The epitope arrangement on flavivirus particles contributes to Mab C10's extraordinary neutralization breadth across Zika and dengue viruses.

The human monoclonal antibody C10 exhibits extraordinary cross-reactivity, potently neutralizing Zika virus (ZIKV) and the four serotypes of dengue virus (DENV1-DENV4). Here we describe a comparative structure-function analysis of C10 bound to the envelope (E) protein dimers of the five viruses it neutralizes. We demonstrate that the C10 Fab has high affinity for ZIKV and DENV1 but not for DENV2, DENV3, and DENV4. We further show that the C10 interaction with the latter viruses requires an E protein conformational landscape that limits binding to only one of the three independent epitopes per virion. This limited affinity is nevertheless counterbalanced by the particle's icosahedral organization, which allows two different dimers to be reached by both Fab arms of a C10 immunoglobulin. The epitopes' geometric distribution thus confers C10 its exceptional neutralization breadth. Our results highlight the importance not only of paratope/epitope complementarity but also the topological distribution for epitope-focused vaccine design.

Travel Surveillance and Genomics Uncover a Hidden Zika Outbreak during the Waning Epidemic.

The Zika epidemic in the Americas has challenged surveillance and control. As the epidemic appears to be waning, it is unclear whether transmission is still ongoing, which is exacerbated by discrepancies in reporting. To uncover locations with lingering outbreaks, we investigated travel-associated Zika cases to identify transmission not captured by reporting. We uncovered an unreported outbreak in Cuba during 2017, a year after peak transmission in neighboring islands. By sequencing Zika virus, we show that the establishment of the virus was delayed by a year and that the ensuing outbreak was sparked by long-lived lineages of Zika virus from other Caribbean islands. Our data suggest that, although mosquito control in Cuba may initially have been effective at mitigating Zika virus transmission, such measures need to be maintained to be effective. Our study highlights how Zika virus may still be "silently" spreading and provides a framework for understanding outbreak dynamics. VIDEO ABSTRACT.

Comparative Flavivirus-Host Protein Interaction Mapping Reveals Mechanisms of Dengue and Zika Virus Pathogenesis.

Mosquito-borne flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), are a growing public health concern. Systems-level analysis of how flaviviruses hijack cellular processes through virus-host protein-protein interactions (PPIs) provides information about their replication and pathogenic mechanisms. We used affinity purification-mass spectrometry (AP-MS) to compare flavivirus-host interactions for two viruses (DENV and ZIKV) in two hosts (human and mosquito). Conserved virus-host PPIs revealed that the flavivirus NS5 protein suppresses interferon stimulated genes by inhibiting recruitment of the transcription complex PAF1C and that chemical modulation of SEC61 inhibits DENV and ZIKV replication in human and mosquito cells. Finally, we identified a ZIKV-specific interaction between NS4A and ANKLE2, a gene linked to hereditary microcephaly, and showed that ZIKV NS4A causes microcephaly in Drosophila in an ANKLE2-dependent manner. Thus, comparative flavivirus-host PPI mapping provides biological insights and, when coupled with in vivo models, can be used to unravel pathogenic mechanisms.

Initiation of Antiviral B Cell Immunity Relies on Innate Signals from Spatially Positioned NKT Cells.

B cells constitute an essential line of defense from pathogenic infections through the generation of class-switched antibody-secreting cells (ASCs) in germinal centers. Although this process is known to be regulated by follicular helper T (TfH) cells, the mechanism by which B cells initially seed germinal center reactions remains elusive. We found that NKT cells, a population of innate-like T lymphocytes, are critical for the induction of B cell immunity upon viral infection. The positioning of NKT cells at the interfollicular areas of lymph nodes facilitates both their direct priming by resident macrophages and the localized delivery of innate signals to antigen-experienced B cells. Indeed, NKT cells secrete an early wave of IL-4 and constitute up to 70% of the total IL-4-producing cells during the initial stages of infection. Importantly, the requirement of this innate immunity arm appears to be evolutionarily conserved because early NKT and IL-4 gene signatures also positively correlate with the levels of neutralizing antibodies in Zika-virus-infected macaques. In conclusion, our data support a model wherein a pre-TfH wave of IL-4 secreted by interfollicular NKT cells triggers the seeding of germinal center cells and serves as an innate link between viral infection and B cell immunity.

Recurrent Potent Human Neutralizing Antibodies to Zika Virus in Brazil and Mexico.

Antibodies to Zika virus (ZIKV) can be protective. To examine the antibody response in individuals who develop high titers of anti-ZIKV antibodies, we screened cohorts in Brazil and Mexico for ZIKV envelope domain III (ZEDIII) binding and neutralization. We find that serologic reactivity to dengue 1 virus (DENV1) EDIII before ZIKV exposure is associated with increased ZIKV neutralizing titers after exposure. Antibody cloning shows that donors with high ZIKV neutralizing antibody titers have expanded clones of memory B cells that express the same immunoglobulin VH3-23/VK1-5 genes. These recurring antibodies cross-react with DENV1, but not other flaviviruses, neutralize both DENV1 and ZIKV, and protect mice against ZIKV challenge. Structural analyses reveal the mechanism of recognition of the ZEDIII lateral ridge by VH3-23/VK1-5 antibodies. Serologic testing shows that antibodies to this region correlate with serum neutralizing activity to ZIKV. Thus, high neutralizing responses to ZIKV are associated with pre-existing reactivity to DENV1 in humans.

Modified mRNA Vaccines Protect against Zika Virus Infection.

The emergence of ZIKV infection has prompted a global effort to develop safe and effective vaccines. We engineered a lipid nanoparticle (LNP) encapsulated modified mRNA vaccine encoding wild-type or variant ZIKV structural genes and tested immunogenicity and protection in mice. Two doses of modified mRNA LNPs encoding prM-E genes that produced virus-like particles resulted in high neutralizing antibody titers (∼1/100,000) that protected against ZIKV infection and conferred sterilizing immunity. To offset a theoretical concern of ZIKV vaccines inducing antibodies that cross-react with the related dengue virus (DENV), we designed modified prM-E RNA encoding mutations destroying the conserved fusion-loop epitope in the E protein. This variant protected against ZIKV and diminished production of antibodies enhancing DENV infection in cells or mice. A modified mRNA vaccine can prevent ZIKV disease and be adapted to reduce the risk of sensitizing individuals to subsequent exposure to DENV, should this become a clinically relevant concern.

Zika Virus Causes Testis Damage and Leads to Male Infertility in Mice.

Zika virus (ZIKV) persists in the semen of male patients, a first for flavivirus infection. Here, we demonstrate that ZIKV can induce inflammation in the testis and epididymidis, but not in the prostate or seminal vesicle, and can lead to damaged testes after 60 days post-infection in mice. ZIKV induces innate immune responses in Leydig, Sertoli, and epididymal epithelial cells, resulting in the production of pro-inflammatory cytokines/chemokines. However, ZIKV does not induce a rapid and abundant cytokine production in peritubular cell and spermatogonia, suggesting that these cells are vulnerable for ZIKV infection and could be the potential repositories for ZIKV. Our study demonstrates a correlation between ZIKV and testis infection/damage and suggests that ZIKV infection, under certain circumstances, can eventually lead to male infertility.

Viperin triggers ribosome collision-dependent translation inhibition to restrict viral replication.

Innate immune responses induce hundreds of interferon-stimulated genes (ISGs). Viperin, a member of the radical S-adenosyl methionine (SAM) superfamily of enzymes, is the product of one such ISG that restricts the replication of a broad spectrum of viruses. Here, we report a previously unknown antiviral mechanism in which viperin activates a ribosome collision-dependent pathway that inhibits both cellular and viral RNA translation. We found that the radical SAM activity of viperin is required for translation inhibition and that this is mediated by viperin's enzymatic product, 3'-deoxy-3',4'-didehydro-CTP (ddhCTP). Viperin triggers ribosome collisions and activates the MAPKKK ZAK pathway that in turn activates the GCN2 arm of the integrated stress response pathway to inhibit translation. The study illustrates the importance of translational repression in the antiviral response and identifies viperin as a translation regulator in innate immunity.

Affinity-Restricted Memory B Cells Dominate Recall Responses to Heterologous Flaviviruses.

Memory B cells (MBCs) can respond to heterologous antigens either by molding new specificities through secondary germinal centers (GCs) or by selecting preexisting clones without further affinity maturation. To distinguish these mechanisms in flavivirus infections and immunizations, we studied recall responses to envelope protein domain III (DIII). Conditional deletion of activation-induced cytidine deaminase (AID) between heterologous challenges of West Nile, Japanese encephalitis, Zika, and dengue viruses did not affect recall responses. DIII-specific MBCs were contained mostly within the plasma-cell-biased CD80+ subset, and few GCs arose following heterologous boosters, demonstrating that recall responses are confined by preexisting clonal diversity. Measurement of monoclonal antibody (mAb) binding affinity to DIII proteins, timed AID deletion, single-cell RNA sequencing, and lineage tracing experiments point to selection of relatively low-affinity MBCs as a mechanism to promote diversity. Engineering immunogens to avoid this MBC diversity may facilitate flavivirus-type-specific vaccines with minimized potential for infection enhancement.

Dengue Virus Immunity Increases Zika Virus-Induced Damage during Pregnancy.

Zika virus (ZIKV) has recently been associated with birth defects and pregnancy loss after maternal infection. Because dengue virus (DENV) and ZIKV co-circulate, understanding the role of antibody-dependent enhancement in the context of pregnancy is critical. Here, we showed that the presence of DENV-specific antibodies in ZIKV-infected pregnant mice significantly increased placental damage, fetal growth restriction, and fetal resorption. This was associated with enhanced viral replication in the placenta that coincided with an increased frequency of infected trophoblasts. ZIKV-infected human placental tissues also showed increased replication in the presence of DENV antibodies, which was reversed by FcγR blocking antibodies. Furthermore, ZIKV-mediated fetal pathogenesis was enhanced in mice in the presence of a DENV-reactive monoclonal antibody, but not in the presence of the LALA variant, indicating a dependence on FcγR engagement. Our data suggest a possible mechanism for the recent increase in severe pregnancy outcomes after ZIKV infection in DENV-endemic areas.

Mouse circulating extracellular vesicles contain virus-derived siRNAs active in antiviral immunity.

Induction and suppression of antiviral RNA interference (RNAi) has been observed in mammals during infection with at least seven distinct RNA viruses, including some that are pathogenic in humans. However, while the cell-autonomous immune response mediated by antiviral RNAi is gradually being recognized, little is known about systemic antiviral RNAi in mammals. Furthermore, extracellular vesicles (EVs) also function in viral signal spreading and host immunity. Here, we show that upon antiviral RNAi activation, virus-derived small-interfering RNAs (vsiRNAs) from Nodamura virus (NoV), Sindbis virus (SINV), and Zika virus (ZIKV) enter the murine bloodstream via EVs for systemic circulation. vsiRNAs in the EVs are biologically active, since they confer RNA-RNA homology-dependent antiviral activity in both cultured cells and infant mice. Moreover, we demonstrate that vaccination with a live-attenuated virus, rendered deficient in RNAi suppression, induces production of stably maintained vsiRNAs and confers protective immunity against virus infection in mice. This suggests that vaccination with live-attenuated VSR (viral suppressor of RNAi)-deficient mutant viruses could be a new strategy to induce immunity.

Host caveolin-1 facilitates Zika virus infection by promoting viral RNA replication.

Zika virus (ZIKV) has gained notoriety in recent years because there are no targeted therapies or vaccines available so far. Caveolin-1 (Cav-1) in host cells plays crucial functions in the invasion of many viruses. However, its specific involvement in ZIKV infection has remained unclear. Here, we reveal that depleting Cav-1 leads to a substantial reduction in ZIKV RNA levels, protein expression and viral particle production, indicating that ZIKV exploits Cav-1 for its infection. By dissecting each stage of the viral life cycle, we unveil that, unlike its invasion role in many other viruses, Cav-1 depletion selectively impairs ZIKV replication, resulting in altered replication dynamics and reduced strand-specific RNA levels, but does not affect viral entry, maturation and release. These results reveal an unforeseen function of Cav-1 in facilitating ZIKV replication, which provides new insights into the intricate interaction between Cav-1 and ZIKV and underscores Cav-1 as a potential candidate for anti-ZIKV approaches.

High-resolution RNA tertiary structures in Zika virus stem-loop A for the development of inhibitory small molecules.

Flaviviruses such as Zika (ZIKV) and dengue virus (DENV) are positive-sense RNA viruses belonging to Flaviviridae The flavivirus genome contains a 5' end stem-loop promoter sequence known as stem-loop A (SLA) that is recognized by the flavivirus polymerase NS5 during viral RNA synthesis and 5' guanosine cap methylation. The crystal structures of ZIKV and DENV SLAs show a well-defined fold, consisting of a bottom stem, side loop, and top stem-loop, providing unique interaction sites for small molecule inhibitors to disrupt the promoter function. To facilitate the identification of small molecule binding sites in flavivirus SLA, we determined high-resolution structures of the bottom and top stems of ZIKV SLA, which contain a single U- or G-bulge, respectively. Both bulge nucleotides exhibit multiple orientations, from folded back on the adjacent nucleotide to flipped out of the helix, and are stabilized by stacking or base triple interactions. These structures suggest that even a single unpaired nucleotide can provide flexibility to RNA structures, and its conformation is mainly determined by the stabilizing chemical environment. To facilitate discovery of small molecule inhibitors that interfere with the functions of ZIKV SLA, we screened and identified compounds that bind to the bottom and top stems of ZIKV SLA.

Pathogenesis of viral infections during pregnancy.

SUMMARYViral infections during pregnancy are associated with significant adverse perinatal and fetal outcomes. Pregnancy is a unique immunologic and physiologic state, which can influence control of virus replication, severity of disease, and vertical transmission. The placenta is the organ of the maternal-fetal interface and provides defense against microbial infection while supporting the semi-allogeneic fetus via tolerogenic immune responses. Some viruses, such as cytomegalovirus, Zika virus, and rubella virus, can breach these defenses, directly infecting the fetus and having long-lasting consequences. Even without direct placental infection, other viruses, including respiratory viruses like influenza viruses and severe acute respiratory syndrome coronavirus 2, still cause placental damage and inflammation. Concentrations of progesterone and estrogens rise during pregnancy and contribute to immunological adaptations, placentation, and placental development and play a pivotal role in creating a tolerogenic environment at the maternal-fetal interface. Animal models, including mice, nonhuman primates, rabbits, and guinea pigs, are instrumental for mechanistic insights into the pathogenesis of viral infections during pregnancy and identification of targetable treatments to improve health outcomes of pregnant individuals and offspring.

Molecular Determinants of Flavivirus Virion Assembly.

Virion assembly is an important step in the life cycle of all viruses. For viruses of the Flavivirus genus, a group of enveloped positive-sense RNA viruses, the assembly step represents one of the least understood processes in the viral life cycle. While assembly is primarily driven by the viral structural proteins, recent studies suggest that several nonstructural proteins also play key roles in coordinating the assembly and packaging of the viral genome. This review focuses on describing recent advances in our understanding of flavivirus virion assembly, including the intermolecular interactions between the viral structural (capsid) and nonstructural proteins (NS2A and NS2B-NS3), host factors, as well as features of the viral genomic RNA required for efficient flavivirus virion assembly.

Actomyosin-dependent cell contractility orchestrates Zika virus infection.

Emerging pathogen infections, such as Zika virus (ZIKV), pose an increasing threat to human health, but the role of mechanobiological attributes of host cells during ZIKV infection is largely unknown. Here, we reveal that ZIKV infection leads to increased contractility of host cells. Importantly, we investigated whether host cell contractility contributes to ZIKV infection efficacy, from both the intracellular and extracellular perspective. By performing drug perturbation and gene editing experiments, we confirmed that disruption of contractile actomyosin compromises ZIKV infection efficiency, viral genome replication and viral particle production. By culturing on compliant matrix, we further demonstrate that a softer substrate, leading to less contractility of host cells, compromises ZIKV infection, which resembles the effects of disrupting intracellular actomyosin organization. Together, our work provides evidence to support a positive correlation between host cell contractility and ZIKV infection efficacy, thus unveiling an unprecedented layer of interplay between ZIKV and the host cell.

The fetal lineage is susceptible to Zika virus infection within days of fertilization.

Adults contracting Zika virus (ZIKV) typically exhibit mild symptoms, yet ZIKV infection of pregnant individuals can cause miscarriage or birth defects in their offspring. Many studies have focused on maternal-to-fetal ZIKV transmission via blood and placenta. Notably, however, ZIKV is also transmitted sexually, raising the possibility that ZIKV could infect the embryo shortly after fertilization, long before the placenta is established. Here, we evaluate the consequences of ZIKV infection in mouse embryos during the first few days of embryogenesis. We show that divergent strains of ZIKV can infect the fetal lineage and can cause developmental arrest, raising concern for the developmental consequences of sexual ZIKV transmission. This article has an associated 'The people behind the papers' interview.

The subgenomic flaviviral RNA suppresses RNA interference through competing with siRNAs for binding RISC components.

During the life cycle of mosquito-borne flaviviruses, substantial subgenomic flaviviral RNA (sfRNA) is produced via incomplete degradation of viral genomic RNA by host XRN1. Zika virus (ZIKV) sfRNA has been detected in mosquito and mammalian somatic cells. Human neural progenitor cells (hNPCs) in the developing brain are the major target cells of ZIKV, and antiviral RNA interference (RNAi) plays a critical role in hNPCs. However, whether ZIKV sfRNA was produced in ZIKV-infected hNPCs as well as its function remains not known. In this study, we demonstrate that abundant sfRNA was produced in ZIKV-infected hNPCs. RNA pulldown and mass spectrum assays showed ZIKV sfRNA interacted with host proteins RHA and PACT, both of which are RNA-induced silencing complex (RISC) components. Functionally, ZIKV sfRNA can antagonize RNAi by outcompeting small interfering RNAs (siRNAs) in binding to RHA and PACT. Furthermore, the 3' stem loop (3'SL) of sfRNA was responsible for RISC components binding and RNAi inhibition, and 3'SL can enhance the replication of a viral suppressor of RNAi (VSR)-deficient virus in a RHA- and PACT-dependent manner. More importantly, the ability of binding to RISC components is conversed among multiple flaviviral 3'SLs. Together, our results identified flavivirus 3'SL as a potent VSR in RNA format, highlighting the complexity in virus-host interaction during flavivirus infection.IMPORTANCEZika virus (ZIKV) infection mainly targets human neural progenitor cells (hNPCs) and induces cell death and dysregulated cell-cycle progression, leading to microcephaly and other central nervous system abnormalities. RNA interference (RNAi) plays critical roles during ZIKV infections in hNPCs, and ZIKV has evolved to encode specific viral proteins to antagonize RNAi. Herein, we first show that abundant sfRNA was produced in ZIKV-infected hNPCs in a similar pattern to that in other cells. Importantly, ZIKV sfRNA acts as a potent viral suppressor of RNAi (VSR) by competing with siRNAs for binding RISC components, RHA and PACT. The 3'SL of sfRNA is responsible for binding RISC components, which is a conserved feature among mosquito-borne flaviviruses. As most known VSRs are viral proteins, our findings highlight the importance of viral non-coding RNAs during the antagonism of host RNAi-based antiviral innate immunity.

Single-cell analysis reveals an antiviral network that controls Zika virus infection in human dendritic cells.

Zika virus (ZIKV) is a mosquito-borne flavivirus that caused an epidemic in the Americas in 2016 and is linked to severe neonatal birth defects, including microcephaly and spontaneous abortion. To better understand the host response to ZIKV infection, we adapted the 10× Genomics Chromium single-cell RNA sequencing (scRNA-seq) assay to simultaneously capture viral RNA and host mRNA. Using this assay, we profiled the antiviral landscape in a population of human monocyte-derived dendritic cells infected with ZIKV at the single-cell level. The bystander cells, which lacked detectable viral RNA, expressed an antiviral state that was enriched for genes coinciding predominantly with a type I interferon (IFN) response. Within the infected cells, viral RNA negatively correlated with type I IFN-dependent and -independent genes (the antiviral module). We modeled the ZIKV-specific antiviral state at the protein level, leveraging experimentally derived protein interaction data. We identified a highly interconnected network between the antiviral module and other host proteins. In this work, we propose a new paradigm for evaluating the antiviral response to a specific virus, combining an unbiased list of genes that highly correlate with viral RNA on a per-cell basis with experimental protein interaction data. IMPORTANCE: Zika virus (ZIKV) remains a public health threat given its potential for re-emergence and the detrimental fetal outcomes associated with infection during pregnancy. Understanding the dynamics between ZIKV and its host is critical to understanding ZIKV pathogenesis. Through ZIKV-inclusive single-cell RNA sequencing (scRNA-seq), we demonstrate on the single-cell level the dynamic interplay between ZIKV and the host: the transcriptional program that restricts viral infection and ZIKV-mediated inhibition of that response. Our ZIKV-inclusive scRNA-seq assay will serve as a useful tool for gaining greater insight into the host response to ZIKV and can be applied more broadly to the flavivirus field.

LINC08148 promotes the caveola-mediated endocytosis of Zika virus through upregulating transcription of Src.

Long non-coding RNAs (lncRNAs) represent a new group of host factors involved in viral infection. Current study identified an intergenic lncRNA, LINC08148, as a proviral factor of Zika virus (ZIKV) and Dengue virus 2 (DENV2). Knockout (KO) or silencing of LINC08148 decreases the replication of ZIKV and DENV2. LINC08148 mainly acts at the endocytosis step of ZIKV but at a later stage of DENV2. RNA-seq analysis reveals that LINC08148 knockout downregulates the transcription levels of five endocytosis-related genes including AP2B1, CHMP4C, DNM1, FCHO1, and Src. Among them, loss of Src significantly decreases the uptake of ZIKV. Trans-complementation of Src in the LINC08148KO cells largely restores the caveola-mediated endocytosis of ZIKV, indicating that the proviral effect of LINC08148 is exerted through Src. Finally, LINC08148 upregulates the Src transcription through associating with its transcription factor SP1. This work establishes an essential role of LINC08148 in the ZIKV entry, underscoring a significance of lncRNAs in the viral infection. IMPORTANCE: Long non-coding RNAs (lncRNAs), like proteins, participate in viral infection. However, functions of most lncRNAs remain unknown. In this study, we performed a functional screen based on microarray data and identified a new proviral lncRNA, LINC08148. Then, we uncovered that LINC08148 is involved in the caveola-mediated endocytosis of ZIKV, rather than the classical clathrin-mediated endocytosis. Mechanistically, LINC08148 upregulates the transcription of Src, an initiator of caveola-mediated endocytosis, through binding to its transcription factor SP1. This study identifies a new lncRNA involved in the ZIKV infection, suggesting lncRNAs and cellular proteins are closely linked and cooperate to regulate viral infection.