Search

Influenza A Virus (IAV) is a recurring respiratory virus with limited availability of antiviral therapies. Understanding host proteins essential for IAV infection can identify targets for alternative host-directed therapies (HDTs). Using affinity purification-mass spectrometry and global phosphoproteomic and protein abundance analyses using three IAV strains (pH1N1, H3N2, H5N1) in three human cell types (A549, NHBE, THP-1), we map 332 IAV-human protein-protein interactions and identify 13 IAV-modulated kinases. Whole exome sequencing of patients who experienced severe influenza reveals several genes, including scaffold protein AHNAK, with predicted loss-of-function variants that are also identified in our proteomic analyses. Of our identified host factors, 54 significantly alter IAV infection upon siRNA knockdown, and two factors, AHNAK and coatomer subunit COPB1, are also essential for productive infection by SARS-CoV-2. Finally, 16 compounds targeting our identified host factors suppress IAV replication, with two targeting CDK2 and FLT3 showing pan-antiviral activity across influenza and coronavirus families. This study provides a comprehensive network model of IAV infection in human cells, identifying functional host targets for pan-viral HDT.

A deficient interferon (IFN) response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been implicated as a determinant of severe coronavirus disease 2019 (COVID-19). To identify the molecular effectors that govern IFN control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human IFN-stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors inhibiting viral entry, RNA binding proteins suppressing viral RNA synthesis, and a highly enriched cluster of endoplasmic reticulum (ER)/Golgi-resident ISGs inhibiting viral assembly/egress. These included broad-acting antiviral ISGs and eight ISGs that specifically inhibited SARS-CoV-2 and SARS-CoV-1 replication. Among the broad-acting ISGs was BST2/tetherin, which impeded viral release and is antagonized by SARS-CoV-2 Orf7a protein. Overall, these data illuminate a set of ISGs that underlie innate immune control of SARS-CoV-2/SARS-CoV-1 infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.

How transcription affects genome 3D organization is not well understood. We found that during influenza A (IAV) infection, rampant transcription rapidly reorganizes host cell chromatin interactions. These changes occur at the ends of highly transcribed genes, where global inhibition of transcription termination by IAV NS1 protein causes readthrough transcription for hundreds of kilobases. In these readthrough regions, elongating RNA polymerase II disrupts chromatin interactions by inducing cohesin displacement from CTCF sites, leading to locus decompaction. Readthrough transcription into heterochromatin regions switches them from the inert (B) to the permissive (A) chromatin compartment and enables transcription factor binding. Data from non-viral transcription stimuli show that transcription similarly affects cohesin-mediated chromatin contacts within gene bodies. Conversely, inhibition of transcription elongation allows cohesin to accumulate at previously transcribed intragenic CTCF sites and to mediate chromatin looping and compaction. Our data indicate that transcription elongation by RNA polymerase II remodels genome 3D architecture.

In clinical trials of two rifamycin antibiotics (rifampin and rifapentine) for treating tuberculosis (TB), patients with cavitary lung lesions did not appear to derive benefit from rifapentine. Rifapentine was found not to outperform rifampin, despite a lower minimum inhibitory concentration against Mycobacterium tuberculosis in mouse models of TB. To understand these findings, we have developed a rabbit model of TB that reliably develops lung cavities with features similar to those of patients with pulmonary cavitary TB. After single or multiple doses of rifampin or rifapentine that produced human-equivalent plasma exposures, rabbits were sacrificed at different time points after dosing. We measured site-of-disease drug pharmacokinetics and tissue drug distribution. We used pharmacokinetic-pharmacodynamic (PK/PD) modeling to estimate drug penetration into different types of tubercular lesions. Both drugs penetrated rabbit lung cellular lesions, as well as the fibrotic cavity wall of cavitary lesions (penetration coefficients ≥1 compared to plasma). For the necrotic liquefied material inside cavitary lesions known as caseum (which contains high numbers of bacteria), the penetration coefficient was 1.0 for rifampin but only 0.25 for rifapentine. When estimates of site-of-disease drug PK were substituted into clinical PK/PD models, the relationship between site-of-action exposure and sputum culture conversion was significant (P < 10-7). We propose that poor penetration of rifapentine into lung cavitary lesions explains, in part, why rifapentine doses required to improve treatment outcomes in two phase 2 clinical trials were four times higher in TB patients with large cavities compared to TB patients without cavitary lung disease.