scholarly journals Inhibition of arenavirus entry and replication by the cell-intrinsic restriction factor ZMPSTE24 is enhanced by IFITM antiviral activity

2021 ◽  
Author(s):  
Robert J Stott ◽  
Toshana L Foster
Keyword(s):  
Virus Infection ◽  
Antiviral Activity ◽  
Viral Entry ◽  
Transmembrane Protein ◽  
Membrane Integrity ◽  
A549 Cells ◽  
Cellular Membrane ◽  
Restriction Factor ◽  
Lassa Virus ◽  
Live Virus

In the absence of effective vaccines and treatments, annual outbreaks of severe human haemorrhagic fever caused by arenaviruses, such as Lassa virus, continue to pose a significant human health threat. Understanding the balance of cellular factors that inhibit or promote arenavirus infection may have important implications for the development of effective antiviral strategies. Here, we identified the cell-intrinsic zinc transmembrane metalloprotease, ZMPSTE24, as a restriction factor against arenaviruses. Notably, CRISPR-Cas9-mediated knockout of ZMPSTE24 in human alveolar epithelial A549 cells increased arenavirus glycoprotein-mediated viral entry in pseudoparticle assays and live virus infection models. As a barrier to viral entry and replication, ZMPSTE24 may act as a downstream effector of interferon-induced transmembrane protein (IFITM) antiviral function; though through a yet poorly understood mechanism. Overexpression of IFITM1, IFITM2 and IFITM3 proteins did not restrict the entry of pseudoparticles carrying arenavirus envelope glycoproteins and live virus infection, yet depletion of IFITM protein expression enhanced virus entry and replication. Furthermore, gain-of-function studies revealed that IFITMs augment the antiviral activity of ZMPSTE24 against arenaviruses, suggesting a cooperative effect of viral restriction. We show that ZMPSTE24 and IFITMs affect the kinetics of cellular endocytosis, suggesting that perturbation of membrane structure and stability is likely the mechanism of ZMPSTE24-mediated restriction and cooperative ZMPSTE24-IFITM antiviral activity. Collectively, our findings define the role of ZMPSTE24 host restriction activity in the early stages of arenavirus infection. Moreover, we provide insight into the importance of cellular membrane integrity for productive fusion of arenaviruses and highlight a novel avenue for therapeutic development.

scholarly journals Human and Murine IFIT1 Proteins Do Not Restrict Infection of Negative-Sense RNA Viruses of the Orthomyxoviridae, Bunyaviridae, and Filoviridae Families

2015 ◽  
Vol 89 (18) ◽  
pp. 9465-9476 ◽  
Author(s):  
Amelia K. Pinto ◽  
Graham D. Williams ◽  
Kristy J. Szretter ◽  
James P. White ◽  
José Luiz Proença-Módena ◽  
...  
Keyword(s):  
Cell Culture ◽  
Antiviral Activity ◽  
Ebola Virus ◽  
Rna Viruses ◽  
Ectopic Expression ◽  
A549 Cells ◽  
Host Protein ◽  
Interferon Response ◽  
Restriction Factor ◽  
Negative Sense

ABSTRACTInterferon-induced protein with tetratricopeptide repeats 1 (IFIT1) is a host protein with reported cell-intrinsic antiviral activity against several RNA viruses. The proposed basis for the activity against negative-sense RNA viruses is the binding to exposed 5′-triphosphates (5′-ppp) on the genome of viral RNA. However, recent studies reported relatively low binding affinities of IFIT1 for 5′-ppp RNA, suggesting that IFIT1 may not interact efficiently with this moiety under physiological conditions. To evaluate the ability of IFIT1 to have an impact on negative-sense RNA viruses, we infectedIfit1−/−and wild-type control mice and primary cells with four negative-sense RNA viruses (influenza A virus [IAV], La Crosse virus [LACV], Oropouche virus [OROV], and Ebola virus) corresponding to three distinct families. Unexpectedly, a lack ofIfit1gene expression did not result in increased infection by any of these viruses in cell culture. Analogously, morbidity, mortality, and viral burdens in tissues were identical betweenIfit1−/−and control mice after infection with IAV, LACV, or OROV. Finally, deletion of the human IFIT1 protein in A549 cells did not affect IAV replication or infection, and reciprocally, ectopic expression of IFIT1 in HEK293T cells did not inhibit IAV infection. To explain the lack of antiviral activity against IAV, we measured the binding affinity of IFIT1 for RNA oligonucleotides resembling the 5′ ends of IAV gene segments. The affinity for 5′-ppp RNA was approximately 10-fold lower than that for non-2′-O-methylated (cap 0) RNA oligonucleotides. Based on this analysis, we conclude that IFIT1 is not a dominant restriction factor against negative-sense RNA viruses.IMPORTANCENegative-sense RNA viruses, including influenza virus and Ebola virus, have been responsible for some of the most deadly outbreaks in recent history. The host interferon response and induction of antiviral genes contribute to the control of infections by these viruses. IFIT1 is highly induced after virus infection and reportedly has antiviral activity against several RNA and DNA viruses. However, its role in restricting infection by negative-sense RNA viruses remains unclear. In this study, we evaluated the ability of IFIT1 to inhibit negative-sense RNA virus replication and pathogenesis bothin vitroandin vivo. Detailed cell culture and animal studies demonstrated that IFIT1 is not a dominant restriction factor against three different families of negative-sense RNA viruses.

scholarly journals Identification of a Broad-Spectrum Arenavirus Entry Inhibitor

2008 ◽  
Vol 82 (21) ◽  
pp. 10768-10775 ◽  
Author(s):  
Ryan A. Larson ◽  
Dongcheng Dai ◽  
Virginia T. Hosack ◽  
Ying Tan ◽  
Tove’ C. Bolken ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Viral Entry ◽  
Transmembrane Domain ◽  
Benzimidazole Derivative ◽  
Genetic Selection ◽  
Hemorrhagic Fever ◽  
Lymphocytic Choriomeningitis ◽  
Site Directed Mutagenesis ◽  
Lassa Virus ◽  
Carboxy Terminal

ABSTRACT Several arenaviruses, including Lassa virus (LASV), are causative agents of hemorrhagic fever, for which effective therapeutic options are lacking. The LASV envelope glycoprotein (GP) gene was used to generate lentiviral pseudotypes to identify small-molecule inhibitors of viral entry. A benzimidazole derivative with potent antiviral activity was identified from a high-throughput screen utilizing this strategy. Subsequent lead optimization for antiviral activity identified a modified structure, ST-193, with a 50% inhibitory concentration (IC50) of 1.6 nM against LASV pseudotypes. ST-193 inhibited pseudotypes generated with other arenavirus envelopes as well, including the remaining four commonly associated with hemorrhagic fever (IC50s for Junín, Machupo, Guanarito, and Sabiá were in the 0.2 to 12 nM range) but exhibited no antiviral activity against pseudotypes incorporating either the GP from the LASV-related arenavirus lymphocytic choriomeningitis virus (LCMV) or the unrelated G protein from vesicular stomatitis virus, at concentrations of up to 10 μM. Determinants of ST-193 sensitivity were mapped through a combination of LASV-LCMV domain-swapping experiments, genetic selection of viral variants, and site-directed mutagenesis. Taken together, these studies demonstrate that sensitivity to ST-193 is dictated by a segment of about 30 amino acids within the GP2 subunit. This region includes the carboxy-terminal region of the ectodomain and the predicted transmembrane domain of the envelope protein, revealing a novel antiviral target within the arenavirus envelope GP.

scholarly journals Cell culture model system utilizing engineered A549 cells to express high levels of ACE2 and TMPRSS2 for investigating SARS-CoV-2 infection and antivirals

2022 ◽  
Author(s):  
Ching-Wen Chang ◽  
Krishna Mohan Parsi ◽  
Mohan Somasundaran ◽  
Emma Vanderleeden ◽  
John Cruz ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Virus Infection ◽  
Viral Entry ◽  
Human Lung ◽  
A549 Cells ◽  
Model System ◽  
Lung Epithelial Cell ◽  
Clinical Feature ◽  
Lung Epithelial

Novel pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose an imminent global threat since its initial outbreak in December 2019. A simple in vitro model system using cell lines highly susceptible to SARS-CoV-2 infection are critical to facilitate the study of the virus cycle and to discover effective antivirals against the virus. Human lung alveolar A549 cells are regarded as a useful and valuable model for respiratory virus infection. However, SARS-CoV-2 uses the ACE2 as receptor for viral entry and the TMPRSS2 to prime the Spike protein, both of which are negligibly expressed in A549 cells. Here, we report the generation of a robust human lung epithelial cell-based model by transducing ACE2 and TMPRSS2 into A549 cells and show that the ACE2 enriched A549ACE2/TMPRSS2 cells (ACE2plus) and its single-cell-derived subclone (ACE2plusC3) are highly susceptible to SARS-CoV-2 infection. These engineered ACE2plus showed higher ACE2 and TMPRSS2 mRNA expression levels than currently used Calu3 and commercial A549ACE2/TMPRSS2 cells. ACE2 and TMPRSS2 proteins were also highly and ubiquitously expressed in ACE2plusC3 cells. Additionally, antiviral drugs like Camostat mesylate, EIDD-1931, and Remdesivir strongly inhibited SARS-CoV-2 replication. Notably, multinucleated syncytia, a clinical feature commonly observed in severe COVID-19 patients was induced in ACE2plusC3 cells either by virus infection or by overexpressing the Spike proteins of different variants of SARS-CoV-2. Syncytial process was effectively blocked by the furin protease inhibitor, Decanoyl-RVKR-CMK. Taken together, we have developed a robust human A549 lung epithelial cell-based model that can be applied to probe SARS-CoV-2 replication and to facilitate the discovery of SARS-CoV-2 inhibitors.

scholarly journals Human Immunodeficiency Viruses Pseudotyped with SARS-CoV-2 Spike Proteins Infect a Broad Spectrum of Human Cell Lines through Multiple Entry Mechanisms

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 953
Author(s):  
Chuan Xu ◽  
Annie Wang ◽  
Ke Geng ◽  
William Honnen ◽  
Xuening Wang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Viral Entry ◽  
Broad Spectrum ◽  
A549 Cells ◽  
Cell Types ◽  
Pseudotyped Virus ◽  
Angiotensin Converting Enzyme 2 ◽  
Human Immunodeficiency Viruses ◽  
Tyrosine Protein Kinase ◽  
Overexpressing Cell

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), the causative agent of coronavirus disease 19 (COVID-19), enters cells through attachment to the human angiotensin converting enzyme 2 (hACE2) via the receptor-binding domain (RBD) in the surface/spike (S) protein. Several pseudotyped viruses expressing SARS-CoV-2 S proteins are available, but many of these can only infect hACE2-overexpressing cell lines. Here, we report the use of a simple, two-plasmid, pseudotyped virus system comprising a SARS-CoV-2 spike-expressing plasmid and an HIV vector with or without vpr to investigate the SARS-CoV-2 entry event in various cell lines. When an HIV vector without vpr was used, pseudotyped SARS-CoV-2 viruses produced in the presence of fetal bovine serum (FBS) were able to infect only engineered hACE2-overexpressing cell lines, whereas viruses produced under serum-free conditions were able to infect a broader range of cells, including cells without hACE2 overexpression. When an HIV vector containing vpr was used, pseudotyped viruses were able to infect a broad spectrum of cell types regardless of whether viruses were produced in the presence or absence of FBS. Infection sensitivities of various cell types did not correlate with mRNA abundance of hACE2, TMPRSS2, or TMPRSS4. Pseudotyped SARS-CoV-2 viruses and replication-competent SARS-CoV-2 virus were equally sensitive to neutralization by an anti-spike RBD antibody in cells with high abundance of hACE2. However, the anti-spike RBD antibody did not block pseudotyped viral entry into cell lines with low abundance of hACE2. We further found that CD147 was involved in viral entry in A549 cells with low abundance of hACE2. Thus, our assay is useful for drug and antibody screening as well as for investigating cellular receptors, including hACE2, CD147, and tyrosine-protein kinase receptor UFO (AXL), for the SARS-CoV-2 entry event in various cell lines.

scholarly journals Phytochemical Characterization of Olea europea Leaf Extracts and Assessment of Their Anti-Microbial and Anti-HSV-1 Activity

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1085
Author(s):  
Ichrak Ben-Amor ◽  
Maria Musarra-Pizzo ◽  
Antonella Smeriglio ◽  
Manuela D’Arrigo ◽  
Rosamaria Pennisi ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Viral Entry ◽  
Antiviral Effect ◽  
Vero Cells ◽  
Biological Properties ◽  
Leaf Extracts ◽  
Gram Positive Bacteria ◽  
Entry Assay ◽  
Olea Europea ◽  
Hsv 1

Owing to the richness of bioactive compounds, Olea europea leaf extracts exhibit a range of health effects. The present research evaluated the antibacterial and antiviral effect of leaf extracts obtained from Olea europea L. var. sativa (OESA) and Olea europea var. sylvestris (OESY) from Tunisia. LC-DAD-ESI-MS analysis allowed the identification of different compounds that contributed to the observed biological properties. Both OESA and OESY were active against Gram-positive bacteria (MIC values between 7.81 and 15.61 μg/mL and between 15.61 and 31.25 μg/mL against Staphylococcus aureus ATCC 6538 for OESY and OESA, respectively). The antiviral activity against the herpes simplex type 1 (HSV-1) was assessed on Vero cells. The results of cell viability indicated that Olea europea leaf extracts were not toxic to cultured Vero cells. The half maximal cytotoxic concentration (CC50) values for OESA and OESY were 0.2 mg/mL and 0.82 mg/mL, respectively. Furthermore, both a plaque reduction assay and viral entry assay were used to demonstrate the antiviral activity. In conclusion, Olea europea leaf extracts demonstrated a bacteriostatic effect, as well as remarkable antiviral activity, which could provide an alternative treatment against resistant strains.

scholarly journals Recombinant proteins of Zaire ebolavirus induce potent humoral and cellular immune responses and protect against live virus infection in mice

Vaccine ◽  
2018 ◽  
Vol 36 (22) ◽  
pp. 3090-3100 ◽  
Author(s):  
Axel T. Lehrer ◽  
Teri-Ann S. Wong ◽  
Michael M. Lieberman ◽  
Tom Humphreys ◽  
David E. Clements ◽  
...  
Keyword(s):  
Virus Infection ◽  
Immune Responses ◽  
Recombinant Proteins ◽  
Live Virus ◽  
Cellular Immune Responses ◽  
Zaire Ebolavirus ◽  
Cellular Immune

Pharmacokinetics and antiviral activity of a novel isonucleoside, BMS-181165, against simian varicella virus infection in African green monkeys

1994 ◽  
Vol 23 (3-4) ◽  
pp. 219-224 ◽  
Author(s):  
K.F. Soike ◽  
J.-L. Huang ◽  
J.W. Russell ◽  
V.J. Whiterock ◽  
J.E. Sundeen ◽  
...  
Keyword(s):  
Virus Infection ◽  
Antiviral Activity ◽  
Simian Varicella Virus ◽  
Varicella Virus ◽  
Green Monkeys
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