The RNA Exosome Syncs IAV-RNAPII Transcription to Promote Viral Ribogenesis and Infectivity.

The nuclear RNA exosome is an essential multi-subunit complex that controls RNA homeostasis. Congenital mutations in RNA exosome genes are associated with neurodegenerative diseases. Little is known about the role of the RNA exosome in the cellular response to pathogens. Here, using NGS and human and mouse genetics, we show that influenza A virus (IAV) ribogenesis and growth are suppressed by impaired RNA exosome activity. Mechanistically, the nuclear RNA exosome coordinates the initial steps of viral transcription with RNAPII at host promoters. The viral polymerase complex co-opts the nuclear RNA exosome complex and cellular RNAs en route to 3' end degradation. Exosome deficiency uncouples chromatin targeting of the viral polymerase complex and the formation of cellular:viral RNA hybrids, which are essential RNA intermediates that license transcription of antisense genomic viral RNAs. Our results suggest that evolutionary arms races have shaped the cellular RNA quality control machinery.

Screening for Novel Small-Molecule Inhibitors Targeting the Assembly of Influenza Virus Polymerase Complex by a Bimolecular Luminescence Complementation-Based Reporter System.

Influenza virus RNA-dependent RNA polymerase consists of three viral protein subunits: PA, PB1, and PB2. Protein-protein interactions (PPIs) of these subunits play pivotal roles in assembling the functional polymerase complex, which is essential for the replication and transcription of influenza virus RNA. Here we developed a highly specific and robust bimolecular luminescence complementation (BiLC) reporter system to facilitate the investigation of influenza virus polymerase complex formation. Furthermore, by combining computational modeling and the BiLC reporter assay, we identified several novel small-molecule compounds that selectively inhibited PB1-PB2 interaction. Function of one such lead compound was confirmed by its activity in suppressing influenza virus replication. In addition, our studies also revealed that PA plays a critical role in enhancing interactions between PB1 and PB2, which could be important in targeting sites for anti-influenza intervention. Collectively, these findings not only aid the development of novel inhibitors targeting the formation of influenza virus polymerase complex but also present a new tool to investigate the exquisite mechanism of PPIs. IMPORTANCE Formation of the functional influenza virus polymerase involves complex protein-protein interactions (PPIs) of PA, PB1, and PB2 subunits. In this work, we developed a novel BiLC assay system which is sensitive and specific to quantify both strong and weak PPIs between influenza virus polymerase subunits. More importantly, by combining in silico modeling and our BiLC assay, we identified a small molecule that can suppress influenza virus replication by disrupting the polymerase assembly. Thus, we developed an innovative method to investigate PPIs of multisubunit complexes effectively and to identify new molecules inhibiting influenza virus polymerase assembly.

Determination of cold-adapted influenza virus (Orthomyxoviridae: Alphainfluenzavirus) polymerase activity by the minigenome method with a fluorescent protein.

INTRODUCTION: Polymerase proteins PB1 and PB2 determine the cold-adapted phenotype of the influenza virus A/Krasnodar/101/35/59 (H2N2), as was shown earlier. OBJECTIVE: The development of the reporter construct to determine the activity of viral polymerase at 33 and 37 °C using the minigenome method. MATERIALS AND METHODS: Co-transfection of Cos-1 cells with pHW2000 plasmids expressing viral polymerase proteins PB1, PB2, PA, NP (minigenome) and reporter construct. RESULTS: Based on segment 8, two reporter constructs were created that contain a direct or inverted NS1-GFP-NS2 sequence for the expression of NS2 and NS1 proteins translationally fused with green fluorescent protein (GFP), which allowed the evaluation the transcriptional and/or replicative activity of viral polymerase. CONCLUSION: Polymerase of virus A/Krasnodar/101/35/59 (H2N2) has higher replicative and transcriptional activity at 33 °C than at 37 °C. Its transcriptional activity is more temperature-dependent than its replicative activity. The replicative and transcriptional activity of polymerase A/Puerto Rico/8/34 virus (H1N1, Mount Sinai variant) have no significant differences and do not depend on temperature.

Exploring the cycloheptathiophene-3-carboxamide scaffold to disrupt the interactions of the influenza polymerase subunits and obtain potent anti-influenza activity.

With the aim to identify small molecules able to disrupt PA-PB1 subunits interaction of influenza virus (flu) RNA-dependent RNA polymerase, and based on previous structural and computational information, in this paper we have designed and synthesized a new series of cycloheptathiophene-3-carboxamide (cHTC) derivatives. Their biological evaluation led to highlight important structural insights along with new interesting compounds, such as the 2-hydroxybenzamido derivatives 29, 31, and 32, and the 4-aminophenyl derivative 54, which inhibited viral growth in the low micromolar range (EC50 = 0.18-1.2 µM) at no toxic concentrations (CC50 > 250 µM). This study permitted to obtain among the most potent anti-flu compounds within the PA-PB1 interaction inhibitors, confirming the cHTC scaffold as particularly suitable to achieve innovative anti-flu agents.