Biology subjects

Moorman, N.

Publications and source records attributed to Moorman, N..

6 recordsLinked to original sources

bioRxiv2026

The nucleoside analog CMX521 inhibits coronavirus RNA-dependent RNA polymerase via a two-pronged mechanism

The SARS-CoV-2 pandemic has underscored the urgent need for broad-spectrum antivirals in pandemic preparedness efforts. Nucleoside analogs targeting viral polymerases are often considered in this context. Here, we employ ensemble biochemical assays and single-molecule magnetic tweezers to characterize the detailed mechanism of action of the adenosine analog CMX521 (developed through Phase 1 clinical studies), a broad-spectrum antiviral against caliciviruses and coronaviruses, against SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). The triphosphate form of CMX521 is efficiently incorporated by RdRp, even against saturating ATP concentrations. Analog incorporation induces only a brief pause in nascent RNA synthesis. When embedded in the template strand, CMX521 causes the polymerase to stall [~]9 s on average due to impaired uridine opposite incorporation. Multiple CMX521 residues in the template strand completely inhibit polymerase elongation. When the coronavirus polymerase is associated with the viral helicase, CMX521 strongly promotes copy-back RNA synthesis suggesting a second inhibitory mechanism for CMX521. Collectively, our findings establish a two-pronged mode of coronavirus polymerase inhibition by CMX521.

microbiology↗

bioRxiv2025

Novel Computational Pipeline to Identify Target Sites for Broad Spectrum Antiviral Drugs

Emerging viruses pose an ongoing threat to human health. While certain viral families are common sources of outbreaks, predicting the specific virus within a family that will cause the next outbreak or pandemic is not possible, creating an urgent need for broad spectrum antiviral drugs that are effective against an array of related viral pathogens. However, broad spectrum drug development is hindered by the lack of detailed knowledge of compound binding sites that are structurally and functionally conserved between viral family members and are essential for virus replication. To overcome this limitation, we developed an in silico approach that combines AI-driven protein structure prediction, computational fragment soaking, multiple sequence alignment, and protein stability calculations to identify highly conserved target sites that are both solvent-accessible and conserved. We applied this approach to the Togaviridae family, which includes emerging pandemic disease threats such as chikungunya and Venezuelan equine encephalitis virus for which there are currently no approved antiviral therapies. Our analysis identified multiple solvent accessible and structurally conserved pockets in the alphavirus non-structural protein 2 (nsP2) protease domain, which is essential for processing of the viral replicase proteins. Mutagenesis of key solvent accessible and conserved residues identified novel pockets that are essential for protease activity and the replication of multiple alphaviruses, validating these pockets as potential antiviral target sites for nsP2 inhibitors. These findings highlight the potential of artificial intelligence-informed modeling for revealing functionally conserved, accessible pockets as a means of identifying potential target binding sites for broadly active direct acting antivirals. Significance StatementHere we present a novel integrative computational approach to identify novel target sites for broadly acting antiviral drugs. We used this technique to identify multiple functionally and structurally conserved protein surface pockets within the alphavirus nsP2 protease and methyl-transferase-like domain. Mutagenesis of these pockets identified that they are essential for protease activity and replication of a genetically diverse group of alphaviruses, validating these sites as potential targets for broadly active small molecule alphavirus inhibitors. This integrative AI-driven approach thus provides an important tool in developing antivirals essential for pandemic preparedness.

microbiology↗

bioRxiv2025

The Host DHX29 RNA Helicase Regulates HCMV Immediate Early Protein Synthesis

The dead box helicase DHX29 plays a critical role in the translation of mRNAs containing complex RNA secondary structure in their 5 untranslated regions. The human cytomegalovirus (HCMV) genome has a high GC content, suggesting the 5UTRs of viral mRNAs may contain significant secondary structure and require DHX29 for their efficient translation initiation. We found that depleting DHX29 from primary human fibroblasts prior to infection reduced viral mRNA and protein levels and decreased HCMV replication. The defect in HCMV replication correlated with decreased expression of the HCMV immediate early proteins IE1 and IE2, which are necessary for the establishment of lytic infection. Analysis of polysome associated mRNAs revealed that the defect in IE1 and IE2 expression is due to decreased mRNA translation efficiency. We found that DHX29 depletion led to reduced levels of the eIF4F translation initiation complex, resulting from decreased translation of the eIF4G mRNA. However, in line with our previous results showing a minimal role for the eIF4F complex in HCMV mRNA translation, we found that depleting eIF4G prior to infection did not impact IE1 and IE2 translation. Together our results define a new role for DHX29 in regulating eIF4F-dependent translation and identify a critical role for DHX29 in the translation of HCMV mRNAs. SignificanceExpression of the HCMV immediate early proteins IE1 and IE2 is critical for the establishment of lytic replication and the reactivation of latent HCMV infections. Defining the mechanisms controlling HCMV IE1 and IE2 protein expression has the potential to identify new strategies for therapeutic interventions that can limit HCMV disease in immune naive and immune compromised individuals. Our finding that the cellular DHX29 helicase is necessary for the efficient translation of mRNAs encoding IE1 and IE2 suggests that therapies that inhibit DHX29 could potentially be useful in treating HCMV disease and adds to the growing body of literature suggesting DHX29 activity is a disease driver in multiple indications including viral disease, inflammation and cancer.

microbiology↗

bioRxiv2023

LETHAL COVID-19 ASSOCIATES WITH RAAS-INDUCED INFLAMMATION FOR MULTIPLE ORGAN DAMAGE INCLUDING MEDIASTINAL LYMPH NODES

Lethal COVID-19 outcomes are most often attributed to classic cytokine storm and attendant excessive immune signaling. We re-visit this question using RNA sequencing in nasopharyngeal and 40 autopsy samples from COVID-19-positive and negative individuals. In nasal swabs, the top 100 genes which significantly correlated with COVID-19 viral load, include many canonical innate immune genes. However, 22 much less studied "non-canonical" genes are found and despite the absence of viral transcripts, subsets of these are upregulated in heart, lung, kidney, and liver, but not mediastinal lymph nodes. An important regulatory potential emerges for the non-canonical genes for over-activating the renin-angiotensin-activation-system (RAAS) pathway, resembling this phenomenon in hereditary angioedema (HAE) and its overlapping multiple features with lethal COVID-19 infections. Specifically, RAAS overactivation links increased fibrin deposition, leaky vessels, thrombotic tendency, and initiating the PANoptosis death pathway, as suggested in heart, lung, and especially mediastinal lymph nodes, with a tightly associated mitochondrial dysfunction linked to immune responses. For mediastinal lymph nodes, immunohistochemistry studies validate the transcriptomic findings showing abnormal architecture, excess fibrin and collagen deposition, and pathogenic fibroblasts. Further, our findings overlap findings in SARS-CoV-2 infected hamsters, C57BL/6 and BALB/c mouse models, and importantly peripheral blood mononuclear cell (PBMC) and whole blood samples from COVID-19 patients infected with early variants and later SARS-CoV-2 strains. We thus present cytokine storm in lethal COVID-19 disease as an interplay between upstream immune gene signaling producing downstream RAAS overactivation with resultant severe organ damage, especially compromising mediastinal lymph node function.

immunology↗

bioRxiv2022

The AKT Forkhead box O transcription factor axis regulates human cytomegalovirus replication

The serine/threonine protein kinase AKT is a critical mediator of growth factor signaling that broadly impacts mRNA translation, metabolism, programmed cell death and stress responses. Human cytomegalovirus (HCMV), a large dsDNA virus in the herpesvirus family, dramatically remodels host cell signaling pathways during lytic infection. Here, we show that AKT accumulates in an inactive form during HCMV infection, as indicated by hypo-phosphorylation at Thr308 and Ser473, as well as nuclear localization of FOXO3a. Moreover, we find that expression of constitutively active myristoylated AKT (myr-AKT) causes a 1-log defect in viral replication, accompanied by defects in viral DNA synthesis and viral late gene expression. These findings suggest that HCMV shutoff of AKT is not merely incidental to viral factors such as pUL38 that maintain high levels of mTORC1 activity independently of extracellular growth factor signals. Short interfering RNA knockdown of FOXO3a, but not FOXO1, phenocopied the replication defects seen during expression of myr-AKT, corroborating a role for FOXO3a during HCMV infection. Accordingly, a chimeric FOXO3a-estrogen receptor fusion protein, whose nuclear localization is regulated by the small molecule ligand 4-hydroxytamoxifen instead of AKT activity, rescues virtually all the replication defects induced by myr-AKT expression. Taken together, our results argue that HCMV dampens AKT signaling to promote nuclear localization of FOXO transcription factors, which are required for efficient viral replication. IMPORTANCEEvidence from a diverse herpesvirus infection models suggests that the PI3K / AKT signaling pathway suppresses reactivation from latency, and that inactivation of the pathway stimulates viral lytic replication. Here, we show that AKT accumulates in an inactive state during HCMV infection of lytically permissive cells while the presence of constitutive AKT activity causes substantial viral replication defects. Although AKT phosphorylates a diverse array of cellular substrates, we identify an important role for the Forkhead box class O transcription factors. Our findings show that when FoxO3a nuclear localization is decoupled from its negative regulation by AKT, the viral replication defects observed in the presence of constitutively active AKT are reversed. Collectively, our results reveal that HCMV inactivates AKT to promote the nuclear localization of FOXO transcription factors, which strongly implies that FOXOs play critical roles in transactivating cellular and/or viral genes during infection.

microbiology↗

bioRxiv2018

Alternative Promoters Drive Human Cytomegalovirus Reactivation from Latency

Reactivation from latency requires reinitiation of viral gene expression and culminates in the production of infectious progeny. The major immediate early promoter (MIEP) of human cytomegalovirus (HCMV) drives the expression of crucial lytic cycle transactivators but is silenced during latency in hematopoietic progenitor cells (HPCs). Because the MIEP is poorly active in HPCs, it is unclear how viral transactivators are expressed during reactivation. We demonstrate that transcripts originating from alternative promoters within the canonical major immediate early locus are abundantly expressed upon reactivation, whereas MIEP-derived transcripts remain undetectable. Further, we show that these promoters are necessary for efficient reactivation in primary CD34+ HPCs. Our findings change the paradigm for HCMV reactivation by demonstrating that promoter switching governs reactivation from viral latency in a context-specific manner.

microbiology↗