The Dengue Virus Replication Complex: From RNA Replication to Protein-Protein Interactions to Evasion of Innate Immunity

Lescar, Julien; Soh, Sherryl Tingjin; Lee, Le Tian; Vasudevan, Subhash G.; Kang, CongBao; Lim, Siew Pheng

doi:10.1007/978-981-10-8727-1_9

Cited by 49 publications

(36 citation statements)

References 80 publications

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“…Flavivirus full-length NS5 is found not only in the cytoplasm but also in the nucleus (11,12,15,17,42). USUV is not an exception, and here we found signal of both USUV NS5 and RdRpD in the cytoplasm as well as in the nucleus (Fig.…”

Section: Discussionsupporting

confidence: 65%

“…Some molecules have already been tested against several members of this virus family (8)(9)(10). NS5 interacts with different host proteins to support viral replication and also, in some cases, to interfere with or modulate cellular functions (11). Specifically, it has been documented that NS5 migrates to the nucleus, where it establishes interactions with cellular factors that affect the normal expression of several host genes to favor viral replication.…”

mentioning

confidence: 99%

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Polymerase Activity, Protein-Protein Interaction, and Cellular Localization of the Usutu Virus NS5 Protein

Albentosa-González¹,

Clemente-Casares

Sabariegos

et al. 2019

Antimicrob Agents Chemother

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Usutu virus (USUV) has become increasingly relevant in recent years, with large outbreaks that sporadically have affected humans being reported in wildlife. Similarly to the rest of flaviviruses, USUV contains a positive-sense single-stranded RNA genome which is replicated by the activity of nonstructural protein 5 (NS5). USUV NS5 shows high sequence identity with the remaining viruses in this genus. This permitted us to identify the predicted methyltransferase domain and the RNA-dependent RNA polymerase domain (RdRpD). Owing to their high degree of conservation, viral polymerases are considered priority targets for the development of antiviral compounds. In the present study, we cloned and expressed the entire NS5 and the RdRpD in a heterologous system and used purified preparations for protein characterizations. We determined the optimal reaction conditions by investigating how variations in different physicochemical parameters, such as buffer concentration, temperature, and pH, affect RNA polymerization activity. We also found that USUV polymerase, but not the full-length NS5, exhibits cooperative activity in the synthesis of RNA and that the RdRp activity is not inhibited by sofosbuvir. To further examine the characteristics of USUV polymerase in a more specifically biological context, we have expressed NS5 and the RdRpD in eukaryotic cells and analyzed their subcellular location. NS5 is predominantly found in the cytoplasm; a significant proportion is directed to the nucleus, and this translocation involves nuclear location signals (NLS) located at least between the MTase and RdRpD domains.

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Section: Discussionsupporting

confidence: 65%

mentioning

confidence: 99%

Polymerase Activity, Protein-Protein Interaction, and Cellular Localization of the Usutu Virus NS5 Protein

Albentosa-González¹,

Clemente-Casares

Sabariegos

et al. 2019

Antimicrob Agents Chemother

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“…In addition, the RHA K417R mutant lacking the ATPase/helicase activity also binds to these proteins, and the expression of RHA K417R in the RHA-KD cells largely rescued the viral protein levels and viral yields. Since NS1, NS3, and NS4B are key elements in the viral replication cycle (1,38), all of which closely interact with each other to form a complex (7,39,40), it was rational to deduce that RHA, a big molecule with RNA and protein binding activities, functions as a scaffold, independent of its helicase activity, thereby connecting the viral RNA and proteins for efficient viral replication.…”

Section: Discussionmentioning

confidence: 99%

RNA Helicase A Is an Important Host Factor Involved in Dengue Virus Replication

Wang

Chen

Xie

et al. 2019

J Virol

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Dengue virus (DENV) utilizes host factors throughout its life cycle. In this study, we identified RNA helicase A (RHA), a member of the DEAD/H helicase family, as an important host factor of DENV. In response to DENV2 infection, nuclear RHA protein was partially redistributed into the cytoplasm. The short interfering RNA-mediated knockdown of RHA significantly reduced the amounts of infectious viral particles in various cells. The RHA knockdown reduced the multistep viral growth of DENV2 and Japanese encephalitis virus but not Zika virus. Further study showed that the absence of RHA resulted in a reduction of both viral RNA and protein levels, and the data obtained from the reporter replicon assay indicated that RHA does not directly promote viral protein synthesis. RHA bound to the DENV RNA and associated with three nonstructural proteins, including NS1, NS2B3, and NS4B. Further study showed that different domains of RHA mediated its interaction with these viral proteins. The expression of RHA or RHA-K417R mutant protein lacking ATPase/helicase activity in RHA-knockdown cells successfully restored DENV2 replication levels, suggesting that the helicase activity of RHA is dispensable for its proviral effect. Overall, our work reveals that RHA is an important factor of DENV and might serve as a target for antiviral agents. IMPORTANCE Dengue, caused by dengue virus, is a rapidly spreading disease, and currently there are no treatments available. Host factors involved in the viral replication of dengue virus are potential antiviral therapeutic targets. Although RHA has been shown to promote the multiplication of several viruses, such as HIV and adenovirus, its role in the flavivirus family, including dengue virus, Japanese encephalitis virus, and emerging Zika virus, remains elusive. The current study revealed that RHA relocalized into the cytoplasm upon DENV infection and associated with viral RNA and nonstructural proteins, implying that RHA was actively engaged in the viral life cycle. We further provide evidence that RHA promoted the viral yields of DENV2 independent of its helicase activity. These findings demonstrated that RHA is a new host factor required for DENV replication and might serve as a target for antiviral drugs.

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“…Dengue virus (DENV) is a member of the Flavivirus genus of the Flaviviridae family with approximately 11,000 nucleotides single-stranded RNA positive-sense genome that encodes three structural proteins (envelope or E; pre-membrane/membrane or pre-M/M; and the capsid, C) and seven nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). These nonstructural proteins appear to play important roles in viral replication [1]. DENV are grouped into four serologically similar but antigenically distinct serotypes, DENV-1, DENV-2, DENV-3, and DENV-4; each serotypes is able to produce the same full spectrum of disease and could be recognized during the diagnostic tests by molecular tools or by specific antibodies raised during infection or a distinct host immune response [2].…”

Section: Virusmentioning

confidence: 99%

Humanized Mice in Dengue Research: A Comparison with Other Mouse Models

et al. 2020

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Dengue virus (DENV) is an arbovirus of the Flaviviridae family and is an enveloped virion containing a positive sense single-stranded RNA genome. DENV causes dengue fever (DF) which is characterized by an undifferentiated syndrome accompanied by fever, fatigue, dizziness, muscle aches, and in severe cases, patients can deteriorate and develop life-threatening vascular leakage, bleeding, and multi-organ failure. DF is the most prevalent mosquito-borne disease affecting more than 390 million people per year with a mortality rate close to 1% in the general population but especially high among children. There is no specific treatment and there is only one licensed vaccine with restricted application. Clinical and experimental evidence advocate the role of the humoral and T-cell responses in protection against DF, as well as a role in the disease pathogenesis. A lot of pro-inflammatory factors induced during the infectious process are involved in increased severity in dengue disease. The advances in DF research have been hampered by the lack of an animal model that recreates all the characteristics of this disease. Experiments in nonhuman primates (NHP) had failed to reproduce all clinical signs of DF disease and during the past decade, humanized mouse models have demonstrated several benefits in the study of viral diseases affecting humans. In DENV studies, some of these models recapitulate specific signs of disease that are useful to test drugs or vaccine candidates. However, there is still a need for a more complete model mimicking the full spectrum of DENV. This review focuses on describing the advances in this area of research.

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The Dengue Virus Replication Complex: From RNA Replication to Protein-Protein Interactions to Evasion of Innate Immunity

Cited by 49 publications

References 80 publications

Polymerase Activity, Protein-Protein Interaction, and Cellular Localization of the Usutu Virus NS5 Protein

Polymerase Activity, Protein-Protein Interaction, and Cellular Localization of the Usutu Virus NS5 Protein

RNA Helicase A Is an Important Host Factor Involved in Dengue Virus Replication

Humanized Mice in Dengue Research: A Comparison with Other Mouse Models

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