Biology subjects

Lopez-Ayllon, B. D.

Publications and source records attributed to Lopez-Ayllon, B. D..

5 recordsLinked to original sources

bioRxiv2026

A single Omicron mutation reshapes ORF3a-driven host-cell remodelling

SARS-CoV-2 ORF3a remodels host membranes, but the structural basis and metabolic consequences of this process remain unclear. Here, we combine complementary imaging approaches to define ORF3a function at nanometric scale, identifying underlying mechanisms, and determining how Omicron variant rewire this activity. ORF3a from the ancestral Wuhan strain disrupts Golgi cisternae, drives the formation of ORF3a dense vesicles, remodels mitochondrial architecture, and promotes lipid droplet expansion. Multi-omics analyses further reveal selective triacylglycerol accumulation linked to DGAT1 upregulation, which we validate pharmacologically through DGAT1 inhibition. In contrast, Omicron ORF3a variant, despite carrying only the Thr223Ile substitution within the {beta}7-{beta}8 loop at the bottom of the cytosolic domain, induced a dramatic phenotypic shift: ORF3a localizes to multivesicular bodies, preserves Golgi architecture, and fails to induce lipid accumulation. All together, these results identify ORF3a as a regulator of membrane organization and lipid homeostasis, showing how minimal sequence variation rewires host-cell remodelling. Graphical TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/742305v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@135032borg.highwire.dtl.DTLVardef@1633d4borg.highwire.dtl.DTLVardef@4d4115org.highwire.dtl.DTLVardef@1ebdf65_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2026

Omicron-Enhanced Immunosuppressive Effects of SARS-CoV-2 ORF3a and ORF9b Accessory Proteins on Monocytic Inflammatory Response

This study investigates the poorly understood roles of SARS-CoV-2 accessory proteins using monocytic THP-1 cells expressing individual viral ORFs. ORF3a, ORF7b, and ORF9b were identified as major immunomodulators that suppress host inflammatory signaling. Specifically, cells expressing ORF3a or ORF9b exhibited reduced Toll-like receptor 4 (TLR4)-mediated production of key proinflammatory molecules--CCL2, CCL4, and IL-1{beta}--resulting in diminished immune cell recruitment. Importantly, Omicron-associated mutations in ORF3a (T223I) and ORF9b (P10S+{Delta}E27N28A29) amplified this immunosuppressive effect, leading to stronger transcriptomic suppression consistent with Omicrons reduced pathogenicity and clinical outcomes. These findings suggest that SARS-CoV-2 accessory proteins, particularly ORF3a and ORF9b, play pivotal roles in modulating monocytic immune responses. Enhanced suppression in Omicron variants highlights an evolutionary adaptation contributing to immune evasion and milder disease manifestations.

immunology↗

bioRxiv2025

SARS-CoV-2 ORF7a Drives Mitochondrial Dysfunction via PDK4 Activation and Complex I Inhibition

SARS-CoV-2 reprograms host metabolism to promote viral replication and evade immune responses. While infection is known to impair mitochondrial function and enhance glycolysis, the role of viral accessory proteins in these alterations remains unclear. Here, we investigate the metabolic impact of the accessory protein ORF7a. Lentiviral expression of ORF7a in human lung epithelial (A549) and monocytic (THP1) cells, coupled with integrated transcriptomic, proteomic, and metabolomic analyses, revealed profound dysregulation of glucose and lipid metabolism. ORF7a impaired oxidative phosphorylation, reducing basal and maximal respiration, inducing mitochondrial depolarization, and increasing reactive oxygen species. Mechanistically, ORF7a upregulated pyruvate dehydrogenase kinase 4 (PDK4), promoting pyruvate dehydrogenase (PDH) complex phosphorylation and suppressing pyruvate oxidation. However, pharmacological PDK4 inhibition did not restore respiration. High-resolution respirometry in frozen samples revealed impaired complex I function, while Blue Native-PAGE demonstrated defective respiratory supercomplex assembly. By linking enzymatic inhibition with structural destabilization, our study uncovers a functional vulnerability of the mitochondrial respiratory chain to viral manipulation. These findings establish ORF7a as a key modulator of host metabolic reprogramming and highlight mitochondrial pathways as potential therapeutic targets in COVID-19.

immunology↗

bioRxiv2024

Dominant induction of the inflammasome by the SARS-CoV-2 accessory protein ORF9b, abrogated by small-molecule ORF9b homodimerization inhibitors

Viral accessory proteins play critical roles in viral escape form host innate immune responses and in viral inflammatory pathogenesis. Here we show that the SARS-CoV-2 accessory protein, ORF9b, but not other SARS-CoV-2 accessory proteins (ORF3a, ORF3b, ORF6, ORF7, ORF8, ORF9c, ORF10), strongly activates inflammasome-dependent caspase-1 in A549 lung carcinoma cells and THP-1 monocyte-macrophage cells. Exposure to lipopolysaccharide (LPS) and ATP additively enhanced the activation of caspase-1 by ORF9b, suggesting that ORF9b and LPS follow parallel pathways in the activation of the inflammasome and caspase-1. Following rational in silico approaches, we have designed small molecules capable of inhibiting the homodimerization of ORF9b, which experimentally inhibited ORF9b-ORF9b homotypic interactions, caused mitochondrial eviction of ORF9b, inhibited ORF9b-induced activation of caspase-1 in A549 and THP-1 cells, cytokine release in THP-1 cells, and restored type I interferon (IFN-I) signaling suppressed by ORF9b in both cell models. These small molecules are first-in-class compounds targeting a viral accessory protein critical for viral-induced exacerbated inflammation and escape from innate immune responses, with the potential of mitigating the severe immunopathogenic damage induced by highly pathogenic coronaviruses and restoring antiviral innate immune responses curtailed by viral infection.

microbiology↗

bioRxiv2023

Metabolic and mitochondria alterations induced by SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10

Antiviral signaling, immune response and cell metabolism in human body are dysregulated by SARS-CoV-2, the causative agent of the COVID-19. Here, we show that SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10 induce a significant mitochondrial and metabolic reprogramming in A549 lung epithelial cells. While all four ORFs caused mitochondrial fragmentation and altered mitochondrial function, only ORF3a and ORF9c induced a marked structural alteration in mitochondrial cristae. ORF9b, ORF9c and ORF10 induced largely overlapping transcriptomes. In contrast, ORF3a induced a distinct transcriptome, including the downregulation of numerous genes for proteins with critical mitochondrial functions and morphology. Genome-Scale Metabolic Models predicted common and private metabolic flux reprogramming, notably a depressed amino acid metabolism, and an enhanced metabolism of specific lipids distinctly induced by ORF3a. These findings reveal metabolic dependencies and vulnerabilities prompted by SARS-CoV-2 accessory proteins that may be exploited to identify new targets for intervention. One-Sentence SummaryMitochondria and metabolic alterations induced by SARS- CoV-2 accessory proteins ORF3a, ORF9b, ORF9c, ORF10 in pulmonary cells unravel new targets of intervention.

immunology↗