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Human herpesviruses 6A and 6B are betaherpesviruses that can integrate their genomes into the telomeres of latently infected cells. Integration can also occur in germ cells, resulting in individuals who harbor the integrated virus in every cell of their body and can pass it on to their offspring. This condition is termed inherited chromosomally integrated HHV-6 (iciHHV-6) and affects about 1% of the human population. The integrated HHV-6A/B genome can reactivate in iciHHV-6 patients and in rare cases can also cause severe diseases including encephalitis and graft-versus-host disease. Until now, it has remained impossible to prevent virus reactivation or remove the integrated virus genome. Therefore, we developed a system that allows the removal of HHV-6A from the host telomeres using the CRISPR/Cas9 system. We used specific guide RNAs (gRNAs) targeting the direct repeat region at the ends of the viral genome to remove the virus from latently infected cells generated in vitro and iciHHV-6A patient cells. Fluorescence-activated cell sorting (FACS), quantitative PCR (qPCR), and fluorescence in situ hybridization (FISH) analyses revealed that the virus genome was efficiently excised and lost in most cells. Efficient excision was achieved with both constitutive and transient expression of Cas9. In addition, reverse transcription-qPCR (RT-qPCR) revealed that the virus genome did not reactivate upon excision. Taken together, our data show that our CRISPR/Cas9 approach allows efficient removal of the integrated virus genome from host telomeres.

Les Herpèsvirus sont des maîtres incontestés du camouflage. En effet, ces virus utilisent divers moyens pour assurer leur persistance chez l’hôte infecté. Les Herpèsvirus humains-6A et -6B (HHV-6A/B) ont ainsi développé une approche unique, en intégrant l’ensemble de leur génome au sein des extrémités des chromosomes des cellules qu’ils infectent. Cette capacité, propre aux HHV-6A/B parmi les Herpèsvirus humains, requiert des interactions étroites entre les régions télomériques des chromosomes de l’hôte et le génome viral. Dans cette revue, la biologie des télomères et les mécanismes responsables de l’intégration virale seront abordés et les conséquences biologiques de l’intégration des HHV-6A/B au sein de l’ADN chromosomique seront discutées.

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 ade novospike 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-2’s tendency to evolve with phenotypic consequences, its evolution varying by lineage, and suggests non-dominant quasi-species contribute.

Platelets and platelet extracellular vesicles (pEV) are at the crossroads of coagulation and immunity. Extracellular vesicles are messengers that not only transmit signals between cells, but also provide information about the status of their cell of origin. Thus, pEVs have potential as both biomarkers of platelet activation and contributors to pathology. Coronavirus Disease‐19 (COVID‐19), caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), is a complex disease affecting multiple organs and is characterized by a high degree of inflammation and risk of thrombosis in some patients. In this review, we introduce pEVs as valuable biomarkers in disease with a special focus on their potential as predictors of and contributors to COVID‐19., Graphical Abstract Review on platelet extracellular vesicles (pEVs); includes a special focus on pEV's potential as predictors of and contributors to COVID‐19.

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 IFNβ 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 IFNα. Using in vitro systems, Ifnβ gene translation inhibition was observed using an Nsp1 expression vector. Conversely, SARS-CoV-2 and its variants induce robust expression of NF-κB-driven genes such as those encoding CCL2 ans CXCL10 chemokines. We also identified Nsp2 as an activator of NF-κB 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-κB-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 IFNΒ1 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-κB responsive genes for which Nsp2 is partially responsible.

A vaccine candidate to SARS-CoV-2 was constructed by coupling the viral receptor binding domain (RBD) to the surface of the papaya mosaic virus (PapMV) nanoparticle (nano) to generate the RBD-PapMV vaccine. Immunization of mice with the coupled RBD-PapMV vaccine enhanced the antibody titers and the T-cell mediated immune response directed to the RBD antigen as compared to immunization with the non-coupled vaccine formulation (RBD + PapMV nano). Anti-RBD antibodies, generated in vaccinated animals, neutralized SARS-CoV-2 infection in vitro against the ancestral, Delta and the Omicron variants. At last, immunization of mice susceptible to the infection by SARS-CoV-2 (K18-hACE2 transgenic mice) with the RBD-PapMV vaccine induced protection to the ancestral SARS-CoV-2 infectious challenge. The induction of the broad neutralization against SARS-CoV-2 variants induced by the RBD-PapMV vaccine demonstrate the potential of the PapMV vaccine platform in the development of efficient vaccines against viral respiratory infections.

Rationale: In addition to the overwhelming lung inflammation that prevails in coronavirus disease 2019 (COVID-19), hypercoagulation and thrombosis contribute to the lethality of subjects infected with severe acute respiratory syndrome coronavirus 2. Platelets are chiefly implicated in thrombosis. Moreover, they can interact with viruses and are an important source of inflammatory mediators. While a lower platelet count is associated with severity and mortality, little is known about platelet function during COVID-19. Objective: To evaluate the contribution of platelets to inflammation and thrombosis in patients with COVID-19. Methods and Results: Blood was collected from 115 consecutive patients with COVID-19 presenting nonsevere (n=71) and severe (n=44) respiratory symptoms. We document the presence of severe acute respiratory syndrome coronavirus 2 RNA associated with platelets of patients with COVID-19. Exhaustive assessment of cytokines in plasma and in platelets revealed the modulation of platelet-associated cytokine levels in both patients with nonsevere and severe COVID-19, pointing to a direct contribution of platelets to the plasmatic cytokine load. Moreover, we demonstrate that platelets release their alpha- and dense-granule contents in both nonsevere and severe forms of COVID-19. In comparison to concentrations measured in healthy volunteers, phosphatidylserine-exposing platelet extracellular vesicles were increased in nonsevere, but not in severe cases of COVID-19. Levels of D-dimers, a marker of thrombosis, failed to correlate with any measured indicators of platelet activation. Functionally, platelets were hyperactivated in COVID-19 subjects presenting nonsevere and severe symptoms, with aggregation occurring at suboptimal thrombin concentrations. Furthermore, platelets adhered more efficiently onto collagen-coated surfaces under flow conditions. Conclusions: Taken together, the data suggest that platelets are at the frontline of COVID-19 pathogenesis, as they release various sets of molecules through the different stages of the disease. Platelets may thus have the potential to contribute to the overwhelming thrombo-inflammation in COVID-19, and the inhibition of pathways related to platelet activation may improve the outcomes during COVID-19.

Platelets are hyperactivated in coronavirus disease 2019 (COVID-19). However, the mechanisms promoting platelet activation by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not well understood. This may be due to inherent challenges in discriminating the contribution of viral vs host components produced by infected cells. This is particularly true for enveloped viruses and extracellular vesicles (EVs), as they are concomitantly released during infection and share biophysical properties. To study this, we evaluated whether SARS-CoV-2 itself or components derived from SARS-CoV-2-infected human lung epithelial cells could activate isolated platelets from healthy donors. Activation was measured by the surface expression of P-selectin and the activated conformation of integrin αIIbβ3, degranulation, aggregation under flow conditions, and the release of EVs. We find that neither SARS-CoV-2 nor purified spike activates platelets. In contrast, tissue factor (TF) produced by infected cells was highly potent at activating platelets. This required trace amounts of plasma containing the coagulation factors FX, FII, and FVII. Robust platelet activation involved thrombin and the activation of protease-activated receptor (PAR)-1 and -4 expressed by platelets. Virions and EVs were identified by electron microscopy. Through size-exclusion chromatography, TF activity was found to be associated with a virus or EVs, which were indistinguishable. Increased TF messenger RNA (mRNA) expression and activity were also found in lungs in a murine model of COVID-19 and plasma of severe COVID-19 patients, respectively. In summary, TF activity from SARS-CoV-2–infected cells activates thrombin, which signals to PARs on platelets. Blockade of molecules in this pathway may interfere with platelet activation and the coagulation characteristic of COVID-19.

Herpesviruses are undisputed masters of disguise. The ability to become invisible to the immune system effectors is a complex process resting on a variety of stealth approaches. Among these, human herpesviruses-6A and -6B (HHV-6A/B) have developed the unique ability to integrate their genome within the ends of chromosomes allowing viral persistence in the absence of viral protein expression. This aptitude, unique to HHV-6A/B among human herpesviruses, requires close interactions between the telomeric regions of chromosomes and the viral genome. In this review article, the biology of telomeres and the mechanisms responsible for viral integration are discussed. In closing, the possible biological consequences of HHV-6A/B integration into chromosomal DNA are discussed.L’importance des télomères dans les infections par les Herpèsvirus humains-6A/B.Les Herpèsvirus sont des maîtres incontestés du camouflage. En effet, ces virus utilisent divers moyens pour assurer leur persistance chez l’hôte infecté. Les Herpèsvirus humains-6A et -6B (HHV-6A/B) ont ainsi développé une approche unique, en intégrant l’ensemble de leur génome au sein des extrémités des chromosomes des cellules qu’ils infectent. Cette capacité, propre aux HHV-6A/B parmi les Herpèsvirus humains, requiert des interactions étroites entre les régions télomériques des chromosomes de l’hôte et le génome viral. Dans cette revue, la biologie des télomères et les mécanismes responsables de l’intégration virale seront abordés et les conséquences biologiques de l’intégration des HHV-6A/B au sein de l’ADN chromosomique seront discutées.

Immune complexes form in systemic disorders such as rheumatological, autoimmune, and allergic diseases or in response to infections or medications. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) adenoviral vector vaccines have been associated with rare yet serious thrombotic complications in the brain due to the formation of immune complexes that activate platelets. There are currently no data visualizing the interplay of platelets with leukocytes and the brain vasculature endothelium in response to immune complexes. This is in part due to the absence of FcγRIIA in mice, a receptor for immune complexes implicated in these thrombotic incidents. Here, we describe and illustrate events at the cellular level that take place in the brain vasculature in response to systemic administration of surrogate immune complexes. We used Ly6gCre+/−::Rosa26-TdT+/−::CD41-YFP+/− mice expressing the FcγRIIA transgene and fluorescence in neutrophils and platelets. Using real-time videomicroscopy to capture high-velocity events in conjunction with unbiased computer-assisted analyses, we provide images and quantifications of the cellular responses downstream of FcγRIIA stimulation. We observed transient and stable platelet–neutrophil interactions, platelets forming thrombi, and neutrophil adhesion to blood vessel walls. This imaging approach in a quadruple transgenic animal model can be used for the study of the pathogenic roles of immune complexes in disease.

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.

Herpesviruses are extremely well adapted to their hosts and cause persistent infections that span over decades. Typically, these viruses establish latency by maintaining their viral genomes as extrachromosomal episomes and restricting their gene expression to a minimum. Human herpesvirus 6 (HHV-6) is perhaps one of the most well-adapted human herpesvirus with a prevalence approaching 100%. Unlike any other human herpesviruses, HHV-6 has developed the ability to integrate its genome into the host chromosomes by targeting the telomeric region. Whether integration represents a true mode of latency for this virus or whether is constitutes a viral dead-end remains uncertain. The present review aims to familiarize the readers with the concept of viral integration and delineates the various biological, pathological and medical consequences associated with HHV-6 chromosomal integration.

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 cell’s 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 host’s telomeres is not strictly dependent on NBS1, challenging current models where integration occurs through homology-directed repair.

The 11th International Conference on Human Herpesviruses (HHV)‐6A, ‐6B, and ‐7 was held in Quebec City, from 23 to 26 June 2019. It attracted 144 basic, translational, and clinical scientists from 20 countries. Important new information was presented regarding: the mechanisms of chromosomal integration for HHV‐6A and ‐6B; the biology of inherited chromosomally integrated HHV‐6A and ‐6B; animal models for, and animal viruses with similarities to, HHV‐6A, ‐6B, and ‐7; established and possible disease associations, including provocative new information suggesting these viruses may be one trigger of Alzheimer's disease, and new treatment strategies. In this review, we summarize presentations that were of particular interest. The full text of the Abstracts cited in the manuscript is available in the online Supporting Information Materials.

Coronavirus disease 19 (COVID-19) is a respiratory illness caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). COVID-19 pathogenesis causes vascular-mediated neurological disorders via elusive mechanisms. SARS-CoV-2 infects host cells via the binding of viral Spike (S) protein to transmembrane receptor, angiotensin-converting enzyme 2 (ACE2). Although brain pericytes were recently shown to abundantly express ACE2 at the neurovascular interface, their response to SARS-CoV-2 S protein is still to be elucidated. Using cell-based assays, we found that ACE2 expression in human brain vascular pericytes was increased upon S protein exposure. Pericytes exposed to S protein underwent profound phenotypic changes associated with an elongated and contracted morphology accompanied with an enhanced expression of contractile and myofibrogenic proteins, such as α-smooth muscle actin (α-SMA), fibronectin, collagen I, and neurogenic locus notch homolog protein-3 (NOTCH3). On the functional level, S protein exposure promoted the acquisition of calcium (Ca2+) signature of contractile ensheathing pericytes characterized by highly regular oscillatory Ca2+ fluctuations. Furthermore, S protein induced lipid peroxidation, oxidative and nitrosative stress in pericytes as well as triggered an immune reaction translated by activation of nuclear factor-kappa-B (NF-κB) signaling pathway, which was potentiated by hypoxia, a condition associated with vascular comorbidities that exacerbate COVID-19 pathogenesis. S protein exposure combined to hypoxia enhanced the production of pro-inflammatory cytokines involved in immune cell activation and trafficking, namely macrophage migration inhibitory factor (MIF). Using transgenic mice expressing the human ACE2 that recognizes S protein, we observed that the intranasal infection with SARS-CoV-2 rapidly induced hypoxic/ischemic-like pericyte reactivity in the brain of transgenic mice, accompanied with an increased vascular expression of ACE2. Moreover, we found that SARS-CoV-2 S protein accumulated in the intranasal cavity reached the brain of mice in which the nasal mucosa is deregulated. Collectively, these findings suggest that SARS-CoV-2 S protein impairs the vascular and immune regulatory functions of brain pericytes, which may account for vascular-mediated brain damage. Our study provides a better understanding for the mechanisms underlying cerebrovascular disorders in COVID-19, paving the way to develop new therapeutic interventions.

Severe acute respiratory syndrome coronavirus 2 is responsible for coronavirus disease 2019 (COVID‐19). While COVID‐19 is often benign, a subset of patients develops severe multilobar pneumonia that can progress to an acute respiratory distress syndrome. There is no cure for severe COVID‐19 and few treatments significantly improved clinical outcome. Dexamethasone and possibly aspirin, which directly/indirectly target the biosynthesis/effects of numerous lipid mediators are among those options. Our objective was to define if severe COVID‐19 patients were characterized by increased bioactive lipids modulating lung inflammation. A targeted lipidomic analysis of bronchoalveolar lavages (BALs) by tandem mass spectrometry was done on 25 healthy controls and 33 COVID‐19 patients requiring mechanical ventilation. BALs from severe COVID‐19 patients were characterized by increased fatty acids and inflammatory lipid mediators. There was a predominance of thromboxane and prostaglandins. Leukotrienes were also increased, notably LTB4, LTE4, and eoxin E4. Monohydroxylated 15‐lipoxygenase metabolites derived from linoleate, arachidonate, eicosapentaenoate, and docosahexaenoate were also increased. Finally yet importantly, specialized pro‐resolving mediators, notably lipoxin A4 and the D‐series resolvins, were also increased, underscoring that the lipid mediator storm occurring in severe COVID‐19 involves pro‐ and anti‐inflammatory lipids. Our data unmask the lipid mediator storm occurring in the lungs of patients afflicted with severe COVID‐19. We discuss which clinically available drugs could be helpful at modulating the lipidome we observed in the hope of minimizing the deleterious effects of pro‐inflammatory lipids and enhancing the effects of anti‐inflammatory and/or pro‐resolving lipid mediators.

RNA sequencing (RNA-seq) is an important tool for studying RNA transcripts, particularly during active viral infection. We made use of RNA-seq to study human herpesvirus 6B (HHV-6B) infection., The “omics” revolution of recent years has simplified the study of RNA transcripts produced during viral infection and under specific defined conditions. In the quest to find new and differentially expressed transcripts during the course of human herpesvirus 6B (HHV-6B) infection, we made use of large-scale RNA sequencing to analyze the HHV-6B transcriptome during productive infection of human Molt-3 T cells. Analyses were performed at different time points following infection, and specific inhibitors were used to classify the kinetic class of each open reading frame (ORF) reported in the annotated genome of the HHV-6B Z29 strain. The initial search focused on HHV-6B-specific reads matching new HHV-6B transcripts. Differential expression of new HHV-6B transcripts was observed in all samples analyzed. The presence of many of these new HHV-6B transcripts was confirmed by reverse transcriptase PCR and Sanger sequencing. Many of these transcripts represented new splice variants of previously reported open reading frames (ORFs), including some transcripts that have yet to be defined. Overall, our work demonstrates the diversity and the complexity of the HHV-6B transcriptome. IMPORTANCE RNA sequencing (RNA-seq) is an important tool for studying RNA transcripts, particularly during active viral infection. We made use of RNA-seq to study human herpesvirus 6B (HHV-6B) infection. Using six different time points, we were able to identify the presence of differentially spliced genes at 6, 9, 12, 24, 48, and 72 h postinfection. Determination of the RNA profiles in the presence of cycloheximide (CHX) or phosphonoacetic acid (PAA) also permitted identification of the kinetic class of each ORF described in the annotated GenBank file. We also identified new spliced transcripts for certain genes and evaluated their relative expression over time. These data and next-generation sequencing (NGS) of the viral DNA have led us to propose a new version of the HHV-6B Z29 GenBank annotated file, without changing ORF names, to facilitate trace-back and correlate our work with previous studies on HHV-6B.

The herpes simplex virus 1 (HSV-1) genome is extremely rich in guanine tracts that fold into G-quadruplexes (G4s), nucleic acid secondary structures implicated in key biological functions. Viral G4s were visualized in HSV-1 infected cells, with massive virus cycle-dependent G4-formation peaking during viral DNA replication. Small molecules that specifically interact with G4s have been shown to inhibit HSV-1 DNA replication. We here investigated the antiviral activity of TMPyP4, a porphyrin known to interact with G4s. The analogue TMPyP2, with lower G4 affinity, was used as control. We showed by biophysical analysis that TMPyP4 interacts with HSV-1 G4s, and inhibits polymerase progression in vitro, in infected cells, it displayed good antiviral activity which, however, was independent of inhibition of virus DNA replication or entry. At low TMPyP4 concentration, the virus released by the cells was almost null, while inside the cell virus amounts were at control levels. TEM analysis showed that virus particles were trapped inside cytoplasmatic vesicles, which could not be ascribed to autophagy, as proven by RT-qPCR, western blot, and immunofluorescence analysis. Our data indicate a unique mechanism of action of TMPyP4 against HSV-1, and suggest the unprecedented involvement of currently unknown G4s in viral or antiviral cellular defense pathways.

BACKGROUNDSevere Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is the infectious agent responsible for Coronavirus disease 2019 (COVID-19). While SARS-CoV-2 infections are often benign, there are also severe COVID-19 cases, characterized by severe bilobar pneumonia that can decompensate to an acute respiratory distress syndrome, notably characterized by increased inflammation and a cytokine storm. While there is no cure against severe COVID-19 cases, some treatments significantly decrease the severity of the disease, notably aspirin and dexamethasone, which both directly or indirectly target the biosynthesis (and effects) of numerous bioactive lipids.OBJECTIVEOur working hypothesis was that severe COVID-19 cases necessitating mechanical ventilation were characterized by increased bioactive lipid levels modulating lung inflammation. We thus quantitated several lung bioactive lipids using liquid chromatography combined to tandem mass spectrometry.RESULTSWe performed an exhaustive assessment of the lipid content of bronchoalveolar lavages from 25 healthy controls and 33 COVID-19 patients necessitating mechanical ventilation. Severe COVID-19 patients were characterized by increased fatty acid levels as well as an accompanying inflammatory lipid storm. As such, most quantified bioactive lipids were heavily increased. There was a predominance of cyclooxygenase metabolites, notably TXB2 >> PGE2 ∼ 12-HHTrE > PGD2. Leukotrienes were also increased, notably LTB4, 20-COOH-LTB4, LTE4, and eoxin E4. 15-lipoxygenase metabolites derived from linoleic, arachidonic, eicosapentaenoic and docosahexaenoic acids were also increased. Finally, yet importantly, specialized pro-resolving mediators, notably lipoxin A4 and the D-series resolvins, were also found at important levels, underscoring that the lipid storm occurring in severe SARS-CoV-2 infections involves pro- and anti-inflammatory lipids.CONCLUSIONSOur data unmask the important lipid storm occurring in the lungs of patients afflicted with severe COVID-19. We discuss which clinically available drugs could be helpful at modulating the lipidome we observed in the hope of minimizing the deleterious effects of pro-inflammatory lipids and enhancing the effects of anti-inflammatory and/or pro-resolving lipids.

RationaleIn addition to the overwhelming lung inflammation that prevails in COVID-19, hypercoagulation and thrombosis contribute to the lethality of subjects infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Platelets are chiefly implicated in thrombosis. Moreover, they can interact with viruses and are an important source of inflammatory mediators. While a lower platelet count is associated with severity and mortality, little is known about platelet function during COVID-19.ObjectiveTo evaluate the contribution of platelets to inflammation and thrombosis in COVID-19 patients.Methods and ResultsWe document the presence of SARS-CoV-2 RNA in platelets of COVID-19 patients. Exhaustive assessment of cytokines in plasma and in platelets revealed the modulation of platelet-associated cytokine levels in COVID-19, pointing to a direct contribution of platelets to the plasmatic cytokine load. Moreover, we demonstrate that platelets release their alpha- and dense-granule contents and phosphatidylserine-exposing extracellular vesicles. Functionally, platelets were hyperactivated in COVID-19 subjects, with aggregation occurring at suboptimal thrombin concentrations. Furthermore, platelets adhered more efficiently onto collagen-coated surfaces under flow conditions.ConclusionsThese data suggest that platelets could participate in the dissemination of SARS-CoV-2 and in the overwhelming thrombo-inflammation observed in COVID-19. Thus, blockade of platelet activation pathways may improve outcomes in this disease.KEY POINTSPlatelets are a source of inflammatory cytokines and degranulate in COVID-19 Platelets contain SARS-CoV-2 RNA molecules and are prone to activation in COVID-19Subject termsInfectious diseases/Emerging infectious diseases, SARS-CoV-2, COVID-19, Hematology, Platelets

Human herpesvirus 6B (HHV-6B) is a betaherpesvirus capable of integrating its genome into the telomeres of host chromosomes. Until now, the cellular and/or viral proteins facilitating HHV-6B integration have remained elusive. Here we show that a cellular protein, the promyelocytic leukemia protein (PML) that forms nuclear bodies (PML-NBs), associates with the HHV-6B immediate early 1 (IE1) protein at telomeres. We report enhanced levels of SUMOylated IE1 in the presence of PML and have identified a putative SUMO Interacting Motif (SIM) within IE1, essential for its nuclear distribution, overall SUMOylation and association with PML to nuclear bodies. Furthermore, using PML knockout cell lines we made the original observation that PML is required for efficient HHV-6B integration into host chromosomes. Taken together, we could demonstrate that PML-NBs are important for IE1 multiSUMOylation and that PML plays an important role in HHV-6B integration into chromosomes, a strategy developed by this virus to maintain its genome in its host over long periods of time., Author summary Human herpesvirus 6B (HHV-6B) is a ubiquitous virus that can be life threatening in immunocompromised patients. HHV-6B is among a few other herpesviruses that integrate their genome in host chromosomes as a mean to establish dormancy. Integration of HHV-6B occurs in host telomeres, a region that protects our genome from deterioration and controls the cellular lifespan. To date, the mechanisms leading to HHV-6B integration remain elusive. Our laboratory has identified that the IE1 protein of HHV-6B associates with PML, a cellular protein that is responsible for the regulation of important cellular mechanisms including DNA recombination and repair. With the objective of understanding how IE1 is brought to PML, we discovered that PML aids the SUMOylation of IE1. This finding led us to identify a putative SUMO interaction motif on IE1 that is essentials for both its SUMOylation and IE1 oligomerization with PML-NBs. We next studied the role of PML on HHV-6B integration and identified that cells that are deficient for PML were less susceptible to HHV-6B integration. These results correlate with the fact that PML influences IE1 localization at telomeres, the site of HHV-6B integration. Our study further contributes to our understanding of the mechanisms leading to HHV-6B chromosomal integration.