scholarly journals miR-122 affects both the initiation and maintenance of Hepatitis C Virus infections

2021 ◽  
Author(s):  
Mamata Panigrahi ◽  
Patricia A Thibault ◽  
Joyce A Wilson
Keyword(s):  
Complex Formation ◽  
Virus Replication ◽  
Positive Impact ◽  
Hcv Infection ◽  
Viral Rna ◽  
Minor Role ◽  
Replication Complex ◽  
Independent Replication ◽  
Rna Accumulation ◽  
A Minor

ABSTRACT: A liver-specific microRNA, miR-122, anneals to the HCV genomic 5’ terminus and is essential for virus replication in cell culture. However, bicistronic HCV replicons and full length RNAs with specific mutations in the 5’ UTR can replicate, albeit to low levels, without miR-122. In this study, we have identified that HCV RNAs lacking the structural gene region or having EMCV IRES-regulated translation had reduced requirements for miR-122. In addition, we found that a smaller proportion of cells supported miR-122-independent replication when compared a population of cells supporting miR-122-dependent replication, while viral protein levels per positive cell were similar. Further, the proportion of cells supporting miR-122-independent replication increased with the amount of viral RNA delivered, suggesting that establishment of miR-122-independent replication in a cell is affected by amount of viral RNA delivered. HCV RNAs replicating independent of miR-122 were not affected by supplementation with miR-122, suggesting that miR-122 is not essential for maintenance of a miR-122-independent HCV infection. However, miR-122 supplementation had a small positive impact on miR-122-dependent replication suggesting a minor role in enhancing ongoing virus RNA accumulation. We suggest that miR-122 functions primarily to initiate an HCV infection but has a minor influence on its maintenance, and we present a model in which miR-122 is required for replication complex formation at the beginning of an infection, and also supports new replication complex formation during ongoing infection and after infected cell division. IMPORTANCE: The mechanism by which miR-122 promotes the HCV life cycle is not well understood, and a role in directly promoting genome amplification is still debated. In this study, we have shown that miR-122 increases the rate of viral RNA accumulation and promotes the establishment of an HCV infection in a greater number of cells than in the absence of miR-122. However, we also confirm a minor role in promoting ongoing virus replication and propose a role in the initiation of new replication complexes throughout a virus infection. This study has implications for the use of anti-miR-122 as potential HCV therapy.

scholarly journals miR-122 affects both the initiation and maintenance of Hepatitis C Virus infections

2021 ◽  
Author(s):  
Mamata Panigrahi ◽  
Patricia A Thibault ◽  
Joyce A Wilson
Keyword(s):  
Complex Formation ◽  
Positive Impact ◽  
Hcv Infection ◽  
Viral Rna ◽  
Minor Role ◽  
Virus Infections ◽  
Replication Complex ◽  
Independent Replication ◽  
Rna Accumulation ◽  
A Minor

A liver-specific microRNA, miR-122, anneals to the HCV genomic 5’ terminus and is essential for virus replication in cell culture. However, bicistronic HCV replicons and full length RNAs with specific mutations in the 5’ UTR can replicate, albeit to low levels, without miR-122. In this study, we have identified that HCV RNAs lacking the structural gene region or having EMCV IRES-regulated translation had reduced requirements for miR-122. In addition, we found that a smaller proportion of cells supported miR-122-independent replication when compared a population of cells supporting miR-122-dependent replication, while viral protein levels per positive cell were similar. Further, the proportion of cells supporting miR-122-independent replication increased with the amount of viral RNA delivered, suggesting that establishment of miR-122-independent replication in a cell is affected by amount of viral RNA delivered. HCV RNAs replicating independent of miR-122 were not affected by supplementation with miR-122, suggesting that miR-122 is not essential for maintenance of a miR-122-independent HCV infection. However, miR-122 supplementation had a small positive impact on miR-122-dependent replication suggesting a minor role in enhancing ongoing virus RNA accumulation. We suggest that miR-122 functions primarily to initiate an HCV infection but has a minor influence on its maintenance, and we present a model in which miR-122 is required for replication complex formation at the beginning of an infection, and also supports new replication complex formation during ongoing infection and after infected cell division.

scholarly journals Non-structural protein-1 is required for West Nile virus replication complex formation and viral RNA synthesis

2013 ◽  
Vol 10 (1) ◽  
pp. 339 ◽  
Author(s):  
Soonjeon Youn ◽  
Rebecca L Ambrose ◽  
Jason M Mackenzie ◽  
Michael S Diamond
Keyword(s):  
West Nile Virus ◽  
Complex Formation ◽  
Virus Replication ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Structural Protein ◽  
West Nile ◽  
Replication Complex

scholarly journals 5′ cis Elements Direct Nodavirus RNA1 Recruitment to Mitochondrial Sites of Replication Complex Formation

2009 ◽  
Vol 83 (7) ◽  
pp. 2976-2988 ◽  
Author(s):  
Priscilla M. Van Wynsberghe ◽  
Paul Ahlquist
Keyword(s):  
Complex Formation ◽  
Mutational Analysis ◽  
Protein A ◽  
Rna Replication ◽  
Viral Rna ◽  
Yeast Cells ◽  
Positive Strand ◽  
Replication Complex ◽  
Negative Strand ◽  
Positive Strand Rna

ABSTRACT Positive-strand RNA viruses replicate their genomes on intracellular membranes, usually in conjunction with virus-induced membrane rearrangements. For the nodavirus flock house virus (FHV), we recently showed that multifunctional FHV replicase protein A induces viral RNA template recruitment to a membrane-associated state, but the site(s) and function of this recruitment were not determined. By tagging viral RNA with green fluorescent protein, we show here in Drosophila cells that protein A recruits FHV RNA specifically to the outer mitochondrial membrane sites of RNA replication complex formation. Using Drosophila cells and yeast cells, which also support FHV replication, we also defined the cis-acting regions that direct replication and template recruitment for FHV genomic RNA1. RNA1 nucleotides 68 to 205 were required for RNA replication and directed efficient protein A-mediated RNA recruitment in both cell types. RNA secondary structure prediction, structure probing, and phylogenetic comparisons in this region identified two stable, conserved stem-loops with nearly identical loop sequences. Further mutational analysis showed that both stem-loops and certain flanking sequences were required for RNA1 recruitment, negative-strand synthesis, and subsequent positive-strand amplification in yeast and Drosophila cells. Thus, we have shown that protein A recruits RNA1 templates to mitochondria, as expected for RNA replication, and identified a new RNA1 cis element that is necessary and sufficient for RNA1 template recognition and recruitment to these mitochondrial membranes for negative-strand RNA1 synthesis. These results establish RNA recruitment to the sites of replication complex formation as an essential, distinct, and selective early step in nodavirus replication.

scholarly journals Phosphatidylserine-Specific Phospholipase A1 Involved in Hepatitis C Virus Assembly through NS2 Complex Formation

2014 ◽  
Vol 89 (4) ◽  
pp. 2367-2377 ◽  
Author(s):  
Min Guo ◽  
Rongjuan Pei ◽  
Qi Yang ◽  
Huang Cao ◽  
Yun Wang ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Complex Formation ◽  
Protein Localization ◽  
Protein Translation ◽  
Viral Assembly ◽  
Host Factor ◽  
Hcv Infection ◽  
Phospholipase A1 ◽  
Replication Complex

ABSTRACTSeveral members of the phospholipase family have been reported to be involved in hepatitis C virus (HCV) replication. Here, we identified another phospholipase, phosphatidylserine-specific phospholipase A1 (PLA1A), as a host factor involved in HCV assembly. PLA1A was upregulated by HCV infection, and PLA1A knockdown significantly reduced J399EM (genotype 2a) HCV propagation at the assembly step but not the entry, RNA replication, and protein translation steps of the life cycle. Protein localization and interaction analysis further revealed a role of PLA1A in the interaction of NS2-E2 and NS2-NS5A, as the formation of the NS2-E2 and NS2-NS5A complexes was weakened in the absence of PLA1A. In addition, PLA1A stabilized the NS2/NS5A dotted structure during infection. These data suggest that PLA1A plays an important role in bridging the membrane-associated NS2-E2 complex and the NS5A-associated replication complex via its interaction with E2, NS2, and NS5A, which leads to a coordinating interaction between the structural and nonstructural proteins and facilitates viral assembly.IMPORTANCEHepatitis C virus (HCV) genomic replication is driven by the replication complex and occurs at the membranous web, while the lipid droplet is the organelle in which virion assembly is initiated. In this study, we identified phosphatidylserine-specific phospholipase A1 (PLA1A), a member of phospholipase A 1 family, as a novel host factor involved in the assembly process of HCV. PLA1A, which is induced by HCV infection at a late infection stage, interacts with HCV E2, NS2, and NS5A proteins and enhances and stabilizes the NS2-E2 and NS2-NS5A complex formation, which is essential for viral assembly. Thus, PLA1A is an important host factor which is involved in the initiation of the viral assembly in close proximity to Core-decorated lipid droplets through bringing together the HCV replication complex and envelope complex.

scholarly journals Endocytic Rab proteins are required for hepatitis C virus replication complex formation

Virology ◽  
2010 ◽  
Vol 398 (1) ◽  
pp. 21-37 ◽  
Author(s):  
David Manna ◽  
Jason Aligo ◽  
Chenjia Xu ◽  
Wei Sun Park ◽  
Hasan Koc ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Complex Formation ◽  
Virus Replication ◽  
Rab Proteins ◽  
Replication Complex

scholarly journals Human Choline Kinase-α Promotes Hepatitis C Virus RNA Replication through Modulation of Membranous Viral Replication Complex Formation

2016 ◽  
Vol 90 (20) ◽  
pp. 9075-9095 ◽  
Author(s):  
Mun-Teng Wong ◽  
Steve S. Chen
Keyword(s):  
Viral Replication ◽  
Rna Replication ◽  
Hcv Infection ◽  
Viral Rna ◽  
Choline Kinase ◽  
Replication Complex ◽  
Active Viral Replication ◽  
Infected Cells ◽  
Viral Replication Complex ◽  
Replication Site

ABSTRACTHepatitis C virus (HCV) infection reorganizes cellular membranes to create an active viral replication site named the membranous web (MW). The role that human choline kinase-α (hCKα) plays in HCV replication remains elusive. Here, we first showed that hCKα activity, not the CDP-choline pathway, promoted viral RNA replication. Confocal microscopy and subcellular fractionation of HCV-infected cells revealed that a small fraction of hCKα colocalized with the viral replication complex (RC) on the endoplasmic reticulum (ER) and that HCV infection increased hCKα localization to the ER. In the pTM-NS3-NS5B model, NS3-NS5B expression increased the localization of the wild-type, not the inactive D288A mutant, hCKα on the ER, and hCKα activity was required for effective trafficking of hCKα and NS5A to the ER. Coimmunoprecipitation showed that hCKα was recruited onto the viral RC presumably through its binding to NS5A domain 1 (D1). hCKα silencing or treatment with CK37, an hCKα activity inhibitor, abolished HCV-induced MW formation. In addition, hCKα depletion hindered NS5A localization on the ER, interfered with NS5A and NS5B colocalization, and mitigated NS5A-NS5B interactions but had no apparent effect on NS5A-NS4B and NS4B-NS5B interactions. Nevertheless, hCKα activity was not essential for the binding of NS5A to hCKα or NS5B. These findings demonstrate that hCKα forms a complex with NS5A and that hCKα activity enhances the targeting of the complex to the ER, where hCKα protein, not activity, mediates NS5A binding to NS5B, thereby promoting functional membranous viral RC assembly and viral RNA replication.IMPORTANCEHCV infection reorganizes the cellular membrane to create an active viral replication site named the membranous web (MW). Here, we report that human choline kinase-α (hCKα) acts as an essential host factor for HCV RNA replication. A fraction of hCKα colocalizes with the viral replication complex (RC) on the endoplasmic reticulum (ER) in HCV-infected cells. NS3-NS5B expression increases ER localization of wild-type, but not D288A mutant, hCKα, and hCKα activity facilitates the transport of itself and NS5A to the ER. Silencing or inactivation of hCKα abrogates MW formation. Moreover, hCKα is recruited by NS5A independent of hCKα activity, presumably through binding to NS5A D1. hCKα activity then mediates the ER targeting of the hCKα-NS5A complex. On the ER membrane, hCKα protein,per se, induces NS5A binding to NS5B, thereby promoting membranous RC formation and viral RNA replication. Our study may benefit the development of hCKα-targeted anti-HCV therapeutics.

Blocking eukaryotic initiation factor 4F complex formation does not inhibit the mTORC1-dependent activation of protein synthesis in cardiomyocytes

2009 ◽  
Vol 296 (2) ◽  
pp. H505-H514 ◽  
Author(s):  
Brandon P. H. Huang ◽  
Yanni Wang ◽  
Xuemin Wang ◽  
Zhuren Wang ◽  
Christopher G. Proud
Keyword(s):  
Protein Synthesis ◽  
Complex Formation ◽  
Mammalian Target Of Rapamycin ◽  
Initiation Factor ◽  
Minor Role ◽  
Eukaryotic Initiation Factor ◽  
Eukaryotic Initiation Factor 4E ◽  
Mtorc1 Signaling ◽  
A Minor ◽  
Rapid Activation

Activation of the mammalian target of rapamycin complex 1 (mTORC1) causes the dissociation of eukaryotic initiation factor 4E complex (eIF4E)-binding protein 1 (4E-BP1) from eIF4E, leading to increased eIF4F complex formation. mTORC1 positively regulates protein synthesis and is implicated in several diseases including cardiac hypertrophy, a potentially fatal disorder involving increased cardiomyocyte size. The importance of 4E-BP1 in mTORC1-regulated protein synthesis was investigated by overexpressing 4E-BP1, which blocks eIF4F formation in isolated primary cardiomyocytes without affecting other targets for mTORC1 signaling. Interestingly, blocking eIF4F formation did not impair the degree of activation of overall protein synthesis by the hypertrophic agent phenylephrine (PE), which, furthermore, remained dependent on mTORC1. Overexpressing 4E-BP1 also only had a small effect on PE-induced cardiomyocyte growth. Overexpressing 4E-BP1 did diminish the PE-stimulated synthesis of luciferase encoded by structured mRNAs, confirming that such mRNAs do require eIF4F for their translation in cardiomyocytes. These data imply that the substantial inhibition of cardiomyocyte protein synthesis and growth caused by inhibiting mTORC1 cannot be attributed to the activation of 4E-BP1 or loss of eIF4F complexes. Our data indicate that increased eIF4F formation plays, at most, only a minor role in the mTORC1-dependent activation of overall protein synthesis in these primary cells but is required for the translation of structured mRNAs. Therefore, other mTORC1 targets are more important in the inhibition by rapamycin of the rapid activation of protein synthesis and of cell growth.

scholarly journals IRAV (FLJ11286), an Interferon-Stimulated Gene with Antiviral Activity against Dengue Virus, Interacts with MOV10

2016 ◽  
Vol 91 (5) ◽  
Author(s):  
Corey A. Balinsky ◽  
Hana Schmeisser ◽  
Alexandra I. Wells ◽  
Sundar Ganesan ◽  
Tengchuan Jin ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Dengue Virus ◽  
Viral Replication ◽  
Virus Replication ◽  
Viral Rna ◽  
Type I ◽  
Dependent Manner ◽  
Content Type ◽  
Replication Complex ◽  
P Bodies

ABSTRACT Dengue virus (DENV) is a member of the genus Flavivirus and can cause severe febrile illness. Here, we show that FLJ11286, which we refer to as IRAV, is induced by DENV in an interferon-dependent manner, displays antiviral activity against DENV, and localizes to the DENV replication complex. IRAV is an RNA binding protein and localizes to cytoplasmic processing bodies (P bodies) in uninfected cells, where it interacts with the MOV10 RISC complex RNA helicase, suggesting a role for IRAV in the processing of viral RNA. After DENV infection, IRAV, along with MOV10 and Xrn1, localizes to the DENV replication complex and associates with DENV proteins. Depletion of IRAV or MOV10 results in an increase in viral RNA. These data serve to characterize an interferon-stimulated gene with antiviral activity against DENV, as well as to propose a mechanism of activity involving the processing of viral RNA. IMPORTANCE Dengue virus, a member of the family Flaviviridae, can result in a life-threatening illness and has a significant impact on global health. Dengue virus has been shown to be particularly sensitive to the effects of type I interferon; however, little is known about the mechanisms by which interferon-stimulated genes function to inhibit viral replication. A better understanding of the interferon-mediated antiviral response to dengue virus may aid in the development of novel therapeutics. Here, we examine the influence of the interferon-stimulated gene IRAV (FLJ11286) on dengue virus replication. We show that IRAV associates with P bodies in uninfected cells and with the dengue virus replication complex after infection. IRAV also interacts with MOV10, depletion of which is associated with increased viral replication. Our results provide insight into a newly identified antiviral gene, as well as broadening our understanding of the innate immune response to dengue virus infection.

scholarly journals A Chikungunya Virustrans-Replicase System Reveals the Importance of Delayed Nonstructural Polyprotein Processing for Efficient Replication Complex Formation in Mosquito Cells

2018 ◽  
Vol 92 (14) ◽  
Author(s):  
Koen Bartholomeeusen ◽  
Age Utt ◽  
Sandra Coppens ◽  
Kai Rausalu ◽  
Katleen Vereecken ◽  
...  
Keyword(s):  
Complex Formation ◽  
Chikungunya Virus ◽  
Rna Replication ◽  
Enzymatic Activities ◽  
Viral Rna ◽  
Nonstructural Proteins ◽  
Content Type ◽  
Replication Complex ◽  
Polyprotein Processing ◽  
Mosquito Cells

ABSTRACTChikungunya virus (CHIKV) is a medically important alphavirus that is transmitted byAedes aegyptiandAedes albopictusmosquitoes. The viral replicase complex consists of four nonstructural proteins (nsPs) expressed as a polyprotein precursor and encompasses all enzymatic activities required for viral RNA replication. nsPs interact with host components of which most are still poorly understood, especially in mosquitos. A CHIKVtrans-replicase system that allows the uncoupling of RNA replication and nsP expression was adapted to mosquito cells and subsequently used for analysis of universal and host-specific effects of 17 different nonstructural polyprotein (ns-polyprotein) mutations. It was found that mutations blocking nsP enzymatic activities as well as insertions of enhanced green fluorescent protein (EGFP) into different nsPs had similar effects ontrans-replicase activity regardless of the host (i.e., mammalian or mosquito). Mutations that slow down or accelerate ns-polyprotein processing generally had no effect or reducedtrans-replicase activity in mammalian cells, while in mosquito cells most of them increasedtrans-replicase activity prominently. Increased RNA replication in mosquito cells was counteracted by an antiviral RNA interference (RNAi) response. Substitution of the W258 residue in the membrane binding peptide of nsP1 resulted in a temperature-sensitive defect, in the context of both thetrans-replicase and infectious CHIKV. The defect was compensated for by secondary mutations selected during passaging of mutant CHIKV. These findings demonstrate the value of alphavirustrans-replicase systems for studies of viral RNA replication and virus-host interactions.IMPORTANCEChikungunya virus is an important mosquito-transmitted human pathogen. This virus actively replicates in mosquitoes, but the underlying molecular mechanisms and interactions of viral and host components are poorly understood. This is partly due to the lack of reliable systems for functional analysis of viral nonstructural polyproteins (ns-polyproteins) and nonstructural proteins (nsPs) in mosquito cells. Adaption of a CHIKVtrans-replicase system allowed study of the effects of mutations in the ns-polyprotein on RNA replication in cells derived from mammalian and mosquito hosts. We found that a slowdown of ns-polyprotein processing facilitates replication complex formation and/or functioning in mosquito cells and that this process is antagonized by the natural RNAi defense system present in mosquito cells. The mosquito-adapted CHIKVtrans-replicase system represents a valuable tool to study alphavirus-mosquito interactions at the molecular level and to develop advanced antiviral strategies.

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