Journal articles: 'Dengue virus genome replication'

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Alcaraz-Estrada, Sofia Lizeth, Martha Yocupicio-Monroy, and Rosa María del Angel. "Insights into dengue virus genome replication." Future Virology 5, no. 5 (September 2010): 575–92. http://dx.doi.org/10.2217/fvl.10.49.

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Wang, Kezhen, Juanjuan Wang, Ta Sun, Gang Bian, Wen Pan, Tingting Feng, Penghua Wang, Yunsen Li, and Jianfeng Dai. "Glycosphingolipid GM3 is Indispensable for Dengue Virus Genome Replication." International Journal of Biological Sciences 12, no. 7 (2016): 872–83. http://dx.doi.org/10.7150/ijbs.15641.

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Alvarez, Diego E., María F. Lodeiro, Silvio J. Ludueña, Lía I. Pietrasanta, and Andrea V. Gamarnik. "Long-Range RNA-RNA Interactions Circularize the Dengue Virus Genome." Journal of Virology 79, no. 11 (June 1, 2005): 6631–43. http://dx.doi.org/10.1128/jvi.79.11.6631-6643.2005.

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ABSTRACT Secondary and tertiary RNA structures present in viral RNA genomes play essential regulatory roles during translation, RNA replication, and assembly of new viral particles. In the case of flaviviruses, RNA-RNA interactions between the 5′ and 3′ ends of the genome have been proposed to be required for RNA replication. We found that two RNA elements present at the ends of the dengue virus genome interact in vitro with high affinity. Visualization of individual molecules by atomic force microscopy reveled that physical interaction between these RNA elements results in cyclization of the viral RNA. Using RNA binding assays, we found that the putative cyclization sequences, known as 5′ and 3′ CS, present in all mosquito-borne flaviviruses, were necessary but not sufficient for RNA-RNA interaction. Additional sequences present at the 5′ and 3′ untranslated regions of the viral RNA were also required for RNA-RNA complex formation. We named these sequences 5′ and 3′ UAR (upstream AUG region). In order to investigate the functional role of 5′-3′ UAR complementarity, these sequences were mutated either separately, to destroy base pairing, or simultaneously, to restore complementarity in the context of full-length dengue virus RNA. Nonviable viruses were recovered after transfection of dengue virus RNA carrying mutations either at the 5′ or 3′ UAR, while the RNA containing the compensatory mutations was able to replicate. Since sequence complementarity between the ends of the genome is required for dengue virus viability, we propose that cyclization of the RNA is a required conformation for viral replication.

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Yamamoto, Kristie A., Kevin Blackburn, Michael B. Goshe, Dennis T. Brown, Edimilson Migoswski, Isabele B. Campanhon, Monica F. Moreira, Davis F. Ferreira, and Marcia R. Soares. "Tizoxanide Antiviral Activity on Dengue Virus Replication." Viruses 15, no. 3 (March 7, 2023): 696. http://dx.doi.org/10.3390/v15030696.

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Dengue virus is an important circulating arbovirus in Brazil responsible for high morbidity and mortality worldwide, representing a huge economic and social burden, in addition to affecting public health. In this study, the biological activity, toxicity, and antiviral activity against dengue virus type 2 (DENV-2) of tizoxanide (TIZ) was evaluated in Vero cell culture. TIZ has a broad spectrum of action in inhibiting different pathogens, including bacteria, protozoa, and viruses. Cells were infected for 1 h with DENV-2 and then treated for 24 h with different concentrations of the drug. The quantification of viral production indicated the antiviral activity of TIZ. The protein profiles in infected Vero cells treated and not treated with TIZ were analyzed using the label-free quantitative proteomic approach. TIZ was able to inhibit virus replication mainly intracellularly after DENV-2 penetration and before the complete replication of the viral genome. Additionally, the study of the protein profile of infected not-treated and infected-treated Vero cells showed that TIZ interferes with cellular processes such as intracellular trafficking and vesicle-mediated transport and post-translational modifications when added after infection. Our results also point to the activation of immune response genes that would eventually lead to a decrease of DENV-2 production. TIZ is a promising therapeutic molecule for the treatment of DENV-2 infections.

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Leitmeyer, Katrin C., David W. Vaughn, Douglas M. Watts, Rosalba Salas, Iris Villalobos, de Chacon, Celso Ramos, and Rebeca Rico-Hesse. "Dengue Virus Structural Differences That Correlate with Pathogenesis." Journal of Virology 73, no. 6 (June 1, 1999): 4738–47. http://dx.doi.org/10.1128/jvi.73.6.4738-4747.1999.

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ABSTRACT The understanding of dengue virus pathogenesis has been hampered by the lack of in vitro and in vivo models of disease. The study of viral factors involved in the production of severe dengue, dengue hemorrhagic fever (DHF), versus the more common dengue fever (DF), have been limited to indirect clinical and epidemiologic associations. In an effort to identify viral determinants of DHF, we have developed a method for comparing dengue type 2 genomes (reverse transcriptase PCR in six fragments) directly from patient plasma. Samples for comparison were selected from two previously described dengue type 2 genotypes which had been shown to be the cause of DF or DHF. When full genome sequences of 11 dengue viruses were analyzed, several structural differences were seen consistently between those associated with DF only and those with the potential to cause DHF: a total of six encoded amino acid charge differences were seen in the prM, E, NS4b, and NS5 genes, while sequence differences observed within the 5′ nontranslated region (NTR) and 3′ NTR were predicted to change RNA secondary structures. We hypothesize that the primary determinants of DHF reside in (i) amino acid 390 of the E protein, which purportedly alters virion binding to host cells; (ii) in the downstream loop (nucleotides 68 to 80) of the 5′ NTR, which may be involved in translation initiation; and (iii) in the upstream 300 nucleotides of the 3′ NTR, which may regulate viral replication via the formation of replicative intermediates. The significance of four amino acid differences in the nonstructural proteins NS4b and NS5, a presumed transport protein and the viral RNA polymerase, respectively, remains unknown. This new approach to the study of dengue virus genome differences should better reflect the true composition of viral RNA populations in the natural host and permit their association with pathogenesis.

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Dethoff, Elizabeth A., Mark A. Boerneke, Nandan S. Gokhale, Brejnev M. Muhire, Darren P. Martin, Matthew T. Sacco, Michael J. McFadden, et al. "Pervasive tertiary structure in the dengue virus RNA genome." Proceedings of the National Academy of Sciences 115, no. 45 (October 19, 2018): 11513–18. http://dx.doi.org/10.1073/pnas.1716689115.

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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.

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Asyura, Muhammad Mikail Athif Zhafir, Ahmad Fauzi, and Fakhru Adlan Ayub. "Potential of Peptide-Based Non-Structural Protein 1 (NS1) Inhibitor in Obstructing Dengue Virus (DENV) Replication." Green Medical Journal 3, no. 1 (April 29, 2021): 1–12. http://dx.doi.org/10.33096/gmj.v3i1.71.

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Introduction: Dengue Virus (DENV) is the pathogen for human dengue fever and is responsible for 390 million infections per year. The viral genome produces about 10 viral protein products, one of them being NS1. The NS1 protein plays a key role in viral replication and stimulation of humoral immune cells, thus being the perfect candidate to create an effective antiviral drug or vaccine for dengue Methods: Dengue Virus (DENV) is the pathogen for human dengue fever and is responsible for 390 million infections per year. The viral genome produces about 10 viral protein products, one of them being NS1. The NS1 protein plays a key role in viral replication and stimulation of humoral immune cells, thus being the perfect candidate to create an effective antiviral drug or vaccine for dengue Conclusion: The review established promising results of using peptide-based intervention on NS1. Further in vivo and randomized controlled trials are advised to solidify the applicability and biosafety of the intervention

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Yang, Siwy Ling, Riccardo Delli Ponti, Yue Wan, and Roland G. Huber. "Computational and Experimental Approaches to Study the RNA Secondary Structures of RNA Viruses." Viruses 14, no. 8 (August 16, 2022): 1795. http://dx.doi.org/10.3390/v14081795.

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Most pandemics of recent decades can be traced to RNA viruses, including HIV, SARS, influenza, dengue, Zika, and SARS-CoV-2. These RNA viruses impose considerable social and economic burdens on our society, resulting in a high number of deaths and high treatment costs. As these RNA viruses utilize an RNA genome, which is important for different stages of the viral life cycle, including replication, translation, and packaging, studying how the genome folds is important to understand virus function. In this review, we summarize recent advances in computational and high-throughput RNA structure-mapping approaches and their use in understanding structures within RNA virus genomes. In particular, we focus on the genome structures of the dengue, Zika, and SARS-CoV-2 viruses due to recent significant outbreaks of these viruses around the world.

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Markoff, Lewis, Xiaou Pang, Huo-shu Houng, Barry Falgout, Raymond Olsen, Estella Jones, and Stephanie Polo. "Derivation and Characterization of a Dengue Type 1 Host Range-Restricted Mutant Virus That Is Attenuated and Highly Immunogenic in Monkeys." Journal of Virology 76, no. 7 (April 1, 2002): 3318–28. http://dx.doi.org/10.1128/jvi.76.7.3318-3328.2002.

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ABSTRACT We recently described the derivation of a dengue serotype 2 virus (DEN2mutF) that exhibited a host range-restricted phenotype; it was severely impaired for replication in cultured mosquito cells (C6/36 cells). DEN2mutF virus had selected mutations in genomic sequences predicted to form a 3′ stem-loop structure (3′-SL) that is conserved among all flavivirus species. The 3′-SL constitutes the downstream terminal ∼95 nucleotides of the 3′ noncoding region in flavivirus RNA. Here we report the introduction of these same mutational changes into the analogous region of an infectious DNA derived from the genome of a human-virulent dengue serotype 1 virus (DEN1), strain Western Pacific (DEN1WP). The resulting DEN1 mutant (DEN1mutF) exhibited a host range-restricted phenotype similar to that of DEN2mutF virus. DEN1mutF virus was attenuated in a monkey model for dengue infection in which viremia is taken as a correlate of human virulence. In spite of the markedly reduced levels of viremia that it induced in monkeys compared to DEN1WP, DEN1mutF was highly immunogenic. In addition, DEN1mutF-immunized monkeys retained high levels of neutralizing antibodies in serum and were protected from challenge with high doses of the DEN1WP parent for as long as 17 months after the single immunizing dose. Phenotypic revertants of DEN1mutF and DEN2mutF were each detected after a total of 24 days in C6/36 cell cultures. Complete nucleotide sequence analysis of DEN1mutF RNA and that of a revertant virus, DEN1mutFRev, revealed that (i) the DEN1mutF genome contained no additional mutations upstream from the 3′-SL compared to the DEN1WP parent genome and (ii) the DEN1mutFRev genome contained de novo mutations, consistent with our previous hypothesis that the defect in DEN2mutF replication in C6/36 cells was at the level of RNA replication. A strategy for the development of a tetravalent dengue vaccine is discussed.

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Sanford, Thomas J., Harriet V. Mears, Teodoro Fajardo, Nicolas Locker, and Trevor R. Sweeney. "Circularization of flavivirus genomic RNA inhibits de novo translation initiation." Nucleic Acids Research 47, no. 18 (August 8, 2019): 9789–802. http://dx.doi.org/10.1093/nar/gkz686.

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Abstract Members of the Flaviviridae family, including dengue virus (DENV) and yellow fever virus, cause serious disease in humans, whilst maternal infection with Zika virus (ZIKV) can induce microcephaly in newborns. Following infection, flaviviral RNA genomes are translated to produce the viral replication machinery but must then serve as a template for the transcription of new genomes. However, the ribosome and viral polymerase proceed in opposite directions along the RNA, risking collisions and abortive replication. Whilst generally linear, flavivirus genomes can adopt a circular conformation facilitated by long-range RNA–RNA interactions, shown to be essential for replication. Using an in vitro reconstitution approach, we demonstrate that circularization inhibits de novo translation initiation on ZIKV and DENV RNA, whilst the linear conformation is translation-competent. Our results provide a mechanism to clear the viral RNA of ribosomes in order to promote efficient replication and, therefore, define opposing roles for linear and circular conformations of the flavivirus genome.

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Fajardo, Teodoro, Thomas J. Sanford, Harriet V. Mears, Annika Jasper, Skye Storrie, Daniel S. Mansur, and Trevor R. Sweeney. "The flavivirus polymerase NS5 regulates translation of viral genomic RNA." Nucleic Acids Research 48, no. 9 (April 20, 2020): 5081–93. http://dx.doi.org/10.1093/nar/gkaa242.

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Abstract Flaviviruses, including dengue virus and Zika virus, contain a single-stranded positive sense RNA genome that encodes viral proteins essential for replication and also serves as the template for new genome synthesis. As these processes move in opposite directions along the genome, translation must be inhibited at a defined point following infection to clear the template of ribosomes to allow efficient replication. Here, we demonstrate in vitro and in cell-based assays that the viral RNA polymerase, NS5, inhibits translation of the viral genome. By reconstituting translation in vitro using highly purified components, we show that this translation block occurs at the initiation stage and that translation inhibition depends on NS5-RNA interaction, primarily through association with the 5′ replication promoter region. This work supports a model whereby expression of a viral protein signals successful translation of the infecting genome, prompting a switch to a ribosome depleted replication-competent form.

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Pugachev, Konstantin V., Farshad Guirakhoo, Simeon W. Ocran, Fred Mitchell, Megan Parsons, Caroline Penal, Soheila Girakhoo, et al. "High Fidelity of Yellow Fever Virus RNA Polymerase." Journal of Virology 78, no. 2 (January 15, 2004): 1032–38. http://dx.doi.org/10.1128/jvi.78.2.1032-1038.2004.

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ABSTRACT Three consecutive plaque purifications of four chimeric yellow fever virus-dengue virus (ChimeriVax-DEN) vaccine candidates against dengue virus types 1 to 4 were performed. The genome of each candidate was sequenced by the consensus approach after plaque purification and additional passages in cell culture. Our data suggest that the nucleotide sequence error rate for SP6 RNA polymerase used in the in vitro transcription step to initiate virus replication was as high as 1.34 × 10−4 per copied nucleotide and that the error rate of the yellow fever virus RNA polymerase employed by the chimeras for genome replication in infected cells was as low as 1.9 × 10−7 to 2.3 × 10−7. Clustering of beneficial mutations that accumulated after multiple virus passages suggests that the N-terminal part of the prM protein, a specific site in the middle of the E protein, and the NS4B protein may be essential for nucleocapsid-envelope interaction during flavivirus assembly.

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Butler, Molly, Nunya Chotiwan, Connie D. Brewster, James E. DiLisio, David F. Ackart, Brendan K. Podell, Randall J. Basaraba, Rushika Perera, Sandra L. Quackenbush, and Joel Rovnak. "Cyclin-Dependent Kinases 8 and 19 Regulate Host Cell Metabolism during Dengue Virus Serotype 2 Infection." Viruses 12, no. 6 (June 17, 2020): 654. http://dx.doi.org/10.3390/v12060654.

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Dengue virus infection is associated with the upregulation of metabolic pathways within infected cells. This effect is common to infection by a broad array of viruses. These metabolic changes, including increased glucose metabolism, oxidative phosphorylation and autophagy, support the demands of viral genome replication and infectious particle formation. The mechanisms by which these changes occur are known to be, in part, directed by viral nonstructural proteins that contact and control cellular structures and metabolic enzymes. We investigated the roles of host proteins with overarching control of metabolic processes, the transcriptional regulators, cyclin-dependent kinase 8 (CDK8) and its paralog, CDK19, as mediators of virally induced metabolic changes. Here, we show that expression of CDK8, but not CDK19, is increased during dengue virus infection in Huh7 human hepatocellular carcinoma cells, although both are required for efficient viral replication. Chemical inhibition of CDK8 and CDK19 with Senexin A during infection blocks virus-induced expression of select metabolic and autophagic genes, hexokinase 2 (HK2) and microtubule-associated protein 1 light chain 3 (LC3), and reduces viral genome replication and infectious particle production. The results further define the dependence of virus replication on increased metabolic capacity in target cells and identify CDK8 and CDK19 as master regulators of key metabolic genes. The common inhibition of CDK8 and CDK19 offers a host-directed therapeutic intervention that is unlikely to be overcome by viral evolution.

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Choi, Kyung H. "The Role of the Stem-Loop A RNA Promoter in Flavivirus Replication." Viruses 13, no. 6 (June 9, 2021): 1107. http://dx.doi.org/10.3390/v13061107.

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An essential challenge in the lifecycle of RNA viruses is identifying and replicating the viral genome amongst all the RNAs present in the host cell cytoplasm. Yet, how the viral polymerase selectively recognizes and copies the viral RNA genome is poorly understood. In flaviviruses, the 5′-end of the viral RNA genome contains a 70 nucleotide-long stem-loop, called stem-loop A (SLA), which functions as a promoter for genome replication. During replication, flaviviral polymerase NS5 specifically recognizes SLA to both initiate viral RNA synthesis and to methylate the 5′ guanine cap of the nascent RNA. While the sequences of this region vary between different flaviviruses, the three-way junction arrangement of secondary structures is conserved in SLA, suggesting that viruses recognize a common structural feature to replicate the viral genome rather than a particular sequence. To better understand the molecular basis of genome recognition by flaviviruses, we recently determined the crystal structures of flavivirus SLAs from dengue virus (DENV) and Zika virus (ZIKV). In this review, I will provide an overview of (1) flaviviral genome replication; (2) structures of viral SLA promoters and NS5 polymerases; and (3) and describe our current model of how NS5 polymerases specifically recognize the SLA at the 5′ terminus of the viral genome to initiate RNA synthesis at the 3′ terminus.

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Lodeiro, María F., Claudia V. Filomatori, and Andrea V. Gamarnik. "Structural and Functional Studies of the Promoter Element for Dengue Virus RNA Replication." Journal of Virology 83, no. 2 (November 12, 2008): 993–1008. http://dx.doi.org/10.1128/jvi.01647-08.

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ABSTRACT The 5′ untranslated region (5′UTR) of the dengue virus (DENV) genome contains two defined elements essential for viral replication. At the 5′ end, a large stem-loop (SLA) structure functions as the promoter for viral polymerase activity. Next to the SLA, there is a short stem-loop that contains a cyclization sequence known as the 5′ upstream AUG region (5′UAR). Here, we analyzed the secondary structure of the SLA in solution and the structural requirements of this element for viral replication. Using infectious DENV clones, viral replicons, and in vitro polymerase assays, we defined two helical regions, a side stem-loop, a top loop, and a U bulge within SLA as crucial elements for viral replication. The determinants for SLA-polymerase recognition were found to be common in different DENV serotypes. In addition, structural elements within the SLA required for DENV RNA replication were also conserved among different mosquito- and tick-borne flavivirus genomes, suggesting possible common strategies for polymerase-promoter recognition in flaviviruses. Furthermore, a conserved oligo(U) track present downstream of the SLA was found to modulate RNA synthesis in transfected cells. In vitro polymerase assays indicated that a sequence of at least 10 residues following the SLA, upstream of the 5′UAR, was necessary for efficient RNA synthesis using the viral 3′UTR as template.

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García-Ariza, Leidy Lorena, Natalia González-Rivillas, Cindy Johanna Díaz-Aguirre, Cristian Rocha-Roa, Leonardo Padilla-Sanabria, and Jhon Carlos Castaño-Osorio. "Antiviral Activity of an Indole-Type Compound Derived from Natural Products, Identified by Virtual Screening by Interaction on Dengue Virus NS5 Protein." Viruses 15, no. 7 (July 17, 2023): 1563. http://dx.doi.org/10.3390/v15071563.

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Dengue is an acute febrile illness caused by the Dengue virus (DENV), with a high number of cases worldwide. There is no available treatment that directly affects the virus or the viral cycle. The objective of this study was to identify a compound derived from natural products that interacts with the NS5 protein of the dengue virus through virtual screening and evaluate its in vitro antiviral effect on DENV-2. Molecular docking was performed on NS5 using AutoDock Vina software, and compounds with physicochemical and pharmacological properties of interest were selected. The preliminary antiviral effect was evaluated by the expression of the NS1 protein. The effect on viral genome replication and/or translation was determined by NS5 production using DENV-2 Huh-7 replicon through ELISA and viral RNA quantification using RT-qPCR. The in silico strategy proved effective in finding a compound (M78) with an indole-like structure and with an effect on the replication cycle of DENV-2. Treatment at 50 µM reduced the expression of the NS5 protein by 70% and decreased viral RNA by 1.7 times. M78 is involved in the replication and/or translation of the viral genome.

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Agis-Juárez, Raúl Azael, Iván Galván, Fernando Medina, Takiko Daikoku, R. Padmanabhan, Juan E. Ludert, and Rosa M. del Angel. "Polypyrimidine tract-binding protein is relocated to the cytoplasm and is required during dengue virus infection in Vero cells." Journal of General Virology 90, no. 12 (December 1, 2009): 2893–901. http://dx.doi.org/10.1099/vir.0.013433-0.

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The 3′ untranslated region (3′UTR) of the dengue virus (DENV) genome contain several sequences required for translation, replication and cyclization processes. This region also binds cellular proteins such as La, polypyrimidine tract-binding protein (PTB), Y box-binding protein 1, poly(A)-binding protein and the translation initiation factor eEF-1α. PTB is a cellular protein that interacts with the regulatory sequences of positive-strand RNA viruses such as several picornaviruses and hepatitis C virus. In the present report, it was demonstrated that PTB translocates from the nucleus to the cytoplasm during DENV infection. At 48 h post-infection, PTB, as well as the DENV proteins NS1 and NS3, were found to co-localize with the endoplasmic reticulum marker calnexin. Silencing of PTB expression inhibited virus translation and replication, whilst overexpression of PTB augmented these processes. Thus, these results provide evidence that, during infection, PTB moves from the nucleus to the cytoplasm and plays an important role in the DENV replicative cycle.

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Friebe, Peter, and Eva Harris. "Interplay of RNA Elements in the Dengue Virus 5′ and 3′ Ends Required for Viral RNA Replication." Journal of Virology 84, no. 12 (March 31, 2010): 6103–18. http://dx.doi.org/10.1128/jvi.02042-09.

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ABSTRACT Dengue virus (DENV) is a member of the Flavivirus genus of positive-sense RNA viruses. DENV RNA replication requires cyclization of the viral genome mediated by two pairs of complementary sequences in the 5′ and 3′ ends, designated 5′ and 3′ cyclization sequences (5′-3′ CS) and the 5′ and 3′ upstream of AUG region (5′-3′ UAR). Here, we demonstrate that another stretch of six nucleotides in the 5′ end is involved in DENV replication and possibly genome cyclization. This new sequence is located downstream of the AUG, designated the 5′ downstream AUG region (5′ DAR); the motif predicted to be complementary in the 3′ end is termed the 3′ DAR. In addition to the UAR, CS and DAR motifs, two other RNA elements are located at the 5′ end of the viral RNA: the 5′ stem-loop A (5′ SLA) interacts with the viral RNA-dependent RNA polymerase and promotes RNA synthesis, and a stem-loop in the coding region named cHP is involved in translation start site selection as well as RNA replication. We analyzed the interplay of these 5′ RNA elements in relation to RNA replication, and our data indicate that two separate functional units are formed; one consists of the SLA, and the other includes the UAR, DAR, cHP, and CS elements. The SLA must be located at the 5′ end of the genome, whereas the position of the second unit is more flexible. We also show that the UAR, DAR, cHP, and CS must act in concert and therefore likely function together to form the tertiary RNA structure of the circularized DENV genome.

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Li, Qingxin, and Congbao Kang. "Structures and Dynamics of Dengue Virus Nonstructural Membrane Proteins." Membranes 12, no. 2 (February 17, 2022): 231. http://dx.doi.org/10.3390/membranes12020231.

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Dengue virus is an important human pathogen threating people, especially in tropical and sub-tropical regions. The viral genome has one open reading frame and encodes one polyprotein which can be processed into structural and nonstructural (NS) proteins. Four of the seven nonstructural proteins, NS2A, NS2B, NS4A and NS4B, are membrane proteins. Unlike NS3 or NS5, these proteins do not harbor any enzymatic activities, but they play important roles in viral replication through interactions with viral or host proteins to regulate important pathways and enzymatic activities. The location of these proteins on the cell membrane and the functional roles in viral replication make them important targets for antiviral development. Indeed, NS4B inhibitors exhibit antiviral activities in different assays. Structural studies of these proteins are hindered due to challenges in crystallization and the dynamic nature of these proteins. In this review, the function and membrane topologies of dengue nonstructural membrane proteins are presented. The roles of solution NMR spectroscopy in elucidating the structure and dynamics of these proteins are introduced. The success in the development of NS4B inhibitors proves that this class of proteins is an attractive target for antiviral development.

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Chatel-Chaix, Laurent, Wolfgang Fischl, Pietro Scaturro, Mirko Cortese, Stephanie Kallis, Marie Bartenschlager, Bernd Fischer, and Ralf Bartenschlager. "A Combined Genetic-Proteomic Approach Identifies Residues within Dengue Virus NS4B Critical for Interaction with NS3 and Viral Replication." Journal of Virology 89, no. 14 (April 29, 2015): 7170–86. http://dx.doi.org/10.1128/jvi.00867-15.

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ABSTRACTDengue virus (DENV) infection causes the most prevalent arthropod-borne viral disease worldwide. Approved vaccines are not available, and targets suitable for the development of antiviral drugs are lacking. One possible drug target is nonstructural protein 4B (NS4B), because it is absolutely required for virus replication; however, its exact role in the DENV replication cycle is largely unknown. With the aim of mapping NS4B determinants critical for DENV replication, we performed a reverse genetic screening of 33 NS4B mutants in the context of an infectious DENV genome. While the majority of these mutations were lethal, for several of them, we were able to select for second-site pseudoreversions, most often residing in NS4B and restoring replication competence. To identify all viral NS4B interaction partners, we engineered a fully viable DENV genome encoding an affinity-tagged NS4B. Mass spectrometry-based analysis of the NS4B complex isolated from infected cells identified the NS3 protease/helicase as a major interaction partner of NS4B. By combining the genetic complementation map of NS4B with a replication-independent expression system, we identified the NS4B cytosolic loop—more precisely, amino acid residue Q134—as a critical determinant for NS4B-NS3 interaction. An alanine substitution at this site completely abrogated the interaction and DENV RNA replication, and both were restored by pseudoreversions A69S and A137V. This strict correlation between the degree of NS4B-NS3 interaction and DENV replication provides strong evidence that this viral protein complex plays a pivotal role during the DENV replication cycle, hence representing a promising target for novel antiviral strategies.IMPORTANCEWith no approved therapy or vaccine against dengue virus infection, the viral nonstructural protein 4B (NS4B) represents a possible drug target, because it is indispensable for virus replication. However, little is known about its precise structure and function. Here, we established the first comprehensive genetic interaction map of NS4B, identifying amino acid residues that are essential for virus replication, as well as second-site mutations compensating for their defects. Additionally, we determined the NS4B viral interactome in infected cells and identified the NS3 protease/helicase as a major interaction partner of NS4B. We mapped residues in the cytosolic loop of NS4B as critical determinants for interaction with NS3, as well as RNA replication. The strong correlation between NS3-NS4B interaction and RNA replication provides strong evidence that this complex plays a pivotal role in the viral replication cycle, hence representing a promising antiviral drug target.

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Yu, Li, and Lewis Markoff. "The Topology of Bulges in the Long Stem of the Flavivirus 3′ Stem-Loop Is a Major Determinant of RNA Replication Competence." Journal of Virology 79, no. 4 (February 15, 2005): 2309–24. http://dx.doi.org/10.1128/jvi.79.4.2309-2324.2005.

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ABSTRACT All flavivirus genomes contain a 3′terminal stem-loop secondary structure (3′SL) formed by the most downstream ∼100 nucleotides (nt) of the viral RNA. The 3′SL is required for virus replication and has been shown to bind both virus-coded and cellular proteins. Results of the present study using an infectious DNA for WN virus strain 956 initially demonstrated that the dengue virus serotype 2 (DEN2) 3′SL nucleotide sequence could not substitute for that of the WN 3′SL to support WN genome replication. To determine what WN virus-specific 3′SL nucleotide sequences were required for WN virus replication, WN virus 3′SL nucleotide sequences were selectively deleted and replaced by analogous segments of the DEN2 3′SL nucleotide sequence such that the overall 3′SL secondary structure was not disrupted. Top and bottom portions of the WN virus 3′SL were defined according to previous studies (J. L. Blackwell and M. A. Brinton, J. Virol. 71:6433-6444, 1997; L. Zeng, L., B. Falgout, and L. Markoff, J. Virol. 72:7510-7522, 1998). A bulge in the top portion of the long stem of the WN 3′SL was essential for replication of mutant WN RNAs, and replication-defective RNAs failed to produce negative strands in transfected cells. Introduction of a second bulge into the bottom portion of the long stem of the wild-type WN 3′SL markedly enhanced the replication competence of WN virus in mosquito cells but had no effect on replication in mammalian cells. This second bulge was identified as a host cell-specific enhancer of flavivirus replication. Results suggested that bulges and their topological location within the long stem of the 3′SL are primary determinants of replication competence for flavivirus genomes.

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Huang, Claire Y. H., Siritorn Butrapet, Dennis J. Pierro, Gwong-Jen J. Chang, Ann R. Hunt, Natth Bhamarapravati, Duane J. Gubler, and Richard M. Kinney. "Chimeric Dengue Type 2 (Vaccine Strain PDK-53)/Dengue Type 1 Virus as a Potential Candidate Dengue Type 1 Virus Vaccine." Journal of Virology 74, no. 7 (April 1, 2000): 3020–28. http://dx.doi.org/10.1128/jvi.74.7.3020-3028.2000.

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ABSTRACT We constructed chimeric dengue type 2/type 1 (DEN-2/DEN-1) viruses containing the nonstructural genes of DEN-2 16681 virus or its vaccine derivative, strain PDK-53, and the structural genes (encoding capsid protein, premembrane protein, and envelope glycoprotein) of DEN-1 16007 virus or its vaccine derivative, strain PDK-13. We previously reported that attenuation markers of DEN-2 PDK-53 virus were encoded by genetic loci located outside the structural gene region of the PDK-53 virus genome. Chimeric viruses containing the nonstructural genes of DEN-2 PDK-53 virus and the structural genes of the parental DEN-1 16007 virus retained the attenuation markers of small plaque size and temperature sensitivity in LLC-MK2 cells, less efficient replication in C6/36 cells, and attenuation for mice. These chimeric viruses elicited higher mouse neutralizing antibody titers against DEN-1 virus than did the candidate DEN-1 PDK-13 vaccine virus or chimeric DEN-2/DEN-1 viruses containing the structural genes of the PDK-13 virus. Mutations in the envelope protein of DEN-1 PDK-13 virus affected in vitro phenotype and immunogenicity in mice. The current PDK-13 vaccine is the least efficient of the four Mahidol candidate DEN virus vaccines in human trials. The chimeric DEN-2/DEN-1 virus might be a potential DEN-1 virus vaccine candidate. This study indicated that the infectious clones derived from the candidate DEN-2 PDK-53 vaccine are promising attenuated vectors for development of chimeric flavivirus vaccines.

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van den Elsen, Kaïn, Jun Ping Quek, and Dahai Luo. "Molecular Insights into the Flavivirus Replication Complex." Viruses 13, no. 6 (May 21, 2021): 956. http://dx.doi.org/10.3390/v13060956.

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Flaviviruses are vector-borne RNA viruses, many of which are clinically relevant human viral pathogens, such as dengue, Zika, Japanese encephalitis, West Nile and yellow fever viruses. Millions of people are infected with these viruses around the world each year. Vaccines are only available for some members of this large virus family, and there are no effective antiviral drugs to treat flavivirus infections. The unmet need for vaccines and therapies against these flaviviral infections drives research towards a better understanding of the epidemiology, biology and immunology of flaviviruses. In this review, we discuss the basic biology of the flavivirus replication process and focus on the molecular aspects of viral genome replication. Within the virus-induced intracellular membranous compartments, flaviviral RNA genome replication takes place, starting from viral poly protein expression and processing to the assembly of the virus RNA replication complex, followed by the delivery of the progeny viral RNA to the viral particle assembly sites. We attempt to update the latest understanding of the key molecular events during this process and highlight knowledge gaps for future studies.

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Faheem, Muhammad, Jônatas Cunha Barbosa Lima, Syed Babar Jamal, Paula Andreia Silva, and João Alexandre Ribeiro Gonçalves Barbosa. "An insight into dengue virus proteins as potential drug/vaccine targets." Future Virology 14, no. 10 (October 2019): 671–91. http://dx.doi.org/10.2217/fvl-2019-0107.

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Dengue virus (DENV) is an arbovirus that belongs to family flaviviridae. Its genome is composed of a single stranded RNA molecule that encodes a single polyprotein. The polyprotein is processed by viral and cellular proteases to generate ten viral proteins. There are four antigenically distinct serotypes of DENV (DENV1, DENV2, DENV3 and DENV4), which are genetically related. Although protein variability is a major problem in dengue treatment, the functional and structural studies of individual proteins are equally important in treatment development. The data accumulated on dengue proteins are significant to provide detailed understanding of viral infection, replication, host-immune evasion and pathogenesis. In this review, we summarized the detailed current knowledge about DENV proteins.

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VADDADI, K., C. GANDIKOTA, and M. VENKATARAMANA. "Complete genome characterization and evolutionary analysis of serotype-4 associated with severe dengue." Epidemiology and Infection 145, no. 7 (February 20, 2017): 1443–50. http://dx.doi.org/10.1017/s0950268817000243.

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SUMMARYDengue virus circulates as four independent serotypes posing a major public health threat around the globe. In the recent years, frequent dengue outbreaks are being reported in many parts of the world including India. Among four serotypes, Den-4 is the least sampled and studied serotype until recent times, but the reported cases with Den-4 infections were mostly known to associate with severe dengue. In the past three decades, only one complete genome sequence of Den-4 has been published from India. Hence there is a deficit in information with reference to this serotype which would be required in deciphering its association with severe dengue. In this study, we have carried out the complete genome characterization of Den-4 virus, isolated from a dengue shock syndrome patient during the 2015 outbreak from Hyderabad, South India. Phylogenetic analysis revealed the circulation of genotype I (lineage C) which showed close relatedness to the reported virulent strains. The data also indicated few unique amino acid substitutions which are known to be important in virus replication and epitope presentation. This is the first report of complete genome characterization of Den-4 from South India, which may assist in shaping the genetic diversity of circulating strains in India.

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SANTOS, JEFFERSON J. S., MARLI T. CORDEIRO, GIOVANI R. BERTANI, ERNESTO T. A. MARQUES, and LAURA H. V. G. GIL. "A two-plasmid strategy for engineering a dengue virus type 3 infectious clone from primary Brazilian isolate." Anais da Academia Brasileira de Ciências 86, no. 4 (December 2014): 1749–59. http://dx.doi.org/10.1590/0001-3765201420130332.

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Dengue infections represent one of the most prevalent arthropod-borne diseases worldwide, causing a wide spectrum of clinical outcomes. Engineered infectious clone is an important tool to study Dengue virus (DENV) biology. Functional full-length cDNA clones have been constructed for many positive-strand RNA viruses and have provided valuable tools for studying the molecular mechanisms involved in viral genome replication, virion assembly, virus pathogenesis and vaccine development. We report herein the successful development of an infectious clone from a primary Brazilian isolate of dengue virus 3 (DENV3) of the genotype III. Using a two-plasmid strategy, DENV3 genome was divided in two parts and cloned separately into a yeast-bacteria shuttle vector. All plasmids were assembled in yeast by homologous recombination technique and a full-length template for transcription was obtained by in vitro ligation of the two parts of the genome. Transcript-derived DENV3 is infectious upon transfection into BHK-21 cells and in vitro characterization confirmed its identity. Growth kinetics of transcript-derived DENV3 was indistinguishable from wild type DENV3. This system is a powerful tool that will help shed light on molecular features of DENV biology, as the relationship of specific mutations and DENV pathogenesis.

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Huang, Claire Y. H., Siritorn Butrapet, Kiyotaka R. Tsuchiya, Natth Bhamarapravati, Duane J. Gubler, and Richard M. Kinney. "Dengue 2 PDK-53 Virus as a Chimeric Carrier for Tetravalent Dengue Vaccine Development." Journal of Virology 77, no. 21 (November 1, 2003): 11436–47. http://dx.doi.org/10.1128/jvi.77.21.11436-11447.2003.

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ABSTRACT Attenuation markers of the candidate dengue 2 (D2) PDK-53 vaccine virus are encoded by mutations that reside outside of the structural gene region of the genome. We engineered nine dengue virus chimeras containing the premembrane (prM) and envelope (E) genes of wild-type D1 16007, D3 16562, or D4 1036 virus within the genetic backgrounds of wild-type D2 16681 virus and the two genetic variants (PDK53-E and PDK53-V) of the D2 PDK-53 vaccine virus. Expression of the heterologous prM-E genes in the genetic backgrounds of the two D2 PDK-53 variants, but not that of wild-type D2 16681 virus, resulted in chimeric viruses that retained PDK-53 characteristic phenotypic markers of attenuation, including small plaque size and temperature sensitivity in LLC-MK2 cells, limited replication in C6/36 cells, and lack of neurovirulence in newborn ICR mice. Chimeric D2/1, D2/3, and D2/4 viruses replicated efficiently in Vero cells and were immunogenic in AG129 mice. Chimeric D2/1 viruses protected adult AG129 mice against lethal D1 virus challenge. Two tetravalent virus formulations, comprised of either PDK53-E- or PDK53-V-vectored viruses, elicited neutralizing antibody titers in mice against all four dengue serotypes. These antibody titers were similar to the titers elicited by monovalent immunizations, suggesting that viral interference did not occur in recipients of the tetravalent formulations. The results of this study demonstrate that the unique attenuation loci of D2 PDK-53 virus make it an attractive vector for the development of live attenuated flavivirus vaccines.

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Yap, Thai Leong, Ting Xu, Yen-Liang Chen, Helene Malet, Marie-Pierre Egloff, Bruno Canard, Subhash G. Vasudevan, and Julien Lescar. "Crystal Structure of the Dengue Virus RNA-Dependent RNA Polymerase Catalytic Domain at 1.85-Angstrom Resolution." Journal of Virology 81, no. 9 (February 14, 2007): 4753–65. http://dx.doi.org/10.1128/jvi.02283-06.

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ABSTRACT Dengue fever, a neglected emerging disease for which no vaccine or antiviral agents exist at present, is caused by dengue virus, a member of the Flavivirus genus, which includes several important human pathogens, such as yellow fever and West Nile viruses. The NS5 protein from dengue virus is bifunctional and contains 900 amino acids. The S-adenosyl methionine transferase activity resides within its N-terminal domain, and residues 270 to 900 form the RNA-dependent RNA polymerase (RdRp) catalytic domain. Viral replication begins with the synthesis of minus-strand RNA from the dengue virus positive-strand RNA genome, which is subsequently used as a template for synthesizing additional plus-strand RNA genomes. This essential function for the production of new viral particles is catalyzed by the NS5 RdRp. Here we present a high-throughput in vitro assay partly recapitulating this activity and the crystallographic structure of an enzymatically active fragment of the dengue virus RdRp refined at 1.85-Å resolution. The NS5 nuclear localization sequences, previously thought to fold into a separate domain, form an integral part of the polymerase subdomains. The structure also reveals the presence of two zinc ion binding motifs. In the absence of a template strand, a chain-terminating nucleoside analogue binds to the priming loop site. These results should inform and accelerate the structure-based design of antiviral compounds against dengue virus.

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de Borba, Luana, Sergio M. Villordo, Nestor G. Iglesias, Claudia V. Filomatori, Leopoldo G. Gebhard, and Andrea V. Gamarnik. "Overlapping Local and Long-Range RNA-RNA Interactions Modulate Dengue Virus Genome Cyclization and Replication." Journal of Virology 89, no. 6 (January 14, 2015): 3430–37. http://dx.doi.org/10.1128/jvi.02677-14.

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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 newcis-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.

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Hodge, Kenneth, Chairat Tunghirun, Maliwan Kamkaew, Thawornchai Limjindaporn, Pa-thai Yenchitsomanus, and Sarin Chimnaronk. "Identification of a Conserved RNA-dependent RNA Polymerase (RdRp)-RNA Interface Required for Flaviviral Replication." Journal of Biological Chemistry 291, no. 33 (June 22, 2016): 17437–49. http://dx.doi.org/10.1074/jbc.m116.724013.

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Dengue virus, an ∼10.7-kb positive-sense RNA virus, is the most common arthropod-communicated pathogen in the world. Despite dengue's clear epidemiological importance, mechanisms for its replication remain elusive. Here, we probed the entire dengue genome for interactions with viral RNA-dependent RNA polymerase (RdRp), and we identified the dominant interaction as a loop-forming ACAG motif in the 3′ positive-stranded terminus, complicating the prevailing model of replication. A subset of interactions coincides with known flaviviral recombination sites inside the viral protein-coding region. Specific recognition of the RNA element occurs via an arginine patch in the C-terminal thumb domain of RdRp. We also show that the highly conserved nature of the consensus RNA motif may relate to its tolerance to various mutations in the interacting region of RdRp. Disruption of the interaction resulted in loss of viral replication ability in cells. This unique RdRp-RNA interface is found throughout flaviviruses, implying possibilities for broad disease interventions.

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Vial, Thomas, Wei-Lian Tan, Eric Deharo, Dorothée Missé, Guillaume Marti, and Julien Pompon. "Mosquito metabolomics reveal that dengue virus replication requires phospholipid reconfiguration via the remodeling cycle." Proceedings of the National Academy of Sciences 117, no. 44 (October 21, 2020): 27627–36. http://dx.doi.org/10.1073/pnas.2015095117.

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Dengue virus (DENV) subdues cell membranes for its cellular cycle by reconfiguring phospholipids in humans and mosquitoes. Here, we determined how and why DENV reconfigures phospholipids in the mosquito vector. By inhibiting and activating the de novo phospholipid biosynthesis, we demonstrated the antiviral impact of de novo–produced phospholipids. In line with the virus hijacking lipids for its benefit, metabolomics analyses indicated that DENV actively inhibited the de novo phospholipid pathway and instead triggered phospholipid remodeling. We demonstrated the early induction of remodeling during infection by using isotope tracing in mosquito cells. We then confirmed in mosquitoes the antiviral impact of de novo phospholipids by supplementing infectious blood meals with a de novo phospholipid precursor. Eventually, we determined that phospholipid reconfiguration was required for viral genome replication but not for the other steps of the virus cellular cycle. Overall, we now propose that DENV reconfigures phospholipids through the remodeling cycle to modify the endomembrane and facilitate formation of the replication complex. Furthermore, our study identified de novo phospholipid precursor as a blood determinant of DENV human-to-mosquito transmission.

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Kinney, Richard M., Claire Y. H. Huang, Becky C. Rose, Andrew D. Kroeker, Theo W. Dreher, Patrick L. Iversen, and David A. Stein. "Inhibition of Dengue Virus Serotypes 1 to 4 in Vero Cell Cultures with Morpholino Oligomers." Journal of Virology 79, no. 8 (April 15, 2005): 5116–28. http://dx.doi.org/10.1128/jvi.79.8.5116-5128.2005.

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ABSTRACT Five dengue (DEN) virus-specific R5F2R4 peptide-conjugated phosphorodiamidate morpholino oligomers (P4-PMOs) were evaluated for their ability to inhibit replication of DEN virus serotype 2 (DEN-2 virus) in mammalian cell culture. Initial growth curves of DEN-2 virus 16681 were obtained in Vero cells incubated with 20 μM P4-PMO compounds. At 6 days after infection, a P4-PMO targeting the 3′-terminal nucleotides of the DEN-2 virus genome and a random-sequence P4-PMO showed relatively little suppression of DEN-2 virus titer (0.1 and 0.9 log10, respectively). P4-PMOs targeting the AUG translation start site region of the single open reading frame and the 5′ cyclization sequence region had moderate activity, generating 1.6- and 1.8-log10 reductions. Two P4-PMO compounds, 5′SL and 3′CS (targeting the 5′-terminal nucleotides and the 3′ cyclization sequence region, respectively), were highly efficacious, each reducing the viral titer by greater than 5.7 log10 compared to controls at 6 days after infection with DEN-2 virus. Further experiments showed that 5′SL and 3′CS inhibited DEN-2 virus replication in a dose-dependent and sequence-specific manner. Treatment with 10 μM 3′CS reduced the titers of all four DEN virus serotypes, i.e., DEN-1 (strain 16007), DEN-2 (16681), DEN-3 (16562), and DEN-4 (1036) viruses by over 4 log10, in most cases to below detectable limits. The extent of 3′CS efficacy was affected by the timing of compound application in relation to viral infection of the cells. The 5′SL and 3′CS P4-PMOs did not suppress the replication of West Nile virus NY99 in Vero cells. These data indicate that further evaluation of the 5′SL and 3′CS compounds as potential DEN virus therapeutics is warranted.

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Cologna, Raymond, and Rebeca Rico-Hesse. "American Genotype Structures Decrease Dengue Virus Output from Human Monocytes and Dendritic Cells." Journal of Virology 77, no. 7 (April 1, 2003): 3929–38. http://dx.doi.org/10.1128/jvi.77.7.3929-3938.2003.

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ABSTRACT The dengue virus type 2 structures probably involved in human virulence were previously defined by sequencing the complete genome of both American and Southeast (SE) Asian genotype templates in patient serum (K. C. Leitmeyer et al., J. Virol. 73:4738-4747, 1999). We have now evaluated the effects of introducing a mutation in the envelope glycoprotein (E) gene and/or replacement of 5′- and 3′-nontranslated regions on dengue virus replication in human primary cell cultures. A series of chimeric infectious clones were generated containing different combinations of American and SE Asian genotype sequences. Some of the chimeric viruses had altered plaque morphology in mammalian cells; however, they replicated at similar rates in mosquito cells as measured by quantitative reverse transcription-PCR and plaque assay. Although susceptibility to virus infection varied from donor to donor in experiments using human macrophage and dendritic cells, we were able to measure consistent differences in viral RNA output per infected cell. Using this measurement, we demonstrated that the chimeric virus containing the E mutation had a lower virus output compared to the parental infectious clone. A larger reduction in virus output was observed for the triple mutant and the wild-type, American genotype virus from which chimeric inserts were derived. It appears that the three changes function synergistically, although the E mutation alone gives a lower output compared to the 5′- and 3′-terminal mutations. The data suggest that these changes may be responsible for decreased dengue virus replication in human target cells and for virulence characteristics during infection.

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Cerikan, Berati, Sarah Goellner, Christopher John Neufeldt, Uta Haselmann, Klaas Mulder, Laurent Chatel-Chaix, Mirko Cortese, and Ralf Bartenschlager. "A Non-Replicative Role of the 3′ Terminal Sequence of the Dengue Virus Genome in Membranous Replication Organelle Formation." Cell Reports 32, no. 1 (July 2020): 107859. http://dx.doi.org/10.1016/j.celrep.2020.107859.

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Hosseini, Samira, Rodrigo B. Muñoz-Soto, Jacqueline Oliva-Ramírez, Patricia Vázquez-Villegas, Nasrin Aghamohammadi, Aida Rodriguez-Garcia, and Sergio O. Martinez-Chapa. "Latest Updates in Dengue Fever Therapeutics: Natural, Marine and Synthetic Drugs." Current Medicinal Chemistry 27, no. 5 (March 16, 2020): 719–44. http://dx.doi.org/10.2174/0929867325666180629124709.

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In this paper, we review the history of Dengue, the mechanism of infection, the molecular characteristics and components of Dengue, the mechanism of entry to the target cells, cyclization of the genome and replication process, as well as translation of the proteins for virus assembly. The major emphasis of this work is on natural products and plant extracts, which were used for as palliative or adjuvant treatment of Dengue. This review article also summarizes the latest findings in regards to the marine products as effective drugs to target different symptoms of Dengue. Furthermore, an update on synthetic drugs for treating Dengue is provided in this review. As a novel alternative, we describe monoclonal antibody therapy for Dengue management and treatment.

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Naik, Nenavath Gopal, and Huey-Nan Wu. "Mutation of Putative N-Glycosylation Sites on Dengue Virus NS4B Decreases RNA Replication." Journal of Virology 89, no. 13 (April 15, 2015): 6746–60. http://dx.doi.org/10.1128/jvi.00423-15.

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ABSTRACTDengue virus (DENV) nonstructural protein 4B (NS4B) is an endoplasmic reticulum (ER) membrane-associated protein, and mutagenesis studies have revealed its significance in viral genome replication. In this work, we demonstrated that NS4B is an N-glycosylated protein in virus-infected cells as well as in recombinant protein expression. NS4B is N glycosylated at residues 58 and 62 and exists in two forms, glycosylated and unglycosylated. We manipulated full-length infectious RNA clones and subgenomic replicons to generate N58Q, N62Q, and N58QN62Q mutants. Each of the single mutants had distinct effects, but the N58QN62Q mutation resulted in dramatic reduction of viral production efficiency without affecting secretion or infectivity of the virion in mammalian and mosquito C6/36 hosts. Real-time quantitative PCR (qPCR), subgenomic replicon, andtrans-complementation assays indicated that the N58QN62Q mutation affected RNA replication possibly by the loss of glycans. In addition, four intragenic mutations (S59Y, S59F, T66A, and A137T) were obtained from mammalian and/or mosquito C6/36 cell culture systems. All of these second-site mutations compensated for the replication defect of the N58QN62Q mutant without creating novel glycosylation sites.In vivoprotein stability analyses revealed that the N58QN62Q mutation alone or plus a compensatory mutation did not affect the stability of NS4B. Overall, our findings indicated that mutation of putative N-glycosylation sites affected the biological function of NS4B in the viral replication complex.IMPORTANCEThis is the first report to identify and reveal the biological significance of dengue virus (DENV) nonstructural protein 4B (NS4B) posttranslation N-glycosylation to the virus life cycle. The study demonstrated that NS4B is N glycosylated in virus-infected cells and in recombinant protein expression. NS4B is modified by glycans at Asn-58 and Asn-62. Functional characterization implied that DENV NS4B utilizes the glycosylation machinery in both mammalian and mosquito hosts. Four intragenic mutations were found to compensate for replication and subsequent viral production deficiencies without creating novel N-glycosylation sites or modulating the stabilities of the protein, suggesting that glycans may be involved in maintaining the NS4B protein conformation. NS4B glycans may be necessary elements of the viral life cycle, but compensatory mutations can circumvent their requirement. This novel finding may have broader implications in flaviviral biology as the most likely glycan at Asn-62 of NS4B is conserved in DENV serotypes and in some related flaviviruses.

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Bagchi, Parikshit, Kaitlyn Speckhart, Andrew Kennedy, Andrew W. Tai, and Billy Tsai. "A specific EMC subunit supports Dengue virus infection by promoting virus membrane fusion essential for cytosolic genome delivery." PLOS Pathogens 18, no. 7 (July 14, 2022): e1010717. http://dx.doi.org/10.1371/journal.ppat.1010717.

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Dengue virus (DENV) represents the most common human arboviral infection, yet its cellular entry mechanism remains unclear. The multi-subunit endoplasmic reticulum membrane complex (EMC) supports DENV infection, in part, by assisting the biosynthesis of viral proteins critical for downstream replication steps. Intriguingly, the EMC has also been shown to act at an earlier step prior to viral protein biogenesis, although this event is not well-defined. Here we demonstrate that the EMC subunit EMC4 promotes fusion of the DENV and endosomal membranes during entry, enabling delivery of the viral genome into the cytosol which is then targeted to the ER for viral protein biosynthesis. We also found that EMC4 mediates ER-to-endosome transfer of phosphatidylserine, a phospholipid whose presence in the endosome facilitates DENV-endosomal membrane fusion. These findings clarify the EMC-dependent DENV early entry step, suggesting a mechanism by which an ER-localized host factor can regulate viral fusion at the endosome.

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Zeng, Lingling, Barry Falgout, and Lewis Markoff. "Identification of Specific Nucleotide Sequences within the Conserved 3′-SL in the Dengue Type 2 Virus Genome Required for Replication." Journal of Virology 72, no. 9 (September 1, 1998): 7510–22. http://dx.doi.org/10.1128/jvi.72.9.7510-7522.1998.

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ABSTRACT The flavivirus genome is a positive-stranded ∼11-kb RNA including 5′ and 3′ noncoding regions (NCR) of approximately 100 and 400 to 600 nucleotides (nt), respectively. The 3′ NCR contains adjacent, thermodynamically stable, conserved short and long stem-and-loop structures (the 3′-SL), formed by the 3′-terminal ∼100 nt. The nucleotide sequences within the 3′-SL are not well conserved among species. We examined the requirement for the 3′-SL in the context of dengue virus type 2 (DEN2) replication by mutagenesis of an infectious cDNA copy of a DEN2 genome. Genomic full-length RNA was transcribed in vitro and used to transfect monkey kidney cells. A substitution mutation, in which the 3′-terminal 93 nt constituting the wild-type (wt) DEN2 3′-SL sequence were replaced by the 96-nt sequence of the West Nile virus (WN) 3′-SL, was sublethal for virus replication. An analysis of the growth phenotypes of additional mutant viruses derived from RNAs containing DEN2-WN chimeric 3′-SL structures suggested that the wt DEN2 nucleotide sequence forming the bottom half of the long stem and loop in the 3′-SL was required for viability. One 7-bp substitution mutation in this domain resulted in a mutant virus that grew well in monkey kidney cells but was severely restricted in cultured mosquito cells. In contrast, transpositions of and/or substitutions in the wt DEN2 nucleotide sequence in the top half of the long stem and in the short stem and loop were relatively well tolerated, provided the stem-loop secondary structure was conserved.

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Tilston-Lunel, Natasha L., Gustavo Olszanski Acrani, Richard E. Randall, and Richard M. Elliott. "Generation of Recombinant Oropouche Viruses Lacking the Nonstructural Protein NSm or NSs." Journal of Virology 90, no. 5 (December 23, 2015): 2616–27. http://dx.doi.org/10.1128/jvi.02849-15.

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ABSTRACTOropouche virus (OROV) is a midge-borne human pathogen with a geographic distribution in South America. OROV was first isolated in 1955, and since then, it has been known to cause recurring outbreaks of a dengue-like illness in the Amazonian regions of Brazil. OROV, however, remains one of the most poorly understood emerging viral zoonoses. Here we describe the successful recovery of infectious OROV entirely from cDNA copies of its genome and generation of OROV mutant viruses lacking either the NSm or the NSs coding region. Characterization of the recombinant viruses carried outin vitrodemonstrated that the NSs protein of OROV is an interferon (IFN) antagonist as in other NSs-encoding bunyaviruses. Additionally, we demonstrate the importance of the nine C-terminal amino acids of OROV NSs in IFN antagonistic activity. OROV was also found to be sensitive to IFN-α when cells were pretreated; however, the virus was still capable of replicating at doses as high as 10,000 U/ml of IFN-α, in contrast to the family prototype BUNV. We found that OROV lacking the NSm protein displayed characteristics similar to those of the wild-type virus, suggesting that the NSm protein is dispensable for virus replication in the mammalian and mosquito cell lines that were tested.IMPORTANCEOropouche virus (OROV) is a public health threat in Central and South America, where it causes periodic outbreaks of dengue-like illness. In Brazil, OROV is the second most frequent cause of arboviral febrile illness after dengue virus, and with the current rates of urban expansion, more cases of this emerging viral zoonosis could occur. To better understand the molecular biology of OROV, we have successfully rescued the virus along with mutants. We have established that the C terminus of the NSs protein is important in interferon antagonism and that the NSm protein is dispensable for virus replication in cell culture. The tools described in this paper are important in terms of understanding this important yet neglected human pathogen.

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St Clair, Laura A., Stephanie A. Mills, Elena Lian, Paul S. Soma, Aritra Nag, Caroline Montgomery, Gabriela Ramirez, Nunya Chotiwan, Rebekah C. Gullberg, and Rushika Perera. "Acyl-Coa Thioesterases: A Rheostat That Controls Activated Fatty Acids Modulates Dengue Virus Serotype 2 Replication." Viruses 14, no. 2 (January 25, 2022): 240. http://dx.doi.org/10.3390/v14020240.

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During infection with dengue viruses (DENVs), the lipid landscape within host cells is significantly altered to assemble membrane platforms that support viral replication and particle assembly. Fatty acyl-CoAs are key intermediates in the biosynthesis of complex lipids that form these membranes. They also function as key signaling lipids in the cell. Here, we carried out loss of function studies on acyl-CoA thioesterases (ACOTs), a family of enzymes that hydrolyze fatty acyl-CoAs to free fatty acids and coenzyme A, to understand their influence on the lifecycle of DENVs. The loss of function of the type I ACOTs 1 (cytoplasmic) and 2 (mitochondrial) together significantly increased DENV serotype 2 (DENV2) viral replication and infectious particle release. However, isolated knockdown of mitochondrial ACOT2 significantly decreased DENV2 protein translation, genome replication, and infectious virus release. Furthermore, loss of ACOT7 function, a mitochondrial type II ACOT, similarly suppressed DENV2. As ACOT1 and ACOT2 are splice variants, these data suggest that functional differences and substrate specificities due to the location (cytosol and mitochondria, respectively) of these proteins may account for the differences in DENV2 infection phenotype. Additionally, loss of mitochondrial ACOT2 and ACOT7 expression also altered the expression of several ACOTs located in multiple organelle compartments within the cell, highlighting a complex relationship between ACOTs in the DENV2 virus lifecycle.

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Wang, Sai, Kitti W. K. Chan, Kishore B. Naripogu, Crystall M. D. Swarbrick, John Aaskov, and Subhash G. Vasudevan. "Subgenomic RNA from Dengue Virus Type 2 Suppresses Replication of Dengue Virus Genomes and Interacts with Virus-Encoded NS3 and NS5 Proteins." ACS Infectious Diseases 6, no. 3 (January 10, 2020): 436–46. http://dx.doi.org/10.1021/acsinfecdis.9b00384.

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Blair, Carol D., Zachary N. Adelman, and Ken E. Olson. "Molecular Strategies for Interrupting Arthropod-Borne Virus Transmission by Mosquitoes." Clinical Microbiology Reviews 13, no. 4 (October 1, 2000): 651–61. http://dx.doi.org/10.1128/cmr.13.4.651.

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SUMMARY Arthropod-borne virus (arbovirus) infections cause a number of emerging and resurgent human and veterinary infectious diseases. Traditional means of controlling arbovirus diseases include vaccination of susceptible vertebrates and mosquito control, but in many cases these have been unavailable or ineffective, and so novel strategies for disease control are needed. One possibility is genetic manipulation of mosquito vectors to render them unable to transmit arboviruses. This review describes recent work to test the concept of pathogen-derived resistance in arthropods by expression of viral genes in mosquito cell cultures and mosquitoes. Sense and antisense genome sequences from La Crosse virus (LAC) (a member of the Bunyaviridae) and dengue viruses serotypes 1 to 4 (DEN-1 to DEN-4) (members of the Flaviviridae) were expressed in mosquito cells from double-subgenomic and replicon vectors based on Sindbis virus (a member of the Togaviridae). The cells were then challenged with homologous or related viruses. For LAC, expression of antisense sequences from the small (S) genome segment, particularly full-length antisense S RNA, effectively interfered with replication of challenge virus, whereas expression of either antisense or sense RNA from the medium (M) segment was completely ineffective in LAC inhibition. Expression of sense and antisense RNA derived from certain regions of the DEN genome also blocked homologous virus replication more effectively than did RNA from other regions. Other parameters of RNA-mediated interference have been defined, such as the time when replication is blocked and the minimum size of effector RNA. The mechanism of RNA inhibition has not been determined, although it resembles double-stranded RNA interference in other nonvertebrate systems. Prospects for application of molecular strategies to control arbovirus diseases are briefly reviewed.

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Lindenbach, Brett D., and Charles M. Rice. "Genetic Interaction of Flavivirus Nonstructural Proteins NS1 and NS4A as a Determinant of Replicase Function." Journal of Virology 73, no. 6 (June 1, 1999): 4611–21. http://dx.doi.org/10.1128/jvi.73.6.4611-4621.1999.

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ABSTRACT Nonstructural protein 1 (NS1) of yellow fever virus (YF) is a glycoprotein localized to extracytoplasmic compartments within infected cells. We have previously shown that NS1 can be supplied intrans and is required for viral RNA replication, a process thought to occur in membrane-bound cytoplasmic complexes. Here we report that the NS1 gene from a related virus, dengue virus (DEN), is unable to function in the process of YF RNA replication. This virus-specific incompatibility leads to a lack of initial minus-strand accumulation, suggesting that DEN NS1 is unable to productively interact with the YF replicase. Based on a YF deletion mutant that requires NS1 in trans, a genetic screen for suppressor mutants was used to select virus variants able to utilize DEN NS1. In three independent selections, a single mutation was mapped to the NS4A gene, which encodes a putative transmembrane replicase component. This mutation, as well as several additional mutations, was engineered into the NS1-deficient genome and confirmed a genetic interaction between NS1 and NS4A. These findings suggest a potential mechanism for integrating NS1 into the cytoplasmic process of RNA replication.

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Edgil, Dianna, Charlotta Polacek, and Eva Harris. "Dengue Virus Utilizes a Novel Strategy for Translation Initiation When Cap-Dependent Translation Is Inhibited." Journal of Virology 80, no. 6 (March 15, 2006): 2976–86. http://dx.doi.org/10.1128/jvi.80.6.2976-2986.2006.

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ABSTRACT Viruses have developed numerous mechanisms to usurp the host cell translation apparatus. Dengue virus (DEN) and other flaviviruses, such as West Nile and yellow fever viruses, contain a 5′ m7GpppN-capped positive-sense RNA genome with a nonpolyadenylated 3′ untranslated region (UTR) that has been presumed to undergo translation in a cap-dependent manner. However, the means by which the DEN genome is translated effectively in the presence of capped, polyadenylated cellular mRNAs is unknown. This report demonstrates that DEN replication and translation are not affected under conditions that inhibit cap-dependent translation by targeting the cap-binding protein eukaryotic initiation factor 4E, a key regulator of cellular translation. We further show that under cellular conditions in which translation factors are limiting, DEN can alternate between canonical cap-dependent translation initiation and a noncanonical mechanism that appears not to require a functional m7G cap. This DEN noncanonical translation is not mediated by an internal ribosome entry site but requires the interaction of the DEN 5′ and 3′ UTRs for activity, suggesting a novel strategy for translation of animal viruses.

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Helt, Anna-Marija, and Eva Harris. "S-Phase-Dependent Enhancement of Dengue Virus 2 Replication in Mosquito Cells, but Not in Human Cells." Journal of Virology 79, no. 21 (November 1, 2005): 13218–30. http://dx.doi.org/10.1128/jvi.79.21.13218-13230.2005.

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ABSTRACT Dengue virus (DEN) is the most prevalent cause of arthropod-borne viral illness in humans. We determined the influence of cellular growth state on DEN type 2 (DEN2) replication in mosquito and human cells, based on the hypothesis that manipulation of cellular growth state will facilitate identification of viral and cellular determinants of productive infection. Comparison of density-arrested and cycling C6/36 Aedes albopictus cells infected with a low-passage DEN2 isolate revealed that cycling cells generated higher virus titers per cell. When C6/36 cells were stalled in S-phase via a thymidine (THY) block, titers of low-passage DEN2 isolates and a high-passage strain, 16681, were increased approximately 30-fold and 10-fold, respectively. Moreover, virus release was earlier in THY-treated cells than in asynchronously cycling cells. Adsorption, entry, genome uncoating, and translation were not responsible for increased titers of virus from S-phase C6/36 cells. In contrast to the 30-fold increase in virus titers, intracellular levels of viral RNA were increased approximately 2-fold, suggesting that the S-phase-responsive step is late in the DEN2 replication cycle. Analysis of viral RNA and protein released from the cells indicated that enhanced DEN2 assembly is largely responsible for increased virus titers produced during S-phase. In contrast to C6/36 cells, DEN2 titers from S-phase human hepatoma cells or primary human fibroblasts were not increased. These results demonstrate a differential response of DEN2 to the mosquito and human cell cycle and provide a framework for detailed studies into the mechanisms mediating virus assembly.

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Villordo, S. M., D. E. Alvarez, and A. V. Gamarnik. "A balance between circular and linear forms of the dengue virus genome is crucial for viral replication." RNA 16, no. 12 (October 27, 2010): 2325–35. http://dx.doi.org/10.1261/rna.2120410.

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Syenina, Ayesa, Dhanasekaran Vijaykrishna, Esther Shuyi Gan, Hwee Cheng Tan, Milly M. Choy, Tanamas Siriphanitchakorn, Colin Cheng, Subhash G. Vasudevan, and Eng Eong Ooi. "Positive epistasis between viral polymerase and the 3′ untranslated region of its genome reveals the epidemiologic fitness of dengue virus." Proceedings of the National Academy of Sciences 117, no. 20 (May 4, 2020): 11038–47. http://dx.doi.org/10.1073/pnas.1919287117.

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Dengue virus (DENV) is a global health threat, causing repeated epidemics throughout the tropical world. While low herd immunity levels to any one of the four antigenic types of DENV predispose populations to outbreaks, viral genetic determinants that confer greater fitness for epidemic spread is an important but poorly understood contributor of dengue outbreaks. Here we report that positive epistasis between the coding and noncoding regions of the viral genome combined to elicit an epidemiologic fitness phenotype associated with the 1994 DENV2 outbreak in Puerto Rico. We found that five amino acid substitutions in the NS5 protein reduced viral genomic RNA (gRNA) replication rate to achieve a more favorable and relatively more abundant subgenomic flavivirus RNA (sfRNA), a byproduct of host 5′-3′ exoribonuclease activity. The resulting increase in sfRNA relative to gRNA levels not only inhibited type I interferon (IFN) expression in infected cells through a previously described mechanism, but also enabled sfRNA to compete with gRNA for packaging into infectious particles. We suggest that delivery of sfRNA to new susceptible cells to inhibit type I IFN induction before gRNA replication and without the need for further de novo sfRNA synthesis could form a “preemptive strike” strategy against DENV.

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Dungdung, Ranjeet, Manikanta Bayal, Lathika Valliyott, Unnikrishnan Unniyampurath, Swapna S. Nair, and Rajendra Pilankatta. "A slow, efficient and safe nanoplatform of tailored ZnS QD-mycophenolic acid conjugates for in vitro drug delivery against dengue virus 2 genome replication." Nanoscale Advances 2, no. 12 (2020): 5777–89. http://dx.doi.org/10.1039/d0na00725k.

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The graphical abstract represents the synthesis of size engineered ZnS QDs for conjugating anti-viral drug (MPA) and its safe and effective delivery against cytoplasmically replicating dengue virus 2.

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Green, Timothy J., Peter Speck, Lu Geng, David Raftos, Michael R. Beard, and Karla J. Helbig. "Oyster viperin retains direct antiviral activity and its transcription occurs via a signalling pathway involving a heat-stable haemolymph protein." Journal of General Virology 96, no. 12 (December 1, 2015): 3587–97. http://dx.doi.org/10.1099/jgv.0.000300.

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Little is known about the response of non-model invertebrates, such as oysters, to virus infection. The vertebrate innate immune system detects virus-derived nucleic acids to trigger the type I IFN pathway, leading to the transcription of hundreds of IFN-stimulated genes (ISGs) that exert antiviral functions. Invertebrates were thought to lack the IFN pathway based on the absence of IFN or ISGs encoded in model invertebrate genomes. However, the oyster genome encodes many ISGs, including the well-described antiviral protein viperin. In this study, we characterized oyster viperin and showed that it localizes to caveolin-1 and inhibits dengue virus replication in a heterologous model. In a second set of experiments, we have provided evidence that the haemolymph from poly(I : C)-injected oysters contains a heat-stable, protease-susceptible factor that induces haemocyte transcription of viperin mRNA in conjunction with upregulation of IFN regulatory factor. Collectively, these results support the concept that oysters have antiviral systems that are homologous to the vertebrate IFN pathway.

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Wahongan, Irma F., Elly J. Suoth, Fatimawali, Saad Alhumaid, Hawra Albayat, Mohammed Aljeldah, Basim R. Al Shammari, et al. "Designing an Epitope-Based Peptide Vaccine Derived from RNA-Dependent RNA Polymerase (RdRp) against Dengue Virus Serotype 2." Vaccines 10, no. 10 (October 17, 2022): 1734. http://dx.doi.org/10.3390/vaccines10101734.

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Dengue fever (DF) continues to be one of the tropical and subtropical health concerns. Its prevalence tends to increase in some places in these regions. This disease is caused by the dengue virus (DENV), which is transmitted through the mosquitoes Aedes aegypti and A. albopictus. The treatment of DF to date is only supportive and there is no definitive vaccine to prevent this disease. The non-structural DENV protein, RNA-dependent RNA Polymerase (RdRp), is involved in viral replication. The RdRp-derived peptides can be used in the construction of a universal dengue vaccine. These peptides can be utilized as epitopes to induce immunity. This study was an in silico evaluation of the affinity of the potential epitope for the universal dengue vaccine to dendritic cells and the bonds between the epitope and the dendritic cell receptor. The peptide sequence MGKREKKLGEFGKAKG generated from dengue virus subtype 2 (DENV-2) RdRp was antigenic, did not produce allergies, was non-toxic, and had no homology with the human genome. The potential epitope-based vaccine MGKREKKLGEFGKAKG binds stably to dendritic cell receptors with a binding free energy of −474,4 kcal/mol. This epitope is anticipated to induce an immunological response and has the potential to serve as a universal dengue virus vaccine candidate.

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Journal articles on the topic 'Dengue virus genome replication'

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