Exploiting Mucosal Immunity for Antiviral Vaccines.

Mucosal surfaces provide a remarkably effective barrier against potentially dangerous pathogens. Therefore, enhancing mucosal immunity through vaccines-strengthening that first line of defense-holds significant promise for reducing the burden of viral diseases. The large and varied class of viral pathogens, however, continues to present thorny challenges to vaccine development. Two primary difficulties exist: Viruses exhibit a stunning diversity of strategies for evading the host immune response, and even when we understand the nature of effective immune protection against a given virus, eliciting that protection is technically challenging. Only a few mucosal vaccines have surmounted these obstacles thus far. Recent developments, however, could greatly improve vaccine design. In this review, we first sketch out our understanding of mucosal immunity and then compare the herpes simplex virus, human immunodeficiency virus, and influenza virus to illustrate the distinct challenges of developing successful vaccines and to outline potential solutions.

An Epstein-Barr virus protein interaction map reveals NLRP3 inflammasome evasion via MAVS UFMylation.

Epstein-Barr virus (EBV) causes infectious mononucleosis, triggers multiple sclerosis, and is associated with 200,000 cancers/year. EBV colonizes the human B cell compartment and periodically reactivates, inducing expression of 80 viral proteins. However, much remains unknown about how EBV remodels host cells and dismantles key antiviral responses. We therefore created a map of EBV-host and EBV-EBV interactions in B cells undergoing EBV replication, uncovering conserved herpesvirus versus EBV-specific host cell targets. The EBV-encoded G-protein-coupled receptor BILF1 associated with MAVS and the UFM1 E3 ligase UFL1. Although UFMylation of 14-3-3 proteins drives RIG-I/MAVS signaling, BILF1-directed MAVS UFMylation instead triggered MAVS packaging into mitochondrial-derived vesicles and lysosomal proteolysis. In the absence of BILF1, EBV replication activated the NLRP3 inflammasome, which impaired viral replication and triggered pyroptosis. Our results provide a viral protein interaction network resource, reveal a UFM1-dependent pathway for selective degradation of mitochondrial cargo, and highlight BILF1 as a novel therapeutic target.

Functional landscape of SARS-CoV-2 cellular restriction.

A deficient interferon (IFN) response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been implicated as a determinant of severe coronavirus disease 2019 (COVID-19). To identify the molecular effectors that govern IFN control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human IFN-stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors inhibiting viral entry, RNA binding proteins suppressing viral RNA synthesis, and a highly enriched cluster of endoplasmic reticulum (ER)/Golgi-resident ISGs inhibiting viral assembly/egress. These included broad-acting antiviral ISGs and eight ISGs that specifically inhibited SARS-CoV-2 and SARS-CoV-1 replication. Among the broad-acting ISGs was BST2/tetherin, which impeded viral release and is antagonized by SARS-CoV-2 Orf7a protein. Overall, these data illuminate a set of ISGs that underlie innate immune control of SARS-CoV-2/SARS-CoV-1 infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.

Enhanced inhibition of MHC-I expression by SARS-CoV-2 Omicron subvariants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) possess mutations that confer resistance to neutralizing antibodies within the Spike protein and are associated with breakthrough infection and reinfection. By contrast, less is known about the escape from CD8+ T cell-mediated immunity by VOC. Here, we demonstrated that all SARS-CoV-2 VOCs possess the ability to suppress major histocompatibility complex class I (MHC-I) expression. We identified several viral genes that contribute to the suppression of MHC I expression. Notably, MHC-I upregulation was strongly inhibited after SARS-CoV-2 but not influenza virus infection in vivo. While earlier VOCs possess similar capacity as the ancestral strain to suppress MHC-I, the Omicron subvariants exhibited a greater ability to suppress surface MHC-I expression. We identified a common mutation in the E protein of Omicron that further suppressed MHC-I expression. Collectively, our data suggest that in addition to escaping from neutralizing antibodies, the success of Omicron subvariants to cause breakthrough infection and reinfection may in part be due to its optimized evasion from T cell recognition.

Viral evasion of the interferon response at a glance.

Re-emerging and new viral pathogens have caused significant morbidity and mortality around the world, as evidenced by the recent monkeypox, Ebola and Zika virus outbreaks and the ongoing COVID-19 pandemic. Successful viral infection relies on tactical viral strategies to derail or antagonize host innate immune defenses, in particular the production of type I interferons (IFNs) by infected cells. Viruses can thwart intracellular sensing systems that elicit IFN gene expression (that is, RIG-I-like receptors and the cGAS-STING axis) or obstruct signaling elicited by IFNs. In this Cell Science at a Glance article and the accompanying poster, we review the current knowledge about the major mechanisms employed by viruses to inhibit the activity of intracellular pattern-recognition receptors and their downstream signaling cascades leading to IFN-based antiviral host defenses. Advancing our understanding of viral immune evasion might spur unprecedented opportunities to develop new antiviral compounds or vaccines to prevent viral infectious diseases.

The crustacean DNA virus tegument protein VP26 binds to SNAP29 to inhibit SNARE complex assembly and autophagic degradation.

Autophagy generally functions as a cellular surveillance mechanism to combat invading viruses, but viruses have evolved various strategies to block autophagic degradation and even subvert it to promote viral propagation. White spot syndrome virus (WSSV) is the most highly pathogenic crustacean virus, but little is currently known about whether crustacean viruses such as WSSV can subvert autophagic degradation for escape. Here, we show that even though WSSV proliferation triggers the accumulation of autophagosomes, autophagic degradation is blocked in the crustacean species red claw crayfish. Interestingly, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex including CqSNAP29, CqVAMP7, and the novel autophagosome SNARE protein CqSyx12 is required for autophagic flux to restrict WSSV replication, as revealed by gene silencing experiments. Simultaneously, the expressed WSSV tegument protein VP26, which likely localizes on autophagic membrane mediated by its transmembrane region, binds the Qb-SNARE domain of CqSNAP29 to competitively inhibit the binding of CqSyx12-Qa-SNARE with CqSNAP29-Qb-SNARE; this in turn disrupts the assembly of the CqSyx12-SNAP29-VAMP7 SNARE complex, which is indispensable for the proposed fusion of autophagosomes and lysosomes. Consequently, the autophagic degradation of WSSV is likely suppressed by the expressed VP26 protein in vivo in crayfish, thus probably protecting WSSV components from degradation via the autophagosome-lysosome pathway, resulting in evasion by WSSV. Collectively, these findings highlight how a DNA virus can subvert autophagic degradation by impairing the assembly of the SNARE complex to achieve evasion, paving the way for understanding host-DNA virus interactions from an evolutionary point of view, from crustaceans to mammals.IMPORTANCEWhite spot syndrome virus (WSSV) is one of the largest animal DNA viruses in terms of its genome size and has caused huge economic losses in the farming of crustaceans such as shrimp and crayfish. Detailed knowledge of WSSV-host interactions is still lacking, particularly regarding viral escape from host immune clearance. Intriguingly, we found that the presence of WSSV-VP26 might inhibit the autophagic degradation of WSSV in vivo in the crustacean species red claw crayfish. Importantly, this study is the first to show that viral protein VP26 functions as a core factor to benefit WSSV escape by disrupting the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which is necessary for the proposed fusion of autophagosomes with lysosomes for subsequent degradation. These findings highlight a novel mechanism of DNA virus evasion by blocking SNARE complex assembly and identify viral VP26 as a key candidate for anti-WSSV targeting.

Viral pathogen-induced mechanisms to antagonize mammalian interferon (IFN) signaling pathway.

Antiviral responses of interferons (IFNs) are crucial in the host immune response, playing a relevant role in controlling viralw infections. Three types of IFNs, type I (IFN-α, IFN-ß), II (IFN-γ) and III (IFN-λ), are classified according to their receptor usage, mode of induction, biological activity and amino acid sequence. Here, we provide a comprehensive review of type I IFN responses and different mechanisms that viruses employ to circumvent this response. In the first part, we will give an overview of the different induction and signaling cascades induced in the cell by IFN-I after virus encounter. Next, highlights of some of the mechanisms used by viruses to counteract the IFN induction will be described. And finally, we will address different mechanism used by viruses to interference with the IFN signaling cascade and the blockade of IFN induced antiviral activities.

Tricks and threats of RNA viruses - towards understanding the fate of viral RNA.

Human innate cellular defence pathways have evolved to sense and eliminate pathogens, of which, viruses are considered one of the most dangerous. Their relatively simple structure makes the identification of viral invasion a difficult task for cells. In the course of evolution, viral nucleic acids have become one of the strongest and most reliable early identifiers of infection. When considering RNA virus recognition, RNA sensing is the central mechanism in human innate immunity, and effectiveness of this sensing is crucial for triggering an appropriate antiviral response. Although human cells are armed with a variety of highly specialized receptors designed to respond only to pathogenic viral RNA, RNA viruses have developed an array of mechanisms to avoid being recognized by human interferon-mediated cellular defence systems. The repertoire of viral evasion strategies is extremely wide, ranging from masking pathogenic RNA through end modification, to utilizing sophisticated techniques to deceive host cellular RNA degrading enzymes, and hijacking the most basic metabolic pathways in host cells. In this review, we aim to dissect human RNA sensing mechanisms crucial for antiviral immune defences, as well as the strategies adopted by RNA viruses to avoid detection and degradation by host cells. We believe that understanding the fate of viral RNA upon infection, and detailing the molecular mechanisms behind virus-host interactions, may be helpful for developing more effective antiviral strategies; which are urgently needed to prevent the far-reaching consequences of widespread, highly pathogenic viral infections.

Species-specific disruption of STING-dependent antiviral cellular defenses by the Zika virus NS2B3 protease.

The limited host tropism of numerous viruses causing disease in humans remains incompletely understood. One example is Zika virus (ZIKV), an RNA virus that has reemerged in recent years. Here, we demonstrate that ZIKV efficiently infects fibroblasts from humans, great apes, New and Old World monkeys, but not rodents. ZIKV infection in human-but not murine-cells impairs responses to agonists of the cGMP-AMP synthase/stimulator of IFN genes (cGAS/STING) signaling pathway, suggesting that viral mechanisms to evade antiviral defenses are less effective in rodent cells. Indeed, human, but not mouse, STING is subject to cleavage by proteases encoded by ZIKV, dengue virus, West Nile virus, and Japanese encephalitis virus, but not that of yellow fever virus. The protease cleavage site, located between positions 78/79 of human STING, is only partially conserved in nonhuman primates and rodents, rendering these orthologs resistant to degradation. Genetic disruption of STING increases the susceptibility of mouse-but not human-cells to ZIKV. Accordingly, expression of only mouse, not human, STING in murine STING knockout cells rescues the ZIKV suppression phenotype. STING-deficient mice, however, did not exhibit increased susceptibility, suggesting that other redundant antiviral pathways control ZIKV infection in vivo. Collectively, our data demonstrate that numerous RNA viruses evade cGAS/STING-dependent signaling and affirm the importance of this pathway in shaping the host range of ZIKV. Furthermore, our results explain-at least in part-the decreased permissivity of rodent cells to ZIKV, which could aid in the development of mice model with inheritable susceptibility to ZIKV and other flaviviruses.

Evasion of the Host Immune Response by Betaherpesviruses.

The human immune system boasts a diverse array of strategies for recognizing and eradicating invading pathogens. Human betaherpesviruses, a highly prevalent subfamily of viruses, include human cytomegalovirus (HCMV), human herpesvirus (HHV) 6A, HHV-6B, and HHV-7. These viruses have evolved numerous mechanisms for evading the host response. In this review, we will highlight the complex interplay between betaherpesviruses and the human immune response, focusing on protein function. We will explore methods by which the immune system first responds to betaherpesvirus infection as well as mechanisms by which viruses subvert normal cellular functions to evade the immune system and facilitate viral latency, persistence, and reactivation. Lastly, we will briefly discuss recent advances in vaccine technology targeting betaherpesviruses. This review aims to further elucidate the dynamic interactions between betaherpesviruses and the human immune system.

Intertwined: SAMHD1 cellular functions, restriction, and viral evasion strategies.

SAMHD1 was initially described for its ability to efficiently restrict HIV-1 replication in myeloid cells and resting CD4+ T cells. However, a growing body of evidence suggests that SAMHD1-mediated restriction is by far not limited to lentiviruses, but seems to be a general concept that applies to most retroviruses and at least a number of DNA viruses. SAMHD1 anti-viral activity was long believed to be solely due to its ability to deplete cellular dNTPs by enzymatic degradation. However, since its discovery, several new functions have been attributed to SAMHD1. It has been demonstrated to bind nucleic acids, to modulate innate immunity, as well as to participate in the DNA damage response and resolution of stalled replication forks. Consequently, it is likely that SAMHD1-mediated anti-viral activity is not or not exclusively mediated through its dNTPase activity. Therefore, in this review, we summarize current knowledge on SAMHD1 cellular functions and systematically discuss how these functions could contribute to the restriction of a broad range of viruses besides retroviruses: herpesviruses, poxviruses and hepatitis B virus. Furthermore, we aim to highlight different ways how viruses counteract SAMHD1-mediated restriction to bypass the SAMHD1-mediated block to viral infection.

GCRV hijacks TBK1 to evade IRF7-mediated antiviral immune responses in grass carp Ctenopharyngodon idella.

TANK-binding kinase 1 (TBK1) is an important kinase that regulates the activation of interferon regulatory factor 3/7 (IRF3/7) to induce type I interferon (IFN-I) production in antiviral immune responses. However, in long-term virus-host crosstalk, viruses have evolved elaborate strategies to evade host immune defense mechanisms. In the present study, we found that grass carp (Ctenopharyngodon idella) reovirus (GCRV) hijacks TBK1 to escape IRF7-IFN-Is signaling activation. In brief, GCRV inhibited TBK1 activation by restaining K63-linked ubiquitination of TBK1 and promoting its K48-linked ubiquitination. This regulation resulted in that under low titer of GCRV infection, TBK1 overexpression specifically supressed promoter activity and phosphorylation of IRF7 and induction of downstream IFN1and IFN3. qRT-PCR data uncovered that TBK1 negatively regulated IRF7, IFN1 and IFN3 transcription levels under low viral titer infection. Along with enhancement of GCRV titers, TBK1 swiched its function to up-regulate IRF7, IFN1 and IFN3 mRNA levels. Accordingly, TBK1 promoted GCRV replication at low infected titer, but inhibited GCRV replication at high infected titer. All these results revealed a viral evasion strategy that GCRV utilizes TBK1 to block cellular IFN responses at low titers or early stages in fish species, which will lay a foundation for further researching on host-virus interactions and developing novel antiviral strategies in lower vertebrates.

The evolution of natural killer cell receptors.

Natural killer (NK) cells are immune cells that play a crucial role against viral infections and tumors. To be tolerant against healthy tissue and simultaneously attack infected cells, the activity of NK cells is tightly regulated by a sophisticated array of germline-encoded activating and inhibiting receptors. The best characterized mechanism of NK cell activation is "missing self" detection, i.e., the recognition of virally infected or transformed cells that reduce their MHC expression to evade cytotoxic T cells. To monitor the expression of MHC-I on target cells, NK cells have monomorphic inhibitory receptors which interact with conserved MHC molecules. However, there are other NK cell receptors (NKRs) encoded by gene families showing a remarkable genetic diversity. Thus, NKR haplotypes contain several genes encoding for receptors with activating and inhibiting signaling, and that vary in gene content and allelic polymorphism. But if missing-self detection can be achieved by a monomorphic NKR system why have these polygenic and polymorphic receptors evolved? Here, we review the expansion of NKR receptor families in different mammal species, and we discuss several hypotheses that possibly underlie the diversification of the NK cell receptor complex, including the evolution of viral decoys, peptide sensitivity, and selective MHC-downregulation.

Serial Llama Immunization with Various SARS-CoV-2 RBD Variants Induces Broad Spectrum Virus-Neutralizing Nanobodies.

The emergence of SARS-CoV-2 mutant variants has posed a significant challenge to both the prevention and treatment of COVID-19 with anti-coronaviral neutralizing antibodies. The latest viral variants demonstrate pronounced resistance to the vast majority of human monoclonal antibodies raised against the ancestral Wuhan variant. Less is known about the susceptibility of the evolved virus to camelid nanobodies developed at the start of the pandemic. In this study, we compared nanobody repertoires raised in the same llama after immunization with Wuhan's RBD variant and after subsequent serial immunization with a variety of RBD variants, including that of SARS-CoV-1. We show that initial immunization induced highly potent nanobodies, which efficiently protected Syrian hamsters from infection with the ancestral Wuhan virus. These nanobodies, however, mostly lacked the activity against SARS-CoV-2 omicron-pseudotyped viruses. In contrast, serial immunization with different RBD variants resulted in the generation of nanobodies demonstrating a higher degree of somatic mutagenesis and a broad range of neutralization. Four nanobodies recognizing distinct epitopes were shown to potently neutralize a spectrum of omicron variants, including those of the XBB sublineage. Our data show that nanobodies broadly neutralizing SARS-CoV-2 variants may be readily induced by a serial variant RBD immunization.

Dengue virus exploits autophagy vesicles and secretory pathways to promote transmission by human dendritic cells.

Dengue virus (DENV), transmitted by infected mosquitoes, is a major public health concern, with approximately half the world's population at risk for infection. Recent decades have increasing incidence of dengue-associated disease alongside growing frequency of outbreaks. Although promising progress has been made in anti-DENV immunizations, post-infection treatment remains limited to non-specific supportive treatments. Development of antiviral therapeutics is thus required to limit DENV dissemination in humans and to help control the severity of outbreaks. Dendritic cells (DCs) are amongst the first cells to encounter DENV upon injection into the human skin mucosa, and thereafter promote systemic viral dissemination to additional human target cells. Autophagy is a vesicle trafficking pathway involving the formation of cytosolic autophagosomes, and recent reports have highlighted the extensive manipulation of autophagy by flaviviruses, including DENV, for viral replication. However, the temporal profiling and function of autophagy activity in DENV infection and transmission by human primary DCs remains poorly understood. Herein, we demonstrate that mechanisms of autophagosome formation and extracellular vesicle (EV) release have a pro-viral role in DC-mediated DENV transmission. We show that DENV exploits early-stage canonical autophagy to establish infection in primary human DCs. DENV replication enhanced autophagosome formation in primary human DCs, and intrinsically-heightened autophagosome biogenesis correlated with relatively higher rates of DC susceptibility to DENV. Furthermore, our data suggest that viral replication intermediates co-localize with autophagosomes, while productive DENV infection introduces a block at the late degradative stages of autophagy in infected DCs but not in uninfected bystander cells. Notably, we identify for the first time that approximately one-fourth of DC-derived CD9/CD81/CD63+ EVs co-express canonical autophagy marker LC3, and demonstrate that DC-derived EV populations are an alternative, cell-free mechanism by which DCs promote DENV transmission to additional target sites. Taken together, our study highlights intersections between autophagy and secretory pathways during viral infection, and puts forward autophagosome accumulation and viral RNA-laden EVs as host determinants of DC-mediated DENV infection in humans. Host-directed therapeutics targeting autophagy and exocytosis pathways thus have potential to enhance DC-driven resistance to DENV acquisition and thereby limit viral dissemination by initial human target cells following mosquito-to-human transmission of DENV.

Downregulation of miRNA-26a by HIV-1 Enhances CD59 Expression and Packaging, Impacting Virus Susceptibility to Antibody-Dependent Complement-Mediated Lysis.

MicroRNAs (miRNAs) play important roles in the control of HIV-1 infection. Here, we performed RNA-seq profiling of miRNAs and mRNAs expressed in CD4+ T lymphocytes upon HIV-1 infection. Our results reveal significant alterations in miRNA and mRNA expression profiles in infected relative to uninfected cells. One of the miRNAs markedly downregulated in infected cells is miRNA-26a. Among the putative targets of miRNA-26a are CD59 receptor transcripts, which are significantly upregulated in infected CD4+ T cells. The addition of miRNA-26a mimics to CD4+ T cells reduces CD59 at both the mRNA and surface protein levels, validating CD59 as a miRNA-26a target. Consistent with the reported inhibitory role of CD59 in complement-mediated lysis (CML), knocking out CD59 in CD4+ T cells renders both HIV-1-infected cells and progeny virions more prone to antibody-dependent CML (ADCML). The addition of miRNA-26a mimics to infected cells leads to enhanced sensitivity of progeny virions to ADCML, a condition linked to a reduction in CD59 packaging into released virions. Lastly, HIV-1-mediated downregulation of miRNA-26a expression is shown to be dependent on integrated HIV-1 expression but does not involve viral accessory proteins. Overall, these results highlight a novel mechanism by which HIV-1 limits ADCML by upregulating CD59 expression via miRNA-26a downmodulation.

ISG15: its roles in SARS-CoV-2 and other viral infections.

Interferon (IFN)-stimulated gene 15 (ISG15), a ubiquitin-like pleiotropic protein and one of the most abundant ISGs, has been studied extensively; however, its roles in SARS-CoV-2 and other viral infections have just begun to be elucidated. Emerging evidence suggests that ISG15 - either in its conjugated or unconjugated 'free' form - acts both intracellularly and extracellularly, and exerts anti- or pro-viral effects. To counteract ISG15's antiviral roles, viruses have evolved sophisticated tactics. Here, we discuss recent advances in ISG15's physiological functions as a post-translational modifier or 'cytokine-like' molecule during SARS-CoV-2 and other viral infections. Furthermore, we highlight the detailed mechanisms viruses use to block ISG15-dependent antiviral defenses. A comprehensive understanding of ISG15 biology in the context of virus infection may spur new therapeutic approaches for a range of viral infectious diseases.

Detection of Chemokine Binding Proteins Association to Cell Surface Glycosaminoglycans by Flow Cell Cytometry and Indirect Immunofluorescence.

Viruses encode secreted proteins that bind chemokines to modulate their activity. Viral proteins may simultaneously interact with glycosaminoglycans allowing these proteins to be anchored at the cell surface to increase their anti-chemokine activity in the proximity of infection. Here we describe methodology to evaluate the interaction of viral secreted proteins with cell-surface glycosaminoglycans by immunofluorescence and detection by flow cytometry or microscopy. These methods could be equally applied to other chemokine binding proteins that do not have viral origin.

Interplay between RNA viruses and cGAS/STING axis in innate immunity.

While the function of cGAS/STING signalling axis in the innate immune response to DNA viruses is well deciphered, increasing evidence demonstrates its significant contribution in the control of RNA virus infections. After the first evidence of cGAS/STING antagonism by flaviviruses, STING activation has been detected following infection by various enveloped RNA viruses. It has been discovered that numerous viral families have implemented advanced strategies to antagonize STING pathway through their evolutionary path. This review summarizes the characterized cGAS/STING escape strategies to date, together with the proposed mechanisms of STING signalling activation perpetrated by RNA viruses and discusses possible therapeutic approaches. Further studies regarding the interaction between RNA viruses and cGAS/STING-mediated immunity could lead to major discoveries important for the understanding of immunopathogenesis and for the treatment of RNA viral infections.

Insights on the cGAS-STING Signaling Pathway During Herpesvirus Infections.

Herpesviruses belong to large double-stranded DNA viruses. They are under a wide range of hosts and establish lifelong infection, which creates a burden on human health and animal health. Innate immunity is the host's innate defense ability. Activating the innate immune signaling pathway and producing type I interferon is the host's first line of defense against infectious pathogens. Emerging evidence indicates that the cGAS-STING signaling pathway plays an important role in the innate immunity in response to herpesvirus infections. In parallel, because of the constant selective pressure imposed by host immunity, herpesvirus also evolves to target the cGAS-STING signaling pathway to inhibit or escape the innate immune responses. In the current review, we insight on the classical cGAS-STING signaling pathway. We describe the activation of cGAS-STING signaling pathway during herpesvirus infections and strategies of herpesvirus targeting this pathway to evade host antiviral response. Furthermore, we outline the immunotherapy boosting cGAS-STING signaling pathway.