino acid motif ESEV, while the corresponding NS1 proteins of seasonal human strains carry the C-terminal sequence RSKV. The “avian” ESEV motif facilitates binding to multiple human PDZ domains in vitro, and increases virulence when introduced into the NS1 protein of mouse–adapted H1N1 influenza virus. In contrast, the RSKV motif binds poorly to PDZ domains and has little effect on virulence. In this study the prototypic H5N1 clade 1 influenza A/VN/1203/04 virus that expresses a truncated NS1 protein lacking the PL motif was examined. To elucidate the role of the C-terminal NS1 motif for the virulence of H5N1 influenza virus, a reverse genetic approach was utilized to generate isogenic recombinant viruses with full-length reconstitution of the NS1 C-terminus, ending with either the ESEV or RSKV motif. Compared to the WT and RSKV viruses, the ESEV variant was slightly attenuated for replication on human alveolar cells and murine fibroblasts, but not on avian cells. However, all three viruses caused highly lethal infections in mice and chickens, with little differences in viral organ titers and lethal dose 50 or intravenous pathogenicity index, respectively. These results suggest that the C-terminal ESEV motif in the NS1 protein contributed little to the virulence of the VN/1203 virus in mice and chickens, but restricted viral replication in human cells. The PL motif in NS1 therefore appeared to modulate viral pathogenicity in a strain- and host-dependent manner. Furthermore it was examined whether the A/VN1203-NS1 protein inhibits the double-stranded (ds) RNA dependent protein kinase R (PKR), which is a key mediator of the innate immune defense. Activation of PKR during infection involves recognition of viral nucleic acids, which induces a structural rearrangement leading to dimerization and autophosphorylation of the kinase. Activated PKR phosphorylates the alpha subunit of the eukaryotic translation initiation factor 2 (eIF2a) and thereby blocks the translation of cellular and viral mRNAs. This strongly reduces the viral replication. Different mechanisms were predicted for PKR inhibition by the A/NS1 protein including RNA sequestration (amino acids R38 and K41) or direct binding (amino acids I123, M124, K126, N127). Interestingly, it was recently shown for the B/NS1 protein that inhibition of PKR depends on its capacity to form a physical complex with PKR. The results of the present study confirm that the VN/1203 NS1 protein functions as PKR inhibitor and show that this inhibition depends on amino acids R38 & K41, which are known to facilitate RNA binding and which were essential for efficient viral replication. In addition, these amino acids were also required for the interaction of the NS1 protein with PKR in a GST pulldown assay, indicating similar mechanisms for Influenza A and B viruses to inhibit PKR activation

Virulence Determinants of Avian H5N1 Influenza A Virus in Mammalian and Avian Hosts: Role of the C-Terminal ESEV Motif in the Viral NS1 Protein ▿

We assessed the prediction that access of the viral NS1 protein to cellular PDZ domain protein networks enhances the virulence of highly pathogenic avian influenza A viruses. The NS1 proteins of most avian influenza viruses bear the C-terminal ligand sequence Glu-Ser-Glu-Val (ESEV) for PDZ domains present in multiple host proteins, whereas no such motif is found in the NS1 homologues of seasonal human virus strains. Previous analysis showed that a C-terminal ESEV motif increases viral virulence when introduced into the NS1 protein of mouse-adapted H1N1 influenza virus. To examine the role of the PDZ domain ligand motif in avian influenza virus virulence, we generated three recombinants, derived from the prototypic H5N1 influenza A/Vietnam/1203/04 virus, expressing NS1 proteins that either have the C-terminal ESEV motif or the human influenza virus RSKV consensus or bear a natural truncation of this motif, respectively. Cell biological analyses showed strong control of NS1 nuclear migration in infected mammalian and avian cells, with only minor differences between the three variants. The ESEV sequence attenuated viral replication on cultured human, murine, and duck cells but not on chicken fibroblasts. However, all three viruses caused highly lethal infections in mice and chickens, with little difference in viral titers in organs, mean lethal dose, or intravenous pathogenicity index. These findings demonstrate that a PDZ domain ligand sequence in NS1 contributes little to the virulence of H5N1 viruses in these hosts, and they indicate that this motif modulates viral replication in a strain- and host-dependent manner

Vacuolar Protein Sorting Pathway Contributes to the Release of Marburg Virus ▿

VP40, the major matrix protein of Marburg virus, is the main driving force for viral budding. Additionally, cellular factors are likely to play an important role in the release of progeny virus. In the present study, we characterized the influence of the vacuolar protein sorting (VPS) pathway on the release of virus-like particles (VLPs), which are induced by Marburg virus VP40. In the supernatants of HEK 293 cells expressing VP40, different populations of VLPs with either a vesicular or a filamentous morphology were detected. While the filaments were almost completely composed of VP40, the vesicular particles additionally contained considerable amounts of cellular proteins. In contrast to that in the vesicles, the VP40 in the filaments was regularly organized, probably inducing the elimination of cellular proteins from the released VLPs. Vesicular particles were observed in the supernatants of cells even in the absence of VP40. Mutation of the late-domain motif in VP40 resulted in reduced release of filamentous particles, and likewise, inhibition of the VPS pathway by expression of a dominant-negative (DN) form of VPS4 inhibited the release of filamentous particles. In contrast, the release of vesicular particles did not respond significantly to the expression of DN VPS4. Like the budding of VLPs, the budding of Marburg virus particles was partially inhibited by the expression of DN VPS4. While the release of VLPs from VP40-expressing cells is a valuable tool with which to investigate the budding of Marburg virus particles, it is important to separate filamentous VLPs from vesicular particles, which contain many cellular proteins and use a different budding mechanism