Biology subjects

Rand, U.

Publications and source records attributed to Rand, U..

6 recordsLinked to original sources

bioRxiv2022

Pharmacological modulators of epithelial immunity uncovered by synthetic genetic tracing of SARS-CoV-2 infection responses

Epithelial immune responses govern tissue homeostasis and offer drug targets against maladaptation. Here, we report a framework to generate drug discovery-ready reporters of cellular responses to viral infection. We reverse engineered epithelial cell responses to SARS-CoV-2, the viral agent fueling the ongoing COVID-19 pandemic and designed synthetic transcriptional reporters whose molecular logic comprises interferon-/{beta}/{gamma}-, and NF-{kappa}B pathways. Such regulatory potential reflected single-cell data from experimental models to severe COVID-19 patient epithelial cells infected by SARS-CoV-2. SARS-CoV-2, type-I interferons, and RIG-I drive reporter activation. Live-cell-image-based phenotypic drug screens identified JAK inhibitors and DNA damage inducers as antagonistic modulators of epithelial cell response to interferons, RIG-I stimulation, and SARS-CoV-2. Synergistic or antagonistic modulation of the reporter by drugs underscored their similar mechanism of action. Thus, this study describes a tool for dissecting antiviral responses to infection and sterile cues, and a rapid approach to other emerging viruses of public health concern in order to discover rational drug combinations.

bioengineering↗

bioRxiv2022

PDGFRα+ fibroblasts are a major site of cytomegalovirus replication in vivo and genome maintenance during latency

To date, no herpesvirus has been shown to latently persist in fibroblastic cells. Here, we demonstrate that mouse CMV (MCMV), a {beta}-herpesvirus, persists for the long term and across organs in PDGFR+ fibroblastic cells, with similar or higher genome loads than in the previously known sites of MCMV latency. Whereas MCMV gene transcription in PDGFR+ fibroblastic cells was almost completely silenced at 5 months post-infection, these cells gave rise to reactivated virus ex vivo, arguing that they supported latent MCMV infection. Notably, PDGFR+ fibroblastic cells also supported productive virus replication during primary MCMV infection. Mechanistically, Stat1-deficiency resulted in increased lytic but abolished latent infection of fibroblastic cells in vivo. In sum, fibroblastic cells have a dual role as a site of lytic MCMV replication and a reservoir of latent MCMV in vivo and Stat1 is critically involved in the regulation of MCMV latency.

microbiology↗

bioRxiv2021

Revealing the host antiviral protein ZAP-S as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting

Programmed ribosomal frameshifting (PRF) is a fundamental gene expression event in many viruses including SARS-CoV-2, which allows production of essential structural and replicative enzymes from an alternative reading frame. Despite the importance of PRF for the viral life cycle, it is still largely unknown how and to what extent cellular factors alter mechanical properties of frameshifting RNA molecules and thereby impact virulence. This prompted us to comprehensively dissect the interplay between the host proteome and the SARS-CoV-2 frameshift element. Here, we reveal that zinc-finger antiviral protein (ZAP-S) is a direct and specific regulator of PRF in SARS-CoV-2 infected cells. ZAP-S overexpression strongly impairs frameshifting and viral replication. Using in vitro ensemble and single-molecule techniques, we further demonstrate that ZAP-S directly interacts with the SARS-CoV-2 RNA and ribosomes and interferes with the folding of the frameshift RNA. Together these data illuminate ZAP-S as de novo host-encoded specific inhibitor of SARS-CoV-2 frameshifting and expand our understanding of RNA-based gene regulation.

molecular biology↗

bioRxiv2021

Antiviral activity of influenza A virus defective interfering particles against SARS-CoV-2 replication in vitro through stimulation of innate immunity

SARS-CoV-2 causing COVID-19 emerged in late 2019 and resulted in a devastating pandemic. Although the first approved vaccines were already administered by the end of 2020, worldwide vaccine availability is still limited. Moreover, immune escape variants of the virus are emerging against which the current vaccines may confer only limited protection. Further, existing antivirals and treatment options against COVID-19 only show limited efficacy. Influenza A virus (IAV) defective interfering particles (DIPs) were previously proposed not only for antiviral treatment of the influenza disease but also for pan-specific treatment of interferon (IFN)-sensitive respiratory virus infections. To investigate the applicability of IAV DIPs as an antiviral for the treatment of COVID-19, we conducted in vitro co-infection experiments with cell culture-derived DIPs and the IFN-sensitive SARS-CoV-2 in human lung cells. We show that treatment with IAV DIPs leads to complete abrogation of SARS-CoV-2 replication. Moreover, this inhibitory effect was dependent on janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling. Further, our results suggest boosting of IFN-induced antiviral activity by IAV DIPs as a major contributor in suppressing SARS-CoV-2 replication. Thus, we propose IAV DIPs as an effective antiviral agent for treatment of COVID-19, and potentially also for suppressing the replication of new variants of SARS-CoV-2.

microbiology↗

bioRxiv2020

A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients by phage display is binding to the ACE2-RBD interface and is tolerant to known RBD mutations

The novel betacoranavirus SARS-CoV-2 causes a form of severe pneumonia disease, termed COVID-19 (coronavirus disease 2019). Recombinant human antibodies are proven potent neutralizers of viruses and can block the interaction of viral surface proteins with their host receptors. To develop neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor binding domain (RBD) of the S1 subunit of the viral spike (S) protein were selected by phage display. The selected antibodies were produced in the scFv-Fc format and 30 showed more than 80% inhibition of spike (S1-S2) binding to cells expressing ACE2, assessed by flow cytometry screening assay. The majority of these inhibiting antibodies are derived from the VH3-66 V-gene. The antibody STE90-C11 showed a sub nM IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody was demonstrated in the Syrian hamster and in the hACE2 mice model using a silenced human IgG1 Fc part. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD was solved at 2.0 [A] resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibtion of STE90-C11 is not blocked by many known RBD mutations including N439K, L452R, E484K or L452R+E484Q (emerging B.1.617). STE90-C11 derived human IgG1 with Fc{gamma}R silenced Fc (COR-101) is currently undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19. In BriefHuman antibodies were selected from convalescent COVID-19 patients using antibody phage display. The antibody STE90-C11 is neutralizing authentic SARS-CoV-2 virus in vitro and in vivo and the crystal structure of STE90-C11 in complex with SARS-CoV-2-RBD revealed that this antibody is binding in the RBD-ACE2 interface. S1 binding of STE90-C11 and inhibition of ACE2 binding is not blocked by many known RBD mutations.

bioengineering↗

bioRxiv2020

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a novel betacoronavirus discovered in December 2019 and closely related to the SARS coronavirus (CoV). Both viruses use the human ACE2 receptor for cell entry, recognizing it with the Receptor Binding Domain (RBD) of the S1 subunit of the viral spike (S) protein. The S2 domain mediates viral fusion with the host cell membrane. Experience with SARS and MERS coronaviruses has shown that potent monoclonal neutralizing antibodies against the RBD can inhibit the interaction with the virus cellular receptor (ACE2 for SARS) and block the virus cell entry. Assuming that a similar strategy would be successful against SARS-CoV-2, we used phage display to select from the human naive universal antibody gene libraries HAL9/10 anti-SARS-CoV-2 spike antibodies capable of inhibiting interaction with ACE2. 309 unique fully human antibodies against S1 were identified. 17 showed more than 75% inhibition of spike binding to cells expressing ACE2 in the scFv-Fc format, assessed by flow cytometry and several antibodies showed even an 50% inhibition at a molar ratio of the antibody to spike protein or RBD of 1:1. All 17 scFv-Fc were able to bind the isolated RBD, four of them with sub-nanomolar EC50. Furthermore, these scFv-Fc neutralized active SARS-CoV-2 virus infection of VeroE6 cells. In a final step, the antibodies neutralizing best as scFv-Fc were converted into the IgG format. The antibody STE73-2E9 showed neutralization of active SARS-CoV-2 with an IC50 0.43 nM and is binding to the ACE2-RBD interface. Universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovered patients in a pandemic situation.

biochemistry↗