Biology subjects

Rajsbaum, R.

Publications and source records attributed to Rajsbaum, R..

11 recordsLinked to original sources

bioRxiv2025

Coronavirus NSP14 Drives Internal m7G Modification to Rewire Host Splicing and Promote Viral Replication

SARS-CoV-2, the causative agent of COVID-19, manipulates host gene expression through multiple mechanisms, including disruption of RNA processing. Here, we identify a novel function of the viral nonstructural protein 14 (NSP14) in inducing N7-methylguanosine (m7G) modification in the internal sequences of host mRNA. We demonstrate that NSP14 catalyzes the conversion of guanosine triphosphate (GTP) to m7GTP, which is subsequently incorporated into mRNA by RNA polymerase II, resulting in widespread internal m7G modification. This activity is dependent on NSP14s N7-methyltransferase (N7-MTase) domain and is enhanced by interaction with NSP10. Internal m7G modification by NSP14 predominantly occurs in the nucleus and is conserved across alpha-, beta- and gamma-coronaviruses. Mechanistically, we show that this RNA modification disrupts normal splicing by promoting intron retention and generating novel splice junctions, particularly in genes regulating genome stability, RNA metabolism and nuclear processes. Importantly, inhibition of m7G modification, through pharmacological targeting of NSP14 or RNA polymerase II, impairs SARS-CoV-2 replication, indicating that the virus hijacks host transcriptomic machinery to support infection. Our findings reveal a previously unrecognized epitranscriptomic mechanism by which coronaviruses reprogram host gene expression and suggest that NSP14-induced m7G modification is a potential therapeutic target.

microbiology↗

bioRxiv2025

IL-4 and TGF-β Regulate Inflammatory cytokines and Cellular Infiltration in the Lung in Mouse-adapted SARS-CoV-2 Infection

The pathology of severe COVID-19 is due to a hyperinflammatory immune response persisting after viral clearance. To understand how the immune response to SARS-CoV-2 is regulated to avoid severe COVID-19, we tested relevant immunoregulatory cytokines. TGF-{beta}, IL-10 and IL-4 were neutralized upon infection with mouse-adapted SARS-CoV-2 (CMA3p20), a model of mild disease; and lung inflammation was quantified by histology and flow cytometry at early and late time points. Mild weight loss, and lung inflammation including consolidation and alveolar thickening were evident 3 days post-infection (dpi) and inflammation persisted to 7 dpi. Coinciding with early monocytic infiltrates, CCL2 and granulocyte-colony stimulating factor (G-CSF) were transiently produced 3 dpi, while IL-12 and CCL5 persisted to 7 dpi, modeling viral and inflammatory phases of disease. Neutralization of TGF-{beta}, but not IL-10 or IL-4, significantly increased lung inflammatory monocytes and elevated serum but not lung IL-6. Neutralization of IL-4 prolonged weight loss and increased early perivascular infiltration without changing viral titer. Anti-IL-4 reduced expression of Arg1, a gene associated with alternative activation of macrophages. Neutralizing TGF-{beta} and IL-4 had differential effects on pathology after virus control. Lung perivascular infiltration was reduced 7 dpi by neutralization of IL-4 or TGF-{beta}, and peri-airway inflammation was affected by anti-TGF-{beta}, while alveolar infiltrates were not affected by either. Anti-IL-4 prolonged IL-12 to 7 dpi along with reduced IL-10 in lungs. Overall, the immunoregulatory cytokines TGF-{beta} and IL-4 dampen initial inflammation in this maSARS-CoV-2 infection, suggesting that promotion of immunoregulation could help patients in early stages of disease. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/653138v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@14c34org.highwire.dtl.DTLVardef@1322a8dorg.highwire.dtl.DTLVardef@8792aborg.highwire.dtl.DTLVardef@ae513e_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Characterization of betacoronavirus HKU-1 and OC43 internal proteins using a prototypic coronavirus

Coronaviruses express a repertoire of accessory proteins for evading host immune responses. A small internal (I) accessory protein is expressed by the genus Betacoronavirus. Previous studies reported that the I proteins of SARS-CoV, MERS-CoV and SARS-CoV-2 inhibit type I interferon (IFN-I) expression through distinct mechanisms and have different roles in pathogenesis. Human coronaviruses (hCoV) HKU1 and OC43 are betacoronaviruses that also encode the I protein as an accessory protein. Although hCoV-HKU1 and hCoV-OC43 predominantly cause common cold in healthy adults, susceptible individuals infected with these viruses can develop severe disease. However, the virulence factors contributing to pathogenesis after infection with common cold CoVs (CCCoVs) have not been fully characterized. In particular, the functions of the hCoV-HKU1 and hCoV-OC43 I proteins have not been previously reported. The lack of robust reverse genetic systems, tissue culture and animal models limit the study of hCoV-HKU1 and hCoV-OC43 pathogenesis. Here, we examine the role of the hCoV-HKU1 and hCoV-OC43 I proteins in pathogenesis using a prototypic coronavirus. We introduce the I proteins of hCoV-HKU1 and hCoV-OC43 independently to a neurotropic strain of mouse hepatitis virus (J2.2). J2.2 infection is well characterized with clearly defined immune responses which allows the study of I proteins in the context of authentic coronavirus infection. We show that the I protein of hCoV-HKU1, but not that of hCoV-OC43, ameliorates MHV-J2.2 pathogenesis while the I protein of MERS-CoV exacerbates disease. The presence of the hCoV-HKU1 I protein decreases virus titers and cytokine expression while the I protein of MERS-CoV leads to increased immune cell infiltration and virus titers in mice after J2.2 infection. Moreover, the I proteins of hCoV-HKU1 and hCoV-OC43 show different patterns of subcellular localization. Overall, our findings suggest that the I protein of different betacoronaviruses play unique roles in pathogenesis. Author SummaryFactors governing the differences in the pathogenicity between highly pathogenic human coronaviruses (SARS-CoV, MERS-CoV and SARSR-CoV-2) and seasonal coronaviruses (hCoV-HKU1 and hCoV-OC43) are not completely understood. These differences are at least in part contributed to by the accessory proteins encoded between these two groups of human coronaviruses. The use of a heterologous coronavirus infection model provides an isogenic background for the direct comparison of viral proteins encoded by various coronaviruses. In this study, we compare the role of one of the accessory proteins encoded by betacoronaviruses, the I protein, in mediating disease outcome using a prototypic coronavirus. We demonstrate that the I protein of the highly pathogenic MERS-CoV but not that of the seasonal coronaviruses HKU1 and OC43 contributes to enhanced disease in the context of MHV-J2.2 infection, highlighting that virus-specific functions of accessory proteins encoded by different hCoVs.

microbiology↗

bioRxiv2024

Comprehensive Analysis of SARS-CoV-2 Spike Evolution: Epitope Classification and Immune Escape Prediction

The evolution of SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has produced unprece-dented numbers of structures of the Spike protein. This study presents a comprehensive analysis of 1,560 published Spike protein structures, capturing most variants that emerged throughout the pandemic and covering diverse heteromerization and interacting complexes. We employ an interaction-energy informed geometric clustering to identify 14 epitopes characterized by their conformational specificity, shared interface with ACE2 binding, and glycosylation patterns. Our per-residue interaction evaluations accurately predict each residues role in antibody recognition and as well as experimental measurements of immune escape, showing strong correlations with DMS data, thus making it possible to predict the behaviour of future variants. We integrate the structural analysis with a longitudinal analysis of nearly 3 million viral sequences. This broad-ranging structural and longitudinal analysis provides insight into the effect of specific mutations on the energetics of interactions and dynamics of the SARS-CoV-2 Spike protein during the course of the pandemic. Specifically, with the emergence of widespread immunity, we observe an enthalpic trade-off in which mutations in the receptor binding motif (RBM) that promote immune escape also weaken the interaction with ACE2. Additionally, we also observe a second mechanism, that we call entropic trade-off, in which mutations outside of the RBM contribute to decrease the occupancy of the open state of SARS-CoV-2 Spike, thus also contributing to immune escape at the expense of ACE2 binding but without changes on the ACE2 binding interface. This work not only highlights the role of mutations across SARS-CoV-2 Spike variants but also reveals the complex interplay of evolutionary forces shaping the evolution of the SARS-CoV-2 Spike protein over the course of the pandemic.

biophysics↗

bioRxiv2024

TRIM7 ubiquitinates SARS-CoV-2 membrane protein to limit apoptosis and viral replication

SARS-CoV-2 is a highly transmissible virus that causes COVID-19 disease. Mechanisms of viral pathogenesis include excessive inflammation and viral-induced cell death, resulting in tissue damage. We identified the host E3-ubiquitin ligase TRIM7 as an inhibitor of apoptosis and SARS-CoV-2 replication via ubiquitination of the viral membrane (M) protein. Trim7-/- mice exhibited increased pathology and virus titers associated with epithelial apoptosis and dysregulated immune responses. Mechanistically, TRIM7 ubiquitinates M on K14, which protects cells from cell death. Longitudinal SARS-CoV-2 sequence analysis from infected patients revealed that mutations on M-K14 appeared in circulating variants during the pandemic. The relevance of these mutations was tested in a mouse model. A recombinant M- K14/K15R virus showed reduced viral replication, consistent with the role of K15 in virus assembly, and increased levels of apoptosis associated with the loss of ubiquitination on K14. TRIM7 antiviral activity requires caspase-6 inhibition, linking apoptosis with viral replication and pathology.

microbiology↗

bioRxiv2023

Ebola Virus VP35 Interacts Non-Covalently with Ubiquitin Chains to Promote Viral Replication Creating New Therapeutic Opportunities

Ebolavirus (EBOV) belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans. EBOV replication requires the activity of the viral polymerase complex, which includes the co-factor and Interferon antagonist VP35. We previously showed that the covalent ubiquitination of VP35 promotes virus replication by regulating interactions with the polymerase complex. In addition, VP35 can also interact non-covalently with ubiquitin (Ub); however, the function of this interaction is unknown. Here, we report that VP35 interacts with free (unanchored) K63-linked polyUb chains. Ectopic expression of Isopeptidase T (USP5), which is known to degrade unanchored polyUb chains, reduced VP35 association with Ub and correlated with diminished polymerase activity in a minigenome assay. Using computational methods, we modeled the VP35-Ub non-covalent interacting complex, identified the VP35-Ub interacting surface and tested mutations to validate the interface. Docking simulations identified chemical compounds that can block VP35-Ub interactions leading to reduced viral polymerase activity that correlated with reduced replication of infectious EBOV. In conclusion, we identified a novel role of unanchored polyUb in regulating Ebola virus polymerase function and discovered compounds that have promising anti-Ebola virus activity. Significance StatementEbola virus infection can result in high mortality rates with extreme risk of person-to-person transmission. Sporadic outbreaks in Africa have resulted in thousands of fatal cases, highlighting that there is still insufficient knowledge to develop effective antiviral therapies. Like other viruses, Ebola utilizes the host machinery to replicate. Understanding how viral and host proteins interact can help identifying targets for the rational design of antiviral drugs. Here, we identified interactions between the cellular ubiquitin machinery and the Ebola virus polymerase cofactor protein VP35, which are important for efficient virus replication. We developed an approach to identify and block these virus-host interactions using small chemical compounds, which provides a useful tool to study functional molecular mechanisms and at the same time a potential approach to antiviral therapies.

immunology↗

bioRxiv2021

A novel mechanism of regulation of the transcription factor GLI3 by toll-like receptor signaling

The transcription factor GLI3 is a member of the GLI family and has been shown to be regulated by canonical hedgehog (HH) signaling through smoothened (SMO). Little is known about SMO-independent regulation of GLI3. Here, we identify TLR signaling as a novel pathway regulating GLI3 expression. We show that GLI3 expression is induced by LPS/TLR4 in human monocyte cell lines and peripheral blood CD14+ cells. Further analysis identified TRIF, but not MyD88, signaling as the adapter used by TLR4 to regulate GLI3. Using pharmacological and genetic tools, we identified IRF3 as the transcription factor regulating GLI3 downstream of TRIF. Furthermore, using additional TLR ligands that signal exclusively through TRIF such as the TLR4 ligand, MPLA and the TLR3 ligand, Poly(I:C), we confirm the role of TRIF-IRF3 in the regulation of GLI3. We found that IRF3 directly binds to the GLI3 promoter region and this binding was increased upon stimulation of TRIF-IRF3 with Poly(I:C). Furthermore, using IRF3-/- MEFs, we found that Poly(I:C) stimulation no longer induced GLI3 expression. Finally, using macrophages from mice lacking Gli3 expression in myeloid cells (M-Gli3-/-), we found that in the absence of Gli3, LPS stimulated macrophages secrete less CCL2 and TNF- compared with macrophages from wild-type (WT) mice. Taken together, these results identify a novel TLR-TRIF-IRF3 pathway that regulates the expression of GLI3 that regulates inflammatory cytokines and expands our understanding of the non-canonical signaling pathways involved in the regulation of GLI transcription factors.

immunology↗

bioRxiv2020

Topoisomerase III-beta is required for efficient replication of positive-sense RNA viruses

Based on genome-scale loss-of-function screens we discovered that Topoisomerase III-{beta} (TOP3B), a human topoisomerase that acts on DNA and RNA, is required for yellow fever virus and dengue virus-2 replication. Remarkably, we found that TOP3B is required for efficient replication of all positive-sense-single stranded RNA viruses tested, including SARS-CoV-2. While there are no drugs that specifically inhibit this topoisomerase, we posit that TOP3B is an attractive anti-viral target.

microbiology↗

bioRxiv2019

VAMP8 contributes to TRIM6-mediated type-I interferon antiviral response during West Nile virus infection

Several members of the tripartite motif (TRIM) family of E3 ubiquitin ligases regulate immune pathways including the antiviral type I interferon (IFN-I) system. Previously, we demonstrated that TRIM6 is involved in IFN-I induction and signaling. In the absence of TRIM6, optimal IFN-I signaling is reduced, allowing increased replication of interferon-sensitive viruses. Despite having evolved numerous mechanisms to restrict the vertebrate hosts IFN-I response, West Nile Virus (WNV) replication is sensitive to pre-treatment with IFN-I. However, the regulators and products of the IFN-I pathway that are important in regulating WNV replication are incompletely defined. Consistent with WNVs sensitivity to IFN-I, we found that in TRIM6 knockout (TRIM6-KO) A549 cells WNV replication is significantly increased and IFN-I induction and signaling is impaired compared to wild-type (wt) cells. IFN{beta} pre-treatment was more effective in protecting against subsequent WNV infection in wt cells as compared to TRIM6-KO, indicating that TRIM6 contributes to the establishment of an IFN-induced antiviral response against WNV. Using next generation sequencing, we identified VAMP8 as a potential factor involved in this TRIM6-mediated antiviral response. VAMP8 knockdown resulted in reduced Jak1 and STAT1 phosphorylation and impaired induction of several ISGs following WNV infection or IFN{beta} treatment. Furthermore, VAMP8-mediated STAT1 phosphorylation required the presence of TRIM6. Therefore, the VAMP8 protein is a novel regulator of IFN-I signaling, and its expression and function is dependent on TRIM6 activity. Overall, these results provide evidence that TRIM6 contributes to the antiviral response against WNV and identified VAMP8 as a novel regulator of the IFN-I system.\n\nIMPORTANCEWNV is a mosquito-borne flavivirus that poses threat to human health across large discontinuous areas throughout the world. Infection with WNV results in febrile illness, which can progress to severe neurological disease. Currently, there are no approved treatment options to control WNV infection. Understanding the cellular immune responses that regulate viral replication is important in diversifying the resources available to control WNV. Here we show that the elimination of TRIM6 in human cells results in an increase in WNV replication and alters the expression and function of other components of the IFN-I pathway through VAMP8. Dissecting the interactions between WNV and host defenses both informs basic molecular virology and promotes the development of host- and viral-targeted antiviral strategies.

immunology↗

bioRxiv2019

Peptidoglycan associated cyclic lipopeptide disrupts viral infectivity

Enteric viruses exploit bacterial components including lipopolysaccharides (LPS) and peptidoglycan (PG) to facilitate infection in humans. With origins in the bat enteric system, we wondered if severe acute respiratory syndrome-coronavirus (SARS-CoV) or Middle East respiratory syndrome-CoV (MERS-CoV) also use bacterial components to modulate infectivity. To test this question, we incubated CoVs with LPS and PG and evaluated infectivity finding no change following LPS treatment. However, PG from B. subtilis reduced infection >10,000-fold while PG from other bacterial species failed to recapitulate this. Treatment with an alcohol solvent transferred inhibitory activity to the wash and mass spectrometry revealed surfactin, a cyclic lipopeptide antibiotic, as the inhibitory compound. This antibiotic had robust dose- and temperature-dependent inhibition of CoV infectivity. Mechanistic studies indicated that surfactin disrupts CoV virion integrity and surfactin treatment of the virus inoculum ablated infection in vivo. Finally, similar cyclic lipopeptides had no effect on CoV infectivity and the inhibitory effect of surfactin extended broadly to enveloped viruses including influenza, Ebola, Zika, Nipah, Chikungunya, Una, Mayaro, Dugbe, and Crimean-Congo hemorrhagic fever viruses. Overall, our results indicate that peptidoglycan-associated surfactin has broad virucidal activity and suggest bacteria byproducts may negatively modulate virus infection.\n\nImportanceIn this manuscript, we considered a role for bacteria in shaping coronavirus infection. Taking cues from studies of enteric viruses, we initially investigated how bacterial surface components might improve CoV infection. Instead, we found that peptidoglycan-associated surfactin is a potent viricidal compound that disrupts virion integrity with broad activity against enveloped viruses. Our results indicate that interactions with commensal bacterial may improve or disrupt viral infections highlighting the importance of understanding these microbial interactions and their implications for viral pathogenesis and treatment.

microbiology↗

bioRxiv2018

VAMP8 contributes to TRIM6-mediated type-I interferon antiviral response during West Nile virus infection

Members of the tripartite motif (TRIM) family of E3 ubiquitin ligases regulate immune pathways including the antiviral type I interferon (IFN-I) system. Previously, we demonstrated that TRIM6 is involved in IFN-I induction and signaling. In absence of TRIM6 function, optimal IFN-I signaling is reduced, allowing increased replication of interferon-sensitive viruses. Despite numerous mechanisms to restrict vertebrate hosts IFN-I response, West Nile Virus (WNV) replication is sensitive to pre-treatment with IFN-I. However, the regulators and products of the IFN-I pathway important in regulating WNV replications are incompletely defined. Consistent with WNVs sensitivity to IFN-I, we found that in TRIM6 knockout (TRIM6 KO) A549 cells WNV replication is significantly increased. Additionally, induction of Ifnb mRNA was delayed and the expression of several IFN-stimulated genes (ISGs) was reduced in TRIM6 KO cells. IFN{beta} pre-treatment was more effective in protecting against subsequent WNV infection in wt cells, indicating that TRIM6 contributes to the establishment of an IFN-induced antiviral response against WNV. Using next generation sequencing, we identified potential factors involved in this TRIM6-mediated antiviral response. One identified gene, VAMP8, is a soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) in the vesicle-associated membrane protein subfamily. Knockdown of VAMP8 resulted in reduced STAT1 phosphorylation and impaired induction of several ISGs following WNV infection or IFN{beta} treatment. Therefore, VAMP8 is a novel gene involved in the regulation of IFN-I signaling, and its expression is dependent on TRIM6 function. Overall, these results indicate that TRIM6 contributes to the antiviral response against WNV by regulating the IFN-I system.\n\nIMPORTANCEWNV is a mosquito-borne flavivirus that poses threat to human health across large discontinuous areas throughout the world. Infection with WNV results in febrile illness, which can progress to severe neurological disease. Currently, there are no approved treatment options to control WNV infection. Understanding the cellular immune responses that regulate viral replication is important in diversifying the resources available to control WNV. Here we show that the elimination of TRIM6 in human cells results in an increase in WNV replication and alters the expression and function of other components of the IFN-I pathway through VAMP8. Dissecting the interactions between WNV and host defenses both informs basic molecular virology and promotes the development of host- and viral-targeted antiviral strategies.

immunology↗