Although a causal relationship between Zika virus (ZIKV) and microcephaly has been established, it remains unclear why ZIKV, but not other pathogenic flaviviruses, causes congenital defects. Here we show that when viruses are produced in mammalian cells, ZIKV, but not the closely related dengue virus (DENV) or West Nile virus (WNV), can efficiently infect key placental barrier cells that directly contact the fetal bloodstream. We show that AXL, a receptor tyrosine kinase, is the primary ZIKV entry cofactor on human umbilical vein endothelial cells (HUVECs), and that ZIKV uses AXL with much greater efficiency than does DENV or WNV. Consistent with this observation, only ZIKV, but not WNV or DENV, bound the AXL ligand Gas6. In comparison, when DENV and WNV were produced in insect cells, they also infected HUVECs in an AXLdependent manner. Our data suggest that ZIKV, when produced from mammalian cells, infects fetal endothelial cells much more efficiently than other pathogenic flaviviruses because it binds Gas6 more avidly, which in turn facilitates its interaction with AXL.Zika virus | Flaviviruses | AXL | placental barrier | fetal endothelial cell Z ika (ZIKV), West Nile (WNV), and dengue (DENV) viruses are closely related, and belong to the Flavivirus genus in the Flaviviridae family. Although a causal relation between ZIKV and microcephaly has been established by human and animal studies (1-7), it remains unclear why only ZIKV, but not other pathogenic flaviviruses, causes congenital diseases. Although WNV is known to infect neuronal cells and results in encephalitis (8), it does not cause microcephaly. DENV is not generally neurotropic and is not linked to congenital defects.To reach the fetal brain, a virus must be transported from the maternal to the fetal circulation, which necessitates crossing of the placental barrier. In the placenta, fetal blood in capillaries is separated from maternal blood by placental barrier cells, namely trophoblasts and fetal endothelial cells. Recent studies indicate that the placenta and its barrier cells are infected by ZIKV, and fetal brain lesions develop in mice, pigtail macaques, and humans (1-6, 9). However, it remains unclear why only ZIKV, and not other neurotropic flaviviruses, results in microcephaly and other congenital disorders.Although bona fide entry receptors for flaviviruses remain unknown, many cell surface-expressed molecules contribute to infection, including C-type lectins dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and DC-SIGN-related protein (L-SIGN) (10, 11) and phosphatidylserine (PS) receptors (12-15) . PS receptors, which serve as entry cofactors for flaviviruses, include members of the TIM (T-cell Ig mucin) family and the TAM (TYRO3, AXL, and MERTK) family. TIMfamily receptors bind PS directly (14, 15), whereas TAM-family members bind PS indirectly, through the soluble intermediates Gas6 (growth arrest-specific 6) and protein S present in serum and other bodily fluids (16, 17). Whereas Gas6 binds to all th...

Phosphatidylserine (PS) receptors contribute to two crucial biological processes: apoptotic clearance and entry of many enveloped viruses. In both cases, they recognize PS exposed on the plasma membrane. Here we demonstrate that phosphatidylethanolamine (PE) is also a ligand for PS receptors and that this phospholipid mediates phagocytosis and viral entry. We show that a subset of PS receptors, including T-cell immunoglobulin (Ig) mucin domain protein 1 (TIM1), efficiently bind PE. We further show that PE is present in the virions of flaviviruses and filoviruses, and that the PE-specific cyclic peptide lantibiotic agent Duramycin efficiently inhibits the entry of West Nile, dengue, and Ebola viruses. The inhibitory effect of Duramycin is specific: it inhibits TIM1-mediated, but not L-SIGNmediated, virus infection, and it does so by blocking virus attachment to TIM1. We further demonstrate that PE is exposed on the surface of apoptotic cells, and promotes their phagocytic uptake by TIM1-expressing cells. Together, our data show that PE plays a key role in TIM1-mediated virus entry, suggest that disrupting PE association with PS receptors is a promising broad-spectrum antiviral strategy, and deepen our understanding of the process by which apoptotic cells are cleared. M embers of the filovirus and flavivirus families are the causative agents of life-threatening diseases. Ebola virus (EBOV), a filovirus, causes hemorrhagic fever with an average case fatality rate as high as 65% (1). Although there are EBOV vaccine candidates (2, 3), there is currently no licensed vaccine or treatment. Dengue virus (DENV) and West Nile virus (WNV) belong to the flavivirus family. Both are transmitted to humans through mosquito bites and can cause lethal hemorrhagic fever (in the case of DENV) or severe neurological diseases (in the case of WNV) (4, 5). Flaviviruses are emerging as major health concerns in tropical and subtropical areas worldwide. More than one third of the world's population is estimated to be at risk for DENV infection, with approximately 400 million people infected yearly (6). There are currently no approved vaccines or therapeutic agents against DENV or WNV.Virus entry into host cells typically initiates with the interaction between viral entry glycoproteins (GPs) and a receptor or coreceptor expressed at the surface of the target cell. Viruses also use less specific mechanisms to localize to target cell membranes, for example through GP association with various attachment factors (7). During the past few years, it has been increasingly recognized that many viruses also use a strategy known as apoptotic mimicry to promote their association with, and internalization into their target cells (8). Receptors for phospholipids, specifically phosphatidylserine (PS), normally involved in the clearance of apoptotic cells, markedly enhance the infection of a number of enveloped viruses. These PS receptors are presumed to engage PS on the virion membrane rather than the viral entry protein (9, 10). Enveloped viruses acquire...

SARS-CoV-2, the causative agent of COVID-191, features a receptor-binding domain (RBD) for binding to the host cell ACE2 protein1–6. Neutralizing antibodies that block RBD-ACE2 interaction are candidates for the development of targeted therapeutics7–17. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages in bioavailability, amenability, and production and storage owing to their small sizes and high stability. Here, we report the rapid selection of 99 synthetic nanobodies (sybodies) against RBD by in vitro selection using three libraries. The best sybody, MR3 binds to RBD with high affinity (KD = 1.0 nM) and displays high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.42 μg mL−1). Structural, biochemical, and biological characterization suggests a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency have been generated by structure-based design, biparatopic construction, and divalent engineering. Two divalent forms of MR3 protect hamsters from clinical signs after live virus challenge and a single dose of the Fc-fusion construct of MR3 reduces viral RNA load by 6 Log10. Our results pave the way for the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid development of targeted medical interventions during an outbreak.

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