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.

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.

Despite current prophylactic strategies, sexually transmitted infections (STIs) remain significant contributors to global health challenges, spurring the development of new multipurpose delivery technologies to protect individuals from and treat virus infections. However, there are few methods currently available to prevent and no method to date that cures human immunodeficiency virus (HIV) infection or combinations of STIs. While current oral and topical preexposure prophylaxes have protected against HIV infection, they have primarily relied on antiretrovirals (ARVs) to inhibit infection. Yet continued challenges with ARVs include user adherence to daily treatment regimens and the potential toxicity and antiviral resistance associated with chronic use. The integration of new biological agents may avert some of these adverse effects while also providing new mechanisms to prevent infection. Of the biologic-based antivirals, griffithsin (GRFT) has demonstrated potent inhibition of HIV-1 (and a multitude of other viruses) by adhering to and inactivating HIV-1 immediately upon contact. In parallel with the development of GRFT, electrospun fibers (EFs) have emerged as a promising platform for the delivery of agents active against HIV infection. In the study described here, our goal was to extend the mechanistic diversity of active agents and electrospun fibers by incorporating the biologic GRFT on the EF surface rather than within the EFs to inactivate HIV prior to cellular entry. We fabricated and characterized GRFT-modified EFs (GRFT-EFs) with different surface modification densities of GRFT and demonstrated their safety and efficacy against HIV-1 infection in vitro. We believe that EFs are a unique platform that may be enhanced by incorporation of additional antiviral agents to prevent STIs via multiple mechanisms. N ewly acquired sexually transmitted infections (STIs) affect 340 million people per year (1-3) and exert a significant impact on global health. Human immunodeficiency virus type 1 (HIV-1) affects ϳ35 million people globally (2-5), while untreated STIs, such as those caused by herpes simplex virus 2 (HSV-2), can enhance both the acquisition and the transmission of HIV and other agents of STIs by 2-to 4-fold (6, 7). In light of the findings of recent clinical trials, a specific, multipurpose prevention technology that has the ability to prevent multiple STIs using one delivery platform and that also increases user adherence urgently needs to be developed (8-18). In the study described here, we sought to shift current topical preexposure prophylaxis (PrEP) paradigms by integrating a multipurpose biological delivery approach to debilitate and inactivate HIV.Our long-term goal is to develop a multipurpose biologically inspired electrospun fiber (EF) prevention technology that takes cues from the innate microenvironment of the female reproductive tract to more strategically narrow the gaps in microbicide efficacy. In this work, we evaluated the potential of polymeric EF scaffolds surface modified with the po...

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.

Sexually transmitted infections affect hundreds of millions of people worldwide. Both human immunodeficiency virus (HIV-1 and -2) and herpes simplex virus-2 (HSV-2) remain incurable, urging the development of new prevention strategies. While current prophylactic technologies are dependent on strict user adherence to achieve efficacy, there is a dearth of delivery vehicles that provide discreet and convenient administration, combined with prolonged-delivery of active agents. To address these needs, we created electrospun fibers (EFs) comprised of FDA-approved polymers, poly(lactic-co-glycolic acid) (PLGA) and poly(DL-lactide-co-ε-caprolactone) (PLCL), to provide sustained-release and in vitro protection against HIV-1 and HSV-2. PLGA and PLCL EFs, incorporating the antiretroviral, tenofovir disoproxil fumarate (TDF), exhibited sustained-release for up to 4 weeks, and provided complete in vitro protection against HSV-2 and HIV-1 for 24 hr and 1 wk, respectively, based on the doses tested. In vitro cell culture and EpiVaginal tissue tests confirmed the safety of fibers in vaginal and cervical cells, highlighting the potential of PLGA and PLCL EFs as multipurpose next-generation drug delivery vehicles.

SARS-CoV-2 utilizes a number of strategies to modulate host responses to ensure efficient propagation. Here, we used ribosome profiling in SARS-CoV-2-infected cells to gain a deeper understanding of the translationally regulated events in infected cells.

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...

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