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 (DENV) affects millions of people, causing more than 20,000 deaths annually. No effective treatment for the disease caused by DENV infection is currently available, partially due to the lack of knowledge on the basic aspects of the viral life cycle, including the molecular basis of the interaction between viral components and cellular compartments. Here, we characterized the properties of the interaction between the DENV capsid (C) protein and hepatic lipid droplets (LDs), which was recently shown to be essential for the virus replication cycle. Zeta potential analysis revealed a negative surface charge of LDs, with an average surface charge of ؊19 mV. The titration of LDs with C protein led to an increase of the surface charge, which reached a plateau at ؉13.7 mV, suggesting that the viral protein-LD interaction exposes the protein cationic surface to the aqueous environment. Atomic force microscopy (AFM)-based force spectroscopy measurements were performed by using C proteinfunctionalized AFM tips. The C protein-LD interaction was found to be strong, with a single (un)binding force of 33.6 pN. This binding was dependent on high intracellular concentrations of potassium ions but not sodium. The inhibition of Na ؉ /K ؉ -ATPase in DENV-infected cells resulted in the dissociation of C protein from LDs and a 50-fold inhibition of infectious virus production but not of RNA replication, indicating a biological relevance for the potassium-dependent interaction. Limited proteolysis of the LD surface impaired the C protein-LD interaction, and force measurements in the presence of specific antibodies indicated that perilipin 3 (TIP47) is the major DENV C protein ligand on the surface of LDs. Dengue virus (DENV) causes the most important arthropodborne human viral disease, with 2.5 billion people at risk, 100 million infections, and more than 20,000 deaths annually, primarily in tropical developing countries (20). Four genetically distinct serotypes (DENV1 to DENV4) have been identified, which are transmitted among humans through the bite of an infected mosquito of the genus Aedes. DENV belongs to the family Flaviviridae, together with other important human pathogens, such as yellow fever virus (YFV), West Nile virus (WNV), and hepatitis C virus (HCV). The clinical manifestations of DENV infection range from a mild illness to a severe and potentially life-threatening disease for which no treatment is available so far, due at least in part to the limited understanding of the molecular mechanisms that underlie the interaction between DENV and its host cells.The DENV genome is a single-stranded positive-sense RNA molecule of approximately 11 kb that is translated from a single open reading frame, generating a polyprotein associated with the endoplasmic reticulum (ER) membrane (34). The polyprotein is cleaved co-and posttranslationally by cellular and viral proteases into three structural proteins (capsid [C], premembrane [prM], and envelope [E]) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B,...

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.

Dengue fever is an emerging viral disease transmitted by arthropods to humans in tropical countries. Dengue hemorrhagic fever (DHF) is escalating in frequency and mortality rates. Here we studied the involvement of macrophage migration inhibitory factor (MIF) in dengue virus (DENV) infection and its pathogenesis. Patients with DHF had elevated plasma concentrations of MIF. Both leukocytes from these patients and macrophages from healthy donors infected in vitro with DENV showed a substantial amount of MIF within lipid droplets. The secretion of MIF by macrophages and hepatocytes required a productive infection and occurred without an increase in gene transcription or cell death, thus indicating active secretion from preformed stocks. In vivo infection of wild-type and mif-deficient (Mif(-/-)) mice demonstrated a role of MIF in dengue pathogenesis. Clinical disease was less severe in Mif(-/-) mice, and they exhibited a significant delay in lethality, lower viremia, and lower viral load in the spleen than wild-type mice. This reduction in all parameters of severity on DENV infection in Mif(-/-) mice correlated with reduced proinflammatory cytokine concentrations. These results demonstrated the contribution of MIF to the pathogenesis of dengue and pointed to a possible beneficial role of neutralizing MIF as an adjunctive therapeutic approach to treat the severe forms of the disease.

Arthritogenic alphaviruses, including Ross River virus (RRV), Chikungunya virus (CHIKV), Sindbis virus (SINV), Mayaro virus (MAYV), O'nyong-nyong virus (ONNV), and Barmah Forest virus (BFV), cause incapacitating and long lasting articular disease/myalgia. Outbreaks of viral arthritis and the global distribution of these diseases point to the emergence of arthritogenic alphaviruses as an important public health problem. This review discusses the molecular mechanisms involved in alphavirus-induced arthritis, exploring the recent data obtained with in vitro systems and in vivo studies using animal models and samples from patients. The factors associated to the extension and persistence of symptoms are highlighted, focusing on (a) virus replication in target cells, and tissues, including macrophages and muscle cells; (b) the inflammatory and immune responses with recruitment and activation of macrophage, NK cells and T lymphocytes to the lesion focus and the increase of inflammatory mediators levels; and (c) the persistence of virus or viral products in joint and muscle tissues. We also discuss the importance of the establishment of novel animal models to test new molecular targets and to develop more efficient and selective drugs to treat these diseases.

Although congenital Zika virus (ZIKV) exposure has been associated with microcephaly and other neurodevelopmental disorders, long-term consequences of perinatal infection are largely unknown. We evaluated short- and long-term neuropathological and behavioral consequences of neonatal ZIKV infection in mice. ZIKV showed brain tropism, causing postnatal-onset microcephaly and several behavioral deficits in adulthood. During the acute phase of infection, mice developed frequent seizures, which were reduced by tumor necrosis factor-α (TNF-α) inhibition. During adulthood, ZIKV replication persisted in neonatally infected mice, and the animals showed increased susceptibility to chemically induced seizures, neurodegeneration, and brain calcifications. Altogether, the results show that neonatal ZIKV infection has long-term neuropathological and behavioral complications in mice and suggest that early inhibition of TNF-α-mediated neuroinflammation might be an effective therapeutic strategy to prevent the development of chronic neurological abnormalities.

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Virus resistance to antiviral therapies is an increasing concern that makes the development of broad-spectrum antiviral drugs urgent. Targeting of the viral envelope, a component shared by a large number of viruses, emerges as a promising strategy to overcome this problem. Natural and synthetic porphyrins are good candidates for antiviral development due to their relative hydrophobicity and pro-oxidant character. In the present work, we characterized the antiviral activities of protoprophyrin IX (PPIX), Znprotoporphyrin IX (ZnPPIX), and mesoporphyrin IX (MPIX) against vesicular stomatitis virus (VSV) and evaluated the mechanisms involved in this activity. Treatment of VSV with PPIX, ZnPPIX, and MPIX promoted dose-dependent virus inactivation, which was potentiated by porphyrin photoactivation. All three porphyrins inserted into lipid vesicles and disturbed the viral membrane organization. In addition, the porphyrins also affected viral proteins, inducing VSV glycoprotein cross-linking, which was enhanced by porphyrin photoactivation. Virus incubation with sodium azide and ␣-tocopherol partially protected VSV from inactivation by porphyrins, suggesting that singlet oxygen ( 1 O 2 ) was the main reactive oxygen species produced by photoactivation of these molecules. Furthermore, 1 O 2 was detected by 9,10-dimethylanthracene oxidation in photoactivated porphyrin samples, reinforcing this hypothesis. These results reveal the potential therapeutic application of PPIX, ZnPPIX, and MPIX as good models for broad antiviral drug design.KEYWORDS photoactivation, porphyrin, singlet oxygen, vesicular stomatitis virus, viral inactivation D espite the increasing number of antiviral drugs available nowadays, therapeutics for viral diseases often fail due to drug resistance development by the target viruses. In the search for antiviral agents with a broad spectrum of activity against viruses, antiviral agents targeting the viral envelope, a component shared by a large number of viruses, emerge as promising compounds able to overcome the drug resistance problem. Porphyrins, amphipathic molecules able to interact with membranes, appear to be good candidates for the development of such antivirals. Porphyrins are formed by four pyrrole rings linked by methine bonds. The nitrogen atoms in the center of the ring form a pocket able to coordinate metallic ions, conferring to porphyrin the ability to absorb light at additional wavelengths and act in catalytic transformations (1). These activities allow porphyrins to be involved in important biological processes, such as respiration (heme) and photosynthesis (chlorophyll and bacteriochlorophyll) (2).