In vivo pair correlation microscopy reveals dengue virus capsid protein nucleocytoplasmic bidirectional movement in mammalian infected cells.

Flaviviruses are major human disease-causing pathogens, including dengue virus (DENV), Zika virus, yellow fever virus and others. DENV infects hundreds of millions of people per year around the world, causing a tremendous social and economic burden. DENV capsid (C) protein plays an essential role during genome encapsidation and viral particle formation. It has been previously shown that DENV C enters the nucleus in infected cells. However, whether DENV C protein exhibits nuclear export remains unclear. By spatially cross-correlating different regions of the cell, we investigated DENV C movement across the nuclear envelope during the infection cycle. We observed that transport takes place in both directions and with similar translocation times (in the ms time scale) suggesting a bidirectional movement of both C protein import and export.Furthermore, from the pair cross-correlation functions in cytoplasmic or nuclear regions we found two populations of C molecules in each compartment with fast and slow mobilities. While in the cytoplasm the correlation times were in the 2-6 and 40-110 ms range for the fast and slow mobility populations respectively, in the cell nucleus they were 1-10 and 25-140 ms range, respectively. The fast mobility of DENV C in cytoplasmic and nuclear regions agreed with the diffusion coefficients from Brownian motion previously reported from correlation analysis. These studies provide the first evidence of DENV C shuttling from and to the nucleus in infected cells, opening new venues for antiviral interventions.

Determinants in Nonstructural Protein 4A of Dengue Virus Required for RNA Replication and Replication Organelle Biogenesis.

Dengue virus (DENV) constitutes one of the most important arboviral pathogens affecting humans. The high prevalence of DENV infections, which cause more than 20,000 deaths annually, and the lack of effective vaccines or direct-acting antiviral drugs make it a global health concern. DENV genome replication occurs in close association with the host endomembrane system, which is remodeled to form the viral replication organelle that originates from endoplasmic reticulum (ER) membranes. To date, the viral and cellular determinants responsible for the biogenesis of DENV replication organelles are still poorly defined. The viral nonstructural protein 4A (NS4A) can remodel membranes and has been shown to associate with numerous host factors in DENV-replicating cells. In the present study, we used reverse and forward genetic screens and identified sites within NS4A required for DENV replication. We also mapped the determinants in NS4A required for interactions with other viral proteins. Moreover, taking advantage of our recently developed polyprotein expression system, we evaluated the role of NS4A in the formation of DENV replication organelles. Together, we report a detailed map of determinants within NS4A required for RNA replication, interaction with other viral proteins, and replication organelle formation. Our results suggest that NS4A might be an attractive target for antiviral therapy. IMPORTANCE DENV is the most prevalent mosquito-borne virus, causing around 390 million infections each year. There are no approved therapies to treat DENV infection, and the only available vaccine shows limited efficacy. The viral nonstructural proteins have emerged as attractive drug targets due to their pivotal role in RNA replication and establishment of virus-induced membranous compartments, designated replication organelles (ROs). The transmembrane protein NS4A, generated by cleavage of the NS4A-2K-4B precursor, contributes to DENV replication by unknown mechanisms. Here, we report a detailed genetic interaction map of NS4A and identify residues required for RNA replication and interaction between NS4A-2K-4B and NS2B-3 as well as NS1. Importantly, by means of an expression-based system, we demonstrate the essential role of NS4A in RO biogenesis and identify determinants in NS4A required for this process. Our data suggest that NS4A is an attractive target for antiviral therapy.

Enhancing sensitivity of QCM for dengue type 1 virus detection using graphene-based polymer composites.

Graphene oxide-molecularly imprinted polymer composites (GO-MIP) have attracted significant attention as recognition materials in sensing due to their outstanding properties in terms of electrical and thermal conductivity, high mechanical modulus, and the comparably straightforward way to functionalize them. The aim of this study was to design a MIP-based sensor recognition material and enhance its sensitivity by blending it with GO for sensing a harmful dengue hemorrhagic fever pathogen, namely the dengue type 1 virus (DENV-1). Polymer composites comprising GO incorporated to an acrylamide (AAM)/methacrylic acid (MAA)/methyl methacrylate (MMA)/N-vinylpyrrolidone (VP) copolymer were synthesized and compared to the "pure" MIP, i.e., the copolymer without GO. The pure polymer revealed a zeta potential of + 9.9 ± 0.5 mV, whereas GO sheets prepared have a zeta potential of - 60.3 ± 2.7 mV. This results in an overall zeta potential of - 11.2 ± 0.2 mV of the composite. Such polymer composites seem appropriate to bind the positively charged DENV-1 particle (+ 42.2 ± 2.8 mV). GO-MIP coated onto 10-MHz quartz crystal microbalance (QCM) sensors indeed revealed two times sensitivity compared to sensors based on the pure MIP. Furthermore, GO-polymer composites revealed imprinting factors of up to 21, compared to 3 of the pure MIP. When plotting the sensor characteristic in a semilogarithmic way, the composite sensor shows the linear response to DENV-1 in the concentration range from 100 to 103 pfu mL-1. The corresponding limits of detection (S/N = 3) and quantification (S/N = 10) are 0.58 and 1.94 pfu mL-1, respectively. Furthermore, imprinted polymer composites selectively bind DENV-1 without significant interference: DENV-2, DENV-3, DENV-4, respectively, yield 13-16% of DENV-1 signal. The sensor requires only about 15-20 min to obtain a result.

Dengue: A Minireview.

Dengue, caused by infection of any of four dengue virus serotypes (DENV-1 to DENV-4), is a mosquito-borne disease of major public health concern associated with significant morbidity, mortality, and economic cost, particularly in developing countries. Dengue incidence has increased 30-fold in the last 50 years and over 50% of the world's population, in more than 100 countries, live in areas at risk of DENV infection. We reviews DENV biology, epidemiology, transmission dynamics including circulating serotypes and genotypes, the immune response, the pathogenesis of the disease as well as updated diagnostic methods, treatments, vector control and vaccine developments.

A Non-Replicative Role of the 3' Terminal Sequence of the Dengue Virus Genome in Membranous Replication Organelle Formation.

Dengue virus (DENV) and Zika virus (ZIKV), members of the Flavivirus genus, rearrange endoplasmic reticulum membranes to induce invaginations known as vesicle packets (VPs), which are the assumed sites for viral RNA replication. Mechanistic information on VP biogenesis has so far been difficult to attain due to the necessity of studying their formation under conditions of viral replication, where perturbations reducing replication will inevitably impact VP formation. Here, we report a replication-independent expression system, designated pIRO (plasmid-induced replication organelle formation) that induces bona fide DENV and ZIKV VPs that are morphologically indistinguishable from those in infected cells. Using this system, we demonstrate that sequences in the 3' terminal RNA region of the DENV, but not the ZIKV genome, contribute to VP formation in a non-replicative manner. These results validate the pIRO system that opens avenues for mechanistically dissecting virus replication from membrane reorganization.

Cell-to-Cell Spread of Dengue Viral RNA in Mosquito Cells.

Dengue virus (DENV) is an important mosquito-borne arbovirus that is particularly prevalent in tropical and subtropical areas of the world. The virus is generally ingested with a blood meal, replicates in host tissues, and disseminates into salivary glands for transmission to the next host. Membrane-bound vacuoles carrying DENV particles have been documented in mosquito cells and play a role in the cell-to-cell transmission of DENV2. C189 is one member of the tetraspanin family and generally increases its expression as one component of the vacuoles (C189-VCs) within C6/36 cells infected with DENV2. In the present study, we have further demonstrated via sucrose gradient centrifugation as well as magnetic immune isolation (MI) that the RNA of DENV2 was eventually carried by C189-VCs. In addition, viral RNA was shown to spread from donor to recipient cells in a coculture assay even when 20 mM NH4Cl was added to inhibit virus replication in the culture. In an alternate assay using the transwell system, viral RNA was only detected in recipient cells in the absence of 40 mM NH4Cl, suggesting that cell-cell contact is required for the intercellular spread of DENV2. In turn, the formation of viral synapse (VS) derived from aggregates of viral particles was frequently observed at sites of cell contact. Taken together, the formation of C189-VCs in C6/36 cells is induced by DENV2 infection, which may serve as a vehicle for transferring virions and also viral RNA to neighboring cells by cell-to-cell transmission after cell-cell contact. This finding provides insight into the understanding of viral spread between mosquito cells. It may also elucidate the benign persistent infection in mosquito cells and efficient dissemination of DENV infection within a mosquito vector.

High flavivirus structural plasticity demonstrated by a non-spherical morphological variant.

Previous flavivirus (dengue and Zika viruses) studies showed largely spherical particles either with smooth or bumpy surfaces. Here, we demonstrate flavivirus particles have high structural plasticity by the induction of a non-spherical morphology at elevated temperatures: the club-shaped particle (clubSP), which contains a cylindrical tail and a disc-like head. Complex formation of DENV and ZIKV with Fab C10 stabilize the viruses allowing cryoEM structural determination to ~10 Å resolution. The caterpillar-shaped (catSP) Fab C10:ZIKV complex shows Fabs locking the E protein raft structure containing three E dimers. However, compared to the original spherical structure, the rafts have rotated relative to each other. The helical tail structure of Fab C10:DENV3 clubSP showed although the Fab locked an E protein dimer, the dimers have shifted laterally. Morphological diversity, including clubSP and the previously identified bumpy and smooth-surfaced spherical particles, may help flavivirus survival and immune evasion.

The Nuclear Pore Complex: A Target for NS3 Protease of Dengue and Zika Viruses.

During flavivirus infection, some viral proteins move to the nucleus and cellular components are relocated from the nucleus to the cytoplasm. Thus, the integrity of the main regulator of the nuclear-cytoplasmic transport, the nuclear pore complex (NPC), was evaluated during infection with dengue virus (DENV) and Zika virus (ZIKV). We found that while during DENV infection the integrity and distribution of at least three nucleoporins (Nup), Nup153, Nup98, and Nup62 were altered, during ZIKV infection, the integrity of TPR, Nup153, and Nup98 were modified. In this work, several lines of evidence indicate that the viral serine protease NS2B3 is involved in Nups cleavage. First, the serine protease inhibitors, TLCK and Leupeptin, prevented Nup98 and Nup62 cleavage. Second, the transfection of DENV and ZIKV NS2B3 protease was sufficient to inhibit the nuclear ring recognition detected in mock-infected cells with the Mab414 antibody. Third, the mutant but not the active (WT) protease was unable to cleave Nups in transfected cells. Thus, here we describe for the first time that the NS3 protein from flavivirus plays novel functions hijacking the nuclear pore complex, the main controller of the nuclear-cytoplasmic transport.

Molecular basis of dengue virus serotype 2 morphological switch from 29°C to 37°C.

The ability of DENV2 to display different morphologies (hence different antigenic properties) complicates vaccine and therapeutics development. Previous studies showed most strains of laboratory adapted DENV2 particles changed from smooth to "bumpy" surfaced morphology when the temperature is switched from 29°C at 37°C. Here we identified five envelope (E) protein residues different between two alternative passage history DENV2 NGC strains exhibiting smooth or bumpy surface morphologies. Several mutations performed on the smooth DENV2 infectious clone destabilized the surface, as observed by cryoEM. Molecular dynamics simulations demonstrated how chemically subtle substitution at various positions destabilized dimeric interactions between E proteins. In contrast, three out of four DENV2 clinical isolates showed a smooth surface morphology at 37°C, and only at high fever temperature (40°C) did they become "bumpy". These results imply vaccines should contain particles representing both morphologies. For prophylactic and therapeutic treatments, this study also informs on which types of antibodies should be used at different stages of an infection, i.e., those that bind to monomeric E proteins on the bumpy surface or across multiple E proteins on the smooth surfaced virus.

Complementary use of mass spectrometry and cryo-electron microscopy to assess the maturity of live attenuated dengue vaccine viruses.

Dengue virus (DENV) infection is a global health threat with the potential to affect at least 3.6 billion people living in areas of risk. No specific curative treatments against dengue disease are available and vaccines are currently the only way to prevent the disease. The tetravalent dengue vaccine developed by Sanofi Pasteur has demonstrated significant efficacy in phase III studies and is now licensed in several countries for the prevention of disease in dengue-seropositives over 9 years of age. The vaccine is composed of four recombinant, live, attenuated vaccines (CYD 1-4) based on a yellow fever vaccine 17D (YFV 17D) backbone, each expressing the pre-membrane (prM) and envelope (E) genes of one of the four DENV serotypes. Virus maturity could impact the biological activity of the vaccine viruses. To address this question, the maturity of the four vaccine viruses used in phase III clinical studies was assessed by two complementary techniques: mass spectrometry (MS) and cryo-electron microscopy (cryoEM). MS assessed viral maturity at the molecular level by quantifying specifically the prM, and M proteins. CryoEM provided information at the particle level, allowing visualizing the different phenotypes of viral particles: spiky (immature), smooth/bumpy (mature), and mixed (partially mature). Results of the two assays used in this study show that all four CYD dengue vaccine viruses present in lots used in phase III efficacy trials, display in the majority a mature phenotype.

Infectivity of Dengue Virus Serotypes 1 and 2 Is Correlated with E-Protein Intrinsic Dynamics but Not to Envelope Conformations.

Dengue is a mosquito-borne virus with dire health and economic impacts. Dengue is responsible for an estimated 390 million infections per year, with dengue 2 (DENV2) being the most virulent strain among the four serotypes. Interestingly, it is also in strains of this serotype that temperature-dependent large-scale morphological changes, termed "breathing," have been observed. Although the structure of these morphologies has been solved to 3.5-Å resolution, the dynamics of the viral envelope are unknown. Here, we combine fluorescence and mass spectrometry with molecular dynamics simulations to provide insights into DENV2 (NGC strain) structural dynamics in comparison with DENV1 (PVP 159). We observe hitherto unseen conformational changes and structural dynamics of the DENV2 envelope that are influenced by both temperature and divalent cations. Our results show that for DENV2 and DENV1 the intrinsic dynamics, but not the specific morphologies, are correlated with viral infectivity.

Epitope resurfacing on dengue virus-like particle vaccine preparation to induce broad neutralizing antibody.

Dengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading cause of worldwide arboviral diseases in humans. Virus-like particles (VLPs) containing flavivirus prM/E proteins have been demonstrated to be a potential vaccine candidate; however, the structure of dengue VLP is poorly understood. Herein VLP derived from DENV serotype-2 were engineered becoming highly matured (mD2VLP) and showed variable size distribution with diameter of ~31 nm forming the major population under cryo-electron microscopy examination. Furthermore, mD2VLP particles of 31 nm diameter possess a T = 1 icosahedral symmetry with a groove located within the E-protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing epitopes. Mice vaccinated with mD2VLP generated higher cross-reactive (CR) neutralization antibodies (NtAbs) and were fully protected against all 4 serotypes of DENV. Our results highlight the potential of 'epitope-resurfaced' mature-form D2VLPs in inducing quaternary structure-recognizing broad CR NtAbs to guide future dengue vaccine design.

Pervasive tertiary structure in the dengue virus RNA genome.

RNA virus genomes are efficient and compact carriers of biological information, encoding information required for replication both in their primary sequences and in higher-order RNA structures. However, the ubiquity of RNA elements with higher-order folds-in which helices pack together to form complex 3D structures-and the extent to which these elements affect viral fitness are largely unknown. Here we used single-molecule correlated chemical probing to define secondary and tertiary structures across the RNA genome of dengue virus serotype 2 (DENV2). Higher-order RNA structures are pervasive and involve more than one-third of nucleotides in the DENV2 genomic RNA. These 3D structures promote a compact overall architecture and contribute to viral fitness. Disrupting RNA regions with higher-order structures leads to stable, nonreverting mutants and could guide the development of vaccines based on attenuated RNA viruses. The existence of extensive regions of functional RNA elements with tertiary folds in viral RNAs, and likely many other messenger and noncoding RNAs, means that there are significant regions with pocket-containing surfaces that may serve as novel RNA-directed drug targets.

Molecular Aspects of the Dengue Virus Infection Process: A Review.

BACKGROUND: At the present time, dengue is one of the most important arboviruses affecting man, becoming a serious global public health problem, especially in subtropical and tropical countries, where environmental conditions favor the development and proliferation of the mosquito Aedes aegypti. Dengue is caused by a type of flavivírus, which is an enveloped virus of spherical geometry. Nowadays, it is one of the diseases with the highest incidence in Brazil, reaching the population of all states, regardless of social class. Several papers address the molecular aspects of infection of human cell by the viruses, which are reviewed in this work. CONCLUSION: Analyzing the three-dimensional structures of the fusion peptide of dengue virus protein E, we observed that the fusion peptide presents a region rich in hydrophobic residues and a "collar" of charged, polar residues. Probably, this hydrophilic collar plays an important role in the fusion process between the dengue virus and the cell membrane. In order for this disease to cease being a serious global public health problem, we must deepen our knowledge about the fusion process between the dengue virus and the cell membrane through further experimental and, especially, computational studies to find ways to inhibit the mechanism of virus infection.

TIM-1 Ubiquitination Mediates Dengue Virus Entry.

Dengue virus (DENV) is a major human pathogen causing millions of infections yearly. Despite intensive investigations, a DENV receptor that directly participates in virus internalization has not yet been characterized. Here, we report that the phosphatidylserine receptor TIM-1 is an authentic DENV entry receptor that plays an active role in virus endocytosis. Genetic ablation of TIM-1 strongly impaired DENV infection. Total internal reflection fluorescence microscopy analyses of live infected cells show that TIM-1 is mostly confined in clathrin-coated pits and is co-internalized with DENV during viral entry. TIM-1 is ubiquitinated at two lysine residues of its cytoplasmic domain, and this modification is required for DENV endocytosis. Furthermore, STAM-1, a component of the ESCRT-0 complex involved in intracellular trafficking of ubiquitinated cargos, interacts with TIM-1 and is required for DENV infection. Overall, our results show that TIM-1 is the first bona fide receptor identified for DENV.

Strand-like structures and the nonstructural proteins 5, 3 and 1 are present in the nucleus of mosquito cells infected with dengue virus.

Dengue virus (DENV) is an arbovirus, which replicates in the endoplasmic reticulum. Although replicative cycle takes place in the cytoplasm, some viral proteins such as NS5 and C are translocated to the nucleus during infection in mosquitoes and mammalian cells. To localized viral proteins in DENV-infected C6/36 cells, an immunofluorescence (IF) and immunoelectron microscopy (IEM) analysis were performed. Our results indicated that C, NS1, NS3 and NS5 proteins were found in the nucleus of DENV-infected C6/36 cells. Additionally, complex structures named strand-like structures (Ss) were observed in the nucleus of infected cells. Interestingly, the NS5 protein was located in these structures. Ss were absent in mock-infected cells, suggesting that DENV induces their formation in the nucleus of infected mosquito cells.

Genome-Wide Mutagenesis of Dengue Virus Reveals Plasticity of the NS1 Protein and Enables Generation of Infectious Tagged Reporter Viruses.

Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that the regions within capsid, NS1, and the 3' untranslated region were the most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector, and ß-ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information, we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high-resolution imaging of its localization to the surface and interior of viral replication vesicles. In addition, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies.IMPORTANCE Regions of genetic flexibility in viral genomes can be exploited in the generation of reporter virus tools and should arguably be avoided in antiviral drug and vaccine design. Here, we subjected the DENV genome to high-throughput insertional mutagenesis to identify regions of genetic flexibility and enable tagged reporter virus generation. In particular, the viral NS1 protein displayed remarkable tolerance of small insertions. This genetic flexibility enabled generation of several novel NS1-tagged reporter viruses, including an APEX2-tagged virus that we used in high-resolution imaging of NS1 localization in infected cells by electron microscopy. For the first time, this analysis revealed the localization of NS1 within viral replication factories known as "vesicle packets" (VPs), in addition to its acknowledged localization to the luminal surface of these VPs. Together, this genetic profile of DENV may be further refined and exploited in the identification of antiviral targets and the generation of reporter virus tools.

Systematic analysis of protein identity between Zika virus and other arthropod-borne viruses.

OBJECTIVE: To analyse the proportions of protein identity between Zika virus and dengue, Japanese encephalitis, yellow fever, West Nile and chikungunya viruses as well as polymorphism between different Zika virus strains. METHODS: We used published protein sequences for the Zika virus and obtained protein sequences for the other viruses from the National Center for Biotechnology Information (NCBI) protein database or the NCBI virus variation resource. We used BLASTP to find regions of identity between viruses. We quantified the identity between the Zika virus and each of the other viruses, as well as within-Zika virus polymorphism for all amino acid k-mers across the proteome, with k ranging from 6 to 100. We assessed accessibility of protein fragments by calculating the solvent accessible surface area for the envelope and nonstructural-1 (NS1) proteins. FINDINGS: In total, we identified 294 Zika virus protein fragments with both low proportion of identity with other viruses and low levels of polymorphisms among Zika virus strains. The list includes protein fragments from all Zika virus proteins, except NS3. NS4A has the highest number (190 k-mers) of protein fragments on the list. CONCLUSION: We provide a candidate list of protein fragments that could be used when developing a sensitive and specific serological test to detect previous Zika virus infections.

Properties and Functions of the Dengue Virus Capsid Protein.

Dengue virus affects hundreds of millions of people each year around the world, causing a tremendous social and economic impact on affected countries. The aim of this review is to summarize our current knowledge of the functions, structure, and interactions of the viral capsid protein. The primary role of capsid is to package the viral genome. There are two processes linked to this function: the recruitment of the viral RNA during assembly and the release of the genome during infection. Although particle assembly takes place on endoplasmic reticulum membranes, capsid localizes in nucleoli and lipid droplets. Why capsid accumulates in these locations during infection remains unknown. In this review, we describe available data and discuss new ideas on dengue virus capsid functions and interactions. We believe that a deeper understanding of how the capsid protein works during infection will create opportunities for novel antiviral strategies, which are urgently needed to control dengue virus infections.

Enhancing dengue virus maturation using a stable furin over-expressing cell line.

Flaviviruses are positive-stranded RNA viruses that incorporate envelope (E) and premembrane (prM) proteins into the virion. Furin-mediated cleavage of prM defines a required maturation step in the flavivirus lifecycle. Inefficient prM cleavage results in structurally heterogeneous virions with unique antigenic and functional characteristics. Recent studies with dengue virus suggest that viruses produced in tissue culture cells are less mature than those produced in primary cells. In this study, we describe a Vero cell line that ectopically expresses high levels of human furin (Vero-furin) for use in the production of more homogenous mature flavivirus populations. Laboratory-adapted and clinical dengue virus isolates grow efficiently in Vero-furin cells. Biochemical and structural techniques demonstrate efficient prM cleavage in Vero-furin derived virus preparations. These virions also were less sensitive to neutralization by antibodies that bind efficiently to immature virions. This furin-expressing cell line will be of considerable utility for flavivirus neutralization and structural studies.