Biology subjects

Connolly, S. A.

Publications and source records attributed to Connolly, S. A..

7 recordsLinked to original sources

bioRxiv2026

Glycoprotein C Mutations Regulate the Plaque Size of Fusion-Defective Herpes Simplex Virus

Herpes simplex virus 1 (HSV-1) entry and virus-induced cell fusion require the coordinated activity of the essential glycoproteins gD, gH/gL, and gB. Mutations in this core fusion machinery reduce viral entry, fusion, and spread. Previously, we showed that introducing three mutations in gB (gB3A) or replacing HSV-1 gB with saimiriine herpesvirus 1 (SaHV-1) gB resulted in fusion-defective, small-plaque viruses. Serial passage of these viruses selected mutations in gH and gC, and we demonstrated that the gH mutations partially restored fusion function. In this study, we examined whether mutations in gC contribute to plaque formation, fusion, and entry. We generated an additional fusion-defective, small-plaque gD chimeric virus by replacing the profusion domain of HSV-1 gD with the corresponding region from SaHV-1 gD (gDchi). Serial passage of this virus partially restored plaque size and also selected for a mutation in gC. This study demonstrates that the selected gC mutants contributed to the partial restoration of plaque size. Plaque size of all three fusion-defective viruses was enhanced by exogenous expression of the corresponding selected gC mutants, whereas exogenous expression of wild-type gC reduced plaque size of the passaged isolates carrying gC mutations. Although gC mutants did not enhance fusion or entry mediated by the wild-type core fusion machinery, both wild-type and mutant gC enhanced cell-cell fusion when coexpressed with fusion-defective gH mutants. These results indicate that gC functions as a conditional accessory regulator that can partially compensate for defects in the HSV-1 core fusion machinery. IMPORTANCEHerpes simplex virus 1 (HSV-1) entry is mediated by a core fusion machinery comprised of glycoproteins gD, gH/gL, and gB. The contributions of additional viral glycoproteins to this process are not well defined. Glycoprotein C (gC) is known to function in cell attachment to heparan sulfate and immune evasion but is dispensable for viral entry under standard conditions. Here, we show that gC can act as a conditional accessory regulator of HSV-1 entry and fusion. Using serial passage of fusion-defective viruses, we demonstrate that mutations in gC are repeatedly selected and can partially compensate for defects in entry and fusion. These findings indicate that gC contributes to viral fusion under suboptimal conditions and highlight a role for this accessory glycoprotein in the adaptability of herpesvirus entry.

microbiology↗

bioRxiv2026

Daily HIV pre-exposure prophylaxis enhances monocyte activation and effector function and reprogrammes cellular metabolism.

BackgroundPre-exposure prophylaxis (PrEP) with tenofovir/emtricitabine (TDF/FTC) is highly effective for HIV prevention. While antiretroviral therapy (ART) is linked to chronic inflammation in people living with HIV, its direct effects on immune phenotype, function, and metabolism in HIV-negative individuals remain unclear. MethodsGay, bisexual, and other men who have sex with men (gbMSM) on daily TDF/FTC PrEP underwent immunophenotyping and single-cell metabolic profiling using SCENITH. Cytokine and chemokine responses were measured ex vivo and after lipopolysaccharide or Mycobacterium tuberculosis stimulation. These were compared with a demographically similar PrEP-naive cohort, with five individuals followed 6-9 months after PrEP initiation. FindingsMonocytes from people taking PrEP (n=15; median 533 days) exhibited higher activation marker expression (HLA-DR, CD14) ex vivo and enhanced IL-1{beta} and TNF after bacterial challenge compared with PrEP-naive individuals (n=11). Longitudinal follow-up indicated that PrEP initiation increased monocyte activation markers (HLA-DR, CD14, CD40, TNFRI/II) and cytokine production (IL-1{beta}, TNF, GM-CSF, IFN-{gamma}, Granzyme B, MIP-1). Reduced glucose dependency was observed in monocytes, CD56dim NK cells and CD4+ T cells 6-9 months after PrEP initiation. InterpretationDaily TDF/FTC promotes monocyte activation, enhances pro-inflammatory responses, and reprogrammes immune cell metabolism, highlighting ARTs potential to modulate immune-mediated inflammatory pathways in HIV-negative individuals. FundingTrinity Translational Medicine Institute, Trinity College Dublin, Dublin, The Association of Physicians of Great Britain and Ireland, Health Research Board Ireland. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/698427v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@d48088org.highwire.dtl.DTLVardef@1428926org.highwire.dtl.DTLVardef@13a4facorg.highwire.dtl.DTLVardef@144d1f1_HPS_FORMAT_FIGEXP M_FIG C_FIG Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSDespite effective viral suppression with antiretroviral therapy (ART), people living with HIV (PLWHIV) experience persistent immune activation and chronic inflammation that increase their risk of age-associated comorbidities. The extent to which ART itself contributes to this dysregulated immune state remains unclear, as HIV infection confounds most studies. Pre-exposure prophylaxis (PrEP) with tenofovir disoproxil fumarate and emtricitabine (TDF/FTC) offers a unique opportunity to examine the direct immunological effects of ART in HIV-negative individuals. However, few studies have explored how PrEP influences immune cell activation, effector function, or cellular metabolism in the absence of viral infection. Added value of this studyThis study is the first to characterise how daily PrEP with TDF/FTC modulates innate immune activation and cellular metabolism independently of HIV infection. We show that PrEP enhances monocyte activation and effector cytokine production while reprogramming metabolic pathways to reduce glucose dependency in monocytes, NK cells, and CD4 T cells. These findings reveal previously unrecognised immunometabolic effects of ART in HIV-negative individuals, providing mechanistic insight into how ART exposure can shape immune homeostasis in the absence of infection. Implications of all the available evidenceOur findings, together with existing data from PLWHIV, suggest that ART itself can remodel immune and metabolic pathways, potentially contributing to both beneficial and detrimental outcomes. ART-induced immunometabolic reprogramming may underlie persistent inflammation and the early onset of comorbidities observed in treated HIV infection, while also enhancing innate responsiveness to bacterial pathogens such as Mycobacterium tuberculosis. Understanding these mechanisms may inform future strategies to optimise ART regimens and mitigate inflammation-associated complications.

immunology↗

bioRxiv2025

Species-Specific gB Ectodomain Interactions and Cytoplasmic Domain Stability Regulate Herpes Simplex Virus Fusion

Entry of herpesviruses into cells requires coordinated action of multiple viral glycoproteins, including gH/gL and gB which comprise the core fusion machinery conserved in herpesviruses. The gH/gL heterodimer activates the gB fusion protein, triggering its refolding from a prefusion to a postfusion form to drive membrane merger. The cytoplasmic tail domain (CTD) of gB is proposed to act as an inhibitory clamp that stabilizes the prefusion state, with interactions between gH and gB CTDs destabilizing this clamp. We previously found that herpes simplex virus 1 (HSV-1) and saimiriine herpesvirus 1 (SaHV-1) gB homologs are functionally interchangeable but mediate reduced fusion when coexpressed with heterotypic gH/gL. To map the regions of gB responsible for species-specific interactions, we generated HSV-1/SaHV-1 gB chimeras by swapping ectodomain, membrane-proximal region (MPR), transmembrane domain (TMD), and CTD segments. Our results show that homotypic CTD interactions alone are insufficient to trigger fusion, suggesting that gH/gL contacts the ectodomain of gB. We show that the HSV-1 gB CTD is hyperfusogenic relative to the SaHV-1 CTD, whereas the HSV-1 MPR is hypofusogenic relative to the SaHV-1 MPR. Together, these findings suggest that functional interaction between gH/gL-gB occur on both sides of the membrane and that gB maintains a balance between promotion and restraint of fusion through coordinated contributions of its domains.

microbiology↗

bioRxiv2025

Immunometabolic Reprogramming of Monocytes in Tuberculosis Infection and Disease

RationaleMonocytes are central to host defence against Mycobacterium tuberculosis (Mtb), yet their functional and metabolic profiles during latent TB infection (TBI) and active TB disease (TBD) remain poorly defined. Immunometabolic dysfunction may underlie ineffective responses in TB, but cell-specific mechanisms are unclear. ObjectivesTo compare the phenotypic, functional, and metabolic profiles of circulating monocytes from individuals with TBI, TBD, and healthy controls (HC), and assess the impact of treatment. MeasurementsPeripheral blood monocytes were profiled using high-dimensional flow cytometry, Luminex cytokine/chemokine assays, and SCENITH, a flow-based metabolic assay. Unstimulated and Mtb-stimulated monocytes from treatment-naive and treated individuals were analysed. Main ResultsMonocytes from TBI and TBD showed distinct phenotypes from HC, marked by elevated CD14 and CD45RA. HLA-DR was reduced in TBI versus HC and further decreased in TBD. TNF receptors were downregulated in TBI but unchanged in TBD. Baseline cytokine and chemokine profiles in TBI and TBD were similar (yet distinct from HC), but Mtb stimulation elicited a stronger cytokine response in TBI. Metabolically, TBI and TBD monocytes exhibited increased glycolysis and reduced mitochondrial dependence versus HC. Treatment partially restored mitochondrial function. Upon Mtb challenge, TBI monocytes had higher glycolytic capacity than TBD. ConclusionsMonocyte metabolic plasticity and cytokine production distinguish latent from active TB and are partially reversible with treatment. Circulating monocyte metabolism reflects TB immune status and may serve as a biomarker or therapeutic target. Reprogrammed glycolytic profiles in TBI contrast with impaired adaptability in TBD, suggesting dysfunctional myeloid activation during disease.

immunology↗

bioRxiv2024

Human airway macrophages are metabolically reprogrammed by IFN-γ resulting in glycolysis dependent functional plasticity.

Airway macrophages (AM) are the predominant immune cell in the lung and play a crucial role in preventing infection, making them a target for host directed therapy. Macrophage effector functions are associated with cellular metabolism. A knowledge gap remains in understanding metabolic reprogramming and functional plasticity of distinct human macrophage subpopulations, especially in lung resident AM. We examined tissue-resident AM and monocyte derived macrophages (MDM; as a model of blood derived macrophages) in their resting state and after priming with IFN-{gamma} or IL-4 to model the Th1/Th2 axis in the lung. Human macrophages, regardless of origin, had a strong induction of glycolysis in response to IFN-{gamma} or upon stimulation. IFN-{gamma} significantly enhanced cellular energetics in both AM and MDM by upregulating both glycolysis and oxidative phosphorylation. Upon stimulation, AM do not decrease oxidative phosphorylation unlike MDM which shift to "Warburg"-like metabolism. IFN-{gamma} priming promoted cytokine secretion in AM. Blocking glycolysis with 2-deoxyglucose significantly reduced IFN-{gamma} driven cytokine production in AM, indicating that IFN-{gamma} induces functional plasticity in human AM, which is mechanistically mediated by glycolysis. Directly comparing responses between macrophages, AM were more responsive to IFN-{gamma} priming and dependent on glycolysis for cytokine secretion than MDM. Interestingly, TNF production was under the control of glycolysis in AM and not in MDM. MDM exhibited glycolysis-dependent upregulation of HLA-DR and CD40, whereas IFN-{gamma} upregulated HLA-DR and CD40 on AM independently of glycolysis. These data indicate that human AM are functionally plastic and respond to IFN-{gamma} in a manner distinct from MDM. These data provide evidence that human AM are a tractable target for inhalable immunomodulatory therapies for respiratory diseases.

immunology↗

bioRxiv2022

Multiple sites on glycoprotein H (gH) functionally interact with the gB fusion protein to promote fusion during herpes simplex virus (HSV) entry

Enveloped virus entry requires fusion of the viral envelope with a host cell membrane. Herpes simplex virus type 1 (HSV-1) entry is mediated by a set of glycoproteins that interact to trigger the viral fusion protein glycoprotein B (gB). In the current model, receptor-binding by gD signals a gH/gL heterodimer to trigger a refolding event in gB that fuses the membranes. To explore functional interactions between gB and gH/gL, we used a bacterial artificial chromosome (BAC) to generate two HSV-1 mutants that show a small plaque phenotype due to changes in gB. We passaged the viruses to select for restoration of plaque size and analyzed second-site mutations that arose in gH. HSV-1 gB was replaced either by gB from saimiriine herpesvirus type 1 (SaHV-1) or by a mutant form of HSV-1 gB with three alanine substitutions in domain V (gB3A). To shift the selective pressure away from gB, the gB3A virus was passaged in cells expressing gB3A. Sequencing of passaged viruses identified two interesting mutations in gH, including gH-H789 in domain IV and gH-S830N in the cytoplasmic tail (CT). Characterization of these gH mutations indicated they are responsible for the enhanced plaque size. Rather than being globally hyperfusogenic, both gH mutations partially rescued function of the specific gB version present during their selection. These sites may represent functional interaction sites on gH/gL for gB. gH-H789 may alter the positioning of a membrane-proximal flap in the gH ectodomain, whereas gH-S830 may contribute to an interaction between the gB and gH CTs. IMPORTANCEEnveloped viruses enter cells by fusing their envelope with the host cell membrane. Herpes simplex virus type 1 (HSV-1) entry requires the coordinated interaction of several viral glycoproteins, including gH/gL and gB. gH/gL and gB are essential for virus replication and both proteins are targets of neutralizing antibodies. gB fuses the membranes after being activated by gH/gL, but the details of how gH/gL activates gB are not known. This study examined the gH/gL-gB interaction using HSV-1 mutants that displayed reduced virus entry due to changes in gB. The mutant viruses were grown over time to select for additional mutations that could partially restore entry. Two mutations in gH (H789Y and S830N) were identified. The positions of the mutations in gH/gL may represent sites that contribute to gB activation during virus entry.

microbiology↗

bioRxiv2018

Natural selection of glycoprotein B (gB) mutations that rescue the small plaque phenotype of a fusion-impaired herpes simplex virus (HSV) mutant

Glycoprotein B (gB) is a conserved viral fusion protein that is required for herpesvirus entry. To mediate fusion with the cellular membrane, gB refolds from a prefusion to a postfusion conformation. We hypothesize that an interaction between the C-terminal arm and the central coiled-coil of the herpes simplex virus 1 (HSV-1) gB ectodomain is critical for fusion. We previously reported that three mutations in the C-terminal arm (I671A/H681A/F683A, called gB3A) greatly reduced cell-cell fusion and that virus carrying these mutations had a small plaque phenotype and delayed entry into cells. By serially passaging gB3A virus, we selected three revertant viruses with larger plaques. These revertant viruses acquired mutations in gB that restore the fusion function of gB3A, including gB-A683V, gB-S383F/G645R/V705I/A855V, and gB-T509M/N709H. V705I and N709H are novel mutations that map to the portion of domain V that enters domain I in the postfusion structure. S383F, G645R, and T509M are novel mutations that map to an intersection of three domains in a prefusion model of gB. We introduced these second-site mutations individually and in combination into wild-type gB and gB3A to examine the impact of the mutations on fusion and expression. V705I and A855V (a known hyperfusogenic mutation) restored the fusion function of gB3A, whereas S383F and G645R dampened fusion and T509M and N709H worked in concert to restore gB3A fusion. The results identify two regions in the gB ectodomain that modulate the fusion activity of gB, potentially by impacting intramolecular interactions and stability of the prefusion and/or postfusion gB trimer.\n\nIMPORTANCEGlycoprotein B (gB) is an essential viral protein that is conserved in all herpesviruses and is required for virus entry. gB is thought to undergo a conformational change that provides the energy to fuse the viral and cellular membranes, however the details of this conformational change and the structure of the prefusion and intermediate conformations of gB are not known. Previously, we demonstrated that mutations in the gB \"arm\" region inhibit fusion and impart a small plaque phenotype. Using serial passage of a virus carrying these mutations, we identified revertants with restored plaque size. The revertant viruses acquired novel mutations in gB that restored fusion function and mapped to two sites in the gB ectodomain. This work supports our hypothesis that an interaction between the gB arm and the core of gB is critical for gB refolding and provides details about the function of gB in herpesvirus mediated fusion and subsequent virus entry.

microbiology↗