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 NS5 protein plays multiple functions in the cytoplasm of infected cells, enabling viral RNA replication and counteracting host antiviral responses. Here, we demonstrate a novel function of NS5 in the nucleus where it interferes with cellular splicing. Using global proteomic analysis of infected cells together with functional studies, we found that NS5 binds spliceosome complexes and modulates endogenous splicing as well as minigene-derived alternative splicing patterns. In particular, we show that NS5 alone, or in the context of viral infection, interacts with core components of the U5 snRNP particle, CD2BP2 and DDX23, alters the inclusion/exclusion ratio of alternative splicing events, and changes mRNA isoform abundance of known antiviral factors. Interestingly, a genome wide transcriptome analysis, using recently developed bioinformatics tools, revealed an increase of intron retention upon dengue virus infection, and viral replication was improved by silencing specific U5 components. Different mechanistic studies indicate that binding of NS5 to the spliceosome reduces the efficiency of pre-mRNA processing, independently of NS5 enzymatic activities. We propose that NS5 binding to U5 snRNP proteins hijacks the splicing machinery resulting in a less restrictive environment for viral replication.

Dengue virus RNA-dependent RNA polymerase specifically binds to the viral genome by interacting with a promoter element known as stem-loop A (SLA). Although a great deal has been learned in recent years about the function of this promoter in dengue virus-infected cells, the molecular details that explain how the SLA interacts with the polymerase to promote viral RNA synthesis remain poorly understood. Using RNA binding and polymerase activity assays, we defined two elements of the SLA that are involved in polymerase interaction and RNA synthesis. Mutations at the top of the SLA resulted in RNAs that retained the ability to bind the polymerase but impaired promoter-dependent RNA synthesis. These results indicate that protein binding to the SLA is not sufficient to induce polymerase activity and that specific nucleotides of the SLA are necessary to render an active polymerase-promoter complex for RNA synthesis. We also report that protein binding to the viral RNA induces conformational changes downstream of the promoter element. Furthermore, we found that structured RNA elements at the 3 end of the template repress dengue virus polymerase activity in the context of a fully active SLA promoter. Using assays to evaluate initiation of RNA synthesis at the viral 3-UTR, we found that the RNA-RNA interaction mediated by 5-3-hybridization was able to release the silencing effect of the 3-stem-loop structure. We propose that the long range RNA-RNA interactions in the viral genome play multiple roles during RNA synthesis. Together, we provide new molecular details about the promoter-dependent dengue virus RNA polymerase activity. Dengue virus (DENV)3 is a member of the Flavivirus genus in the Flaviviridae family, together with other important human pathogens such as yellow fever virus, West Nile virus (WNV), Saint Luis encephalitis virus, and Japanese encephalitis virus (1). DENV is the most significant mosquito-borne human viral pathogen worldwide and is responsible for the highest rates of disease and mortality among the members of the Flavivirus genus. The lack of vaccines and antivirals against DENV leaves two billion people at risk, mainly in poor countries.DENV genome is a single-stranded RNA molecule of positive polarity of ϳ11 kb in length that encodes a long polyprotein that is co-and post-translationally processed by host and viral proteases to yield three structural proteins (C, prM, and E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The coding sequence is flanked by highly structured 5Ј-and 3Ј-UTRs. In recent years, a number of cis-acting RNA elements have been identified in the viral 5Ј-and 3Ј-UTRs that are essential for viral RNA amplification (for review see Ref.2). A model for DENV RNA synthesis has been proposed previously (3). This model involves binding of the viral RNA-dependent RNA polymerase (RdRp) to an RNA element present at the 5Ј end of the genome known as stemloop A (SLA). This element of 70 nucleotides folds into a Yshaped structure that is conserved among diff...

The process of genome release or uncoating after viral entry is one of the least-studied steps in the flavivirus life cycle. Flaviviruses are mainly arthropod-borne viruses, including emerging and reemerging pathogens such as dengue, Zika, and West Nile viruses. Currently, dengue virus is one of the most significant human viral pathogens transmitted by mosquitoes and is responsible for about 390 million infections every year around the world. Here, we examined for the first time molecular aspects of dengue virus genome uncoating. We followed the fate of the capsid protein and RNA genome early during infection and found that capsid is degraded after viral internalization by the host ubiquitin-proteasome system. However, proteasome activity and capsid degradation were not necessary to free the genome for initial viral translation. Unexpectedly, genome uncoating was blocked by inhibiting ubiquitination. Using different assays to bypass entry and evaluate the first rounds of viral translation, a narrow window of time during infection that requires ubiquitination but not proteasome activity was identified. In this regard, ubiquitin E1-activating enzyme inhibition was sufficient to stabilize the incoming viral genome in the cytoplasm of infected cells, causing its retention in either endosomes or nucleocapsids. Our data support a model in which dengue virus genome uncoating requires a nondegradative ubiquitination step, providing new insights into this crucial but understudied viral process.

The dengue virus genome is a dynamic molecule that adopts different conformations in the infected cell. Here, using RNA folding predictions, chemical probing analysis, RNA binding assays, and functional studies, we identified new cis-acting elements present in the capsid coding sequence that facilitate cyclization of the viral RNA by hybridization with a sequence involved in a local dumbbell structure at the viral 3= untranslated region (UTR). The identified interaction differentially enhances viral replication in mosquito and mammalian cells. Dengue virus (DENV) is a member of the Flaviviridae family that includes other important pathogens such as yellow fever virus (YFV), West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV). The DENV genome is a plus-stranded RNA molecule that contains a single open reading frame flanked by highly structured 5= and 3= untranslated regions (UTRs) (1-3). RNA elements located within these regions are responsible for translation initiation and genome replication (4-7). The 5= UTR is about 100 nucleotides (nt) long and includes three different elements: (i) stem-loop A (SLA), which is the promoter for viral polymerase binding and activation (8-10); (ii) stem-loop B (SLB), which contains a sequence known as 5= upstream of the AUG region (5= UAR) that is complementary to a sequence present at the 3= UTR (3= UAR) and mediates longrange RNA-RNA interactions between the ends of the genome (11); and (iii) a spacer sequence between SLA and SLB rich in U's, which functions as an enhancer of viral replication (10). The viral 3= UTR is about 450 nucleotides long and comprises four defined domains: domain A1, which features a variable region (VR) (12); domains A2 and A3, which present two almost-identical dumbbell-like secondary structures (DB1 and DB2), which appear to work as enhancers for viral RNA replication (13-15); and domain A4, which contains a small hairpin (sHP) and the 3= stem-loop (3= SL), which are essential elements for viral replication (3,16). In addition to RNA structures defined in the UTRs that play different roles during infection, important RNA elements have been described in the protein coding region. In this regard, essential sequences that mediate long-range RNA-RNA interactions known as 5= cyclization sequence (5= CS) and 5= downstream of AUG region (5= DAR) are located within the capsid coding sequence (11,13,(17)(18)(19)(20)(21). Also, a hairpin known as cHP, located between 5= CS and 5= DAR, has been shown to be necessary for efficient RNA replication (22). The current model for viral RNA synthesis includes the interaction of the viral polymerase NS5 with the 5=-end SLA promoter and its transfer to the 3=-end initiation site by cyclization of the viral genome (9). Despite great advances in knowledge of cis-acting RNA elements in the flavivirus genomes, the molecular details and mechanisms by which many of them function during viral replication are still not well understood.Intrigued by dual roles of RNA sequences in v...

The mechanism by which viral RNA-dependent RNA polymerases (RdRp) specifically amplify viral genomes is still unclear. In the case of flaviviruses, a model has been proposed that involves the recognition of an RNA element present at the viral 5 untranslated region, stem-loop A (SLA), that serves as a promoter for NS5 polymerase binding and activity. Here, we investigated requirements for specific promoter-dependent RNA synthesis of the dengue virus NS5 protein. Using mutated purified NS5 recombinant proteins and infectious viral RNAs, we analyzed the requirement of specific amino acids of the RdRp domain on polymerase activity and viral replication. A battery of 19 mutants was designed and analyzed. By measuring polymerase activity using nonspecific poly(rC) templates or specific viral RNA molecules, we identified four mutants with impaired polymerase activity. Viral full-length RNAs carrying these mutations were found to be unable to replicate in cell culture. Interestingly, one recombinant NS5 protein carrying the mutations K456A and K457A located in the F1 motif lacked RNA synthesis dependent on the SLA promoter but displayed high activity using a poly(rC) template. Promoter RNA binding of this NS5 mutant was unaffected while de novo RNA synthesis was abolished. Furthermore, the mutant maintained RNA elongation activity, indicating a role of the F1 region in promoter-dependent initiation. In addition, four NS5 mutants were selected to have polymerase activity in the recombinant protein but delayed or impaired virus replication when introduced into an infectious clone, suggesting a role of these amino acids in other functions of NS5. This work provides new molecular insights on the specific RNA synthesis activity of the dengue virus NS5 polymerase.Dengue virus (DENV) is the single most significant arthropod-borne virus pathogen in humans. It belongs to the Flaviviridae family together with other important pathogens such as yellow fever virus (YFV), West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV). The World Health Organization continues reporting dengue outbreaks every year in the Americas and Asia. In spite of the urgent medical need to control DENV infections, vaccines and antivirals are still unavailable. Although a model for DENV RNA synthesis was previously proposed (17), molecular aspects of the mechanism by which the polymerase specifically amplifies the viral genome are still unclear for DENV and other flaviviruses. To further understand this viral process, we investigated functional properties of the viral polymerase NS5.NS5 is the largest of the flavivirus proteins (105 kDa); it contains an N-terminal methyltransferase domain (MTase) and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. The MTase is responsible for methylation of the cap structure present at the 5Ј end of the viral genome. This process involves methylation in two positions, guanine N-7 and ribose 2Ј-O (12, 13, 28, 38). The RdRp domain has primer independent (de novo) RNA synth...

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Abstract RNA-seq experiments previously performed by our laboratories showed enrichment in intronic sequences and alterations in alternative splicing in dengue-infected human cells. The transcript of the SAT1 gene, of well-known antiviral action, displayed higher inclusion of exon 4 in infected cells, leading to an mRNA isoform that is degraded by non-sense mediated decay. SAT1 is a spermidine/spermine acetyl-transferase enzyme that decreases the reservoir of cellular polyamines, limiting viral replication. Delving into the molecular mechanism underlying SAT1 pre-mRNA splicing changes upon viral infection, we observed lower protein levels of RBM10, a splicing factor responsible for SAT1 exon 4 skipping. We found that the dengue polymerase NS5 interacts with RBM10 and its sole expression triggers RBM10 proteasome-mediated degradation. RBM10 over-expression in infected cells prevents SAT1 splicing changes and limits viral replication, while its knock-down enhances the splicing switch and also benefits viral replication, revealing an anti-viral role for RBM10. Consistently, RBM10 depletion attenuates expression of interferon and pro-inflammatory cytokines. In particular, we found that RBM10 interacts with viral RNA and RIG-I, and even promotes the ubiquitination of the latter, a crucial step for its activation. We propose RBM10 fulfills diverse pro-inflammatory, anti-viral tasks, besides its well-documented role in splicing regulation of apoptotic genes.