Biology subjects

de la Torre, J. C.

Publications and source records attributed to de la Torre, J. C..

At least 19 recordsLinked to original sources

bioRxiv2026

A bivalent Lassa virus mRNA vaccine protects guinea pigs from lethal infection with Lassa virus

Lassa virus (LASV), the causative agent of the life-threatening febrile disease, Lassa fever, remains a major public health concern in West Africa, where hundreds of thousands of infections occur annually. However, no licensed vaccine is currently available, highlighting the urgent need for effective preventive measures. We previously demonstrated that an mRNA-lipid nanoparticle (LNP) vaccine expressing LASV glycoprotein precursor (GPC; LASgpc-mRNA-LNP) conferred protection against lethal infection with a recombinant lymphocytic choriomeningitis virus expressing LASV GPC in mice. In this study, we extended our previous work with the aim of evaluating the efficacy of an mRNA-LNP vaccine expressing LASV nucleoprotein (NP; LASnp-mRNA-LNP) and a bivalent vaccine combining LASgpc-mRNA-LNP and LASnp-mRNA-LNP. Using a lethal CBA/N mouse model of infection with ML29, an LASV live-attenuated vaccine candidate encoding LASV GPC and NP, we found that LASgpc-mRNA-LNP and LASnp-mRNA-LNP each conferred protection in the absence of detectable neutralizing antibody responses. We identified LASV GPC- and NP-derived CD4 and CD8 T-cell epitope peptides in CBA/N mice and demonstrated that vaccination induced robust antigen-specific T-cell responses. Importantly, the bivalent vaccine provided complete protection against lethal challenge with doses at which monovalent vaccines failed to protect all mice, suggesting a potential dose-sparing benefit of the bivalent formulation. Furthermore, the bivalent vaccine fully protected guinea pigs against lethal challenge with authentic LASV. Together, these findings establish LASV NP, in addition to GPC, as a protective antigen in the mRNA vaccine platform and provide a rationale for developing bivalent mRNA-LNP vaccines to improve protection against Lassa fever.

microbiology↗

bioRxiv2026

Sirtuin 1 Is Required for Optimal Mammarenavirus Multiplication

Mammarenaviruses (MaAv) cause persistent infections in diverse rodent reservoirs worldwide and several are zoonotic pathogens with an important public-health burden in their endemic regions. Moreover, the globally distributed MaAv lymphocytic choriomeningitis virus (LCMV) is an underrecognized pathogen of clinical significance in congenital infections and immunocompromised individuals. The lack of FDA-approved vaccines or antivirals for MaAv infections underscores the urgent need for novel anti-MaAv therapeutic strategies. Neutral sphingomyelinase 2 (nSMase2) was recently identified as a host factor contributing to LCMV multiplication, and its inhibitor cambinol exhibits dose-dependent antiviral activity against LCMV but the underlying mechanisms remain undefined. Here, we show that cambinol disrupts multiple stages of the LCMV life cycle. Cambinol inhibits the pH-dependent fusion event mediated by MaAv glycoprotein, a step required for completion of virus cell entry. It also reduces viral ribonucleoprotein (vRNP)-directed genome replication and transcription and impairs the budding activity of the virus matrix Z protein. Cambinol also inhibits sirtuins 1 and 2 (Sirt-1 and Sirt-2), two NAD+-dependent protein deacetylases with pleiotropic roles in cellular metabolism and stress responses, raising the question of whether cambinol anti-LCMV activity reflects nSMase2 inhibition alone or also involves sirtuin-dependent pathways. LCMV multiplication was significantly reduced in SIRT1, but not SIRT2, knockout (KO) cells, uncovering a pro-viral role for Sirt-1 in the LCMV life cycle. Consistent with this finding, LCMV vRNP activity and production of infectious progeny were reduced in SIRT1 KO cells. These findings identify Sirt-1 as a host factor required for optimal LCMV multiplication. Sirt-1 inhibitors are in clinical development for oncological and neurological indications, raising the possibility of repurposing Sirt-1 inhibitors as host-directed antivirals (HDAs) against human pathogenic MaAv. Abstract figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/740332v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@d53c5forg.highwire.dtl.DTLVardef@16ea861org.highwire.dtl.DTLVardef@1f09addorg.highwire.dtl.DTLVardef@1472e6b_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2026

Mammarenavirus-Induced Remodeling of the Cellular Lipid Landscape Reveals Sphingolipid Metabolism as a Novel Target for Antiviral Intervention

Several mammarenaviruses (MaAv) cause severe and often life-threatening disease in humans and represent major public health threats in their endemic regions. Lassa (LASV) and Junin (JUNV) MaAv, endemic to Western Africa and the Argentine Pampas, respectively, are etiologic agents of viral hemorrhagic fevers associated with high morbidity and mortality. In addition, the globally distributed MaAv lymphocytic choriomeningitis virus (LCMV) is an underrecognized human pathogen capable of causing severe congenital disease and fatal infections in immunocompromised individuals. Despite their public health importance, no FDA-approved vaccines or virus-specific antiviral therapies exist to prevent and treat human MaAv infections. Current treatment relies on the off-label use of ribavirin whose therapeutic efficacy remains controversial. These findings underscore the urgent need to develop effective antiviral strategies against human pathogenic MaAv. Here, we investigated the impact of LCMV infection on host lipid metabolism using an integrated transcriptomic and lipidomic approach. Our data reveal extensive time-dependent remodeling of the cellular lipid landscape, with particularly prominent alterations in sphingolipid and fatty acid metabolic pathways. Functional interrogation of these pathways using pharmacological inhibitors identified acetyl-CoA carboxylase (ACC) and neutral sphingomyelinase 2 (nSMase2) as host factors contributing to efficient viral replication. Notably, inhibition of nSMase2 reduced infectious virus production by 2 logs of infectious virus. Our findings showed that LCMV reprograms host lipid metabolism to facilitate infection and identified sphingolipid turnover as a promising target for host-directed antiviral strategies against MaAv infections.

microbiology↗

bioRxiv2026

Prolyl tRNA Synthetase Is Required for Mammarenavirus Multiplication

Several mammarenaviruses (MaAv), chiefly Lassa virus (LASV) in Western Africa and Junin virus (JUNV) in the Argentinean Pampas, cause severe disease in humans and pose important public health problems in their endemic regions. In addition, the globally distributed MaAv lymphocytic choriomeningitis virus (LCMV) is an underrecognized human pathogen of clinical significance especially in congenital infections and LCMV poses a serious risk for immunocompromised individuals. There are no FDA-approved MaAv vaccines or antivirals and current anti-MaAv therapy is limited to an off-label use of ribavirin whose efficacy remains controversial. This highlights an urgent unmet need for developing antivirals against human pathogenic MaAv. Halofuginone (HF), a derivative of the natural alkaloid febrifugine, has been shown to exhibit antiviral activity against several RNA viruses. Here, we present evidence that HF exhibits a potent dose-dependent antiviral activity against LCMV, and the hemorrhagic fever causing MaAv LASV and JUNV. HF binds to the bifunctional enzyme glutamyl-prolyl-tRNA synthetase 1 (EPRS1) and specifically inhibits its prolyl-tRNA synthetase (PRS) activity, resulting in translation inhibition via the amino acid starvation (AAS) response with preferential impact on proline-rich proteins. HF anti-LCMV activity was prevented by the addition of exogenous proline supporting that inhibition of PRS activity plays a critical role on the anti-MaAv activity of HF. We found that HF did not affect LCMV cell entry, modestly (twofold) reduced the activity of the virus ribonucleoprotein (vRNP) but strongly inhibited (>90%) Z budding activity, a process involving the Z proline-rich late domain motifs.

microbiology↗

bioRxiv2025

Pandemic Response Box Screening Identified CRM1/XPO1 as an Anti-Mammarenavirus Druggable Target

Mammarenaviruses (MaAv) cause persistent infection in their natural rodent hosts across the world and via zoonotic events can cause severe disease in humans. Thus, the MaAv Lassa virus (LASV) in Western Africa and Junin virus (JUNV) in the Argentinean Pampas cause hemorrhagic fever diseases with significant case fatality rates in their endemic regions. In addition, the globally distributed MaAv lymphocytic choriomeningitis virus (LCMV) is an underrecognized human pathogen of clinical significance capable of causing devastating infections in neonates and immunocompromised individuals. Despite their impact on human health, there are currently no FDA-approved vaccines or specific antiviral treatments for MaAv infections. Existing anti-MaAv therapies are limited to the off-label use of ribavirin whose efficacy remains controversial, hence, the significance of developing novel therapeutics to combat human pathogenic MaAv. We employed a high-throughput cell-based infection assay to screen the Pandemic Response Box, a collection of 400 diverse compounds with established antimicrobial activity, for MaAv inhibitors. We identified Ro-24-7429, an antagonist of the HIV-1 Tat protein and RUNX Family Transcription Factor 1 inhibitor; WO 2006118607 A2, a dihydroorotate dehydrogenase inhibitor; and verdinexor, a novel selective inhibitor of nuclear export (SINE) targeting the CRM1/XPO1, as potent anti-MaAv compounds. Consistent with their distinct validated targets, verdinexor and WO 2006118607 A2 exhibited a very strong synergistic antiviral activity when used in combination therapy. Our findings pave the way for the development of verdinexor as a potent host-directed antiviral against MaAv, which could be integrated into the development of combination therapy with direct-or host-acting antivirals to combat human pathogenic MaAv.

microbiology↗

bioRxiv2025

Integrated phenotypic screening and chemical proteomics identifies ETF1 ligands that modulate viral translation and replication

Emerging and re-emerging viruses pose a significant threat to global health. Although direct-acting antivirals have shown success, their efficacy is limited by the rapid emergence of drug-resistant viral variants. Hence, there is an urgent need for additional broad spectrum antiviral therapeutic strategies. Here, we identify by phenotypic screening a set of stereochemically defined photoreactive small molecules (photo-stereoprobes) that stereoselectively suppress SARS-CoV-2 replication in human lung epithelial cells. Structure-activity relationship-guided chemical proteomics identified the eukaryotic translation termination factor 1 (ETF1) as a target of the photo-stereoprobes, and this interaction was recapitulated with recombinant purified ETF1. We found that the photo-stereoprobes modulate programmed ribosomal frameshifting mechanisms essential for SARS-CoV-2 infection without causing ETF1 degradation, thus distinguishing the photo-stereoprobes from other known ETF1-directed small molecules. We finally show that the photo-stereoprobes also inhibit the replication of additional viruses with non-canonical ribosomal frameshifting mechanisms. Our findings thus identify a mechanistically distinct class of ETF1 ligands that implicate host translation termination processes as a potential target for antiviral development. SIGNIFICANCE STATEMENTThe identification of broad-spectrum antivirals that target host proteins is a desirable strategy to combat emerging viral infections given the rapid escape potential of viruses and the need to develop new countermeasures for clinically significant pathogens. Here, we integrate phenotypic screening and chemical proteomics to identify photo-stereoprobe small molecules that inhibit the replication of multiple viruses. We show that these compounds bind the protein eukaryotic peptide chain release factor subunit 1 (ETF1) and modulate programmed ribosomal frameshifting. Unlike previously described ligands for ETF1, which lead to proteasomal destruction of the protein, we did not find that the photo-stereoprobes altered ETF1 content in cells. Our findings thus point to an opportunity to pharmacologically modulate a host protein implicated in programmed ribosomal frameshifting as a strategy to combat infection of viruses from different families.

microbiology↗

bioRxiv2025

N-myristoyltransferase Inhibitors as Candidate Broad-Spectrum Antivirals to Treat Viral Infections Promoted by Immunosuppression Associated with JAK Inhibitors Therapy

The use of Janus kinase (JAK) inhibitors in the clinic has been expanded significantly during the last decade. However, the immunosuppressive effects of JAK inhibitors, via modulation of key innate cellular signaling pathways, can predispose treated patients to infections, and can also result in reduced control of silent infections and increased risk of reactivation of opportunistic infections. Thus, the JAK inhibitor ruxolitinib, approved for the treatment of myelofibrosis and polycythemia vera, has been shown to exerts a proviral activity during infection with different viruses. Therefore, the clinical relevance of developing antiviral treatments that can be effective in the presence of JAK inhibitors. N-terminal myristoyl transferase (NMT) inhibitors have been shown to exhibit potent antiviral activity against different viruses. Here we document that in the presence of ruxolitinib, NMT inhibitors retain their potent antiviral activity against different viruses, including HSV-1. Our findings support that NMT inhibitors should be explored as therapeutics to treat viral infections associated with immunosuppression caused by treatments with JAK inhibitors.

microbiology↗

bioRxiv2025

Mammarenavirus Z protein myristoylation and oligomerization are not required for its dose-dependent inhibitory effect on vRNP activity

We have recently documented the use of N-Myristoyltransferase inhibitors (NMTi) as an antiviral strategy against human pathogenic mammarenaviruses, including Lassa and Junin viruses, responsible for the hemorrhagic fever (HF) diseases Lassa fever (LF) and Argentine HF (AHF), respectively. Mammarenavirus Z matrix protein has been shown to exert a dose-dependent inhibitory effect in the activity of the virus ribonucleoprotein (vRNP) complex responsible for directing replication and transcription of the viral genome. Prevention of Z myristoylation by NMTi targeted Z protein for degradation, which resulted in inhibition of virus multiplication. Here, we review the recent findings of the use NMTi as antiviral, and reconcile the mechanistic process behind this inhibitory effect, more importantly, we used NMTi and G2A-mutated Z protein as a control of myristoylation function of wild type Z protein to elucidate that the vRNP suppression is monomeric-dependent activity of Z protein abundancy and it is independent of its myristoylation function.

microbiology↗

bioRxiv2024

Cellular N-myristoyl transferases Are Required for Mammarenavirus Multiplication

The mammarenavirus matrix Z protein plays critical roles in virus assembly and cell egress, whereas heterotrimer complexes of a stable signal peptide (SSP) together with glycoprotein subunits GP1 and GP2, generated via co-and post-translational processing of the surface glycoprotein precursor GPC, form the spikes that decorate the virion surface and mediate virus cell entry via receptor-mediated endocytosis. The Z protein and SSP undergo N-terminal myristoylation by host cell N-myristoyltransferases (NMT1 and NMT2), and G2A mutations that prevent myristoylation of Z or SSP have been shown to affect Z mediated virus budding and GP2 mediated fusion activity required to complete the virus cell entry process. In the present work, we present evidence that the validated on-target specific pan NMT inhibitor DDD85464 exerts a potent antiviral activity against the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV) that correlated with reduced Z budding activity and GP2 mediated fusion activity, as well as proteasome mediated degradation of the Z protein. The potent anti-mammarenaviral activity of DDD85646 was also observed with the hemorrhagic fever causing mammarenaviruses Junin (JUNV) and Lassa (LASV) viruses. Our results support exploration of NMT inhibition as a broad-spectrum antiviral against human pathogenic mammarenaviruses.

microbiology↗

bioRxiv2024

Functional impairment of "helpless" CD8+ memory T cells is transient and driven by prolonged but finite cognate antigen presentation

Generation of functional CD8+ T cell memory typically requires engagement of CD4+ T cells. However, in certain scenarios, such as acutely-resolving viral infections, effector (TE) and subsequent memory (TM) CD8+ T cell formation appear impervious to a lack of CD4+ T cell help during priming. Nonetheless, such "helpless" CD8+ TM respond poorly to pathogen rechallenge. At present, the origin and long-term evolution of helpless CD8+ T cell memory remain incompletely understood. Here, we demonstrate that helpless CD8+ TE differentiation is largely normal but a multiplicity of helpless CD8 TM defects, consistent with impaired memory maturation, emerge as a consequence of prolonged yet finite exposure to cognate antigen. Importantly, these defects resolve over time leading to full restoration of CD8+ TM potential and recall capacity. Our findings provide a unified explanation for helpless CD8+ T cell memory and emphasize an unexpected CD8+ TM plasticity with implications for vaccination strategies and beyond.

immunology↗

bioRxiv2023

Activation of Protein Kinase R (PKR) Plays a Pro-Viral Role in Mammarenavirus Infected Cells

Many viruses, including mammarenaviruses, have evolved mechanisms to counteract different components of the host cell innate immunity, which is required to facilitate robust virus multiplication. The double strand (ds)RNA sensor protein kinase receptor (PKR) pathway plays a critical role in the cell antiviral response. Whether PKR can restrict the multiplication of the Old World mammarenavirus lymphocytic choriomeningitis virus (LCMV) and the mechanisms by which LCMV may counteract the antiviral functions of PKR have not yet been investigated. Here we present evidence that LCMV infection results in very limited levels of PKR activation, but LCMV multiplication is enhanced in the absence of PKR. In contrast, infection with a recombinant LCMV with a mutation affecting the 3-5 exonuclease (ExoN) activity of the viral nucleoprotein (NP) resulted in robust PKR activation in the absence of detectable levels of dsRNA, which was associated with severely restricted virus multiplication that was alleviated in the absence of PKR. However, pharmacological inhibition of PKR activation resulted in reduced levels of LCMV multiplication. These findings uncovered a complex role of the PKR pathway in LCMV-infected cells involving both pro-and anti- viral activities. IMPORTANCEAs with many other viruses, the prototypic Old World mammarenavirus lymphocytic choriomeningitis virus (LCMV) can interfere with the host cell innate immune response to infection, which includes the double strand (ds)RNA sensor protein kinase receptor (PKR) pathway. A detailed understanding of LCMV-PKR interactions can provide novel insights about mammarenavirus-host cell interactions and facilitate the development of effective antiviral strategies against human pathogenic mammarenaviruses. In the present work, we present evidence that LCMV multiplication is enhanced in PKR- deficient cells, but pharmacological inhibition of PKR activation unexpectedly resulted in severely restricted propagation of LCMV. Likewise, we document a robust PKR activation in LCMV-infected cells in the absence of detectable levels of dsRNA. Our findings have revealed a complex role of the PKR pathway during LCMV infection and uncovered the activation of PKR as a druggable target for the development of antiviral drugs against human pathogenic mammarenaviruses.

microbiology↗

bioRxiv2023

Antivirals against monkeypox infections

Monkeypox virus (MPXV) infection in humans are historically restricted to endemic regions in Africa. However, in 2022, an alarming number of MPXV cases have been reported globally with evidence of person-to-person transmission. Because of this, the World Health Organization (WHO) declared the MPXV outbreak a public health emergency of international concern. MPXV vaccines are limited and only two antivirals, tecovirimat and brincidofovir, approved by the United States (US) Food and Drug Administration (FDA) for the treatment of smallpox, are currently available for the treatment of MPXV infection. Here, we evaluated 19 compounds previously shown to inhibit different RNA viruses for their ability to inhibit Orthopoxvirus infections. We first used recombinant vaccinia virus (rVACV) expressing fluorescence (Scarlet or GFP) and luciferase (Nluc) reporter genes to identify compounds with anti-Orthopoxvirus activity. Seven compounds from the ReFRAME library (antimycin A, mycophenolic acid, AVN- 944, pyrazofurin, mycophenolate mofetil, azaribine, and brequinar) and six compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) showed antiviral activity against rVACV. Notably, the anti-VACV activity of some of the compounds in the ReFRAME library (antimycin A, mycophenolic acid, AVN- 944, mycophenolate mofetil, and brequinar) and all the compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) were confirmed with MPXV, demonstrating the broad-spectrum antiviral activity against Orthopoxviruses and their potential to be used for the antiviral treatment of MPXV, or other Orthopoxvirus, infections. IMPORTANCEDespite the eradication of smallpox, some Orthopoxviruses remain important human pathogens, as exemplified by the recent 2022 monkeypox virus (MPXV) outbreak. Although smallpox vaccines are effective against MPXV, there is presently limited access to those vaccines. In addition, current antiviral treatment against MPXV infections is limited to the use of the FDA-approved drugs tecovirimat and brincidofovir. Thus, there is an urgent need to identify novel antivirals for the treatment of MPXV, and other potentially zoonotic Orthopoxvirus infections. Here, we show that thirteen compounds, derived from two different libraries, previously found to inhibit several RNA viruses, exhibit also antiviral activity against VACV. Notably, eleven compounds also displayed antiviral activity against MPXV, demonstrating their potential to be incorporated into the therapeutic armamentarium to combat Orthopoxvirus infections.

microbiology↗

bioRxiv2021

Inhibitors of anti-apoptotic Bcl-2 family proteins exhibit potent and broad-spectrum anti-mammarenavirus activity via cell cycle arrest at G0/G1 phase

Targeting host factors is a promising strategy to develop broad-spectrum antiviral drugs. Drugs targeting anti-apoptotic Bcl-2 family proteins that were originally developed as tumor suppressors have been reported to inhibit multiplication of different types of viruses. However, the mechanisms whereby Bcl-2 inhibitors exert their antiviral activity remain poorly understood. In this study, we have investigated the mechanisms by which obatoclax (OLX) and ABT-737 Bcl-2 inhibitors exhibited a potent antiviral activity against the mammarenavirus lymphocytic choriomeningitis virus (LCMV). OLX and ABT-737 potent anti-LCMV activity was not associated with their pro-apoptotic properties, but rather their ability of inducing cell arrest at G0/G1 phase. OLX and ABT-737 mediated inhibition of Bcl-2 correlated with reduced expression levels of thymidine kinase 1 (TK1), cyclin A2 (CCNA2), and cyclin B1 (CCNB1) cell cycle regulators. In addition, siRNA-mediated knock down of TK1, CCNA2, and CCNB1 resulted in reduced levels of LCMV multiplication. The antiviral activity exerted by Bcl-2 inhibitors correlated with reduced levels of viral RNA synthesis at early times of infection. Importantly, ABT-737 exhibited moderate efficacy in a mouse model of LCMV infection, and Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and SARS-CoV-2. Our results suggest that Bcl-2 inhibitors, actively being explored as anti-cancer therapeutics, might be repositioned as broad-spectrum antivirals. IMPORTANCEAnti-apoptotic Bcl-2 inhibitors have been shown to exert potent antiviral activities against various types of viruses via mechanisms that are currently poorly understood. This study has revealed that Bcl-2 inhibitors mediated cell cycle arrest at the G0/G1 phase, rather than their pro-apoptotic activity, plays a critical role in blocking mammarenavirus multiplication in cultured cells. In addition, we show that Bcl-2 inhibitor ABT-737 exhibited moderate anti-mammarenavirus activity in vivo, and that Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and SARS-CoV-2. Our results suggest that Bcl-2 inhibitors, actively being explored as anti-cancer therapeutics, might be repositioned as broad-spectrum antivirals.

microbiology↗

bioRxiv2021

Functional interactomes of the Ebola virus polymerase identified by proximity proteomics in the context of viral replication

Ebola virus (EBOV) critically depends on the viral polymerase to replicate and transcribe the viral RNA genome in the cytoplasm of host cells, where cellular factors can antagonize or facilitate the virus life cycle. Here we leveraged proximity proteomics and conducted an siRNA screen to define the functional interactome of EBOV polymerase. As proof-of-principle, we validated two cellular mRNA decay factors from 35 identified host factors: eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1) and up-frameshift protein 1 (UPF1). Our data suggest that EBOV can subvert restrictions of cellular mRNA decay and repurpose both GSPT1 and UPF1 to promote viral replication. Treating EBOV-infected human hepatocytes with a drug candidate that targets GSPT1 for degradation significantly reduced viral RNA load and particle production. Our work demonstrates the utility of proximity proteomics to capture the functional host-interactome of the EBOV polymerase and to illuminate host-dependent regulations of viral RNA synthesis.

microbiology↗

bioRxiv2021

Proximity interactome analysis of Lassa polymerase reveals eRF3a/GSPT1 as a druggable target for host directed antivirals.

Completion of the Lassa virus (LASV) life cycle critically depends on the activities of the virally encoded RNA-dependent RNA polymerase in replication and transcription of the viral RNA genome in the cytoplasm of infected cells. The contribution of cellular proteins to these processes remains unclear. Here, we applied proximity proteomics to define the interactome of LASV polymerase in cells, under conditions that recreate LASV RNA synthesis. We engineered a LASV polymerase-biotin ligase (TurboID) fusion protein that retained polymerase activity and successfully biotinylated the proximal proteome, which allowed the identification of 42 high-confidence LASV polymerase interactors. We subsequently performed an siRNA screen to identify those interactors that have functional roles in authentic LASV infection. As proof-of-principle, we characterized eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1), which we found to be a proviral factor that physically associates with LASV polymerase. Targeted degradation of GSPT1 by a small molecule drug candidate, CC-90009, resulted in strong inhibition of LASV infection in cultured cells. Our work demonstrates the feasibility of using proximity proteomics to illuminate and characterize yet to be defined, host-pathogen interactome, which can reveal new biology and uncover novel targets for the development of antivirals against highly pathogenic RNA viruses. Significance StatementLassa virus (LASV), the causative agent of Lassa fever (LF), represents an important public health problem in Western Africa. There is no FDA-approved therapeutic intervention to treat LF. Due to its limited genome coding capacity, LASV proteins are often multifunctional and orchestrate complex interactions with cellular factors to execute steps required to complete the viral life cycle. LASV polymerase is essential for replication and expression of the viral genome, and thus is an attractive target for antiviral intervention. Here we present the first host interactome of the LASV polymerase, which can guide identification of novel druggable host cellular targets for the development of cost-effective antiviral therapies for LF.

microbiology↗

bioRxiv2021

A SARS-CoV-2 nucleocapsid protein TR-FRET assay amenable to high-throughput screening

Drug development for specific antiviral agents against coronavirus disease 2019 (COVID-19) is still an unmet medical need as the pandemic continues to spread globally. Although huge efforts for drug repurposing and compound screens have put forth, only few compounds remain in late stage clinical trials. New approaches and assays are needed to accelerate COVID-19 drug discovery and development. Here we report a time-resolved fluorescence resonance energy transfer-based assay that detects the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP) produced in infected cells. It uses two specific anti-NP monoclonal antibodies (MAbs) conjugated to donor and acceptor fluorophores that produces a robust ratiometric signal for high throughput screening of large compound collections. Using this assay, we measured a half maximal inhibitory concentration (IC50) for Remdesivir of 9.3 M against infection with SARS-CoV-2 USA/WA1/2020 (WA-1). The assay also detected SARS-CoV-2 South African (Beta, {beta}), Brazilian/Japanese variant P.1 (Gamma, {gamma}), and Californian (Epsilon, {varepsilon}), variants of concern or interest (VoC). Therefore, this homogeneous SARS-CoV-2 NP detection assay can be used for accelerating lead compound discovery for drug development and for evaluating drug efficacy against emerging SARS-CoV-2 VoC.

pharmacology and toxicology↗

bioRxiv2021

A bifluorescent-based assay for the identification of neutralizing antibodies against SARS-CoV-2 variants of concern in vitro and in vivo

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and has been responsible for the still ongoing coronavirus disease 2019 (COVID-19) pandemic. Prophylactic vaccines have been authorized by the United States (US) Food and Drug Administration (FDA) for the prevention of COVID-19. Identification of SARS-CoV-2 neutralizing antibodies (NAbs) is important to assess vaccine protection efficacy, including their ability to protect against emerging SARS- CoV-2 variants of concern (VoC). Here we report the generation and use of a recombinant (r)SARS-CoV-2 USA/WA1/2020 (WA-1) strain expressing Venus and a rSARS-CoV-2 expressing mCherry and containing mutations K417N, E484K, and N501Y found in the receptor binding domain (RBD) of the spike (S) glycoprotein of the South African (SA) B.1.351 (beta, {beta}) VoC, in bifluorescent-based assays to rapidly and accurately identify human monoclonal antibodies (hMAbs) able to neutralize both viral infections in vitro and in vivo. Importantly, our bifluorescent-based system accurately recapitulated findings observed using individual viruses. Moreover, fluorescent- expressing rSARS-CoV-2 and the parental wild-type (WT) rSARS-CoV-2 WA-1 had similar viral fitness in vitro, as well as similar virulence and pathogenicity in vivo in the K18 human angiotensin converting enzyme 2 (hACE2) transgenic mouse model of SARS-CoV-2 infection. We demonstrate that these new fluorescent-expressing rSARS- CoV-2 can be used in vitro and in vivo to easily identify hMAbs that simultaneously neutralize different SARS-CoV-2 strains, including VoC, for the rapid assessment of vaccine efficacy or the identification of prophylactic and/or therapeutic broadly NAbs for the treatment of SARS-CoV-2 infection.

microbiology↗

bioRxiv2021

Visualization of SARS-CoV-2 infection dynamic

Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing rSARS-CoV-2 have jeopardized their use to monitor the dynamics of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the viral nucleocapsid gene followed by a 2A cleavage peptide. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and K18 hACE2 transgenic mice. Importantly, real-time viral infection was readily tracked using a non-invasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2 retained wild-type virus like pathogenicity in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis and therapeutic interventions for the treatment of SARS-CoV-2 in vivo.

microbiology↗