Production of Infectious Dengue Virus in Aedes aegypti Is Dependent on the Ubiquitin Proteasome Pathway

Dengue virus (DENV) relies on host factors to complete its life cycle in its mosquito host for subsequent transmission to humans. DENV first establishes infection in the midgut of Aedes aegypti and spreads to various mosquito organs for lifelong infection. Curiously, studies have shown that infectious DENV titers peak and decrease thereafter in the midgut despite relatively stable viral genome levels. However, the mechanisms that regulate this decoupling of infectious virion production from viral RNA replication have never been determined. We show here that the ubiquitin proteasome pathway (UPP) plays an important role in regulating infectious DENV production. Using RNA interference studies, we show in vivo that knockdown of selected UPP components reduced infectious virus production without altering viral RNA replication in the midgut. Furthermore, this decoupling effect could also be observed after RNAi knockdown in the head/thorax of the mosquito, which otherwise showed direct correlation between infectious DENV titer and viral RNA levels. The dependence on the UPP for successful DENV production is further reinforced by the observed up-regulation of key UPP molecules upon DENV infection that overcome the relatively low expression of these genes after a blood meal. Collectively, our findings indicate an important role for the UPP in regulating DENV production in the mosquito vector.     

An Integrated Systems Biology Approach Identifies the Proteasome as A Critical Host Machinery for ZIKV and DENV Replication

The Zika virus (ZIKV) and dengue virus (DENV) flaviviruses exhibit similar replicative processes but have distinct clinical outcomes. A systematic understanding of virus–host protein–pro-tein interaction networks can reveal cellular pathways critical to viral replication and disease patho-genesis. Here we employed three independent systems biology approaches toward this goal. First, protein array analysis of direct interactions between individual ZIKV/DENV viral proteins and 20,240 human proteins revealed multiple conserved cellular pathways and protein complexes, including proteasome complexes. Second, an RNAi screen of 10,415 druggable genes identified the host proteins required for ZIKV infection and uncovered that proteasome proteins were crucial in this process. Third, high-throughput screening of 6016 bioactive compounds for ZIKV inhibition yielded 134 effective compounds, including six proteasome inhibitors that suppress both ZIKV and DENV replication. Integrative analyses of these orthogonal datasets pinpoint proteasomes as crit-ical host machinery for ZIKV/DENV replication. Our study provides multi-omics datasets for fur-ther studies of flavivirus–host interactions, disease pathogenesis, and new drug targets.     

Adaptor Protein 1A Facilitates Dengue Virus Replication

Rearrangement of membrane structure induced by dengue virus (DENV) is essential for replication, and requires host cellular machinery. Adaptor protein complex (AP)-1 is a host component, which can be recruited to components required for membrane rearrangement. Therefore, dysfunction of AP-1 may affect membrane organization, thereby decreasing replication of virus in infected cells. In the present study, AP-1-dependent traffic inhibitor inhibited DENV protein expression and virion production. We further clarified the role of AP-1A in the life cycle of DENV by RNA interference. AP-1A was not involved in DENV entry into cells. However, it facilitated DENV RNA replication. Viral RNA level was reduced significantly in Huh7 cells transfected with AP-1A small interfering RNA (siRNA) compared with control siRNA. Transfection of naked DENV viral RNA into Huh7 cells transfected with AP-1A siRNA resulted in less viral RNA and virion production than transfection into Huh7 cells transfected with control siRNA. Huh7 cells transfected with AP-1A siRNA showed greater modification of membrane structures and fewer vesicular packets compared with cells transfected with control siRNA. Therefore, AP-1A may partly control DENV-induced rearrangement of membrane structures required for viral replication.     

G Protein-Coupled Receptor Kinase 2 Promotes Flaviviridae Entry and Replication

Flaviviruses cause a wide range of severe diseases ranging from encephalitis to hemorrhagic fever. Discovery of host factors that regulate the fate of flaviviruses in infected cells could provide insight into the molecular mechanisms of infection and therefore facilitate the development of anti-flaviviral drugs. We performed genome-scale siRNA screens to discover human host factors required for yellow fever virus (YFV) propagation. Using a 2×2 siRNA pool screening format and a duplicate of the screen, we identified a high confidence list of YFV host factors. To find commonalities between flaviviruses, these candidates were compared to host factors previously identified for West Nile virus (WNV) and dengue virus (DENV). This comparison highlighted a potential requirement for the G protein-coupled receptor kinase family, GRKs, for flaviviral infection. The YFV host candidate GRK2 (also known as ADRBK1) was validated both in siRNA-mediated knockdown HuH-7 cells and in GRK^−/− mouse embryonic fibroblasts. Additionally, we showed that GRK2 was required for efficient propagation of DENV and Hepatitis C virus (HCV) indicating that GRK2 requirement is conserved throughout the Flaviviridae. Finally, we found that GRK2 participates in multiple distinct steps of the flavivirus life cycle by promoting both entry and RNA synthesis. Together, our findings identified GRK2 as a novel regulator of flavivirus infection and suggest that inhibition of GRK2 function may constitute a new approach for treatment of flavivirus associated diseases.     

Dengue virus type 3 isolated from a fatal case with visceral complications induces enhanced proinflammatory responses and apoptosis of human dendritic cells

A recent (2007 to 2009) dengue outbreak caused by dengue virus (DENV) in Paraguay presented unusual severe clinical outcomes associated with 50% mortality rates. Although it has been reported that inflammatory responses influence the severity of dengue virus infection (T. Pang, M. J. Cardosa, and M. G. Guzman, Immunol. Cell Biol. 85:43-45, 2007), there remains a paucity of information on virus-innate immunity interactions influencing clinical outcome. Using human dendritic cells from a major innate immune cell population as an in vitro model, we have investigated signature cytokine responses as well as infectivity-replicative profiles of DENV clinical isolates from either a nonfatal case of classical dengue fever (strain DENV3/290; isolated in Brazil in 2002) or a fatal case of dengue fever with visceral complications isolated in Paraguay in 2007 (strain DENV3/5532). Strain DENV3/5532 was found to display significantly higher replicative ability than DENV3/290 in monocyte-derived dendritic cells (mdDCs). In addition, compared to DENV3/290 results, mdDCs exposed to DENV3/5532 showed increased production of proinflammatory cytokines associated with higher rates of programmed cell death, as shown by annexin V staining. The observed phenotype was due to viral replication, and tumor necrosis factor alpha (TNF-α) appears to exert a protective effect on virus-induced mdDC apoptosis. These results suggest that the DENV3/5532 strain isolated from the fatal case replicates within human dendritic cells, modulating cell survival and synthesis of inflammatory mediators.     

Dengue and Zika viruses subvert reticulophagy by NS2B3-mediated cleavage of FAM134B

The endoplasmic reticulum (ER) is exploited by several diverse viruses during their infectious life cycles. Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), utilize the ER as a source of membranes to establish their replication organelles and to facilitate their assembly and eventual maturation along the secretory pathway. To maintain normal homeostasis, host cells have evolved highly efficient processes to dynamically regulate the ER, such as through reticulophagy, a selective form of autophagy that leads to ER degradation. Here, we identify the ER-localized reticulophagy receptor FAM134B as a host cell restriction factor for both DENV and ZIKV. We show that RNAi-mediated depletion of FAM134B significantly enhances both DENV and ZIKV replication at an early stage of the viral life cycle. Consistent with its role as an antiviral host factor, we found that several flaviviruses including DENV, ZIKV, and West Nile virus (WNV), utilize their NS3 virally-encoded proteases to directly cleave FAM134B at a single site within its reticulon homology domain (RHD). Mechanistically, we show that NS3-mediated cleavage of FAM134B blocks the formation of ER and viral protein-enriched autophagosomes, suggesting that the cleavage of FAM134B serves to specifically suppress the reticulophagy pathway. These findings thus point to an important role for FAM134B and reticulophagy in the regulation of flavivirus infection and suggest that these viruses specifically target these pathways to promote viral replication.     

Recent progress in dengue vaccine development

Dengue virus(DENV) has four distinct serotypes. DENV infection can result in classic dengue fever and life-threatening dengue hemorrhagic fever/dengue shock syndrome. In recent decades, DENV infection has become an important public health concern in epidemic-prone areas. Vaccination is the most effective measure to prevent and control viral infections. However, several challenges impede the development of effective DENV vaccines, such as the lack of suitable animal models and the antibody-dependent enhancement phenomenon. Although no licensed DENV vaccine is available, significant progress has been made. This review summarizes candidate DENV vaccines from recent investigations.     

Small G Rac1 is involved in replication cycle of dengue serotype 2 virus in EAhy926 cells via the regulation of actin cytoskeleton

Bleeding is a clinical characteristic of severe dengue and may be due to increased vascular permeability. However, the pathogenesis of severe dengue remains unclear. In this study, we showed that the Rac1-microfilament signal pathway was involved in the process of DENV serotype 2 (DENV2) infection in EAhy926 cells. DENV2 infection induced dynamic changes in actin organization, and treatment with Cytochalasin D or Jasplakinolide disrupted microfilament dynamics, reduced DENV2 entry, and inhibited DENV2 assembly and maturation. Rac1 activities decreased during the early phase and gradually increased by the late phase of infection. Expression of the dominant-negative form of Rac1 promoted DENV2 entry but inhibited viral assembly, maturation and release. Our findings demonstrated that Rac1 plays an important role in the DENV2 life cycle by regulating actin reorganization in EAhy926 cells. This finding provides further insight into the pathogenesis of severe dengue.     

Oleic acid Enhances Dengue Virus But Not Dengue Virus-Like Particle Production from Mammalian Cells

Despite the recent introduction of a commercial vaccine, the mosquito-transmitted dengue virus is still a worldwide public health problem. Based on the live attenuated vaccine strategy, the commercial vaccine has a less than optimal protective profile. Virus-like particles (VLPs) offer an attractive alternate vaccination strategy due to the effectively native presentation of epitopes in the absence of any infectious genetic material. However, the production of amounts of VLP in a platform that can support commercial development remains a major obstacle. This study generated two DENV 2 VLPs [codon-optimized and chimeric DENV/Japanese encephalitis virus (JEV)] and directly compared yields of these constructs by western blotting and dot blot hybridization. The effect of oleic acid supplementation, a process known to increase DENV production in natural infection, was also investigated. Results showed that the chimeric construct gave a two- to threefold higher yield than the codon-optimized construct and that while oleic acid increased DENV virion production in natural infection, it inhibited VLP production. These results suggest that further optimization of DENV VLP expression is possible, but it will require more understanding of how native DENV infection remodels the host cell machinery.     

Appraising the roles of CBLL1 and the ubiquitin/proteasome system for flavivirus entry and replication

The ubiquitin ligase CBLL1 (also known as HAKAI) has been proposed to be a critical cellular factor exploited by West Nile virus (WNV) for productive infection. CBLL1 has emerged as a major hit in a recent RNA interference screen designed to identify cellular factors required for the early stages of the WNV life cycle. Follow-up experiments showed that HeLa cells knocked down for CBLL1 by a small interfering RNA (siRNA) failed to internalize WNV particles and resisted infection. Furthermore, depletion of a free-ubiquitin pool by the proteasome inhibitor MG132 abolished WNV endocytosis, suggesting that CBLL1 acts in concert with the ubiquitin proteasome system to mediate virus internalization. Here, we examined the effect of CBLL1 knockdown and proteasome inhibitors on infection by WNV and other flaviviruses. We identified new siRNAs that repress the CBLL1 protein and strongly inhibit the endocytosis of Listeria monocytogenes, a bacterial pathogen known to require CBLL1 to invade host cells. Strikingly, however, we detected efficient WNV, dengue virus, and yellow fever virus infection of human cells, despite potent downregulation of CBLL1 by RNA interference. In addition, we found that the proteasome inhibitors MG132 and lactacystin did not affect WNV internalization but strongly repressed flavivirus RNA translation and replication. Together, these data do not support a requirement for CBLL1 during flavivirus entry and rather suggest an essential role of the ubiquitin/proteasome pathway for flavivirus genome amplification.     

Interaction of dengue virus nonstructural protein 5 with Daxx modulates RANTES production

Dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), caused by dengue virus (DENV) infection, are important public health problems in the tropical and subtropical regions. Abnormal hemostasis and plasma leakage are the main patho-physiological changes in DHF/DSS. A remarkably increased production of cytokines, the so called 'cytokine storm', is observed in the patients with DHF/DSS. A complex interaction between DENV proteins and the host immune response contributes to cytokine production. However, the molecular mechanism(s) by which DENV nonstructural protein 5 (NS5) mediates these responses has not been fully elucidated. In the present study, yeast two-hybrid assay was performed to identify host proteins interacting with DENV NS5 and a death-domain-associate protein (Daxx) was identified. The in vivo relevance of this interaction was suggested by co-immunoprecipitation and nuclear co-localization of these two proteins in HEK293 cells expressing DENV NS5. HEK293 cells expressing DENV NS5-K/A, which were mutated at the nuclear localization sequences (NLS), were created to assess its functional roles in nuclear translocation, Daxx interaction, and cytokine production. In the absence of NLS, DENV NS5 could neither translocate into the nucleus nor interact with Daxx to increase the DHF-associated cytokine, RANTES (CCL5) production. This work demonstrates the interaction between DENV NS5 and Daxx and the role of the interaction on the modulation of RANTES production.     

Current progress in dengue vaccines

Dengue is one of the most important emerging vector-borne viral diseases. There are four serotypes of dengue viruses (DENV), each of which is capable of causing self-limited dengue fever (DF) or even life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The major clinical manifestations of severe DENV disease are vascular leakage, thrombocytopenia, and hemorrhage, yet the detailed mechanisms are not fully resolved. Besides the direct effects of the virus, immunopathological aspects are also involved in the development of dengue symptoms. Although no licensed dengue vaccine is yet available, several vaccine candidates are under development, including live attenuated virus vaccines, live chimeric virus vaccines, inactivated virus vaccines, and live recombinant, DNA and subunit vaccines. The live attenuated virus vaccines and live chimeric virus vaccines are undergoing clinical evaluation. The other vaccine candidates have been evaluated in preclinical animal models or are being prepared for clinical trials. For the safety and efficacy of dengue vaccines, the immunopathogenic complications such as antibody-mediated enhancement and autoimmunity of dengue disease need to be considered.     

Galectin-1 Exerts Inhibitory Effects during DENV-1 Infection

Dengue virus (DENV) is an enveloped RNA virus that is mosquito-transmitted and can infect a variety of immune and non-immune cells. Response to infection ranges from asymptomatic disease to a severe disorder known as dengue hemorrhagic fever. Despite efforts to control the disease, there are no effective treatments or vaccines. In our search for new antiviral compounds to combat infection by dengue virus type 1 (DENV-1), we investigated the role of galectin-1, a widely-expressed mammalian lectin with functions in cell-pathogen interactions and immunoregulatory properties. We found that DENV-1 infection of cells in vitro exhibited caused decreased expression of Gal-1 in several different human cell lines, suggesting that loss of Gal-1 is associated with virus production. In test of this hypothesis we found that exogenous addition of human recombinant Gal-1 (hrGal-1) inhibits the virus production in the three different cell types. This inhibitory effect was dependent on hrGal-1 dimerization and required its carbohydrate recognition domain. Importantly, the inhibition was specific for hrGal-1, since no effect was observed using recombinant human galectin-3. Interestingly, we found that hrGal-1 directly binds to dengue virus and acts, at least in part, during the early stages of DENV-1 infection, by inhibiting viral adsorption and its internalization to target cells. To test the in vivo role of Gal-1 in DENV infection, Gal-1-deficient-mice were used to demonstrate that the expression of endogenous Galectin-1 contributes to resistance of macrophages to in vitro-infection with DENV-1 and it is also important to physiological susceptibility of mice to in vivo infection with DENV-1. These results provide novel insights into the functions of Gal-1 in resistance to DENV infection and suggest that Gal-1 should be explored as a potential antiviral compound.     

Mechanisms of monocyte cell death triggered by dengue virus infection

Arthropod-borne viral diseases caused by dengue virus (DENV) are major re-emerging public health problem worldwide. In spite of intense research, DENV pathogenesis is not fully understood and remains enigmatic; however, current evidence suggests that dengue progression is associated with an inflammatory response, mainly in patients suffering from a second DENV infection. Monocytes are one of the main target cells of DENV infection and play an important role in pathogenesis since they are known to produce several inflammatory cytokines that can lead to endothelial dysfunction and therefore vascular leak. In addition, monocytes play an important role in antibody dependent enhancement, infection with consequences in viral load and immune response. Despite the physiological functions of monocytes in immune response, their life span in the bloodstream is very short, and activation of monocytes by DENV infection can trigger different types of cell death. For example, DENV can induce apoptosis in monocytes related with the production of Tumor necrosis factor alpha (TNF-α). Additionally, recent studies have shown that DENV-infected monocytes also exhibit a cell death process mediated by caspase-1 activation together with IL-1 production, referred to as pyroptosis. Taken together, the aforementioned studies strongly depict that multiple cell death pathways may be occurring in monocytes upon DENV-2 infection. This review provides insight into mechanisms of DENV-induced death of both monocytes and other cell types for a better understanding of this process. Further knowledge in cell death induced by DENV will help in the developing novel strategies to prevent disease progression.     

Aggressive organ penetration and high vector transmissibility of epidemic dengue virus-2 Cosmopolitan genotype in a transmission mouse model

Dengue virus (DENV) causes dengue fever and severe hemorrhagic fever in humans and is primarily transmitted by Aedes aegypti and A. albopictus mosquitoes. The incidence of DENV infection has been gradually increasing in recent years due to global urbanization and international travel. Understanding the virulence determinants in host and vector transmissibility of emerging epidemic DENV will be critical to combat potential outbreaks. The DENV serotype 2 (DENV-2), which caused a widespread outbreak in Taiwan in 2015 (TW2015), is of the Cosmopolitan genotype and is phylogenetically related to the virus strain linked to another large outbreak in Indonesia in 2015. We found that the TW2015 virus was highly virulent in type I and type II interferon-deficient mice, with robust replication in spleen, lung, and intestine. The TW2015 virus also had high transmissibility to Aedes mosquitoes and could be effectively spread in a continuous mosquitoes-mouse-mosquitoes-mouse transmission cycle. By making 16681-based mutants carrying different segments of the TW2015 virus, we identified the structural pre-membrane (prM) and envelope (E) genes as key virulence determinants in the host, with involvement in the high transmissibility of the TW2015 virus in mosquitoes. The transmission mouse model will make a useful platform for evaluation of DENV with high epidemic potential and development of new strategies against dengue outbreaks.     

Ubiquitin-proteasome pathway and cellular responses to oxidative stress

The ubiquitin-proteasome pathway (UPP) is the primary cytosolic proteolytic machinery for the selective degradation of various forms of damaged proteins. Thus, the UPP is an important protein quality control mechanism. In the canonical UPP, both ubiquitin and the 26S proteasome are involved. Substrate proteins of the canonical UPP are first tagged by multiple ubiquitin molecules and then degraded by the 26S proteasome. However, in noncanonical UPP, proteins can be degraded by the 26S or the 20S proteasome without being ubiquitinated. It is clear that a proteasome is responsible for selective degradation of oxidized proteins, but the extent to which ubiquitination is involved in this process remains a subject of debate. Whereas many publications suggest that the 20S proteasome degrades oxidized proteins independent of ubiquitin, there is also solid evidence indicating that ubiquitin and ubiquitination are involved in degradation of some forms of oxidized proteins. A fully functional UPP is required for cells to cope with oxidative stress and the activity of the UPP is also modulated by cellular redox status. Mild or transient oxidative stress up-regulates the ubiquitination system and proteasome activity in cells and tissues and transiently enhances intracellular proteolysis. Severe or sustained oxidative stress impairs the function of the UPP and decreases intracellular proteolysis. Both the ubiquitin-conjugating enzymes and the proteasome can be inactivated by sustained oxidative stress, especially the 26S proteasome. Differential susceptibilities of the ubiquitin-conjugating enzymes and the 26S proteasome to oxidative damage lead to an accumulation of ubiquitin conjugates in cells in response to mild oxidative stress. Thus, increased levels of ubiquitin conjugates in cells seem to be an indicator of mild oxidative stress.     

Identification of RNA Binding Proteins Associated with Dengue Virus RNA in Infected Cells Reveals Temporally Distinct Host Factor Requirements

BackgroundThere are currently no vaccines or antivirals available for dengue virus infection, which can cause dengue hemorrhagic fever and death. A better understanding of the host pathogen interaction is required to develop effective therapies to treat DENV. In particular, very little is known about how cellular RNA binding proteins interact with viral RNAs. RNAs within cells are not naked; rather they are coated with proteins that affect localization, stability, translation and (for viruses) replication.Conclusions/SignificanceThe method for identification of host factors described here is robust and broadly applicable to all RNA viruses, providing an avenue to determine the conserved or distinct mechanisms through which diverse viruses manage the viral RNA within cells. This study significantly increases the number of cellular factors known to interact with DENV and reveals how DENV modulates and usurps cellular proteins for efficient amplification.