Sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) restricts a broad spectrum of viruses through multifaceted mechanisms. It also limits spontaneous- and virus-induced innate immune responses by suppressing proinflammatory cytokine and type-I interferon (IFN-I) production. Some viruses escape SAMHD1 restriction and utilize SAMHD1-mediated innate immune suppression to establish effective infection through IFN antagonism. Our previous studies showed that SAMHD1 is a proviral factor facilitating replication of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in human macrophages, monocytic THP-1 and epithelial-like HEK293T cell lines by suppressing IFN responses. However, it is unclear about the function of SAMHD1 in lung epithelial cells during SARS-CoV-2 infection. Here, we report that SAMHD1 knockout (KO) restricts SARS-CoV-2 replication in lung epithelial Calu-3 cells by suppressing endogenous expression of the viral receptor angiotensin-converting enzyme 2 (ACE2) via hepatocyte nuclear factor 1-alpha (HNF1α) and HNF1β. Using pseudotyped SARS-CoV-2 and lentiviral vectors, we found that SARS-CoV-2 spike protein-mediated viral entry was suppressed in SAMHD1 KO Calu-3 cells. SAMHD1 KO repressed ACE2 expression in Calu-3 cells at mRNA and protein levels. Functional analyses revealed that HNF1α and HNF1β were crucial for the endogenous ACE2 expression in Calu-3 cells. Additionally, SAMHD1 KO led to a reduction in the expression levels and ACE2-promoting function of HNF1α and HNF1β. Inhibition of IFN antiviral response by baricitinib, a Janus kinase 1 and 2 (JAK 1/2) inhibitor, did not revert the suppression of SARS-CoV-2 in SAMHD1 KO Calu-3 cells. SAMHD1 knock-in and deoxynucleoside supplementation experiments indicated that SAMHD1 expression and dNTP pool balance collectively regulated HNF1-mediated ACE2 expression in Calu-3 cells. Our findings demonstrate that SAMHD1 depletion hinders HNF1-mediated ACE2 expression and SARS-CoV-2 replication in Calu-3 cells via a novel mechanism beyond its IFN-suppressive function.

Platinum agents are cornerstone therapies for many cancers but often cause neurotoxicity in post-mitotic tissues, for which effective interventions are lacking. This limitation reflects an incomplete understanding of neuronal responses to DNA damage. We show that nucleotide excision repair (NER) mediates cisplatin lesion removal in neurons; however, unlike its protective role in dividing cells, NER promotes neuronal death in response to cisplatin. This vulnerability arises because neurons possess low deoxynucleoside triphosphate (dNTP) pools. dNTPs are initially consumed during transcription-coupled NER to resolve transcription-blocking lesions. As dNTP levels become depleted, repair fails to complete, leading to accumulation of double-strand breaks, particularly during global-genome NER. Supplementation with deoxynucleosides or genetic upregulation of dNTP synthesis restores nucleotide pools, protects neurons from cell death, and reduces cisplatin-induced neuropathic pain. These findings identify limited dNTP availability as a key vulnerability in post-mitotic cells and suggest nucleoside supplementation as a potential strategy to mitigate chemotherapy-induced neurotoxicity.

Respiratory syncytial virus (RSV) is a major global cause of severe lower respiratory tract infections in infants and older adults. RSV has 2 subgroups, A (RSV-A) and B (RSV-B), which circulate together with different patterns of dominance. A vaccine must protect against both. Reccently, prefusion F protein-based vaccines for maternal and older adult populations achieved ∼70% efficacy, and Moderna's mRNA vaccine for older adults further underscores the potential of mRNA platforms. Here, we report preclinical evaluation of VER-027, a novel mRNA vaccine encoding prefusion F proteins from both RSV-A and RSV-B. A 2-dose intramuscular regimen induced high pre-F-specific immunoglobulin G (IgG) titers, potent neutralizing activity against both subgroups, and robust F85-93-specific CD8+ T cell responses. All mice vaccinated with this mRNA vaccine were fully protected against RSV-A and RSV-B challenge. These findings support VER-027 as a strong candidate for clinical development as a dual-subgroup RSV vaccine.

Cellular metabolism supports all viral replication steps and the metabolic state of infected cells is therefore a key factor influencing viral infections. Human Immunodeficiency virus (HIV) remains latent in resting CD4 T lymphocytes but actively replicates in activated CD4 T cells due to enhanced energy metabolism. Here, using the recently developed Human Plasma-Like Medium (HPLM) that mimics physiological plasma concentration of metabolites, we investigated how this near-physiologic environment modulates HIV-1 infection in primary CD4 T cells. Compared to the conventional culture medium (RPMI), HPLM enhanced HIV-1 infection in CD4 T cells despite similar levels of cell activation, proliferation and expression of viral receptor. In contrast with previous studies in RPMI, HPLM increased infection while decreasing energy metabolism and affecting other non-energetic metabolic pathways. Adjusting levels of several metabolites in RPMI and HPLM, we uncovered that the amino acids balance rather than the energy metabolism favoured HIV-1 replication in this system. Overall, our study used near-physiological conditions to better define metabolic dependencies of viral infections and highlights previously overlooked non-energetic metabolism pathways important for HIV-1 infection.

Molnupiravir, a prodrug of β-D-N4-hydroxycytidine (NHC), is an antiviral RNA mutagen that is incorporated by viral RNA-dependent RNA polymerases (RdRp) during replication of viral RNA genomes, ultimately driving target viruses such as SARS-CoV-2 toward lethal mutagenesis. In this study, first, we biochemically tested whether DNA-dependent RNA polymerases (DdRps) including T7 RNA polymerase and host RNA polymerase II, can also incorporate NHC-triphosphate (NHC-TP) during RNA synthesis from double-stranded DNA (dsDNA) templates. In vitro transcription (IVT) by T7 RNA polymerase was evaluated under two conditions: (1) all four natural ribonucleoside triphosphates (rNTPs) and (2) three natural rNTPs (ATP, GTP, and UTP) supplemented with NHC-TP. Full-length RNA products were generated in both conditions, indicating that T7 RNA polymerase incorporates NHC-TP during DNA-dependent RNA synthesis. Second, these IVT-derived RNA products were subsequently reverse-transcribed into single-stranded DNA (ssDNA) using HIV-1 reverse transcriptase (RT). Comparable ssDNA yields were also obtained from both RNA templates, suggesting that NHC-monophosphates embedded in RNA template do not affect the RNA-dependent DNA polymerase (RdDp) activity of HIV-1 RT under the experimental conditions tested. Third, next-generation sequencing (NGS) analysis of the reverse-transcribed products revealed the expected NHC-mediated C to T transition mutations, confirming the mutagenic impact of NHC during the HIV-1 RT-mediated RdDp reactions. Finally, incorporation of NHC-TP by human RNA polymerase II was further confirmed using IVT reactions performed with HeLa cell nuclear extracts. Overall, these biochemical investigations establish both the capacity of DdRps to incorporate NHC-TP and the characteristic mutagenic signature induced by NHC during HIV-1 RT-mediated RNA-dependent DNA synthesis.

Ribonucleoside monophosphates (rNMPs) are the most abundant non-canonical nucleotides in DNA, yet their distribution and function in the human nuclear genome remain unclear. We present high-resolution maps of ∼1 million rNMPs per genome across diverse human cells, revealing a non-random nuclear "ribome," the genome-wide landscape of embedded rNMPs, enriched in GC-rich regions, regulatory elements, and telomeres. Ribonucleotide-enriched zones (REZs) cluster near transcription start sites (TSSs), coincide with C-phosphate-G (CpG) islands, R-loops, and G4 structures, and scale with gene expression. Ribonuclease (RNase) H2 deficiency increases rGMP levels and is associated with topoisomerase 1 (Top1)-dependent, strand-biased rNMP enrichment near TSSs, while Top1 depletion further enhances rGMP accumulation. RNase H2-mediated nicking at rNMPs alters DNA supercoiling in vitro, and RNH2A-deficient cells show altered supercoiling at rNMP-enriched TSSs. Our findings identify embedded rNMPs as epigenetic modulators of DNA supercoiling linked to DNA sequence and transcription, revealing a connection between ribonucleotide processing and transcription-associated DNA topology in human cells.

p4E-BP1 protein levels upon MET inhibition: Whole-cell lysates were subjected to Western blotting using a specific antibody against p4E-BP1 following the treatment either with vehicle or 50nM tepotinib (EMD1214063, shortly EMD) for either 4 or 24h as indicated. Western blots representative of N=3 independent experiments are shown.

Influenza viruses cause mild to severe lower respiratory infections, sometimes resulting in hospitalization and death. Vaccination remains the primary prophylactic strategy. Live attenuated influenza vaccines (LAIVs) efficiently induce antiviral immune responses and contain temperature-sensitive and cold-adapted mutations that render them safe. These mutations are principally located in the PB1 and PB2 subunits of the viral RNA polymerase, but the mechanism by which they attenuate the virus is unclear. We introduced the PB1 and PB2 mutations from two LAIV backbones, A/Ann Arbor/6/1960 H2N2 (AA) and A/Leningrad/134/17/1957 H2N2 (Len), into the model influenza strain A/Puerto Rico/8/1934 H1N1 (PR8). In contrast to the wild-type (WT) PR8 polymerase, the two “PR8-LAIV” polymerase complexes demonstrated maximal activity at cold temperatures (30–32 °C) and greatly reduced activity at elevated temperatures (>37 °C). To further understand the impact of the LAIV mutations, we infected MDCK cells with WT and mutated PR8 viruses that contain the Len and AA LAIV mutations in PB1 and PB2. The PR8-LAIV mutant viruses exhibited a selective, temperature-dependent defect in the replicase activity of the viral RNA polymerase relative to WT PR8, while also demonstrating a temperature-dependent enhancement in the transcriptional activity of the enzyme. In addition, the PR8-LAIV mutant viruses produced similar levels of viral proteins to WT PR8 at 37 °C, but greatly (2–3 log10) reduced levels of infectious viral progeny. Collectively, these data show that LAIV mutations selectively alter influenza viral RNA polymerase function, favoring transcription over genome synthesis at 37 °C, thereby preserving viral antigen production while also contributing to viral attenuation.

The early events of the HIV-1 life cycle, such as reverse transcription, and capsid shedding commonly known as uncoating, are interdependent and tightly regulated, enabling HIV-1 to adapt to diverse host cells. Here, we explored how host cell dNTP pool size modulates the kinetics and dynamics of HIV-1 reverse transcription and uncoating. We optimized an easy-to-use tool to inhibit the ribonucleotide reductase (RNR) catalyzed de novo pathway of dNTP biosynthesis in CHOpgsA-745, HeLa (TZMbl), and owl monkey kidney (OMK) cells. RNR inhibitor rapidly reduced the cellular dNTP pool size, thereby restricting HIV-1 infectivity in a dose-dependent manner. This restriction was reversible upon inhibitor removal, and nucleoside supplementation partially restored infection by enhancing salvage pathways. We find that RNR inhibition slows reverse transcription kinetics and delays the initiation of uncoating in both the capsid integrity and TRIM-CypA restriction assays. Besides, the depletion of intracellular dNTP pools by RNR inhibition leads to significant reductions in both early and late HIV-1 reverse transcription products, with late-stage inhibition comparable to that observed with Nevirapine treatment. To demonstrate the impact of RNR inhibitors on capsid shedding, rather than an off-target effect, we resumed the RNR inhibition-induced delayed initiation of uncoating by reintroducing external dNTPs. This induced recommencement of rapid core integrity loss demonstrating its interplay with the progression of reverse transcription. Therefore, by inhibiting the RNR-catalyzed de novo pathway of dNTP biosynthesis, we have reduced the dNTP pool of the host cells to an extent that delays the kinetics and dynamics of HIV-1 early life events.IMPORTANCECellular dNTP pool homeostasis is maintained by the interplay between the biosynthetic (de novo and salvage) pathways and hydrolyzing networks such as SAMHD1. Inhibiting de novo pathway using RNR inhibitors reduces the host cell dNTP pool size, thereby restricting HIV-1 infectivity reversibly. Whereas the salvage pathways cannot rescue HIV-1 infectivity to the full extent without the de novo pathway. This work correlates HIV-1 infectivity with the dynamic nature of dNTP turnover due to RNR small subunit switching between RRM2 & RRM2B and the action of SAMHD1. The observed modulation of HIV-1 reverse transcription and uncoating in response to RNR inhibition demonstrates the flexibility and adaptability of the virus to replicate in hostile internal cellular environments, which attempt to starve the virus of essential metabolites such as dNTPs. These findings provide insights into how RNR inhibition may impact subsequent steps, such as nuclear localization and integration, offering a foundation for future studies.

Differential abundance analysis and ion annotation matches for non-targeted metabolomics measurements.

Supplementations with nucleosides and hypoxanthine: (A) Proliferation of GTL-16 and EBC-1 cells upon supplementation with nucleosides or hypoxanthine. (B) Apoptosis (caspase-3 activation) in GTL-16 and EBC-1 upon supplementation with nucleosides, hypoxanthine or their combination.

Differential expression analysis of the GTL-16 and EBC-1 transcriptomics data.

Summary of the significance analysis for the survival analyses.

GART protein expression upon MAPK and PI3K targeting: GART protein levels in untreated GTL-16 and EBC-1 cells and after MAPK (AZD6244) and PI3K (LY294002) pathways inhibition. ß Actin was used as a loading control.

GART and E2F1 mRNA levels following METi combined with IR: mRNA levels of GART (left) and E2F1 (right) following METi (50nM, 24 hr), IR (10 Gy, 1 hr) and their combination in GTL-16 and EBC-1 cells.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts a board spectrum of viruses through multifaceted mechanisms. It also limits spontaneous- and virus-induced innate immune responses by suppressing proinflammatory cytokine and type-I interferon (IFN-I) production. Some viruses escape SAMHD1 restriction by utilizing SAMHD1-mediated innate immune suppression to establish effective infection through viral antagonism. Our previous studies showed that SAMHD1 is a proviral factor facilitating replication of severe acute respiratory syndrome coronavirus (SARS-CoV-2) in human macrophages, monocytic THP-1 and epithelial-like HEK293T cell lines by suppressing IFN responses. However, it is unclear about the function of SAMHD1 in lung epithelial cells during SARS-CoV-2 infection. Here, we report that SAMHD1 facilitates SARS-CoV-2 replication in lung epithelial Calu-3 cells by enhancing endogenous expression of the viral receptor angiotensin-converting enzyme 2 (ACE2) via hepatocyte nuclear factor 1-alpha (HNF1α) and HNF1β. Using pseudotyped SARS-CoV-2 and lentiviral vectors, we found that SARS-CoV-2 spike protein-mediated viral entry was suppressed in Calu-3 cells with SAMHD1 knockout (KO). SAMHD1 KO repressed ACE2 expression in Calu-3 cells at mRNA and protein levels. Functional analyses revealed that HNF1α and HNF1β were crucial for the endogenous ACE2 expression in Calu-3 cells. Additionally, SAMHD1 KO led to a reduction in the expression levels and ACE2-promoting function of HNF1α and HNF1β. Inhibition of IFN antiviral response by baricitinib, a Janus kinase 1 and 2 (JAK 1/2) inhibitor, did not revert the suppression of SARS-CoV-2 in SAMHD1 KO Calu-3 cells. Our findings demonstrate that SAMHD1 facilitates HNF1-mediated ACE2 expression and SARS-CoV-2 replication in Calu-3 cells via a novel mechanism beyond its IFN-suppressive function.

Ribonucleoside monophosphates (rNMPs) are abundant in DNA, but their distribution and function in human nuclear genomes remain unknown. Here, we mapped nearly one million rNMPs per genome across diverse human cell types, defining a nuclear “ ribome ” with non-random distribution patterns. rNMPs are enriched in C/G-rich sequences, epigenetically marked regions, and telomeres. Conserved ribonucleotide-enriched zones (REZs) overlap with CpG islands and R-loops. rNMP concentration near transcription start sites (TSSs) correlates positively with gene expression. Wild-type cells display a broader gene-expression range than ribonuclease H2A (RNH2A) knockouts, in which loss of rNMP cleavage causes pronounced retention of embedded rG and strand-biased rC near TSSs, both increasing with gene expression. These findings establish DNA-embedded rNMPs as a novel epigenetic mark that modulates human gene expression.

Supplementations with glutamine, serine, and folic acid: (A) Proliferation of GTL-16 and EBC-1 cells upon supplementation with glutamine, serine or folic acid. (B) Apoptosis (caspase-3 activation) in GTL-16 and EBC-1 cells upon supplementation with glutamine, serine or folic acid.