Biology subjects

Kutluay, S. B.

Publications and source records attributed to Kutluay, S. B..

12 recordsLinked to original sources

bioRxiv2026

HIV-1 Integrase C-terminal Domain Mutations Regulate ALLINI Sensitivity

HIV-1 integrase (IN) performs two essential functions in virus replication: catalyzing the integration of reverse-transcribed viral cDNA into host DNA and promoting proper virion maturation. The latter is mediated by IN binding to the genomic RNA (gRNA) through basic residues in its C-terminal domain (CTD), thereby ensuring correct repackaging of the gRNA inside the remodeled capsid lattice. Allosteric integrase inhibitors (ALLINIs) bind within a V-shaped pocket at the interface of the catalytic core domain (CCD) of IN dimers, promote the engagement of the CTDs from adjacent dimers, and thereby occlude gRNA binding. However, virological evidence supporting a role for the IN-CTD in ALLINI mechanism of action is lacking, as mutations in CTD residues implicated in ALLINI-induced multimerization typically yield non-infectious viruses. Here we demonstrate that IN-CTD mutants bearing compensatory substitutions that restore infectivity exhibit markedly altered sensitivity to ALLINIs. Specifically, we show that R269A/K273A substitutions within the IN-CTD confer ~100-fold enhanced sensitivity to ALLINIs. This correlates with a loss of IN-gRNA binding at low ALLINI concentrations, while exerting minimal effects on IN multimerization in vitro and in virions. In contrast, we found that the IN I268T substitution which arose as a suppressor mutation of I268A confers partial resistance to ALLINI-induced inhibition of gRNA binding. Collectively, these findings provide the first direct genetic evidence that CTD residues, despite being distal to the primary drug binding site at the CCD dimer, modulate sensitivity to ALLINIs and highlights IN-gRNA binding as a central target of ALLINIs.

microbiology↗

bioRxiv2026

TMEM106B mediates ACE2-independent replication of the SARS-CoV-2 S-E484D variant in airway-derived cell models

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to cause significant respiratory disease, particularly in vulnerable populations. Although ACE2 is the primary receptor for viral entry, previous studies have identified a naturally occurring, ACE2-independent entry pathway in certain airway-derived cell lines. Utilization of this pathway depends on surface heparan sulfates and requires the E484D substitution within the receptor-binding domain of the viral Spike (S) protein. In this study, we expand the panel of airway-derived cell lines that support ACE2-independent, S-E484D-dependent replication and identify the host lysosomal transmembrane protein TMEM106B as a critical host factor for infection. Knockout of TMEM106B completely abolishes infection by SARS-CoV-2 SE484D in NCI-H522 and NCI-H661 cells. Moreover, ectopic expression of the luminal C-terminal domain (CTD) of TMEM106B-either alone or redirected to the plasma membrane - is sufficient to enable viral entry and infection in otherwise non-permissive cells. We further show that Fc-TMEM106B-CTD decoy protein blocks infection by SARS-CoV-2 SE484D, supporting a direct interaction between the S-E484D protein and TMEM106B-CTD. Finally, passaging experiments with a chimeric VSV-SARS-CoV-2 SE484D identify additional mutations within the heptad repeat 1 that enhance TMEM106B utilization and viral spread in the ACE2-independent cell models. Together, these findings demonstrate that TMEM106B is a key mediator of a naturally occurring ACE2-independent pathway in multiple airway-derived cells lines and suggest that variation in the Spike protein can expand receptor usage by SARS-CoV-2. IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to acquire mutations in the viral spike (S) protein as it circulates in humans. How these mutations influence the cell and tissue tropism of SARS-CoV-2 remains poorly understood. While ACE2 is the canonical host cell receptor for SARS-CoV-2, previous studies revealed the presence of a naturally occurring ACE2-independent entry pathway in multiple airway-derived cell lines that can be utilized only by a SARS-CoV-2 variant bearing the E484D substitution within the viral S protein. Here we expand the airway-derived cell line models that support ACE2-independent and S-E484D-dependent entry and show that the host lysosomal transmembrane protein, TMEM106B, is an essential viral entry factor. Although the E484D substitution is currently rare in human isolates, our findings raise the possibility that small mutations in SARS-CoV-2 S can allow the utilization of alternative entry pathways that broadens its cell and tissue tropism.

microbiology↗

bioRxiv2025

Efficient packaging of HIV-1 genomes via recognition of its adenosine-rich content by a heterologous RNA-binding domain

The HIV-1 genome (gRNA) has an unusually biased nucleotide content and is rich in adenosines. Selective packaging of the gRNA is thought to be driven by specific binding of the nucleocapsid (NC) domain of the viral Gag protein to the packaging signal ({Psi}) in the host cell cytosol. However, deletion of regions within {Psi} reduces--but does not completely abolish--genome packaging. To probe whether another feature of the gRNA may contribute to the selective gRNA packaging process, we replaced NC with heterologous RNA-binding domains (RBDs) with distinct RNA-binding properties. Surprisingly, despite disparate RNA binding specificities, all Gag-RBD chimeras successfully recruited the gRNA to the plasma membrane, suggesting that the initial gRNA recognition in the cytosol is not rate limiting. Notwithstanding, many chimeras exhibiting G/C binding specificity were arrested at the assembly stage. Only the Gag-SRSF5 chimera, which multimerized efficiently on adenosine-rich sequences on the gRNA, assembled efficiently and packaged gRNA at near wild-type levels. Importantly, rationally designed mutations that altered the A/G-rich binding specificity of Gag-SRSF5 decreased genome encapsidation efficiency. Furthermore, many Gag chimeras displayed potent dominant negative activities, highlighting NC functions as a targetable step in virus replication. Together, our findings reveal an unexpected aspect of the HIV-1 gRNA, its biased nucleotide content, as a key driver of selective genome packaging. SIGNIFICANCEHow HIV-1 selectively packages its genome (gRNA) into virions is poorly understood. To probe this, we replaced the viral nucleocapsid (NC) protein with heterologous RNA-binding domains (RBDs) from cellular hnRNP and SR protein families. Remarkably, despite their distinct RNA-binding specificities, all Gag-RBD chimeras successfully recruited the gRNA to the plasma membrane, suggesting that the initial gRNA recognition is not rate limiting. Only the Gag-SRSF5 chimera packaged gRNA efficiently which correlated with its capacity to multimerize on adenosine-rich sequences on the gRNA. Notably, several Gag chimeras exhibited strong dominant negative effects, underscoring NC functions as a targetable step in virus replication. Together, these findings uncover that the biased nucleotide content of the HIV-1 gRNA facilitates its selective packaging into virus particles.

microbiology↗

bioRxiv2024

A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models

Host factors that define the cellular tropism of SARS-CoV-2 beyond the cognate ACE2 receptor are poorly defined. Here we report that SARS-CoV-2 replication is restricted at a post-entry step in a number of ACE2-positive airway-derived cell lines due to tonic activation of the cGAS-STING pathway mediated by mitochondrial DNA leakage and naturally occurring cGAS and STING variants. Genetic and pharmacological inhibition of the cGAS-STING and type I/III IFN pathways as well as ACE2 overexpression overcome these blocks. SARS-CoV-2 replication in STING knockout cell lines and primary airway cultures induces ISG expression but only in uninfected bystander cells, demonstrating efficient antagonism of the type I/III IFN-pathway in productively infected cells. Pharmacological inhibition of STING in primary airway cells enhances SARS-CoV-2 replication and reduces virus-induced innate immune activation. Together, our study highlights that tonic activation of the cGAS-STING and IFN pathways can impact SARS-CoV-2 cellular tropism in a manner dependent on ACE2 expression levels.

microbiology↗

bioRxiv2023

Defining distinct RNA-protein interactomes of SARS-CoV-2 genomic and subgenomic RNAs

Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral defense mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of viral replication. Here, for the first time, we demonstrate the successful isolation of SARS-CoV-2 genomic RNA and three distinct sgRNAs (N, S, and ORF8) from a single population of infected cells and characterize their protein interactomes. Over 500 protein interactors (including 260 previously unknown) were identified as associated with one or more target RNA at either of two time points. These included protein interactors unique to a single RNA pool and others present in multiple pools, highlighting our ability to discriminate between distinct viral RNA interactomes despite high sequence similarity. The interactomes indicated viral associations with cell response pathways including regulation of cytoplasmic ribonucleoprotein granules and posttranscriptional gene silencing. We validated the significance of five protein interactors predicted to exhibit antiviral activity (APOBEC3F, TRIM71, PPP1CC, LIN28B, and MSI2) using siRNA knockdowns, with each knockdown yielding increases in viral production. This study describes new technology for studying SARS-CoV-2 and reveals a wealth of new viral RNA-associated host factors of potential functional significance to infection.

molecular biology↗

bioRxiv2022

Calpain-2 mediates SARS-CoV-2 entry and represents a therapeutic target

Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, much effort has been dedicated to identifying effective antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A number of calpain inhibitors show excellent antiviral activities against SARS-CoV-2 by targeting the viral main protease (Mpro), which plays an essential role in processing viral polyproteins. In this study, we found that calpain inhibitors potently inhibited the infection of a chimeric vesicular stomatitis virus (VSV) encoding the SARS-CoV-2 spike protein, but not Mpro. In contrast, calpain inhibitors did not exhibit antiviral activities towards the wild-type VSV with its native glycoprotein. Genetic knockout of calpain-2 by CRISPR/Cas9 conferred resistance of the host cells to the chimeric VSV-SARS-CoV-2 virus and a clinical isolate of wild-type SARS-CoV-2. Mechanistically, calpain-2 facilitates SARS-CoV-2 spike protein-mediated cell attachment by positively regulating the cell surface levels of ACE2. These results highlight an Mpro-independent pathway targeted by calpain inhibitors for efficient viral inhibition. We also identify calpain-2 as a novel host factor and a potential therapeutic target responsible for SARS-CoV-2 infection at the entry step.

microbiology↗

bioRxiv2021

Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell

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.

microbiology↗

bioRxiv2020

Amilorides inhibit SARS-CoV-2 replication in vitro by targeting RNA structures

The SARS-CoV-2 pandemic, and the likelihood of future coronavirus pandemics, has rendered our understanding of coronavirus biology more essential than ever. Small molecule chemical probes offer to both reveal novel aspects of virus replication and to serve as leads for antiviral therapeutic development. The RNA-biased amiloride scaffold was recently tuned to target a viral RNA structure critical for translation in enterovirus 71, ultimately uncovering a novel mechanism to modulate positive-sense RNA viral translation and replication. Analysis of CoV RNA genomes reveal many conserved RNA structures in the 5-UTR and proximal region critical for viral translation and replication, including several containing bulge-like secondary structures suitable for small molecule targeting. Following phylogenetic conservation analysis of this region, we screened an amiloride-based small molecule library against a less virulent human coronavirus, OC43, to identify lead ligands. Amilorides inhibited OC43 replication as seen in viral plaque assays. Select amilorides also potently inhibited replication competent SARS-CoV-2 as evident in the decreased levels of cell free virions in cell culture supernatants of treated cells. Reporter screens confirmed the importance of RNA structures in the 5-end of the viral genome for small molecule activity. Finally, NMR chemical shift perturbation studies of the first six stem loops of the 5-end revealed specific amiloride interactions with stem loops 4, 5a, and 6, all of which contain bulge like structures and were predicted to be strongly bound by the lead amilorides in retrospective docking studies. Taken together, the use of multiple orthogonal approaches allowed us to identify the first small molecules aimed at targeting RNA structures within the 5-UTR and proximal region of the CoV genome. These molecules will serve as chemical probes to further understand CoV RNA biology and can pave the way for the development of specific CoV RNA-targeted antivirals.

microbiology↗

bioRxiv2020

The translational landscape of SARS-CoV-2 and infected cells

SARS-CoV-2 utilizes a number of strategies to modulate viral and host mRNA translation. Here, we used ribosome profiling in SARS-CoV-2 infected model cell lines and primary airway cells grown at the air-liquid interface to gain a deeper understanding of the translationally regulated events in response to virus replication. We find that SARS-CoV-2 mRNAs dominate the cellular mRNA pool but are not more efficiently translated than cellular mRNAs. SARS-CoV-2 utilized a highly efficient ribosomal frameshifting strategy in comparison to HIV-1, suggesting utilization of distinct structural elements. In the highly permissive cell models, although SARS-CoV-2 infection induced the transcriptional upregulation of numerous chemokines, cytokines and interferon stimulated genes, many of these mRNAs were not translated efficiently. Impact of SARS-CoV-2 on host mRNA translation was more subtle in primary cells, with marked transcriptional and translational upregulation of inflammatory and innate immune responses and downregulation of processes involved in ciliated cell function. Together, these data reveal the key role of mRNA translation in SARS-CoV-2 replication and highlight unique mechanisms for therapeutic development.

microbiology↗

bioRxiv2020

CARD8 inflammasome mediates pyroptosis of HIV-1-infected cells by sensing viral protease activity

HIV-1 has high mutation rates and exists as mutant swarms in the host. Rapid evolution of HIV-1 allows the virus to outpace host immune system, leading to viral persistence. Novel approaches to target immutable components are needed to clear HIV-1 infection. Here we report a pattern-recognition receptor CARD8 that senses enzymatic activity of the HIV-1 protease, which is indispensable for the virus. All subtypes of HIV-1 can be sensed by CARD8 despite substantial viral diversity. HIV-1 evades CARD8 sensing because the viral protease remains inactive in infected cells prior to viral budding. Induction of premature intracellular activation of the viral protease triggers CARD8 inflammasome-mediated pyroptosis of HIV-1-infected cells. This strategy leads to clearance of latent HIV-1 in patient CD4+ T cells after virus reactivation. Taken together, our study identifies CARD8 as an inflammasome sensor of HIV-1 that holds promise as a strategy for clearance of persistent HIV-1 infection.

immunology↗

bioRxiv2020

A facile Q-RT-PCR assay for monitoring SARS-CoV-2 growth in cell culture

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the ongoing COVID-19 pandemic, has infected millions within just a few months and is continuing to spread around the globe causing immense respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here we developed a facile Q-RT-PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 replication kinetics from a small amount of cell culture supernatants. Using this assay, we screened the activities of a number of entry, SARS-CoV-2- and HIV-1-specific inhibitors in a proof of concept study. In line with previous studies which has shown that processing of the viral Spike protein by cellular proteases and endosomal fusion are required for entry, we found that E64D and apilimod potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that macropinocytosis inhibitor EIPA similarly decreased viral RNA in supernatants suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (an NNRTI), amprenavir (a protease inhibitor), and ALLINI-2 (an allosteric integrase inhibitor) modestly inhibited SARS-CoV-2 replication, albeit the IC50 values were much higher than that required for HIV-1. Taken together, this facile assay will undoubtedly expedite basic SARS-CoV-2 research, be amenable to mid-throughput screens to identify chemical inhibitors of SARS-CoV-2, and be applicable to a broad number of RNA and DNA viruses. ImportanceSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the COVID-19 pandemic, has quickly become a major global health problem causing 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 facile Q-RT-PCR assay to monitor SARS-CoV-2 growth in cell culture supernatants that does not necessitate RNA extraction, and 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. This facile approach will undoubtedly expedite basic SARS-CoV-2 research, be amenable to screening platforms to identify therapeutics against SARS-CoV-2 and can be adapted to numerous other RNA and DNA viruses.

microbiology↗

bioRxiv2019

Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis

A large number of HIV-1 integrase (IN) alterations, referred to as class II substitutions, exhibit pleotropic effects during virus replication. However, the underlying mechanism for the class II phenotype is not known. Here we demonstrate that all tested class II IN substitutions compromised IN-RNA binding in virions by one of three distinct mechanisms: i) markedly reducing IN levels thus precluding formation of IN complexes with viral RNA; ii) adversely affecting functional IN multimerization and consequently impairing IN binding to viral RNA; iii) directly compromising IN-RNA interactions without substantially affecting IN levels or functional IN multimerization. Inhibition of IN-RNA interactions resulted in mislocalization of the viral ribonucleoprotein complexes outside the capsid lattice, which led to premature degradation of the viral genome and IN in target cells. Collectively, our studies uncover causal mechanisms for the class II phenotype and highlight an essential role of IN-RNA interactions for accurate virion maturation.

microbiology↗