Biology subjects

Flamand, L.

Publications and source records attributed to Flamand, L..

11 recordsLinked to original sources

bioRxiv2026

The orphan receptor IL-17RD is a negative regulator of RIG-I-like receptor-dependent antiviral innate immunity and restrains SARS-CoV-2-induced lung inflammation

Detection of viral RNA by the RIG-I-like receptors (RLRs) RIG-I and MDA5 triggers assembly of a MAVS-dependent signalosome that activates the TBK1-IRF3 and IKK{beta}-NF-{kappa}B axes together with the JNK and p38 MAPK modules, driving type I and type III interferon (IFN) and inflammatory cytokine production. Because unrestrained activity of this pathway is a major cause of immunopathology, host-encoded negative regulators are essential, yet the full complement of these brakes remains incompletely defined. Here we identify interleukin-17 receptor D (IL-17RD, also known as SEF), an orphan member of the IL-17 receptor family previously characterized as an antagonist of FGF and Toll-like receptor signaling, as a negative regulator of RLR-driven antiviral innate immunity. Using a CRISPR-engineered and shRNA-depleted human airway epithelial-derived lung carcinoma A549 cell line, we show that loss of IL-17RD amplifies and prolongs phosphorylation of TBK1 and IRF3 in response to poly I:C transfection and to infection with encephalomyocarditis virus (EMCV) or Sendai virus (SeV), and likewise potentiates the IKK{beta}-I{kappa}B module and the TAK1-JNK1/2 and p38 MAPK branches. This translates into increased nuclear accumulation of IRF3 and p65, and markedly elevated induction of IFNB1, IFNL1-3, CCL5, IL6, and NFKBIA transcripts, as well as secreted IFN-{beta} and IL-6. Silencing IL-17RD in ACE2-expressing A549 cells similarly derepresses the antiviral and inflammatory transcriptional programme following SARS-CoV-2 infection. Epistasis experiments place IL-17RD at the level of MAVS, downstream of the RLR sentinels. Mechanistically, IL-17RD localizes to the ER-to-Golgi intermediate compartment (ERGIC), the membrane platform on which the MAVS signalosome is present, and associates with RIG-I, MDA5, MAVS, TBK1 and IRF3. Its re-expression in depleted cells redistributes RLR effectors and TRAF proteins across low-molecular-weight signalosome fractions, reducing the amount of IRF3 recruited to the 670 kDa MAVS signalosome complex. Complementation of IL-17RD-deficient cells also indicates that the intracellular TIR subdomain is sufficient to confer this antagonistic activity. Finally, Il17rd-/- mice display a splenic transcriptome enriched for antiviral response signatures, and, following intranasal infection with a moderate dose of SARS-CoV-2, they mount an exaggerated pulmonary cytokine response and develop significantly greater lung inflammation and fibrosis than wild-type littermates. Together, these data establish IL-17RD as a bona fide brake on the RLR-MAVS axis that limits virus-induced immunopathology, and identify the SEFIR/TIR subdomain as the module responsible for this activity.

microbiology↗

bioRxiv2026

SARS-CoV-2 Nsp2 reprograms host immunity to drive pathogenic inflammation

Despite the end of the COVID-19 pandemic, SARS-CoV-2 continues to circulate endemically, highlighting the need to better understand the viral determinants of pathogenesis. Non-structural protein 2 (Nsp2) has been implicated in host-virus interactions, yet its function remains poorly defined in the context of infection. Here, we generated a recombinant SARS-CoV-2 lacking Nsp2 ({Delta}Nsp2) to investigate its role in viral replication and disease. While {Delta}Nsp2 replicated comparably to wild-type virus in vitro and in vivo, its deletion resulted in markedly attenuated disease in K18-hACE2 mice. Wild-type infection induced a strong pro-inflammatory response associated with increased recruitment of monocytes and macrophages, whereas {Delta}Nsp2 infection promoted a more balanced antiviral response characterized by enhanced lymphocyte and NK cell recruitment. This was accompanied by reduced pulmonary and systemic inflammation and distinct transcriptional programs, including downregulation of pathways related to RNA processing and translation. Mechanistically, CLIP-seq and proximity labeling suggest that Nsp2 interacts with host RNA and components of the translational machinery. Together, our findings identify Nsp2 as a key virulence factor that drives immunopathology by skewing host immune responses, highlighting its role as a regulator of host-pathogen interactions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/723222v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@540e40org.highwire.dtl.DTLVardef@73c663org.highwire.dtl.DTLVardef@e5c535org.highwire.dtl.DTLVardef@f62d7f_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Delayed viral clearance and altered inflammatory responses resulted in increased severity of SARS-CoV-2 infection in aged mice.

Since the onset of the COVID-19 pandemic, advanced age has emerged as a major predictor of disease severity. Epidemiological investigations consistently demonstrate an overrepresentation of the elderly in COVID-19 hospitalizations and fatalities. Despite this, a comprehensive understanding of the molecular mechanisms explaining how old age constitutes a critical risk factor remains elusive. To unravel this, we designed an animal study, juxtaposing the course of COVID-19 in young adults (2 months) and geriatric (15-22 months) mice. Both groups of K18(hACE2) mice were intranasally exposed to 500 TCID50 of the SARS-CoV-2 Delta variant with a variety of outcomes assessed on days 3, 5, and 7 post-infections (DPI). Analyses included pulmonary cytokines, RNA, viral loads, lipidomic profiles, and histological assessments, with a concurrent evaluation of the percentage of mice reaching humane endpoints. The findings unveiled notable distinctions between the two groups, with aged mice exhibiting impaired viral clearance at 7 DPI, correlating with diminished survival rates together with an absence of weight loss recovery at 6-7 DPI. Additionally, elderly-infected mice exhibited a deficient Th1 response characterized by diminished productions of IFNg, CCL2, CCL3, and CXCL9 relative to younger mice. Furthermore, mass-spectrometry analysis of the lung lipidome indicated altered expression of several lipids with immunomodulatory and pro-resolution effects in aged mice such as Resolvin, HOTrEs, and NeuroP, but also DiHOMEs-related ARDS. Collectively, disease severity implies a dysregulation of the antiviral response in elderly-infected mice relative to younger mice, resulting in compromised viral clearance and a more unfavorable prognosis. This underscores the potential efficacy of immunomodulatory treatments for elderly subjects experiencing symptoms of severe COVID-19. Author summaryIn this study, we investigated why older age is linked to more severe COVID-19 outcomes by comparing the progression of the disease in young (2 months) and elderly (15-22 months) K18(hACE2) mice infected with the SARS-CoV-2 Delta variant. After exposing both groups to the virus, we assessed various factors such as viral loads, immune responses, and lipid profiles in the lungs at different time points. Our findings revealed that elderly mice struggled to clear the virus by day 7 post-infection, leading to higher mortality rates and poorer recovery compared to younger mice. Aged mice showed weaker immune responses, with reduced production of key antiviral proteins like IFNg and certain chemokines. Lipid analysis also highlighted differences in molecules involved in immune regulation and lung protection, such as decreased levels of pro-resolving lipids and increased lipids associated with lung injury. These results suggest that older mice have a compromised antiviral defense, which could inform new therapeutic approaches for elderly patients with severe COVID-19.

microbiology↗

bioRxiv2023

Natural evolution of SARS-CoV-2 variants in K18-ACE2 mice gives rise to more virulent virus and variant alleles associated with treatment resistance.

The persistence of COVID-19 is partly due to viral evolution reducing vaccine and treatment efficacy. Serial infections of Wuhan-like SARS-CoV-2 in Balb/c mice yielded mouse-adapted strains with greater infectivity and mortality. We investigated if passaging unmodified B.1.351 (Beta) and B.1.617.2 (Delta) 20 times in K18-ACE2 mice, expressing human ACE2 receptor, in a BSL-3 laboratory without selective pressures, would drive human health-relevant evolution and if evolution was lineage-dependent. Late-passage virus caused more severe disease, at organism and lung tissue scales, with late-passage Delta demonstrating antibody resistance and interferon suppression. This resistance co-occurred with a de novo spike S371F mutation, linked with both traits. S371F, an Omicron-characteristic mutation, was co-inherited at times with spike E1182G per Nanopore sequencing, existing in different quasi-species at others. Both are linked to mammalian GOLGA7 and ZDHHC5 interactions, which mediate viral-cell entry and antiviral response. This study demonstrates SARS-CoV-2s tendency to evolve with phenotypic consequences, its evolution varying by lineage, and suggests non-dominant quasi-species contribute.

genetics↗

bioRxiv2022

SARS-CoV-2 type I Interferon modulation by non structural proteins 1 and 2

Since the beginning of the COVID-19 pandemic, enormous efforts were devoted to understanding how SARS-CoV-2 escapes the antiviral response. Yet, modulation of type I interferons (IFNs) by this virus is not completely understood. Using in vitro and in vivo approaches, we have characterized the type I IFN response during SARS-CoV-2 infection as well as immune evasion mechanisms. The transcriptional and translational expression of IFNs, cytokines and chemokines were measured in lung homogenates of Wuhan-like, Beta, and Delta SARS-CoV-2 K18-ACE2 transgenic mice. Using in vitro experiments, we measured SARS-CoV-2 and its non-structural proteins 1 and 2 (Nsp1-2) to modulate expression of IFN{beta} and interferon-stimulated genes (ISG). Our data show that infection of mice with Wuhan-like virus induces robust expression of Ifna and Ifnb1 mRNA and limited type I production. In contrast, Beta and Delta variant infected mice failed to activate and produce IFN. Using in vitro systems, Ifn{beta} gene translation inhibition was observed using an Nsp1 expression vector. Conversely, SARS-CoV-2 and its variants induce robust expression of NF-{kappa}B-driven genes such as those encoding CCL2 ans CXCL10 chemokines. We also identified Nsp2 as an activator of NF-{kappa}B that partially counteracts the inhibitory actions of Nsp1. In summary, our work indicates that SARS-CoV-2 skews the antiviral response in favor of an NF-{kappa}B-driven inflammatory response, a hallmark of acute COVID-19, and that Nsp2 is partly responsible for this effect. ImportanceSeveral studies suggest that SARS-CoV-2 possess multiple mechanisms aimed shunting the type I interferon response. However, few studies have studied type I IFN modulation in the context of infection. Our work indicates that mice and human cells infected with SARS-CoV-2 produce sufficient type I IFN to activate an antiviral response, despite Nsp1 translational blockade of IFNB1 mRNA. In contrast to Wuhan-like virus, Beta and Delta variants failed to induce Ifna gene expression. Our work also showcases the importance of studying protein functions in the context of infection, as demonstrated by the partial antagonizing properties of the Nsp2 protein on the activities of Nsp1. Our studies also highlight that the innate immune response triggered by SARS-CoV-2 is chiefly driven by NF-{kappa}B responsive genes for which Nsp2 is partially responsible.

microbiology↗

bioRxiv2021

The immediate early protein 1 of the human herpesvirus 6B counteracts NBS1 and prevents homologous recombination repair pathways

Viral infection often trigger an ATM-dependent DNA damage response (DDR) in host cells that suppresses viral replication. To counteract this antiviral surveillance system, viruses evolved different strategies to induce the degradation of the MRE11/RAD50/NBS1 (MRN) complex and prevent subsequent DDR signaling. Here, we report that human herpesvirus 6B (HHV-6B) infection causes genomic instability by suppressing the host cells ability to induce ATM-dependent signaling pathways. Expression of immediate early protein 1 (IE1) phenocopies this phenotype and blocks further homology-directed double-strand break (DSB) repair. In contrast to other viruses, IE1 does not affect the stability of the MRN complex. Instead, it uses two distinct domains to inhibit ATM serine/threonine kinase (ATM) activation at DSBs. Structure-based analyses revealed that the N-terminal domain of IE1 interacts with the BRCA1 C-terminal domain 2 of nibrin (NBN, also known as NBS1), while ATM inhibition is attributable to on its C-terminal domain. Consistent with the role of the MRN complex in antiviral responses, NBS1 depletion resulted in increased HHV-6B replication in infected cells. However, in semi-permissive cells, viral integration of HHV-6B into the telomeres was not strictly dependent on NBS1, supporting models where this process occurs via telomere elongation rather than through DNA repair. Interestingly, as IE1 expression has been detected in cells of subjects with inherited chromosomally-integrated form of HHV-6B (iciHHV-6B), a condition associated with several health conditions, our results raise the possibility of a link between genomic instability and the development of iciHHV-6-associated diseases. Significance StatementMany viruses have evolved ways to inhibit DNA damage signaling, presumably to prevent infected cells from activating an antiviral response. Here, we show that this is also true for human herpesvirus 6B (HHV-6B), through its immediate early protein 1 (IE1). However, in contrast to adenovirus immediate early proteins, HHV-6B IE1 is recruited to double-strand breaks in an NBS1-dependent manner and inhibits ATM serine/threonine kinase activation. Characterizing this phenotype revealed a unique mechanism by which HHV-6B manipulates DNA damage signaling in infected cells. Consistently, viral replication is restricted by the MRN complex in HHV-6B infected cells. Viral integration of HHV-6B into the hosts telomeres is not strictly dependent on NBS1, challenging current models where integration occurs through homology-directed repair.

microbiology↗

bioRxiv2021

Variation in Human Herpesvirus 6B telomeric integration, excision and transmission between tissues and individuals

Human herpesviruses 6A and 6B (HHV-6A/6B) are ubiquitous pathogens that persist lifelong in latent form and can cause severe conditions upon reactivation. They are spread by community-acquired infection of free virus (acqHHV6A/6B) and by germline transmission of inherited chromosomally- integrated HHV-6A/6B (iciHHV-6A/6B) in telomeres. We exploited a hypervariable region of the HHV- 6B genome to investigate the relationship between acquired and inherited virus and revealed predominantly maternal transmission of acqHHV-6B in families. Remarkably, we demonstrate that some copies of acqHHV-6B in saliva from healthy adults gained a telomere, indicative of integration and latency, and that the frequency of viral genome excision from telomeres in iciHHV-6B carriers is surprisingly high and varies between tissues. In addition, newly formed short telomeres generated by partial viral genome release are frequently lengthened, particularly in telomerase-expressing pluripotent cells. Consequently, iciHHV-6B carriers are mosaic for different iciHHV-6B structures, including circular extra-chromosomal forms that have the potential to reactivate. Finally, we show transmission of an HHV-6B strain from an iciHHV-6B mother to her non-iciHHV-6B son. Altogether we demonstrate that iciHHV-6B can readily transition between telomere-integrated and free virus forms.

genetics↗

bioRxiv2019

Increased serological response against human herpesvirus 6A is associated with risk for multiple sclerosis

Human herpesvirus (HHV)-6A or HHV-6B involvement in multiple sclerosis (MS) etiology has remained controversial mainly due to the lack of serological methods that can distinguish the two viruses.\n\nA novel multiplex serological assay measuring IgG reactivity against the immediate-early protein 1 from HHV-6A (IE1A) and HHV-6B (IE1B) was used in a MS cohort (8742 persons with MS and 7215 matched controls), and a pre-MS cohort (478 individuals and 476 matched controls) to investigate this further.\n\nThe IgG response against IE1A was positively associated with MS (OR = 1.55, p = 9x10-22), and increased risk of future MS (OR = 2.22, p = 2x10-5). An interaction was observed between IE1A and Epstein-Barr virus (EBV) antibody responses for MS risk (attributable proportion = 0.24, p = 6x10-6). In contrast, the IgG response against IE1B was negatively associated with MS (OR = 0.74, p = 6x10-11). The association did not differ between MS subtypes or vary with severity of disease. The genetic control of HHV-6A/B antibody responses were located to the Human Leukocyte Antigen (HLA) region and the strongest association for IE1A was the DRB1*13:01-DQA1*01:03-DQB1*06:03 haplotype while the main association for IE1B was DRB1*13:02-DQA1*01:02-DQB1*06:04.\n\nIn conclusion a role for HHV-6A in MS etiology is supported by an increased serological response against HHV-6A IE1 protein, an interaction with EBV, and an association to HLA genes.

immunology↗

bioRxiv2019

Inherited chromosomally integrated HHV-6 demonstrates tissue-specific RNA expression in vivo that correlates with increased antibody immune response.

Human herpesvirus-6A and 6B (HHV-6A, HHV-6B) are human viruses capable of chromosomal integration. Approximately 1% of the human population carry one copy of HHV-6A/B integrated into every cell in their body, referred to as inherited chromosomally integrated human herpesvirus 6A/B (iciHHV-6A/B). Whether iciHHV-6A/B is transcriptionally active in vivo and how it shapes the immunological response is still unclear. Here, we screened DNA-Seq and RNA-Seq data for 650 individuals available through the Genotype-Tissue Expression (GTEx) project and identified 2 iciHHV-6A and 4 iciHHV-6B positive candidates. When corresponding tissue-specific gene expression signatures were analyzed, low levels HHV-6A/B gene expression was found across multiple tissues, with the highest levels of gene expression in the brain (specifically for iciHHV-6A), testis, esophagus, and adrenal gland. U90 and U100 were the most highly expressed HHV-6 genes in both iciHHV-6A and iciHHV-6B individuals. To assess whether tissue-specific gene expression from iciHHV-6A/B influences the immune response, a cohort of 15,498 subjects was screened and 85 iciHHV-6A/B+ subjects were identified. Plasma samples from iciHHV-6A/B+ and age and sex matched controls were analyzed for antibodies to control antigens (CMV, EBV, FLU) or HHV-6A/B antigens. Our results indicate that iciHHV-6A/B+ subjects have significantly more antibodies against the U90 gene product (IE1) relative to non-iciHHV-6 individuals. Antibody responses against EBV and FLU antigens or HHV-6A/B gene products either not expressed or expressed at low levels, such as U47, U57 or U72, were identical between controls and iciHHV-6A/B+ subjects. CMV seropositive individuals with iciHHV-6A/B+ have more antibodies against CMV pp150, relative to CMV seropositive controls. These results argue that spontaneous gene expression from integrated HHV-6A/B leads to an increase in antigenic burden that translates into a more robust HHV-6A/B specific antibody response.\n\nImportanceHHV-6A/B are human herpesviruses that have the unique property of being able to integrate into the subtelomeric regions of human chromosomes. Approximately 1% of the worlds population carries integrated HHV-6A/B genome in every cell of their body. Whether viral genes are transcriptionally active in these individuals is unclear. By taking advantage of a unique tissue-specific gene expression dataset, we show the majority of tissues from iciHHV-6 individuals do not show HHV-6 gene expression. Brain and testes showed the highest tissue-specific expression of HHV-6 genes in two separate datasets. Two HHV-6 genes, U90 (immediate early 1 protein) and U100 (glycoproteins Q1 and Q2), were found to be selectively and consistently expressed across several human tissues. Expression of U90 translates into an increase in antigen-specific antibody response in iciHHV-6A/B+ subjects relative to controls. Future studies will be needed to determine the mechanism of gene expression, the effects of these genes on human gene transcription networks and the pathophysiological impact of having increased viral protein expression in tissue in conjunction with increased antigen-specific antibody production.

genomics↗

bioRxiv2019

Modulation and recruitment of TRF2 at viral telomeres during human herpesvirus 6A/B infection

Human herpesviruses 6A and 6B (HHV-6A/B) can integrate their genomes into the telomeres of host chromosomes. The HHV-6A/B genomes contain telomeric repeats essential for integration. Whether HHV-6A/B infections impact telomere homeostasis remains to be studied. We report that during infection, a massive increase in telomeric signals is observed. Such telomeric signals are detected in viral replication compartments (VRC) that colocalize with the viral IE2 and P41 proteins. Infection with HHV-6A mutants lacking telomeric repeats did not reproduce this phenotype. HHV-6A/B infections lead to increased expression of three shelterin genes, TRF1, TRF2 and TPP1. TRF2 was recruited to VRC and binding to the HHV-6A/B telomeric repeats demonstrated by chromatin immunoprecipitation and ELISA. Lastly, the HHV-6A IE2 protein colocalized with shelterin proteins at telomeres during infection. In summary, HHV-6A/B infections results in an excess of telomeric repeats that stimulates the expression of shelterin genes. TRF2 binds to viral telomeres during infection and localizes with HHV-6A IE2 protein. Our results highlight a potential role for shelterin complex proteins and IE2 during infection and possibly during integration of HHV-6A/B into host chromosomes.

microbiology↗

bioRxiv2018

Role of PML-Nuclear Bodies in Human Herpesvirus 6A and 6B Genome Integration.

Human herpesviruses 6A and 6B (HHV-6A/B) are two betaherpesviruses that readily integrate their genomes into the telomeres of human chromosomes. To date, the cellular or viral proteins that facilitate HHV-6A/B integration remain elusive. In the present study, we demonstrate that the immediate early protein 1 (IE1) of HHV-6A/B colocalizes with telomeres during infection. Moreover, IE1 associates with PML-NBs, a nuclear complex that regulates multiples cellular mechanism including DNA repair and antiviral responses. Furthermore, we could demonstrate that IE1 targets all PML isoforms and that both proteins colocalize at telomeres. To determine the role of PML in HHV-6A/B integration, we generated PML knockout cell lines using CRISPR/Cas9. Intriguingly, in the absence of PML, the IE1 protein could still localize to telomeres albeit less frequently. More importantly, HHV-6A/B integration was impaired in the absence of PML, indicating that it plays a role in the integration process. Taken together, we identified the first cellular protein that aids in the integration of HHV-6A/B and shed light on this targeted integration mechanism.\n\nAuthor summaryHuman herpesviruses type 6A and 6B are relatively common viruses whose infections can be life threatening in patients with a compromised immune system. A rather unique feature of these viruses is their ability to integrate their genome in human chromosomes. Integration takes place is a specialized region of the chromosomes known as telomeres, a region that controls cellular lifespan. To date, the mechanisms leading to HHV-6A and HHV-6B integration remain elusive. Our laboratory has identified that the IE1 protein of HHV-6A and HHV-6B target the telomeres. Moreover, we have shown that IE1 associates with a cellular protein, PML, that is responsible for the regulation of important cellular mechanisms such as the life span of cells and DNA repair. Hence, we studied the role of PML in HHV-6 integration. Our study demonstrates that in absence of PML, the HHV-6A and HHV-6B integrate 50-70% less frequently. Thus, our study unveils the first cellular protein involved in HHV-6A and HHV-6 chromosomal integration.

microbiology↗