Dengue virus is responsible for the highest rates of disease and mortality among the members of the Flavivirus genus. Dengue epidemics are still occurring around the world, indicating an urgent need of prophylactic vaccines and antivirals. In recent years, a great deal has been learned about the mechanisms of dengue virus genome amplification. However, little is known about the process by which the capsid protein recruits the viral genome during encapsidation. Here, we found that the mature capsid protein in the cytoplasm of dengue virus infected cells accumulates on the surface of ER-derived organelles named lipid droplets. Mutagenesis analysis using infectious dengue virus clones has identified specific hydrophobic amino acids, located in the center of the capsid protein, as key elements for lipid droplet association. Substitutions of amino acid L50 or L54 in the capsid protein disrupted lipid droplet targeting and impaired viral particle formation. We also report that dengue virus infection increases the number of lipid droplets per cell, suggesting a link between lipid droplet metabolism and viral replication. In this regard, we found that pharmacological manipulation of the amount of lipid droplets in the cell can be a means to control dengue virus replication. In addition, we developed a novel genetic system to dissociate cis-acting RNA replication elements from the capsid coding sequence. Using this system, we found that mislocalization of a mutated capsid protein decreased viral RNA amplification. We propose that lipid droplets play multiple roles during the viral life cycle; they could sequester the viral capsid protein early during infection and provide a scaffold for genome encapsidation. Dengue virus is the single most significant arthropod-borne virus pathogen in humans. In spite of the urgent medical need to control dengue infections, vaccines are still unavailable, and many aspects of dengue virus biology and pathogenesis remain elusive. We discovered a link between dengue virus replication and ER-derived organelles known as lipid droplets (LDs). Dengue infection increases the amount of LDs per cell and pharmacological inhibition of LD formation greatly reduces dengue virus replication. In addition, we have found that the viral capsid protein in infected cells accumulates on the surface of LDs. Manipulation of infectious clones and generation of new reporter dengue viruses allowed us to define the molecular basis of capsid protein association to LDs. Specific amino acids on the α2 helix, located in the center of the capsid protein, were found to be crucial for both accumulation of capsid protein on LDs and dengue virus infectious particle formation. We propose that LDs facilitate viral replication providing a platform for nucleocapsid formation during encapsidation. Our findings begin to unravel the complex mechanism by which dengue virus usurps cellular organelles to coordinate different steps of the viral life cycle.

Key Points: Dengue infection triggers functional inflammasome assembly in platelets. Platelets may contribute to increased vascular permeability in dengue virus infection by synthesis and release of IL-1β.

Dengue is the most prevalent arthropod-borne viral disease, caused by dengue virus, a member of the Flaviviridae family. Its worldwide incidence is now a major health problem, with 2.5 billion people living in risk areas. In this review, we integrate the structural rearrangements of each viral protein and their functions in all the steps of virus entry into the host cells. We describe in detail the putative receptors and attachment factors in mammalian and mosquito cells, and the recognition of viral immunocomplexes via Fcγ receptor in immune cells. We also discuss that virus internalization might occur through distinct entry pathways, including clathrin-mediated or non-classical clathrin-independent endocytosis, depending on the host cell and virus serotype or strain. The implications of viral maturation in virus entry are also explored. Finally, we discuss the mechanisms of viral genome access to the cytoplasm. This includes the role of low pH-induced conformational changes in the envelope protein that mediate membrane fusion, and original insights raised by our recent work that supports the hypothesis that capsid protein would also be an active player in this process, acting on viral genome translocation into the cytoplasm.

Neurological complications affecting the central nervous system have been reported in adult patients infected by Zika virus (ZIKV) but the underlying mechanisms remain unknown. Here, we report that ZIKV replicates in human and mouse adult brain tissue, targeting mature neurons. ZIKV preferentially targets memory-related brain regions, inhibits hippocampal long-term potentiation and induces memory impairment in adult mice. TNF-α upregulation, microgliosis and upregulation of complement system proteins, C1q and C3, are induced by ZIKV infection. Microglia are found to engulf hippocampal presynaptic terminals during acute infection. Neutralization of TNF-α signaling, blockage of microglial activation or of C1q/C3 prevent synapse and memory impairment in ZIKV-infected mice. Results suggest that ZIKV induces synapse and memory dysfunction via aberrant activation of TNF-α, microglia and complement. Our findings establish a mechanism by which ZIKV affects the adult brain, and point to the need of evaluating cognitive deficits as a potential comorbidity in ZIKV-infected adults.

Background Worldwide, dengue is the most prevalent human arbovirus disease. Dengue infection may cause a range of clinical manifestations from self‐limiting febrile illness through to a life‐threatening syndrome accompanied by both bleeding and shock. Thrombocytopenia is frequently observed in mild and severe disease; however, the mechanisms involved in DENV‐induced platelet activation and thrombocytopenia are incompletely understood. Patients and methods Freshly isolated platelets from patients with dengue were evaluated for markers of activation, mitochondrial alteration and activation of cell death pathways. In parallel, we examined direct DENV‐induced activation and apoptosis of platelets obtained from healthy subjects. Results We found that platelets from DENV‐infected patients exhibited increased activation by comparison to control subjects. Moreover, platelets from DENV‐infected patients exhibited classic signs of the intrinsic pathway of apoptosis that include increased surface phosphatidylserine exposure, mitochondrial depolarization and activation of caspase‐9 and ‐3. Indeed, thrombocytopenia was shown to strongly associate with enhanced platelet activation and cell death in DENV‐infected patients. Platelet activation, mitochondrial dysfunction and caspase‐dependent phosphatidylserine exposure on platelets were also observed when platelets from healthy subjects were directly exposed to DENV in vitro. DENV‐induced platelet activation was shown to occur through mechanisms largely dependent on DC‐SIGN. Conclusions Together our results demonstrate that platelets from patients with dengue present signs of activation, mitochondrial dysfunction and activation of the apoptosis caspase cascade, which may contribute to the development of thrombocytopenia in patients with dengue. Our results also suggest the involvement of DC‐SIGN as a critical receptor in DENV‐dependent platelet activation.

Cognitive dysfunction is often reported in patients with post-coronavirus disease 2019 (COVID-19) syndrome, but its underlying mechanisms are not completely understood. Evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein or its fragments are released from cells during infection, reaching different tissues, including the CNS, irrespective of the presence of the viral RNA. Here, we demonstrate that brain infusion of Spike protein in mice has a late impact on cognitive function, recapitulating post-COVID-19 syndrome. We also show that neuroinflammation and hippocampal microgliosis mediate Spike-induced memory dysfunction via complement-dependent engulfment of synapses. Genetic or pharmacological blockage of Toll-like receptor 4 (TLR4) signaling protects animals against synapse elimination and memory dysfunction induced by Spike brain infusion. Accordingly, in a cohort of 86 patients who recovered from mild COVID-19, the genotype GG TLR4-2604G>A (rs10759931) is associated with poor cognitive outcome. These results identify TLR4 as a key target to investigate the long-term cognitive dysfunction after COVID-19 infection in humans and rodents.