Single-chain intracellular antibodies inhibit influenza virus replication by disrupting interaction of proteins involved in viral replication and transcription

ABSTRACTThe positions of host factors required for viral replication within a human protein-protein interaction (PPI) network can be exploited to identify drug targets that are robust to drug-mediated selective pressure. Host factors can physically interact with viral proteins, be a component of virus-regulated pathways (where proteins do not interact with viral proteins), or be required for viral replication but unregulated by viruses. Here, we demonstrate a method of combining human PPI networks with virus-host PPI data to improve antiviral drug discovery for influenza viruses by identifying target host proteins. Analysis shows that influenza virus proteins physically interact with host proteins in network positions significant for information flow, even after the removal of known abundance-degree bias within PPI data. We have isolated a subnetwork of the human PPI network that connects virus-interacting host proteins to host factors that are important for influenza virus replication without physically interacting with viral proteins. The subnetwork is enriched for signaling and immune processes distinct from those associated with virus-interacting proteins. Selecting proteins based on subnetwork topology, we performed an siRNA screen to determine whether the subnetwork was enriched for virus replication host factors and whether network position within the subnetwork offers an advantage in prioritization of drug targets to control influenza virus replication. We found that the subnetwork is highly enriched for target host proteins—more so than the set of host factors that physically interact with viral proteins. Our findings demonstrate that network positions are a powerful predictor to guide antiviral drug candidate prioritization.IMPORTANCEIntegrating virus-host interactions with host protein-protein interactions, we have created a method using these established network practices to identify host factors (i.e., proteins) that are likely candidates for antiviral drug targeting. We demonstrate that interaction cascades between host proteins that directly interact with viral proteins and host factors that are important to influenza virus replication are enriched for signaling and immune processes. Additionally, we show that host proteins that interact with viral proteins are in network locations of power. Finally, we demonstrate a new network methodology to predict novel host factors and validate predictions with an siRNA screen. Our results show that integrating virus-host proteins interactions is useful in the identification of antiviral drug target candidates.

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Cardiac glycosides decrease influenza virus replication by inhibiting cell protein translational machinery

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00173.2018 ◽

2019 ◽

Vol 316 (6) ◽

pp. L1094-L1106 ◽

Cited By ~ 11

Author(s):

Luciano Amarelle ◽

Jeremy Katzen ◽

Masahiko Shigemura ◽

Lynn C. Welch ◽

Héctor Cajigas ◽

...

Keyword(s):

Influenza Virus ◽

Viral Replication ◽

Virus Replication ◽

Survival Benefit ◽

Influenza A ◽

Cardiac Glycosides ◽

Cell Protein ◽

Translational Machinery ◽

Influenza Virus Replication ◽

Concentration Changes

Cardiac glycosides (CGs) are used primarily for cardiac failure and have been reported to have other effects, including inhibition of viral replication. Here we set out to study mechanisms by which CGs as inhibitors of the Na-K-ATPase decrease influenza A virus (IAV) replication in the lungs. We found that CGs inhibit influenza virus replication in alveolar epithelial cells by decreasing intracellular potassium, which in turn inhibits protein translation, independently of viral entry, mRNA transcription, and protein degradation. These effects were independent of the Src signaling pathway and intracellular calcium concentration changes. We found that short-term treatment with ouabain prevented IAV replication without cytotoxicity. Rodents express a Na-K-ATPase-α1 resistant to CGs. Thus we utilized Na-K-ATPase-α1-sensitive mice, infected them with high doses of influenza virus, and observed a modest survival benefit when treated with ouabain. In summary, we provide evidence that the inhibition of the Na-K-ATPase by CGs decreases influenza A viral replication by modulating the cell protein translational machinery and results in a modest survival benefit in mice.

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Nuclear Factor 90 Negatively Regulates Influenza Virus Replication by Interacting with Viral Nucleoprotein

Journal of Virology ◽

10.1128/jvi.00735-09 ◽

2009 ◽

Vol 83 (16) ◽

pp. 7850-7861 ◽

Cited By ~ 52

Author(s):

Pui Wang ◽

Wenjun Song ◽

Bobo Wing-Yee Mok ◽

Pengxi Zhao ◽

Kun Qin ◽

...

Keyword(s):

Influenza Virus ◽

Viral Replication ◽

Virus Replication ◽

Early Phase ◽

Rna Binding ◽

Direct Interaction ◽

Mass Spectrometry Analysis ◽

Nuclear Factor ◽

Infected Cells ◽

Influenza Virus Replication

ABSTRACT Interactions between host factors and the viral replication complex play important roles in host adaptation and regulation of influenza virus replication. A cellular protein, nuclear factor 90 (NF90), was copurified with H5N1 viral nucleoprotein (NP) from human cells in which NP was transiently expressed and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. In vitro coimmunoprecipitation of NF90 and NP coexpressed in HEK 293T cells or individually expressed in bacterial and HEK 293T cells, respectively, confirmed a direct interaction between NF90 and NP, independent of other subunits of the ribonucleoprotein complex. This interaction was prevented by a mutation, F412A, in the C-terminal region of the NP, indicating that the C-terminal of NP is required for NF90 binding. RNase V treatment did not prevent coprecipitation of NP and NF90, which demonstrates that the interaction is RNA binding independent. After small interfering RNA knockdown of NF90 expression in A549 and HeLa cells, viral polymerase complex activity and virus replication were significantly increased, suggesting that NF90 negatively affects viral replication. Both NP and NF90 colocalized in the nucleus of virus-infected cells during the early phase of infection, suggesting that the interaction between NF90 and NP is an early event in virus replication. Quantitative reverse transcription-PCR showed that NF90 downregulates both viral genome replication and mRNA transcription in infected cells. These results suggest that NF90 inhibits influenza virus replication during the early phase of infection through direct interaction with viral NP.

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