Nuclear accumulation of host transcripts during Zika Virus Infection.

Zika virus (ZIKV) infects fetal neural progenitor cells (NPCs) causing severe neurodevelopmental disorders in utero. Multiple pathways involved in normal brain development are dysfunctional in infected NPCs but how ZIKV centrally reprograms these pathways remains unknown. Here we show that ZIKV infection disrupts subcellular partitioning of host transcripts critical for neurodevelopment in NPCs and functionally link this process to the up-frameshift protein 1 (UPF1). UPF1 is an RNA-binding protein known to regulate decay of cellular and viral RNAs and is less expressed in ZIKV-infected cells. Using infrared crosslinking immunoprecipitation and RNA sequencing (irCLIP-Seq), we show that a subset of mRNAs loses UPF1 binding in ZIKV-infected NPCs, consistent with UPF1's diminished expression. UPF1 target transcripts, however, are not altered in abundance but in subcellular localization, with mRNAs accumulating in the nucleus of infected or UPF1 knockdown cells. This leads to diminished protein expression of FREM2, a protein required for maintenance of NPC identity. Our results newly link UPF1 to the regulation of mRNA transport in NPCs, a process perturbed during ZIKV infection.

Dengue virus induces and requires glycolysis for optimal replication.

UNLABELLED: Viruses rely on host cellular metabolism to provide the energy and biosynthetic building blocks required for their replication. Dengue virus (DENV), a member of the Flaviviridae family, is one of the most important arthropod-borne human pathogens worldwide. We analyzed global intracellular metabolic changes associated with DENV infection of primary human cells. Our metabolic profiling data suggested that central carbon metabolism, particularly glycolysis, is strikingly altered during a time course of DENV infection. Glucose consumption is increased during DENV infection and depriving DENV-infected cells of exogenous glucose had a pronounced impact on viral replication. Furthermore, the expression of both glucose transporter 1 and hexokinase 2, the first enzyme of glycolysis, is upregulated in DENV-infected cells. Pharmacologically inhibiting the glycolytic pathway dramatically reduced DENV RNA synthesis and infectious virion production, revealing a requirement for glycolysis during DENV infection. Thus, these experiments suggest that DENV induces the glycolytic pathway to support efficient viral replication. This study raises the possibility that metabolic inhibitors, such as those that target glycolysis, could be used to treat DENV infection in the future. IMPORTANCE: Approximately 400 million people are infected with dengue virus (DENV) annually, and more than one-third of the global population is at risk of infection. As there are currently no effective vaccines or specific antiviral therapies for DENV, we investigated the impact DENV has on the host cellular metabolome to identify metabolic pathways that are critical for the virus life cycle. We report an essential role for glycolysis during DENV infection. DENV activates the glycolytic pathway, and inhibition of glycolysis significantly blocks infectious DENV production. This study provides further evidence that viral metabolomic analyses can lead to the discovery of novel therapeutic targets to block the replication of medically important human pathogens.

Vaccinia virus requires glutamine but not glucose for efficient replication.

UNLABELLED: Viruses require host cell metabolism to provide the necessary energy and biosynthetic precursors for successful viral replication. Vaccinia virus (VACV) is a member of the Poxviridae family, and its use as a vaccine enabled the eradication of variola virus, the etiologic agent of smallpox. A global metabolic screen of VACV-infected primary human foreskin fibroblasts suggested that glutamine metabolism is altered during infection. Glutamine and glucose represent the two main carbon sources for mammalian cells. Depriving VACV-infected cells of exogenous glutamine led to a substantial decrease in infectious virus production, whereas starving infected cells of exogenous glucose had no significant impact on replication. Viral yield in glutamine-deprived cells or in cells treated with an inhibitor of glutaminolysis, the pathway of glutamine catabolism, could be rescued by the addition of multiple tricarboxylic acid (TCA) cycle intermediates. Thus, VACV infection induces a metabolic alteration to fully rely on glutamine to anaplerotically maintain the TCA cycle. VACV protein synthesis, but not viral transcription, was decreased in glutamine-deprived cells, which corresponded with a dramatic reduction in all VACV morphogenetic intermediates. This study reveals the unique carbon utilization program implemented during poxvirus infection and provides a potential metabolic pathway to target viral replication. IMPORTANCE: Viruses are dependent on the metabolic machinery of the host cell to supply the energy and molecular building blocks needed for critical processes including genome replication, viral protein synthesis, and membrane production. This study investigates how vaccinia virus (VACV) infection alters global cellular metabolism, providing the first metabolomic analysis for a member of the poxvirus family. Unlike most viruses examined to date, VACV does not activate glycolysis, and exogenous glucose is not required for maximal virus production. Instead, VACV requires exogenous glutamine for efficient replication, and inhibition of glutamine metabolism effectively blocks VACV protein synthesis. This study defines a major metabolic perturbation essential for the replication of a poxvirus and may lead to the discovery of novel antiviral therapies based on metabolic inhibitors.

NF-κB inhibitor alpha controls SARS-CoV-2 infection in ACE2-overexpressing human airway organoids.

As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor (ACE2-OE) that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. Interferon-lambda was upregulated in cells with low-level infection while the NF-kB inhibitor alpha gene (encoding IkBa) was consistently upregulated in infected cells, and its expression positively correlated with infection levels. Confocal microscopy showed more IkBa expression in infected than bystander cells, but found concurrent nuclear translocation of NF-kB that IkBa usually prevents. Overexpressing a nondegradable IkBa mutant reduced NF-kB translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and underscore that the strength of the NF-kB feedback loop in infected cells controls viral replication.

NF-κB inhibitor alpha has a cross-variant role during SARS-CoV-2 infection in ACE2-overexpressing human airway organoids.

As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that accurately recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. NF-κB inhibitor alpha was consistently upregulated in infected epithelial cells, and its mRNA expression positively correlated with infection levels. Confocal microscopy showed more IκBα expression in infected than bystander cells, but found concurrent nuclear translocation of NF-κB that IκBα usually prevents. Overexpressing a nondegradable IκBα mutant reduced NF-κB translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and identify an incomplete NF-κB feedback loop as a rheostat of viral infection that may promote inflammation and severe disease.

Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins.

Genome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloid-lineage cells have remained largely genetically intractable. We present a method for the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14+ human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically edited cells that retain transcript and protein markers of myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection >50-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible, and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate the development of myeloid cellular therapies.

Neutralizing and non-neutralizing monoclonal antibodies against dengue virus E protein derived from a naturally infected patient.

BACKGROUND: Antibodies produced in response to infection with any of the four serotypes of dengue virus generally provide homotypic immunity. However, prior infection or circulating maternal antibodies can also mediate a non-protective antibody response that can enhance the course of disease in a subsequent heterotypic infection. Naturally occurring human monoclonal antibodies can help us understand the protective and pathogenic roles of the humoral immune system in dengue virus infection. RESULTS: Epstein-Barr Virus (EBV) transformation of B cells isolated from the peripheral blood of a human subject with previous dengue infection was performed. B cell cultures were screened by ELISA for antibodies to dengue (DENV) envelope (E) protein. ELISA positive cultures were cloned by limiting dilution. Three IgG1 human monoclonal antibodies (HMAbs) were purified and their binding specificity to E protein was verified by ELISA and biolayer interferometry. Neutralization and enhancement assays were conducted in epithelial and macrophage-like cell lines, respectively. All three HMAbs bound to E from at least two of the four DENV serotypes, one of the HMAbs was neutralizing, and all were able to enhance DENV infection. CONCLUSIONS: HMAbs against DENV can be successfully generated by EBV transformation of B cells from patients at least two years after naturally acquired DENV infections. These antibodies show different patterns of cross-reactivity, neutralizing, and enhancement activity.

Low expression of RNA sensors impacts Zika virus infection in the lower female reproductive tract.

Innate immune responses to Zika virus (ZIKV) are dampened in the lower female reproductive tract (LFRT) compared to other tissues, but the mechanism that underlies this vulnerability is poorly understood. Using tissues from uninfected and vaginally ZIKV-infected macaques and mice, we show that low basal expression of RNA-sensing pattern recognition receptors (PRRs), or their co-receptors, in the LFRT contributes to high viral replication in this tissue. In the LFRT, ZIKV sensing provides limited protection against viral replication, and the sensors are also minimally induced after vaginal infection. While IFNα/ß receptor signaling offers minimal protection in the LFRT, it is required to prevent dissemination of ZIKV to other tissues, including the upper FRT. Our findings support a role for RNA-sensing PRRs in the dampened innate immunity against ZIKV in the LFRT compared to other tissues and underlie potential implications for systemic dissemination upon heterosexual transmission of ZIKV in women.

The Cellular NMD Pathway Restricts Zika Virus Infection and Is Targeted by the Viral Capsid Protein.

Zika virus (ZIKV) infection of neural progenitor cells (NPCs) in utero is associated with neurological disorders, such as microcephaly, but a detailed molecular understanding of ZIKV-induced pathogenesis is lacking. Here we show that in vitro ZIKV infection of human cells, including NPCs, causes disruption of the nonsense-mediated mRNA decay (NMD) pathway. NMD is a cellular mRNA surveillance mechanism that is required for normal brain size in mice. Using affinity purification-mass spectrometry, we identified multiple cellular NMD factors that bind to the viral capsid protein, including the central NMD regulator up-frameshift protein 1 (UPF1). Endogenous UPF1 interacted with the ZIKV capsid protein in coimmunoprecipitation experiments, and capsid expression posttranscriptionally downregulated UPF1 protein levels, a process that we confirmed occurs during ZIKV infection. Cellular fractionation studies show that the ZIKV capsid protein specifically targets nuclear UPF1 for degradation via the proteasome. A further decrease in UPF1 levels by RNAi significantly enhanced ZIKV infection in NPC cultures, consistent with a model in which NMD restricts ZIKV infection in the fetal brain. We propose that ZIKV, via the capsid protein, has evolved a strategy to lower UPF1 levels and dampen antiviral activities of NMD, which in turn contributes to neuropathology in vivoIMPORTANCE Zika virus (ZIKV) is a significant global health threat, as infection has been linked to serious neurological complications, including microcephaly. Using a human stem cell-derived neural progenitor model system, we find that a critical cellular quality control process called the nonsense-mediated mRNA decay (NMD) pathway is disrupted during ZIKV infection. Importantly, disruption of the NMD pathway is a known cause of microcephaly and other neurological disorders. We further identify an interaction between the capsid protein of ZIKV and up-frameshift protein 1 (UPF1), the master regulator of NMD, and show that ZIKV capsid targets UPF1 for degradation. Together, these results offer a new mechanism for how ZIKV infection can cause neuropathology in the developing brain.

Entangled in a membranous web: ER and lipid droplet reorganization during hepatitis C virus infection.

Hepatitis C virus (HCV) is a major cause of liver disease worldwide. To establish and maintain chronic infection, HCV extensively rearranges cellular organelles to generate distinct compartments for viral RNA replication and virion assembly. Here, we review our current knowledge of how HCV proliferates and remodels ER-derived membranes while preserving and expanding associated lipid droplets during viral infection. Unraveling the molecular mechanisms responsible for HCV-induced membrane reorganization will enhance our understanding of the HCV life-cycle, the associated liver pathology, and the biology of the ER:lipid droplet interface in general.

Dampened antiviral immunity to intravaginal exposure to RNA viral pathogens allows enhanced viral replication.

Understanding the host immune response to vaginal exposure to RNA viruses is required to combat sexual transmission of this class of pathogens. In this study, using lymphocytic choriomeningitis virus (LCMV) and Zika virus (ZIKV) in wild-type mice, we show that these viruses replicate in the vaginal mucosa with minimal induction of antiviral interferon and inflammatory response, causing dampened innate-mediated control of viral replication and a failure to mature local antigen-presenting cells (APCs). Enhancement of innate-mediated inflammation in the vaginal mucosa rescues this phenotype and completely inhibits ZIKV replication. To gain a better understanding of how this dampened innate immune activation in the lower female reproductive tract may also affect adaptive immunity, we modeled CD8 T cell responses using vaginal LCMV infection. We show that the lack of APC maturation in the vaginal mucosa leads to a delay in CD8 T cell activation in the draining lymph node and hinders the timely appearance of effector CD8 T cells in vaginal mucosa, thus further delaying viral control in this tissue. Our study demonstrates that vaginal tissue is exceptionally vulnerable to infection by RNA viruses and provides a conceptual framework for the male to female sexual transmission observed during ZIKV infection.

Release of dengue virus genome induced by a peptide inhibitor.

Dengue virus infects approximately 100 million people annually, but there is no available therapeutic treatment. The mimetic peptide, DN59, consists of residues corresponding to the membrane interacting, amphipathic stem region of the dengue virus envelope (E) glycoprotein. This peptide is inhibitory to all four serotypes of dengue virus, as well as other flaviviruses. Cryo-electron microscopy image reconstruction of dengue virus particles incubated with DN59 showed that the virus particles were largely empty, concurrent with the formation of holes at the five-fold vertices. The release of RNA from the viral particle following incubation with DN59 was confirmed by increased sensitivity of the RNA genome to exogenous RNase and separation of the genome from the E protein in a tartrate density gradient. DN59 interacted strongly with synthetic lipid vesicles and caused membrane disruptions, but was found to be non-toxic to mammalian and insect cells. Thus DN59 inhibits flavivirus infectivity by interacting directly with virus particles resulting in release of the genomic RNA.

Structural optimization and de novo design of dengue virus entry inhibitory peptides.

Viral fusogenic envelope proteins are important targets for the development of inhibitors of viral entry. We report an approach for the computational design of peptide inhibitors of the dengue 2 virus (DENV-2) envelope (E) protein using high-resolution structural data from a pre-entry dimeric form of the protein. By using predictive strategies together with computational optimization of binding "pseudoenergies", we were able to design multiple peptide sequences that showed low micromolar viral entry inhibitory activity. The two most active peptides, DN57opt and 1OAN1, were designed to displace regions in the domain II hinge, and the first domain I/domain II beta sheet connection, respectively, and show fifty percent inhibitory concentrations of 8 and 7 microM respectively in a focus forming unit assay. The antiviral peptides were shown to interfere with virus:cell binding, interact directly with the E proteins and also cause changes to the viral surface using biolayer interferometry and cryo-electron microscopy, respectively. These peptides may be useful for characterization of intermediate states in the membrane fusion process, investigation of DENV receptor molecules, and as lead compounds for drug discovery.

In vitro inhibition of dengue virus entry by p-sulfoxy-cinnamic acid and structurally related combinatorial chemistries.

The anti-adhesive compound p-sulfoxy-cinnamic acid, zosteric acid (ZA), is derived from the temperate marine eelgrass, Zostera marina. ZA and five combinatorial chemistries based on ZA were evaluated for their anti-viral properties against dengue virus in a focus forming unit reduction assay. None of the compounds showed evidence of toxicity to the monkey kidney cell line LLCMK-2 over the concentration ranges tested. ZA showed a modest IC(50) of approximately 2.3 mM against DENV-2. Three other compounds showed IC(50) values of 2.5, 2.4, 0.3 mM, with a fourth not achieving a 50% inhibitory concentration against DENV-2. The most active compound, CF 238, showed IC(50) values of 24, 46, 14 and 47 microM against DENV-1, DENV-2, DENV-3 and DENV-4, respectively. CF 238 showed evidence of inhibition at an entry step in the viral life cycle and enhanced virus:cell binding as evidenced by a quantitative RT-PCR assay system. CF 238 may promote inappropriate virus:cell attachments common to all DENV strains that interfere with receptor interactions required for viral entry. These and other related chemistries may be useful as reagents for studying DENV entry, capturing and detecting DENV, and development of pharmaceuticals.