Innate immune response to SARS-CoV-2 infection contributes to neuronal damage in human iPSC-derived peripheral neurons.

Severe acute respiratory coronavirus 2 (SARS-CoV-2) causes neurological disease in the peripheral and central nervous system (PNS and CNS, respectively) of some patients. It is not clear whether SARS-CoV-2 infection or the subsequent immune response are the key factors that cause neurological disease. Here, we addressed this question by infecting human induced pluripotent stem cell-derived CNS and PNS neurons with SARS-CoV-2. SARS-CoV-2 infected a low number of CNS neurons and did not elicit a robust innate immune response. On the contrary, SARS-CoV-2 infected a higher number of PNS neurons. This resulted in expression of interferon (IFN) λ1, several IFN-stimulated genes and proinflammatory cytokines. The PNS neurons also displayed alterations characteristic of neuronal damage, as increased levels of sterile alpha and Toll/interleukin receptor motif-containing protein 1, amyloid precursor protein and α-synuclein, and lower levels of cytoskeletal proteins. Interestingly, blockade of the Janus kinase and signal transducer and activator of transcription pathway by Ruxolitinib did not increase SARS-CoV-2 infection, but reduced neuronal damage, suggesting that an exacerbated neuronal innate immune response contributes to pathogenesis in the PNS. Our results provide a basis to study coronavirus disease 2019 (COVID-19) related neuronal pathology and to test future preventive or therapeutic strategies.

Viral modulation of type II interferon increases T cell adhesion and virus spread.

During primary infection, varicella zoster virus (VZV) infects epithelial cells in the respiratory lymphoid organs and mucosa. Subsequent infection of lymphocytes, T cells in particular, causes primary viremia allowing systemic spread throughout the host, including the skin. This results in the expression of cytokines, including interferons (IFNs) which partly limit primary infection. VZV also spreads from skin keratinocytes to lymphocytes prior to secondary viremia. How VZV infects lymphocytes from epithelial cells while evading the cytokine response has not been fully established. Here, we show that VZV glycoprotein C (gC) binds IFN-γ and modifies its activity. Transcriptomic analysis revealed that gC in combination with IFN-γ increased the expression of a small subset of IFN-stimulated genes (ISGs), including intercellular adhesion molecule 1 (ICAM1), as well as several chemokines and immunomodulatory genes. The higher ICAM1 protein level at the plasma membrane of epithelial cells resulted in lymphocyte function-associated antigen 1 (LFA-1)-dependent T cell adhesion. This gC activity required a stable interaction with IFN-γ and signalling through the IFN-γ receptor. Finally, the presence of gC during infection increased VZV spread from epithelial cells to peripheral blood mononuclear cells. This constitutes the discovery of a novel strategy to modulate the activity of IFN-γ, inducing the expression of a subset of ISGs, leading to enhanced T cell adhesion and virus spread.

Colonization of peripheral ganglia by herpes simplex virus type 1 and 2.

Herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) infect and establish latency in neurons of the peripheral nervous system to persist lifelong in the host and to cause recurrent disease. During primary infection, HSV replicates in epithelial cells in the mucosa and skin and then infects neurites, highly dynamic structures that grow or retract in the presence of attracting or repelling cues, respectively. Following retrograde transport in neurites, HSV establishes latency in the neuronal nucleus. Viral and cellular proteins participate in the chromatinization of the HSV genome that regulates gene expression, persistence, and reactivation. HSV-2 modulates neurite outgrowth during primary infection and upon reactivation, probably to facilitate infection and survival of neurons. Whether HSV-1 modulates neurite outgrowth and the underlying mechanism is currently under investigation. This review deals with HSV-1 and HSV-2 colonization of peripheral neurons, with a focus on the modulation of neurite outgrowth by these viruses.

Generation of hiPSC-derived low threshold mechanoreceptors containing axonal termini resembling bulbous sensory nerve endings and expressing Piezo1 and Piezo2.

Somatosensory low threshold mechanoreceptors (LTMRs) sense innocuous mechanical forces, largely through specialized axon termini termed sensory nerve endings, where the mechanotransduction process initiates upon activation of mechanotransducers. In humans, a subset of sensory nerve endings is enlarged, forming bulb-like expansions, termed bulbous nerve endings. There is no in vitro human model to study these neuronal endings. Piezo2 is the main mechanotransducer found in LTMRs. Recent evidence shows that Piezo1, the other mechanotransducer considered absent in dorsal root ganglia (DRG), is expressed at low level in somatosensory neurons. We established a differentiation protocol to generate, from iPSC-derived neuronal precursor cells, human LTMR recapitulating bulbous sensory nerve endings and heterogeneous expression of Piezo1 and Piezo2. The derived neurons express LTMR-specific genes, convert mechanical stimuli into electrical signals and have specialized axon termini that morphologically resemble bulbous nerve endings. Piezo2 is concentrated within these enlarged axon termini. Some derived neurons express low level Piezo1, and a subset co-express both channels. Thus, we generated a unique, iPSCs-derived human model that can be used to investigate the physiology of bulbous sensory nerve endings, and the role of Piezo1 and 2 during mechanosensation.

Combination of long- and short-read sequencing fully resolves complex repeats of herpes simplex virus 2 strain MS complete genome.

Herpes simplex virus serotype 2 (HSV-2) is a ubiquitous human pathogen that causes recurrent genital infections and ulcerations. Many HSV-2 strains with different biological properties have been identified, but only the genomes of HSV-2 strains HG52, SD90e and 333 have been reported as complete and fully characterized sequences. We de novo assembled, annotated and manually curated the complete genome sequence of HSV-2 strain MS, a highly neurovirulent strain, originally isolated from a multiple sclerosis patient. We resolved both DNA ends, as well as the complex inverted repeats regions present in HSV genomes, usually undisclosed in previous published partial herpesvirus genomes, using long reads from Pacific Biosciences (PacBio) technology. Additionally, we identified isomeric genomes by determining the alternative relative orientation of unique fragments in the genome of the sequenced viral population. Illumina short-read sequencing was crucial to examine genetic variability, such as nucleotide polymorphisms, insertion/deletions and sequence determinants of strain-specific virulence factors. We used Illumina data to fix two disrupted open reading frames found in coding homopolymers after PacBio assembly. These results support the combination of long- and short-read sequencing technologies as a precise and effective approach for the accurate de novo assembly and curation of complex microbial genomes.

Identification of the Cleavage Domain within Glycoprotein G of Herpes Simplex Virus Type 2.

Glycoprotein G (gG) from herpes simplex virus type 1 and 2 (HSV-1 and HSV-2, respectively) functions as a viral chemokine binding protein (vCKBP). Soluble recombinant forms of gG of HSV-1 and HSV-2 (SgG1 and SgG2, respectively) enhance chemokine-mediated leukocyte migration, in contrast to most known vCKBPs, including those from animal alpha-herpesviruses. Furthermore, both proteins bind to nerve growth factor (NGF), but only SgG2 enhances NGF-dependent neurite outgrowth. The basis and implications of this functional difference between the two proteins are still unknown. While gG1 and gG2 are positional homologues in the genome, they share very limited sequence homology. In fact, US4, the open reading frame encoding gG is the most divergent genetic locus between these viruses. Full-length gG1 and gG2 are type I transmembrane proteins located on the plasma membrane of infected cells and at the viral envelope. However, gG2 is larger than gG1 and is cleaved during protein maturation, secreting the N-terminal domain to the supernatant of infected cells, whereas gG1 is not. The enzyme involved in gG2 cleavage and the functional relevance of gG2 cleavage and secretion are unknown. We aim to identify the gG2 sequence required for cleavage to determine its functional role in future experiments. Our results prove the existence of at least two cleavage motifs in gG2 within the amino acid region 314-343. Transfer of this sequence to a fusion protein results in cleavage. Finally, we show that propeptide convertases like furin are responsible for gG2 cleavage.

Herpes Simplex Virus 2 Counteracts Neurite Outgrowth Repulsion during Infection in a Nerve Growth Factor-Dependent Manner.

During primary infection, herpes simplex virus 2 (HSV-2) replicates in epithelial cells and enters neurites to infect neurons of the peripheral nervous system. Growth factors and attractive and repulsive directional cues influence neurite outgrowth and neuronal survival. We hypothesized that HSV-2 modulates the activity of such cues to increase neurite outgrowth. To test this hypothesis, we exposed sensory neurons to nerve growth factor (NGF) and mock- or HSV-2-infected HEK-293T cells, since they express repellents of neurite outgrowth. We show that HEK-293T cells secrete factors that inhibit neurite outgrowth, while infection with HSV-2 strains MS and 333 reduces this repelling phenotype, increasing neurite numbers. The HSV-2-mediated restoration of neurite outgrowth required the activity of NGF. In the absence of infection, however, NGF did not overcome the repulsion mediated by HEK-293T cells. We previously showed that recombinant, soluble glycoprotein G of HSV-2 (rSgG2) binds and enhances NGF activity, increasing neurite outgrowth. However, the effect of gG2 during infection has not been investigated. Therefore, we addressed whether gG2 contributes to overcoming neurite outgrowth repulsion. To do so, we generated viruses lacking gG2 expression and complemented them by exogenous expression of gG2. Overall, our results suggest that HSV-2 infection of nonneuronal cells reduces their repelling effect on neurite outgrowth in an NGF-dependent manner. gG2 contributed to this phenotype, but it was not the only factor. The enhanced neurite outgrowth may facilitate HSV-2 spread from epithelial cells into neurons expressing NGF receptors and increase HSV-2-mediated pathogenesis.IMPORTANCE Herpes simplex virus 2 (HSV-2) is a prevalent human pathogen that establishes lifelong latency in neurons of the peripheral nervous system. Colonization of neurons is required for HSV-2 persistence and pathogenesis. The viral and cellular factors required for efficient infection of neurons are not fully understood. We show here that nonneuronal cells repel neurite outgrowth of sensory neurons, while HSV-2 infection overcomes this inhibition and, rather, stimulates neurite outgrowth. HSV-2 glycoprotein G and nerve growth factor contribute to this phenotype, which may attract neurites to sites of infection and facilitate virus spread to neurons. Understanding the mechanisms that modulate neurite outgrowth and facilitate HSV-2 infection of neurons might foster the development of therapeutics to reduce HSV-2 colonization of the nervous system and provide insights on neurite outgrowth and regeneration.

Varicella zoster virus glycoprotein C increases chemokine-mediated leukocyte migration.

Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV spreads within the host by hijacking leukocytes, including T cells, in the tonsils and other regional lymph nodes, and modifying their activity. In spite of its importance in pathogenesis, the mechanism of dissemination remains poorly understood. Here we addressed the influence of VZV on leukocyte migration and found that the purified recombinant soluble ectodomain of VZV glycoprotein C (rSgC) binds chemokines with high affinity. Functional experiments show that VZV rSgC potentiates chemokine activity, enhancing the migration of monocyte and T cell lines and, most importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation of chemokine activity occurs through the C-terminal part of gC ectodomain, containing predicted immunoglobulin-like domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the chemokine receptor. Finally, we show that VZV viral particles enhance chemokine-dependent T cell migration and that gC is partially required for this activity. We propose that VZV gC activity facilitates the recruitment and subsequent infection of leukocytes and thereby enhances VZV systemic dissemination in humans.

Chemokine binding proteins: An immunomodulatory strategy going viral.

Chemokines are chemotactic cytokines whose main function is to direct cell migration. The chemokine network is highly complex and its deregulation is linked to several diseases including immunopathology, cancer and chronic pain. Chemokines also play essential roles in the antiviral immune response. Viruses have therefore developed several counter strategies to modulate chemokine activity. One of these is the expression of type I transmembrane or secreted proteins with the ability to bind chemokines and modulate their activity. These proteins, termed viral chemokine binding proteins (vCKBP), do not share sequence homology with host proteins and are immunomodulatory in vivo. In this review we describe the discovery and characterization of vCKBP, explain their role in the context of infection in vivo and discuss relevant novel findings.

A temporal gate for viral enhancers to co-opt Toll-like-receptor transcriptional activation pathways upon acute infection.

Viral engagement with macrophages activates Toll-Like-Receptors (TLRs) and viruses must contend with the ensuing inflammatory responses to successfully complete their replication cycle. To date, known counter-strategies involve the use of viral-encoded proteins that often employ mimicry mechanisms to block or redirect the host response to benefit the virus. Whether viral regulatory DNA sequences provide an opportunistic strategy by which viral enhancer elements functionally mimic innate immune enhancers is unknown. Here we find that host innate immune genes and the prototypical viral enhancer of cytomegalovirus (CMV) have comparable expression kinetics, and positively respond to common TLR agonists. In macrophages but not fibroblasts we show that activation of NFκB at immediate-early times of infection is independent of virion-associated protein, M45. We find upon virus infection or transfection of viral genomic DNA the TLR-agonist treatment results in significant enhancement of the virus transcription-replication cycle. In macrophage time-course infection experiments we demonstrate that TLR-agonist stimulation of the viral enhancer and replication cycle is strictly delimited by a temporal gate with a determined half-maximal time for enhancer-activation of 6 h; after which TLR-activation blocks the viral transcription-replication cycle. By performing a systematic siRNA screen of 149 innate immune regulatory factors we identify not only anticipated anti-viral and pro-viral contributions but also new factors involved in the CMV transcription-replication cycle. We identify a central convergent NFκB-SP1-RXR-IRF axis downstream of TLR-signalling. Activation of the RXR component potentiated direct and indirect TLR-induced activation of CMV transcription-replication cycle; whereas chromatin binding experiments using wild-type and enhancer-deletion virus revealed IRF3 and 5 as new pro-viral host transcription factor interactions with the CMV enhancer in macrophages. In a series of pharmacologic, siRNA and genetic loss-of-function experiments we determined that signalling mediated by the TLR-adaptor protein MyD88 plays a vital role for governing the inflammatory activation of the CMV enhancer in macrophages. Downstream TLR-regulated transcription factor binding motif disruption for NFκB, AP1 and CREB/ATF in the CMV enhancer demonstrated the requirement of these inflammatory signal-regulated elements in driving viral gene expression and growth in cells as well as in primary infection of neonatal mice. Thus, this study shows that the prototypical CMV enhancer, in a restricted time-gated manner, co-opts through DNA regulatory mimicry elements, innate-immune transcription factors to drive viral expression and replication in the face of on-going pro-inflammatory antiviral responses in vitro and in vivo and; suggests an unexpected role for inflammation in promoting acute infection and has important future implications for regulating latency.

Cronobacter sakazakii: stress survival and virulence potential in an opportunistic foodborne pathogen.

A characteristic feature of the opportunistic foodborne pathogen Cronobacter sakazakii is its ability to survive in extremely arid environments, such as powdered infant formula, making it a dangerous opportunistic pathogen of individuals of all age groups, especially infants and neonates. Herein, we provide a brief overview of the pathogen; clinical manifestations, environmental reservoirs and our current understanding of stress response mechanisms and virulence factors which allow it to cause disease.

The transcription factor STAT-1 couples macrophage synthesis of 25-hydroxycholesterol to the interferon antiviral response.

Recent studies suggest that the sterol metabolic network participates in the interferon (IFN) antiviral response. However, the molecular mechanisms linking IFN with the sterol network and the identity of sterol mediators remain unknown. Here we report a cellular antiviral role for macrophage production of 25-hydroxycholesterol (cholest-5-en-3ß,25-diol, 25HC) as a component of the sterol metabolic network linked to the IFN response via Stat1. By utilizing quantitative metabolome profiling of all naturally occurring oxysterols upon infection or IFN-stimulation, we reveal 25HC as the only macrophage-synthesized and -secreted oxysterol. We show that 25HC can act at multiple levels as a potent paracrine inhibitor of viral infection for a broad range of viruses. We also demonstrate, using transcriptional regulatory-network analyses, genetic interventions and chromatin immunoprecipitation experiments that Stat1 directly coupled Ch25h regulation to IFN in macrophages. Our studies describe a physiological role for 25HC as a sterol-lipid effector of an innate immune pathway.

Viral latency drives 'memory inflation': a unifying hypothesis linking two hallmarks of cytomegalovirus infection.

Low public awareness of cytomegalovirus (CMV) results from the only mild and transient symptoms that it causes in the healthy immunocompetent host, so that primary infection usually goes unnoticed. The virus is not cleared, however, but stays for the lifetime of the host in a non-infectious, replicatively dormant state known as 'viral latency'. Medical interest in CMV results from the fact that latent virus can reactivate to cytopathogenic, tissue-destructive infection causing life-threatening end-organ disease in immunocompromised recipients of solid organ transplantation (SOT) or hematopoietic cell transplantation (HCT). It is becoming increasingly clear that CMV latency is not a static state in which the viral genome is silenced at all its genetic loci making the latent virus immunologically invisible, but rather is a dynamic state characterized by stochastic episodes of transient viral gene desilencing. This gene expression can lead to the presentation of antigenic peptides encoded by 'antigenicity-determining transcripts expressed in latency (ADTELs)' sensed by tissue-patrolling effector-memory CD8 T cells for immune surveillance of latency [In Reddehase et al., Murine model of cytomegalovirus latency and reactivation, Current Topics in Microbiology and Immunology, vol 325. Springer, Berlin, pp 315-331, 2008]. A hallmark of the CD8 T cell response to CMV is the observation that with increasing time during latency, CD8 T cells specific for certain viral epitopes increase in numbers, a phenomenon that has gained much attention in recent years and is known under the catchphrase 'memory inflation.' Here, we provide a unifying hypothesis linking stochastic viral gene desilencing during latency to 'memory inflation.'

Reversible inhibition of murine cytomegalovirus replication by gamma interferon (IFN-γ) in primary macrophages involves a primed type I IFN-signaling subnetwork for full establishment of an immediate-early antiviral state.

Activated macrophages play a central role in controlling inflammatory responses to infection and are tightly regulated to rapidly mount responses to infectious challenge. Type I interferon (alpha/beta interferon [IFN-α/ß]) and type II interferon (IFN-γ) play a crucial role in activating macrophages and subsequently restricting viral infections. Both types of IFNs signal through related but distinct signaling pathways, inducing a vast number of interferon-stimulated genes that are overlapping but distinguishable. The exact mechanism by which IFNs, particularly IFN-γ, inhibit DNA viruses such as cytomegalovirus (CMV) is still not fully understood. Here, we investigate the antiviral state developed in macrophages upon reversible inhibition of murine CMV by IFN-γ. On the basis of molecular profiling of the reversible inhibition, we identify a significant contribution of a restricted type I IFN subnetwork linked with IFN-γ activation. Genetic knockout of the type I-signaling pathway, in the context of IFN-γ stimulation, revealed an essential requirement for a primed type I-signaling process in developing a full refractory state in macrophages. A minimal transient induction of IFN-ß upon macrophage activation with IFN-γ is also detectable. In dose and kinetic viral replication inhibition experiments with IFN-γ, the establishment of an antiviral effect is demonstrated to occur within the first hours of infection. We show that the inhibitory mechanisms at these very early times involve a blockade of the viral major immediate-early promoter activity. Altogether our results show that a primed type I IFN subnetwork contributes to an immediate-early antiviral state induced by type II IFN activation of macrophages, with a potential further amplification loop contributed by transient induction of IFN-ß.

Reverse genetics modification of cytomegalovirus antigenicity and immunogenicity by CD8 T-cell epitope deletion and insertion.

The advent of cloning herpesviral genomes as bacterial artificial chromosomes (BACs) has made herpesviruses accessible to bacterial genetics and has thus revolutionised their mutagenesis. This opened all possibilities of reverse genetics to ask scientific questions by introducing precisely accurate mutations into the viral genome for testing their influence on the phenotype under study or to create phenotypes of interest. Here, we report on our experience with using BAC technology for a designed modulation of viral antigenicity and immunogenicity with focus on the CD8 T-cell response. One approach is replacing an intrinsic antigenic peptide in a viral carrier protein with a foreign antigenic sequence, a strategy that we have termed "orthotopic peptide swap". Another approach is the functional deletion of an antigenic peptide by point mutation of its C-terminal MHC class-I anchor residue. We discuss the concepts and summarize recently published major scientific results obtained with immunological mutants of murine cytomegalovirus.

Enhancerless cytomegalovirus is capable of establishing a low-level maintenance infection in severely immunodeficient host tissues but fails in exponential growth.

Major immediate-early transcriptional enhancers are genetic control elements that act, through docking with host transcription factors, as a decisive regulatory unit for efficient initiation of the productive virus cycle. Animal models are required for studying the function of enhancers paradigmatically in host organs. Here, we have sought to quantitatively assess the establishment, maintenance, and level of in vivo growth of enhancerless mutants of murine cytomegalovirus in comparison with those of an enhancer-bearing counterpart in models of the immunocompromised or immunologically immature host. Evidence is presented showing that enhancerless viruses are capable of forming restricted foci of infection but fail to grow exponentially.

Synergism between the components of the bipartite major immediate-early transcriptional enhancer of murine cytomegalovirus does not accelerate virus replication in cell culture and host tissues.

Major immediate-early (MIE) transcriptional enhancers of cytomegaloviruses are key regulators that are regarded as determinants of virus replicative fitness and pathogenicity. The MIE locus of murine cytomegalovirus (mCMV) shows bidirectional gene-pair architecture, with a bipartite enhancer flanked by divergent core promoters. Here, we have constructed recombinant viruses mCMV-DeltaEnh1 and mCMV-DeltaEnh2 to study the impact of either enhancer component on bidirectional MIE gene transcription and on virus replication in cell culture and various host tissues that are relevant to CMV disease. The data revealed that the two unipartite enhancers can operate independently, but synergize in enhancing MIE gene expression early after infection. Kick-start transcription facilitated by the bipartite enhancer configuration, however, did not ultimately result in accelerated virus replication. We conclude that virus replication, once triggered, proceeds with a fixed speed and we propose that synergism between the components of the bipartite enhancer may rather increase the probability for transcription initiation.