Broad spectrum post-entry inhibitors of coronavirus replication: Cardiotonic steroids and monensin.

A small molecule screen identified several cardiotonic steroids (digitoxin and ouabain) and the ionophore monensin as potent inhibitors of HCoV-229E, HCoV-OC43, and SARS-CoV-2 replication with EC50s in the low nM range. Subsequent tests confirmed antiviral activity in primary cell models including human nasal epithelial cells and lung organoids. Addition of digitoxin, ouabain, or monensin strongly reduced viral gene expression as measured by both viral protein and RNA accumulation. Furthermore, the compounds acted post virus entry. While the antiviral activity of digitoxin was dependent upon activation of the MEK and JNK signaling pathways but not signaling through GPCRs, the antiviral effect of monensin was reversed upon inhibition of several signaling pathways. Together, the data demonstrates the potent anti-coronavirus properties of two classes of FDA approved drugs that function by altering the properties of the infected cell, rendering it unable to support virus replication.

Autophagy in Tumor Immunity and Viral-Based Immunotherapeutic Approaches in Cancer.

Autophagy is a fundamental catabolic process essential for the maintenance of cellular and tissue homeostasis, as well as directly contributing to the control of invading pathogens. Unsurprisingly, this process becomes critical in supporting cellular dysregulation that occurs in cancer, particularly the tumor microenvironments and their immune cell infiltration, ultimately playing a role in responses to cancer therapies. Therefore, understanding "cancer autophagy" could help turn this cellular waste-management service into a powerful ally for specific therapeutics. For instance, numerous regulatory mechanisms of the autophagic machinery can contribute to the anti-tumor properties of oncolytic viruses (OVs), which comprise a diverse class of replication-competent viruses with potential as cancer immunotherapeutics. In that context, autophagy can either: promote OV anti-tumor effects by enhancing infectivity and replication, mediating oncolysis, and inducing autophagic and immunogenic cell death; or reduce OV cytotoxicity by providing survival cues to tumor cells. These properties make the catabolic process of autophagy an attractive target for therapeutic combinations looking to enhance the efficacy of OVs. In this article, we review the complicated role of autophagy in cancer initiation and development, its effect on modulating OVs and immunity, and we discuss recent progress and opportunities/challenges in targeting autophagy to enhance oncolytic viral immunotherapy.

Embryonic Origins of Virus-Induced Hearing Loss: Overview of Molecular Etiology.

Hearing loss, one of the most prevalent chronic health conditions, affects around half a billion people worldwide, including 34 million children. The World Health Organization estimates that the prevalence of disabling hearing loss will increase to over 900 million people by 2050. Many cases of congenital hearing loss are triggered by viral infections during different stages of pregnancy. However, the molecular mechanisms by which viruses induce hearing loss are not sufficiently explored, especially cases that are of embryonic origins. The present review first describes the cellular and molecular characteristics of the auditory system development at early stages of embryogenesis. These developmental hallmarks, which initiate upon axial specification of the otic placode as the primary root of the inner ear morphogenesis, involve the stage-specific regulation of several molecules and pathways, such as retinoic acid signaling, Sonic hedgehog, and Wnt. Different RNA and DNA viruses contributing to congenital and acquired hearing loss are then discussed in terms of their potential effects on the expression of molecules that control the formation of the auditory and vestibular compartments following otic vesicle differentiation. Among these viruses, cytomegalovirus and herpes simplex virus appear to have the most effect upon initial molecular determinants of inner ear development. Moreover, of the molecules governing the inner ear development at initial stages, SOX2, FGFR3, and CDKN1B are more affected by viruses causing either congenital or acquired hearing loss. Abnormalities in the function or expression of these molecules influence processes like cochlear development and production of inner ear hair and supporting cells. Nevertheless, because most of such virus-host interactions were studied in unrelated tissues, further validations are needed to confirm whether these viruses can mediate the same effects in physiologically relevant models simulating otic vesicle specification and growth.

Zika Infection Disrupts Proteins Involved in the Neurosensory System.

Newly re-emerging viruses are of great global concern, especially when there are no therapeutic interventions available during the time of an outbreak. There are still no therapeutic interventions for the prevention of Zika virus (ZIKV) infections despite its resurgence more than a decade ago. Newborns infected with ZIKV suffer from microcephaly and delayed neurodevelopment, but the underlying causes are largely unknown. All viruses hijack the host cellular machinery to undergo successful replication. Our tandem mass tag mass spectrometry-based proteomic monitoring of cells infected with ZIKV revealed that among the thousands of host proteins dysregulated over time, many protein candidates were linked to neurodevelopmental processes, including the development of the auditory and visual/retinal system. The role of these dysregulated neurodevelopmental-associated host proteins for ZIKV propagation in eukaryotic cells remains elusive. For the first time, we present temporal neurodevelopmental proteomic responses in cells undergoing ZIKV infection. The future goal is to identify host proteins whose dysregulation results in neurosensory alterations reported in children born to ZIKV-infected mothers.

Zika virus dysregulates human Sertoli cell proteins involved in spermatogenesis with little effect on tight junctions.

Zika virus (ZIKV), a neglected tropical disease until its re-emergence in 2007, causes microcephaly in infants and Guillain-Barré syndrome in adults. Its re-emergence and spread to more than 80 countries led the World Health Organization in 2016 to declare a Public Health Emergency. ZIKV is mainly transmitted by mosquitos, but can persist in infected human male semen for prolonged periods and may be sexually transmitted. Testicular Sertoli cells support ZIKV replication and may be a reservoir for persistent ZIKV infection. Electrical impedance analyses indicated ZIKV infection rapidly disrupted Vero cell monolayers but had little effect upon human Sertoli cells (HSerC). We determined ZIKV-induced proteomic changes in HSerC using an aptamer-based multiplexed technique (SOMAscan) targeting >1300 human proteins. ZIKV infection caused differential expression of 299 proteins during three different time points, including 5 days after infection. Dysregulated proteins are involved in different bio-functions, including cell death and survival, cell cycle, maintenance of cellular function, cell signaling, cellular assembly, morphology, movement, molecular transport, and immune response. Many signaling pathways important for maintenance of HSerC function and spermatogenesis were highly dysregulated. These included IL-6, IGF1, EGF, NF-κB, PPAR, ERK/MAPK, and growth hormone signaling. Down-regulation of the PPAR signaling pathway might impact cellular energy supplies. Upstream molecule analysis also indicated microRNAs involved in germ cell development were downregulated by infection. Overall, this study leads to a better understanding of Sertoli cellular mechanisms used by ZIKV during persistent infection and possible ZIKV impacts on spermatogenesis.

Aptamer Profiling of A549 Cells Infected with Low-Pathogenicity and High-Pathogenicity Influenza Viruses.

Influenza A viruses (IAVs) are important animal and human emerging and re-emerging pathogens that are responsible for yearly seasonal epidemics and sporadic pandemics. IAVs cause a wide range of clinical illnesses, from relatively mild infections by seasonal strains, to acute respiratory distress during infections with highly pathogenic avian IAVs (HPAI). For this study, we infected A549 human lung cells with lab prototype A/PR/8/34 (H1N1) (PR8), a seasonal H1N1 (RV733), the 2009 pandemic H1N1 (pdm09), or with two avian strains, an H5N1 HPAI strain or an H7N9 strain that has low pathogenicity in birds but high pathogenicity in humans. We used a newly-developed aptamer-based multiplexed technique (SOMAscan®) to examine >1300 human lung cell proteins affected by the different IAV strains, and identified more than 500 significantly dysregulated cellular proteins. Our analyses indicated that the avian strains induced more profound changes in the A549 global proteome compared to all tested low-pathogenicity H1N1 strains. The PR8 strain induced a general activation, primarily by upregulating many immune molecules, the seasonal RV733 and pdm09 strains had minimal effect upon assayed molecules, and the avian strains induced significant downregulation, primarily in antimicrobial response, cardiovascular and post-translational modification systems.

Influenza a virus-triggered autophagy decreases the pluripotency of human-induced pluripotent stem cells.

Maternal influenza infection during pregnancy was reported multiple times as the possible cause of many defects and congenital anomalies. Apart from several cases of influenza-related miscarriage during various trimesters of pregnancy, some epidemiological data suggest a link between maternal influenza infection and genetic abnormalities in offspring. However, there are no reports yet describing how maternal influenza alters cellular pathways at early stages of development to result in congenital defects in the fetus. In the present study, using proteomic approaches, we utilized human-induced pluripotent stem cells (hiPSCs) for modeling intrablastocyst infection with influenza virus to not only investigate the vulnerability and responses of pluripotent stem cells to this virus but also to determine the possible impacts of influenza on pluripotency and signaling pathways controlling differentiation and embryogenesis. Our data indicated viral protein production in influenza A virus (IAV)-infected hiPSCs. However, viral replication was restricted in these cells, but cell viability and pluripotency were negatively affected. These events occurred simultaneously with an excessive level of IAV-induced autophagy as well as cytopathic effects. Quantitative SOMAscan screening also indicated that changes in the proteome of hiPSCs corresponded to abnormal differentiation in these cells. Taken together, our results showed that IAV-modulated reduction in hiPSC pluripotency is associated with significant activation of autophagy. Further investigations are required to explore the role of IAV-induced autophagy in leading pluripotent stem cells toward abnormal differentiation and impaired development in early stages of embryogenesis.

Vero Cell Proteomic Changes Induced by Zika Virus Infection.

The re-emergence and the recent spread of the Zika virus (ZIKV) has raised significant global concerns due to lack of information in patient diagnosis and management. Thus, in addition to gaining more basic information about ZIKV biology, appropriate interventions and management strategies are being sought to control ZIKV-associated diseases and its spread. This study's objective is to identify host cell proteins that are significantly dysregulated during ZIKV infection. SOMAScan, a novel aptamer-based assay, is used to simultaneously screen >1300 host proteins to detect ZIKV-induced host protein dysregulation at multiple time points during infection. A total of 125 Vero cell host proteins, including cytokines such as CXCL11 and CCL5, interferon stimulated gene 15, and translation initiation factors EIF5A and EIF4G2, are significantly dysregulated after ZIKV infection. Bioinformatic analyses of 77 host proteins, that are significantly dysregulated ≥1.25-fold, identify several activated biological processes, including the JAK/STAT, Tec kinase, and complement cascade pathways.

Global Interactomics Connect Nuclear Mitotic Apparatus Protein NUMA1 to Influenza Virus Maturation.

Influenza A virus (IAV) infections remain a major human health threat. IAV has enormous genetic plasticity and can rapidly escape virus-targeted anti-viral strategies. Thus, there is increasing interest to identify host proteins and processes the virus requires for replication and maturation. The IAV non-structural protein 1 (NS1) is a critical multifunctional protein that is expressed to high levels in infected cells. Host proteins that interact with NS1 may serve as ideal targets for attenuating IAV replication. We previously developed and characterized broadly cross-reactive anti-NS1 monoclonal antibodies. For the current study, we used these mAbs to co-immunoprecipitate native IAV NS1 and interacting host proteins; 183 proteins were consistently identified in this NS1 interactome study, 124 of which have not been previously reported. RNAi screens identified 11 NS1-interacting host factors as vital for IAV replication. Knocking down one of these, nuclear mitotic apparatus protein 1 (NUMA1), dramatically reduced IAV replication. IAV genomic transcription and translation were not inhibited but transport of viral structural proteins to the cell membrane was hindered during maturation steps in NUMA1 knockdown (KD) cells.