Zika virus alters centrosome organization to suppress the innate immune response.

Zika virus (ZIKV) is a flavivirus transmitted via mosquitoes and sex to cause congenital neurodevelopmental defects, including microcephaly. Inherited forms of microcephaly (MCPH) are associated with disrupted centrosome organization. Similarly, we found that ZIKV infection disrupted centrosome organization. ZIKV infection disrupted the organization of centrosomal proteins including CEP63, a MCPH-associated protein. The ZIKV nonstructural protein NS3 bound CEP63, and expression of NS3 was sufficient to alter centrosome architecture and CEP63 localization. Loss of CEP63 suppressed ZIKV-induced centrosome disorganization, indicating that ZIKV requires CEP63 to disrupt centrosome organization. ZIKV infection or CEP63 loss decreased the centrosomal localization and stability of TANK-binding kinase 1 (TBK1), a regulator of the innate immune response. ZIKV infection also increased the centrosomal accumulation of the CEP63 interactor DTX4, a ubiquitin ligase that degrades TBK1. Therefore, we propose that ZIKV disrupts CEP63 function to increase centrosomal DTX4 localization and destabilization of TBK1, thereby tempering the innate immune response.

Zika virus cell tropism in the developing human brain and inhibition by azithromycin.

The rapid spread of Zika virus (ZIKV) and its association with abnormal brain development constitute a global health emergency. Congenital ZIKV infection produces a range of mild to severe pathologies, including microcephaly. To understand the pathophysiology of ZIKV infection, we used models of the developing brain that faithfully recapitulate the tissue architecture in early to midgestation. We identify the brain cell populations that are most susceptible to ZIKV infection in primary human tissue, provide evidence for a mechanism of viral entry, and show that a commonly used antibiotic protects cultured brain cells by reducing viral proliferation. In the brain, ZIKV preferentially infected neural stem cells, astrocytes, oligodendrocyte precursor cells, and microglia, whereas neurons were less susceptible to infection. These findings suggest mechanisms for microcephaly and other pathologic features of infants with congenital ZIKV infection that are not explained by neural stem cell infection alone, such as calcifications in the cortical plate. Furthermore, we find that blocking the glia-enriched putative viral entry receptor AXL reduced ZIKV infection of astrocytes in vitro, and genetic knockdown of AXL in a glial cell line nearly abolished infection. Finally, we evaluate 2,177 compounds, focusing on drugs safe in pregnancy. We show that the macrolide antibiotic azithromycin reduced viral proliferation and virus-induced cytopathic effects in glial cell lines and human astrocytes. Our characterization of infection in the developing human brain clarifies the pathogenesis of congenital ZIKV infection and provides the basis for investigating possible therapeutic strategies to safely alleviate or prevent the most severe consequences of the epidemic.

Distinct neural stem cell tropism, early immune activation, and choroid plexus pathology following coxsackievirus infection in the neonatal central nervous system.

Coxsackievirus B3 (CVB3) and lymphocytic choriomeningitis virus (LCMV) are both neurotropic RNA viruses, which can establish a persistent infection and cause meningitis and encephalitis in the neonatal host. Utilizing our neonatal mouse model of infection, we evaluated the consequences of early viral infection upon the host central nervous system (CNS) by comparing CVB3 and LCMV infection. Both viruses expressed high levels of viral protein in the choroid plexus and subventricular zone (SVZ), a region of neurogenesis. LCMV infected a greater number of cells in the SVZ and targeted both nestin(+) (neural progenitor cell marker) and olig2(+) (glial progenitor marker) cells at a relatively equal proportion. In contrast, CVB3 preferentially infected nestin(+) cells within the SVZ. Microarray analysis revealed differential kinetics and unique host gene expression changes for each infection. MHC class I gene expression, several developmental-related Hox genes, and transthyretin (TTR), a protein secreted in the cerebrospinal fluid by the choroid plexus, were specifically downregulated following CVB3 infection. Also, we identified severe pathology in the choroid plexus of CVB3-infected animals at 48 h post infection accompanied by a decrease in the level of TTR and carbonic anhydrase II. These results demonstrate broader neural progenitor and stem cell (NPSC) tropism for LCMV in the neonatal CNS, whereas CVB3 targeted a more specific subset of NPSCs, stimulated a distinct early immune response, and induced significant acute damage in the choroid plexus.

Genome-Wide Knockout Screen Identifies Human Sialomucin CD164 as an Essential Entry Factor for Lymphocytic Choriomeningitis Virus.

Lymphocytic choriomeningitis virus (LCMV) is a well-studied mammarenavirus that can be fatal in congenital infections. However, our understanding of LCMV and its interactions with human host factors remains incomplete. Here, host determinants affecting LCMV infection were investigated through a genome-wide CRISPR knockout screen in A549 cells, a human lung adenocarcinoma line. We identified and validated a variety of novel host factors that play a functional role in LCMV infection. Among these, knockout of the sialomucin CD164, a heavily glycosylated transmembrane protein, was found to ablate infection with multiple LCMV strains but not other hemorrhagic mammarenaviruses in several cell types. Further characterization revealed a dependency of LCMV entry on the cysteine-rich domain of CD164, including an N-linked glycosylation site at residue 104 in that region. Given the documented role of LCMV with respect to transplacental human infections, CD164 expression was investigated in human placental tissue and placental cell lines. CD164 was found to be highly expressed in the cytotrophoblast cells, an initial contact site for pathogens within the placenta, and LCMV infection in placental cells was effectively blocked using a monoclonal antibody specific to the cysteine-rich domain of CD164. Together, this study identifies novel factors associated with LCMV infection of human tissues and highlights the importance of CD164, a sialomucin that previously had not been associated with viral infection. IMPORTANCE Lymphocytic choriomeningitis virus (LCMV) is a human-pathogenic mammarenavirus that can be fatal in congenital infections. Although frequently used in the study of persistent infections in the field of immunology, aspects of this virus's life cycle remain incomplete. For example, while viral entry has been shown to depend on a cell adhesion molecule, DAG1, genetic knockout of this gene allows for residual viral infection, implying that additional receptors can mediate cell entry. The significance of our study is the identification of host factors important for successful infection, including the sialomucin CD164, which had not been previously associated with viral infection. We demonstrated that CD164 is essential for LCMV entry into human cells and can serve as a possible therapeutic target for treatment of congenital infection.

A Four-Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotypes C and D.

Human botulism can be caused by botulinum neurotoxin (BoNT) serotypes A to G. Here, we present an antibody-based antitoxin composed of four human monoclonal antibodies (mAbs) against BoNT/C, BoNT/D, and their mosaic toxins. This work built on our success in generating protective mAbs to BoNT /A, B and E serotypes. We generated mAbs from human immune single-chain Fv (scFv) yeast-display libraries and isolated scFvs with high affinity for BoNT/C, BoNT/CD, BoNT/DC and BoNT/D serotypes. We identified four mAbs that bound non-overlapping epitopes on multiple serotypes and mosaic BoNTs. Three of the mAbs underwent molecular evolution to increase affinity. A four-mAb combination provided high-affinity binding and BoNT neutralization of both serotypes and their mosaic toxins. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing and neutralizing BoNT/C and BoNT/D serotypes and their mosaic toxins. A derivative of the four-antibody combination (NTM-1634) completed a Phase 1 clinical trial (Snow et al., Antimicrobial Agents and Chemotherapy, 2019) with no drug-related serious adverse events.

Phage wrapping with cationic polymers eliminates nonspecific binding between M13 phage and high pI target proteins.

M13 phage have provided scaffolds for nanostructure synthesis based upon self-assembled inorganic and hard materials interacting with phage-displayed peptides. Additionally, phage display has been used to identify binders to plastic, TiO(2), and other surfaces. However, synthesis of phage-based materials through the hybridization of soft materials with the phage surface remains unexplored. Here, we present an efficient "phage wrapping" strategy for the facile synthesis of phage coated with soluble, cationic polymers. Polymers bearing high positive charge densities demonstrated the most effective phage wrapping, as shown by assays for blocking nonspecific binding of the anionic phage coat to a high pI target protein. The results establish the functional group requirements for hybridizing phage with soft materials and solve a major problem in phage display-nonspecific binding by the phage to high pI target proteins.

Illuminating uveitis: metagenomic deep sequencing identifies common and rare pathogens.

BACKGROUND: Ocular infections remain a major cause of blindness and morbidity worldwide. While prognosis is dependent on the timing and accuracy of diagnosis, the etiology remains elusive in ~50 % of presumed infectious uveitis cases. The objective of this study is to determine if unbiased metagenomic deep sequencing (MDS) can accurately detect pathogens in intraocular fluid samples of patients with uveitis. METHODS: This is a proof-of-concept study, in which intraocular fluid samples were obtained from five subjects with known diagnoses, and one subject with bilateral chronic uveitis without a known etiology. Samples were subjected to MDS, and results were compared with those from conventional diagnostic tests. Pathogens were identified using a rapid computational pipeline to analyze the non-host sequences obtained from MDS. RESULTS: Unbiased MDS of intraocular fluid produced results concordant with known diagnoses in subjects with (n = 4) and without (n = 1) uveitis. Samples positive for Cryptococcus neoformans, Toxoplasma gondii, and herpes simplex virus 1 as tested by a Clinical Laboratory Improvement Amendments-certified laboratory were correctly identified with MDS. Rubella virus was identified in one case of chronic bilateral idiopathic uveitis. The subject's strain was most closely related to a German rubella virus strain isolated in 1992, one year before he developed a fever and rash while living in Germany. The pattern and the number of viral identified mutations present in the patient's strain were consistent with long-term viral replication in the eye. CONCLUSIONS: MDS can identify fungi, parasites, and DNA and RNA viruses in minute volumes of intraocular fluid samples. The identification of chronic intraocular rubella virus infection highlights the eye's role as a long-term pathogen reservoir, which has implications for virus eradication and emerging global epidemics.

Single nucleoprotein residue modulates arenavirus replication complex formation.

UNLABELLED: The Arenaviridae are enveloped, negative-sense RNA viruses with several family members that cause hemorrhagic fevers. This work provides immunofluorescence evidence that, unlike those of New World arenaviruses, the replication and transcription complexes (RTC) of lymphocytic choriomeningitis virus (LCMV) colocalize with eukaryotic initiation factor 4E (eIF4E) and that eIF4E may participate in the translation of LCMV mRNA. Additionally, we identify two residues in the LCMV nucleoprotein (NP) that are conserved in every mammalian arenavirus and are required for recombinant LCMV recovery. One of these sites, Y125, was confirmed to be phosphorylated by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). NP Y125 is located in the N-terminal region of NP that is disordered when RNA is bound. The other site, NP T206, was predicted to be a phosphorylation site. Immunofluorescence analysis demonstrated that NP T206 is required for the formation of the punctate RTC that are typically observed during LCMV infection. A minigenome reporter assay using NP mutants, as well as Northern blot analysis, demonstrated that although NP T206A does not form punctate RTC, it can transcribe and replicate a minigenome. However, in the presence of matrix protein (Z) and glycoprotein (GP), translation of the minigenome message with NP T206A was inhibited, suggesting that punctate RTC formation is required to regulate viral replication. Together, these results highlight a significant difference between New and Old World arenaviruses and demonstrate the importance of RTC formation and translation priming in RTC for Old World arenaviruses. IMPORTANCE: Several members of the Arenaviridae cause hemorrhagic fevers and are classified as category A pathogens. Arenavirus replication-transcription complexes (RTC) are nucleated by the viral nucleoprotein. This study demonstrates that the formation of these complexes is required for virus viability and suggests that RTC nucleation is regulated by the phosphorylation of a single nucleoprotein residue. This work adds to the body of knowledge about how these key viral structures are formed and participate in virus replication. Additionally, the fact that Old World arenavirus complexes colocalize with the eukaryotic initiation factor 4E, while New World arenaviruses do not, is only the second notable difference observed between New and Old World arenaviruses, the first being the difference in the glycoprotein receptor.

LCMV glycosylation modulates viral fitness and cell tropism.

The glycoprotein (GP) of arenaviruses is glycosylated at 11 conserved N-glycosylation sites. We constructed recombinant lymphocytic choriomeningitis virus (rLCMV) featuring either additions or deletions of these N-glycans to investigate their role in the viral life cycle. N-glycosylation at two sites, T87 and S97, were found to be necessary to rescue rLCMV. Three of nine successfully rescued mutants, S116A, T234A, and S373A, under selective pressures in either epithelial, neuronal, or macrophage cells reverted to WT sequence. Of the seven stable N-glycan deletion mutants, five of these led to altered viral fitness and cell tropism, assessed as growth in either mouse primary cortical neurons or bone marrow derived macrophages. These results demonstrate that the deletion of N-glycans in LCMV GP may confer an advantage to the virus for infection of neurons but a disadvantage in macrophages.

Glycosylation modulates arenavirus glycoprotein expression and function.

The glycoprotein of lymphocytic choriomeningitis virus (LCMV) contains nine potential N-linked glycosylation sites. We investigated the function of these N-glycosylations by using alanine-scanning mutagenesis. All the available sites were occupied on GP1 and two of three on GP2. N-linked glycan mutations at positions 87 and 97 on GP1 resulted in reduction of expression and absence of cleavage and were necessary for downstream functions, as confirmed by the loss of GP-mediated fusion activity with T87A and S97A mutants. In contrast, T234A and E379N/A381T mutants impaired GP-mediated cell fusion without altered expression or processing. Infectivity via virus-like particles required glycans and a cleaved glycoprotein. Glycosylation at the first site within GP2, not normally utilized by LCMV, exhibited increased VLP infectivity. We also confirmed the role of the N-linked glycan at position 173 in the masking of the neutralizing epitope GP-1D. Taken together, our results indicated a strong relationship between fusion and infectivity.