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

2018 ◽  
Vol 93 (4) ◽  
Author(s):  
Yi Wang ◽  
Xiaoyan Chen ◽  
Jiong Xie ◽  
Shili Zhou ◽  
Yanxia Huang ◽  
...  
Keyword(s):  
Life Cycle ◽  
Dengue Virus ◽  
Zika Virus ◽  
Rna Helicase ◽  
Host Factor ◽  
Host Factors ◽  
Helicase Activity ◽  
Nonstructural Proteins ◽  
Rna Helicase A ◽  
Important Host

ABSTRACT 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.

scholarly journals A Genome-Wide CRISPR-Cas9 Screen Identifies the Dolichol-Phosphate Mannose Synthase Complex as a Host Dependency Factor for Dengue Virus Infection

2020 ◽  
Vol 94 (7) ◽  
Author(s):  
Athena Labeau ◽  
Etienne Simon-Loriere ◽  
Mohamed-Lamine Hafirassou ◽  
Lucie Bonnet-Madin ◽  
Sarah Tessier ◽  
...  
Keyword(s):  
Life Cycle ◽  
Dengue Virus ◽  
Zika Virus ◽  
Denv Infection ◽  
Host Factors ◽  
Viral Rna ◽  
Health Concern ◽  
Dengue Disease ◽  
Genome Wide ◽  
A Genome

ABSTRACT Dengue virus (DENV) is a mosquito-borne flavivirus responsible for dengue disease, a major human health concern for which no specific therapies are available. Like other viruses, DENV relies heavily on the host cellular machinery for productive infection. In this study, we performed a genome-wide CRISPR-Cas9 screen using haploid HAP1 cells to identify host genes important for DENV infection. We identified DPM1 and -3, two subunits of the endoplasmic reticulum (ER) resident dolichol-phosphate mannose synthase (DPMS) complex, as host dependency factors for DENV and other related flaviviruses, such as Zika virus (ZIKV). The DPMS complex catalyzes the synthesis of dolichol-phosphate mannose (DPM), which serves as mannosyl donor in pathways leading to N-glycosylation, glycosylphosphatidylinositol (GPI) anchor biosynthesis, and C- or O-mannosylation of proteins in the ER lumen. Mutation in the DXD motif of DPM1, which is essential for its catalytic activity, abolished DPMS-mediated DENV infection. Similarly, genetic ablation of ALG3, a mannosyltransferase that transfers mannose to lipid-linked oligosaccharide (LLO), rendered cells poorly susceptible to DENV. We also established that in cells deficient for DPMS activity, viral RNA amplification is hampered and truncated oligosaccharides are transferred to the viral prM and E glycoproteins, affecting their proper folding. Overall, our study provides new insights into the host-dependent mechanisms of DENV infection and supports current therapeutic approaches using glycosylation inhibitors to treat DENV infection. IMPORTANCE Dengue disease, which is caused by dengue virus (DENV), has emerged as the most important mosquito-borne viral disease in humans and is a major global health concern. DENV encodes only few proteins and relies on the host cell machinery to accomplish its life cycle. The identification of the host factors important for DENV infection is needed to propose new targets for antiviral intervention. Using a genome-wide CRISPR-Cas9 screen, we identified DPM1 and -3, two subunits of the DPMS complex, as important host factors for the replication of DENV as well as other related viruses such as Zika virus. We established that DPMS complex plays dual roles during viral infection, both regulating viral RNA replication and promoting viral structural glycoprotein folding/stability. These results provide insights into the host molecules exploited by DENV and other flaviviruses to facilitate their life cycle.

Current view on the helicase activity of RNA helicase A and its role in gene expression

2020 ◽  
Vol 21 ◽  
Author(s):  
Yuan-Qing Pan ◽  
Li Xing
Keyword(s):  
Gene Expression ◽  
Rna Splicing ◽  
Rna Helicase ◽  
Circular Rna ◽  
Nucleoside Triphosphate ◽  
Current View ◽  
Helicase Activity ◽  
Rna Helicase A ◽  
Cellular Processes

: RNA helicase A (RHA) is a DExH-box helicase that plays regulatory roles in a variety of cellular processes including transcription, translation, RNA splicing, editing, transport, and processing, microRNA genesis and maintenance of genomic stability. It is involved in virus replication, oncogenesis, and innate immune response. RHA can unwind nucleic acid duplex by nucleoside triphosphate hydrolysis. The insight into molecular mechanism of helicase activity is fundamental to understanding the role of RHA in the cell. Herein, we reviewed the current advances on the helicase activity of RHA and its relevance to gene expression, particularly, to genesis of circular RNA.

scholarly journals Dengue Virus Hijacks a Noncanonical Oxidoreductase Function of a Cellular Oligosaccharyltransferase Complex

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
David L. Lin ◽  
Natalia A. Cherepanova ◽  
Leonia Bozzacco ◽  
Margaret R. MacDonald ◽  
Reid Gilmore ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Viral Infection ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Denv Infection ◽  
Host Factors ◽  
Nonstructural Proteins ◽  
Oxidoreductase Activity ◽  
A Genome ◽  
Insight Into

ABSTRACT Dengue virus (DENV) is the most common arboviral infection globally, infecting an estimated 390 million people each year. We employed a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen to identify host dependency factors required for DENV propagation and identified the oligosaccharyltransferase (OST) complex as an essential host factor for DENV infection. Mammalian cells express two OSTs containing either STT3A or STT3B. We found that the canonical catalytic function of the OSTs as oligosaccharyltransferases is not necessary for DENV infection, as cells expressing catalytically inactive STT3A or STT3B are able to support DENV propagation. However, the OST subunit MAGT1, which associates with STT3B, is also required for DENV propagation. MAGT1 expression requires STT3B, and a catalytically inactive STT3B also rescues MAGT1 expression, supporting the hypothesis that STT3B serves to stabilize MAGT1 in the context of DENV infection. We found that the oxidoreductase CXXC active site motif of MAGT1 was necessary for DENV propagation, as cells expressing an AXXA MAGT1 mutant were unable to support DENV infection. Interestingly, cells expressing single-cysteine CXXA or AXXC mutants of MAGT1 were able to support DENV propagation. Utilizing the engineered peroxidase APEX2, we demonstrate the close proximity between MAGT1 and NS1 or NS4B during DENV infection. These results reveal that the oxidoreductase activity of the STT3B-containing OST is necessary for DENV infection, which may guide the development of antiviral agents targeting DENV. IMPORTANCE The host oligosaccharyltransferase (OST) complexes have been identified as essential host factors for dengue virus (DENV) replication; however, their functions during DENV infection are unclear. A previous study showed that the canonical OST activity was dispensable for DENV replication, suggesting that the OST complexes serve as scaffolds for DENV replication. However, our work demonstrates that one function of the OST complex during DENV infection is to provide oxidoreductase activity via the OST subunit MAGT1. We also show that MAGT1 associates with DENV NS1 and NS4B during viral infection, suggesting that these nonstructural proteins may be targets of MAGT1 oxidoreductase activity. These results provide insight into the cell biology of DENV infection, which may guide the development of antivirals against DENV. IMPORTANCE The host oligosaccharyltransferase (OST) complexes have been identified as essential host factors for dengue virus (DENV) replication; however, their functions during DENV infection are unclear. A previous study showed that the canonical OST activity was dispensable for DENV replication, suggesting that the OST complexes serve as scaffolds for DENV replication. However, our work demonstrates that one function of the OST complex during DENV infection is to provide oxidoreductase activity via the OST subunit MAGT1. We also show that MAGT1 associates with DENV NS1 and NS4B during viral infection, suggesting that these nonstructural proteins may be targets of MAGT1 oxidoreductase activity. These results provide insight into the cell biology of DENV infection, which may guide the development of antivirals against DENV.

scholarly journals GRP78 Is an Important Host Factor for Japanese Encephalitis Virus Entry and Replication in Mammalian Cells

2017 ◽  
Vol 91 (6) ◽  
Author(s):  
Minu Nain ◽  
Sriparna Mukherjee ◽  
Sonali Porey Karmakar ◽  
Adrienne W. Paton ◽  
James C. Paton ◽  
...  
Keyword(s):  
Life Cycle ◽  
Japanese Encephalitis ◽  
Envelope Protein ◽  
Viral Encephalitis ◽  
Virus Entry ◽  
Host Factor ◽  
Potential Therapeutic Target ◽  
Novel Host ◽  
Neuro2a Cells ◽  
Important Host

ABSTRACT Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is the leading cause of viral encephalitis in Southeast Asia with potential to become a global pathogen. Here, we identify glucose-regulated protein 78 (GRP78) as an important host protein for virus entry and replication. Using the plasma membrane fractions from mouse neuronal (Neuro2a) cells, mass spectroscopy analysis identified GRP78 as a protein interacting with recombinant JEV envelope protein domain III. GRP78 was found to be expressed on the plasma membranes of Neuro2a cells, mouse primary neurons, and human epithelial Huh-7 cells. Antibodies against GRP78 significantly inhibited JEV entry in all three cell types, suggesting an important role of the protein in virus entry. Depletion of GRP78 by small interfering RNA (siRNA) significantly blocked JEV entry into Neuro2a cells, further supporting its role in virus uptake. Immunofluorescence studies showed extensive colocalization of GRP78 with JEV envelope protein in virus-infected cells. This interaction was also confirmed by immunoprecipitation studies. Additionally, GRP78 was shown to have an important role in JEV replication, as treatment of cells post-virus entry with subtilase cytotoxin that specifically cleaved GRP78 led to a substantial reduction in viral RNA replication and protein synthesis, resulting in significantly reduced extracellular virus titers. Our results indicate that GRP78, an endoplasmic reticulum chaperon of the HSP70 family, is a novel host factor involved at multiple steps of the JEV life cycle and could be a potential therapeutic target. IMPORTANCE Recent years have seen a rapid spread of mosquito-borne diseases caused by flaviviruses. The flavivirus family includes West Nile, dengue, Japanese encephalitis, and Zika viruses, which are major threats to public health with potential to become global pathogens. JEV is the major cause of viral encephalitis in several parts of Southeast Asia, affecting a predominantly pediatric population with a high mortality rate. This study is focused on identification of crucial host factors that could be targeted to cripple virus infection and ultimately lead to development of effective antivirals. We have identified a cellular protein, GRP78, that plays a dual role in virus entry and virus replication, two crucial steps of the virus life cycle, and thus is a novel host factor that could be a potential therapeutic target.

scholarly journals A Novel System to Study Dengue Virus Replication Organelle Formation Independent from Viral RNA Replication

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 139
Author(s):  
Berati Cerikan ◽  
Sarah Goellner ◽  
Christopher John Neufeldt ◽  
Uta Haselmann ◽  
Mirko Cortese ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Rna Replication ◽  
Host Factors ◽  
Viral Rna ◽  
Replication Machinery ◽  
Nonstructural Proteins ◽  
Independent System ◽  
Specific Host ◽  
Infected Cells ◽  
Positive Strand Rna

Positive-strand RNA viruses, such as dengue virus (DENV), induce the extensive rearrangement of intracellular membranes that serve as a scaffold for the assembly of the viral replication machinery. In the case of DENV, the main endomembrane ultrastructure produced in infected cells consists of invaginations of the endoplasmic reticulum, designated vesicle packets (VPs), which are the assumed sites of viral RNA replication. VPs are observed as arrays of vesicles surrounded by an outer membrane, the formation of which is induced by the viral nonstructural proteins, presumably in conjunction with specific host factors. However, little is known about the mechanisms governing VP formation, which is mainly due to the lack of a replication-independent system supporting the biogenesis of these membranous structures. Here we describe an expression-based, viral RNA replication-independent, DENV polyprotein system, designated as pIRO (plasmid-induced replication organelle), which is sufficient to induce VP formation. We show that VPs induced by pIRO expression are morphologically indistinguishable from those found in infected cells, suggesting that DENV replication organelle formation does not require RNA replication. We conclude that the pIRO system is a novel and valuable tool that can be used to dissect the mechanisms underlying DENV replication organelle formation.

Helicase associated 2 domain is essential for helicase activity of RNA helicase A

2014 ◽  
Vol 1844 (10) ◽  
pp. 1757-1764 ◽  
Author(s):  
Li Xing ◽  
Xia Zhao ◽  
Meijuan Niu ◽  
Lawrence Kleiman
Keyword(s):  
Rna Helicase ◽  
Helicase Activity ◽  
Rna Helicase A

scholarly journals Heat Shock Protein 90 Ensures the Integrity of Rubella Virus p150 Protein and Supports Viral Replication

2019 ◽  
Vol 93 (22) ◽  
Author(s):  
Masafumi Sakata ◽  
Hiroshi Katoh ◽  
Noriyuki Otsuki ◽  
Kiyoko Okamoto ◽  
Yuichiro Nakatsu ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Molecular Chaperone ◽  
Rubella Virus ◽  
Heat Shock Protein 90 ◽  
Host Factor ◽  
Host Factors ◽  
Genome Replication ◽  
Nonstructural Proteins ◽  
Infected Cells

ABSTRACT Two viral nonstructural proteins, p150 and p90, are expressed in rubella virus (RUBV)-infected cells and mediate viral genome replication, presumably using various host machineries. Molecular chaperones are critical host factors for the maintenance of cellular proteostasis, and certain viral proteins use this chaperone system. The RUBV p150 and p90 proteins are generated from a precursor polyprotein, p200, via processing by the protease activity of its p150 region. This processing is essential for RUBV genome replication. Here we show that heat shock protein 90 (HSP90), a molecular chaperone, is an important host factor for RUBV genome replication. The treatment of RUBV-infected cells with the HSP90 inhibitors 17-allylamino-17-desmethoxygeldanamycin (17-AAG) and ganetespib suppressed RUBV genome replication. HSP90α physically interacted with p150, but not p90. Further analyses into the mechanism of action of the HSP90 inhibitors revealed that HSP90 activity contributes to p150 functional integrity and promotes p200 processing. Collectively, our data demonstrate that RUBV p150 is a client of the HSP90 molecular chaperone and that HSP90 functions as a key host factor for RUBV replication. IMPORTANCE Accumulating evidence indicates that RNA viruses use numerous host factors during replication of their genomes. However, the host factors involved in rubella virus (RUBV) genome replication are largely unknown. In this study, we demonstrate that the HSP90 molecular chaperone is needed for the efficient replication of the RUBV genome. Further, we reveal that HSP90 interacts with RUBV nonstructural protein p150 and its precursor polyprotein, p200. HSP90 contributes to the stability of p150 and the processing of p200 via its protease domain in the p150 region. We conclude that the cellular molecular chaperone HSP90 is a key host factor for functional maturation of nonstructural proteins for RUBV genome replication. These findings provide novel insight into this host-virus interaction.

Dengue virus life cycle: viral and host factors modulating infectivity

2010 ◽  
Vol 67 (16) ◽  
pp. 2773-2786 ◽  
Author(s):  
Izabela A. Rodenhuis-Zybert ◽  
Jan Wilschut ◽  
Jolanda M. Smit
Keyword(s):  
Life Cycle ◽  
Dengue Virus ◽  
Host Factors ◽  
Virus Life Cycle

scholarly journals Revisiting dengue virus-mosquito interactions: molecular insights into viral fitness

2021 ◽  
Vol 102 (11) ◽  
Author(s):  
Tanamas Siriphanitchakorn ◽  
R. Manjunatha Kini ◽  
Eng Eong Ooi ◽  
Milly M. Choy
Keyword(s):  
Life Cycle ◽  
Dengue Virus ◽  
Denv Infection ◽  
Viral Fitness ◽  
Host Factors ◽  
Mammalian Host ◽  
Mosquito Vectors ◽  
Host Interactions ◽  
Cellular Processes ◽  
Natural Hosts

Dengue virus (DENV), like other viruses, closely interacts with the host cell machinery to complete its life cycle. Over the course of infection, DENV interacts with several host factors with pro-viral activities to support its infection. Meanwhile, it has to evade or counteract host factors with anti-viral activities which inhibit its infection. These molecular virus-host interactions play a crucial role in determining the success of DENV infection. Deciphering such interactions is thus paramount to understanding viral fitness in its natural hosts. While DENV-mammalian host interactions have been extensively studied, not much has been done to characterize DENV-mosquito host interactions despite its importance in controlling DENV transmission. Here, to provide a snapshot of our current understanding of DENV-mosquito interactions, we review the literature that identified host factors and cellular processes related to DENV infection in its mosquito vectors, Aedes aegypti and Aedes albopictus, with a particular focus on DENV-mosquito omics studies. This knowledge provides fundamental insights into the DENV life cycle, and could contribute to the development of novel antiviral strategies.

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