SARS-CoV-2 spike (S) variants govern transmissibility, responsiveness to vaccination and disease severity. In a screen for new models of SARS-CoV-2 infection, we identified human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of ACE2 expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Though the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type-I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.

HIV-1 has high mutation rates and exists as mutant swarms within the host. Rapid evolution of HIV-1 allows the virus to outpace the host immune system, leading to viral persistence. Approaches to target immutable components are needed to clear HIV-1 infection. Here, we report that the CARD8 inflammasome senses HIV-1 protease activity. HIV-1 can evade CARD8 sensing because its protease remains inactive in infected cells prior to viral budding. Premature intracellular activation of the viral protease triggered CARD8 inflammasome-mediated pyroptosis of HIV-1-infected cells. This strategy led to the clearance of latent HIV-1 in patient CD4+ T cells after viral reactivation. Thus, our study identifies CARD8 as an inflammasome sensor of HIV-1, which holds promise as a strategy for clearance of persistent HIV-1 infection.

Established in vitro models for SARS-CoV-2 infection are limited and include cell lines of non-human origin and those engineered to overexpress ACE2, the cognate host cell receptor. We identified human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of ACE2. Infection of H522 cells required the SARS-CoV-2 spike protein, though in contrast to ACE2-dependent models, spike alone was not sufficient for H522 infection. Temporally resolved transcriptomic and proteomic profiling revealed alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type-I interferon signaling. Focused chemical screens point to important roles for clathrin-mediated endocytosis and endosomal cathepsins in SARS-CoV-2 infection of H522 cells. These findings imply the utilization of an alternative SARS-CoV-2 host cell receptor which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.

Small-molecule targeting of RNA stem loops in 5′-end of SARS-CoV-2 reveals novel RNA targets for viral inhibition.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth in vitro depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here, we developed a simplified quantitative real-time PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 growth from a small amount of cell culture supernatants. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. Using this assay, we screened the activities of a number of compounds that were predicted to alter SARS-CoV-2 entry and replication as well as HIV-1-specific drugs in a proof-of-concept study. We found that E64D (inhibitor of endosomal proteases cathepsin B and L) and apilimod (endosomal trafficking inhibitor) potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) similarly decreased SARS-CoV-2 RNA levels in supernatants, suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (a nonnucleoside reverse transcriptase inhibitor [NNRTI]), amprenavir (a protease inhibitor), and allosteric integrase inhibitor 2 (ALLINI-2) modestly inhibited SARS-CoV-2 replication, albeit the 50% inhibitory concentration (IC50) values were much higher than that required for HIV-1. Taking the data together, this simplified assay will expedite basic SARS-CoV-2 research, be amenable to mid-throughput screening assays (i.e., drug, CRISPR, small interfering RNA [siRNA], etc.), and be applicable to a broad number of RNA and DNA viruses. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, is continuing to cause immense respiratory disease and social and economic disruptions. Conventional assays that monitor SARS-CoV-2 growth in cell culture rely on costly and time-consuming RNA extraction procedures, hampering progress in basic SARS-CoV-2 research and development of effective therapeutics. Here, we developed a simple quantitative real-time PCR assay to monitor SARS-CoV-2 growth in cell culture supernatants that does not necessitate RNA extraction and that is as accurate and sensitive as existing methods. In a proof-of-concept screen, we found that E64D, apilimod, EIPA, and remdesivir can substantially impede SARS-Cov-2 replication, providing novel insight into viral entry and replication mechanisms. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. This simplified assay will undoubtedly expedite basic SARS-CoV-2 and virology research and be amenable to use in drug screening platforms to identify therapeutics against SARS-CoV-2.

19Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the 20 ongoing COVID-19 pandemic, has infected millions within just a few months and is continuing to 21 spread around the globe causing immense respiratory disease and mortality. Assays to monitor 22 SARS-CoV-2 growth depend on time-consuming and costly RNA extraction steps, hampering 23 progress in basic research and drug development efforts. Here we developed a facile Q-RT- 24PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 replication 25 kinetics from a small amount of cell culture supernatants. Using this assay, we screened the 26 activities of a number of entry, SARS-CoV-2-and HIV-1-specific inhibitors in a proof of concept 27 study. In line with previous studies which has shown that processing of the viral Spike protein by 28 cellular proteases and endosomal fusion are required for entry, we found that E64D and 29 apilimod potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with 30 minimal cytotoxicity. Surprisingly, we found that macropinocytosis inhibitor EIPA similarly 31 decreased viral RNA in supernatants suggesting that entry may additionally be mediated by an 32 alternative pathway. HIV-1-specific inhibitors nevirapine (an NNRTI), amprenavir (a protease 33 inhibitor), and ALLINI-2 (an allosteric integrase inhibitor) modestly inhibited SARS-CoV-2 34 replication, albeit the IC 50 values were much higher than that required for HIV-1. Taken together, 35this facile assay will undoubtedly expedite basic SARS-CoV-2 research, be amenable to mid-36 throughput screens to identify chemical inhibitors of SARS-CoV-2, and be applicable to a broad 37 number of RNA and DNA viruses. 38 39 40 41 Importance 42 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the 43 COVID-19 pandemic, has quickly become a major global health problem causing immense 44 respiratory disease and social and economic disruptions. Conventional assays that monitor 45 SARS-CoV-2 growth in cell culture rely on costly and time-consuming RNA extraction 46 procedures, hampering progress in basic SARS-CoV-2 research and development of effective 47 therapeutics. Here we developed a facile Q-RT-PCR assay to monitor SARS-CoV-2 growth in 48 cell culture supernatants that does not necessitate RNA extraction, and is as accurate and 49 sensitive as existing methods. In a proof-of-concept screen, we found that E64D, apilimod, EIPA 50 and remdesivir can substantially impede SARS-Cov-2 replication providing novel insight into 51 viral entry and replication mechanisms. This facile approach will undoubtedly expedite basic 52 SARS-CoV-2 research, be amenable to screening platforms to identify therapeutics against 53 SARS-CoV-2 and can be adapted to numerous other RNA and DNA viruses.54 55 56 57 58 59 60 61 62 Observation 63 Severe acute respiratory syndrome coronavirus, SARS-CoV-2, the causative agent of the 64 ongoing COVID-19 pandemic, is continuing to cause substantial morbidity an...

No abstract

In addition to its catalytic function, HIV-1 integrase (IN) binds to the viral RNA genome (gRNA) through positively charged residues (i.e., R262, R263, R269, K273) within its C-terminal domain (CTD) and regulates proper virion maturation. Mutation of these residues results in the formation of morphologically aberrant viruses blocked at an early reverse transcription stage in cells.