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.

scholarly journals Hepatitis C Virus Subverts Human Choline Kinase-α To Bridge Phosphatidylinositol-4-Kinase IIIα (PI4KIIIα) and NS5A and Upregulates PI4KIIIα Activation, Thereby Promoting the Translocation of the Ternary Complex to the Endoplasmic Reticulum for Viral Replication

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
Mun-Teng Wong ◽  
Steve S. Chen
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Replication ◽  
Ternary Complex ◽  
Choline Kinase ◽  
Replication Complex ◽  
Viral Replication Complex ◽  
Phosphatidylinositol 4

ABSTRACT In this study, we elucidated the mechanism by which human choline kinase-α (hCKα) interacts with nonstructural protein 5A (NS5A) and phosphatidylinositol-4-kinase IIIα (PI4KIIIα), the lipid kinase crucial for maintaining the integrity of virus-induced membranous webs, and modulates hepatitis C virus (HCV) replication. hCKα activity positively modulated phosphatidylinositol-4-phosphate (PI4P) levels in HCV-expressing cells, and hCKα-mediated PI4P accumulation was abolished by AL-9, a PI4KIIIα-specific inhibitor. hCKα colocalized with NS5A and PI4KIIIα or PI4P; NS5A expression increased hCKα and PI4KIIIα colocalization; and hCKα formed a ternary complex with PI4KIIIα and NS5A, supporting the functional interplay of hCKα with PI4KIIIα and NS5A. PI4KIIIα inactivation by AL-9 or hCKα inactivation by CK37, a specific hCKα inhibitor, impaired the endoplasmic reticulum (ER) localization and colocalization of these three molecules. Interestingly, hCKα knockdown or inactivation inhibited PI4KIIIα-NS5A binding. In an in vitro PI4KIIIα activity assay, hCKα activity slightly increased PI4KIIIα basal activity but greatly augmented NS5A-induced PI4KIIIα activity, supporting the essential role of ternary complex formation in robust PI4KIIIα activation. Concurring with the upregulation of PI4P production and viral replication, overexpression of active hCKα-R (but not the D288A mutant) restored PI4KIIIα and NS5A translocation to the ER in hCKα stable knockdown cells. Furthermore, active PI4KIIIα overexpression restored PI4P production, PI4KIIIα and NS5A translocation to the ER, and viral replication in CK37-treated cells. Based on our results, hCKα functions as an indispensable regulator that bridges PI4KIIIα and NS5A and potentiates NS5A-stimulated PI4KIIIα activity, which then facilitates the targeting of the ternary complex to the ER for viral replication. IMPORTANCE The mechanisms by which hCKα activity modulates the transport of the hCKα-NS5A complex to the ER are not understood. In the present study, we investigated how hCKα interacts with PI4KIIIα (a key element that maintains the integrity of the “membranous web” structure) and NS5A to regulate viral replication. We demonstrated that HCV hijacks hCKα to bridge PI4KIIIα and NS5A, forming a ternary complex, which then stimulates PI4KIIIα activity to produce PI4P. Pronounced PI4P synthesis then redirects the translocation of the ternary complex to the ER-derived, PI4P-enriched membrane for assembly of the viral replication complex and viral replication. Our study provides novel insights into the indispensable modulatory role of hCKα in the recruitment of PI4KIIIα to NS5A and in NS5A-stimulated PI4P production and reveals a new perspective for understanding the impact of profound PI4KIIIα activation on the targeting of PI4KIIIα and NS5A to the PI4P-enriched membrane for viral replication complex formation.

scholarly journals Transition from Acute to Persistent Theiler's Virus Infection Requires Active Viral Replication That Drives Proinflammatory Cytokine Expression and Chronic Demyelinating Disease

2004 ◽  
Vol 78 (22) ◽  
pp. 12480-12488 ◽  
Author(s):  
Mark Trottier ◽  
Brian P. Schlitt ◽  
Aisha Y. Kung ◽  
Howard L. Lipton
Keyword(s):  
Spinal Cord ◽  
Viral Replication ◽  
Proinflammatory Cytokine ◽  
Viral Genome ◽  
Demyelinating Disease ◽  
Rna Replication ◽  
Viral Rna ◽  
Mrna Levels ◽  
Active Viral Replication ◽  
The Brain

ABSTRACT The dynamics of Theiler's murine encephalomyelitis virus (TMEV) RNA replication in the central nervous systems of susceptible and resistant strains of mice were examined by quantitative real-time reverse transcription-PCR and were found to correlate with host immune responses. During the acute phase of infection in both susceptible and resistant mice, levels of viral replication were high in the brain and brain stem, while levels of viral genome equivalents were 10- to 100-fold lower in the spinal cord. In the brain, viral RNA replication decreased after a peak at 5 days postinfection (p.i.), in parallel with the appearance of virus-specific antibody responses; however, by 15 days p.i., viral RNA levels began to increase in the spinal cords of susceptible mice. During the transition to and the persistent phase of infection, the numbers of viral genome equivalents in the spinal cord varied substantially for individual mice, but high levels were consistently associated with high levels of proinflammatory Th1 cytokine and chemokine mRNAs. Moreover, a large number of viral genome equivalents and high proinflammatory cytokine mRNA levels in spinal cords were only observed for susceptible SJL/J mice who developed demyelinating disease. These results suggest that TMEV persistence requires active viral replication beginning about day 11 p.i. and that active viral replication with high viral genome loads leads to increased levels of Th1 cytokines that drive disease progression in infected mice.

scholarly journals Expression of Hepatitis C Virus Proteins Induces Distinct Membrane Alterations Including a Candidate Viral Replication Complex

2002 ◽  
Vol 76 (12) ◽  
pp. 5974-5984 ◽  
Author(s):  
Denise Egger ◽  
Benno Wölk ◽  
Rainer Gosert ◽  
Leonardo Bianchi ◽  
Hubert E. Blum ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Replication ◽  
Replication Complex ◽  
Hepatitis C Virus Proteins ◽  
Infected Cells ◽  
Viral Replication Complex ◽  
Membrane Budding ◽  
Matrix Expression ◽  
Distinct Membrane

ABSTRACT Plus-strand RNA viruses characteristically replicate their genome in association with altered cellular membranes. In the present study, the capacity of hepatitis C virus (HCV) proteins to elicit intracellular membrane alterations was investigated by expressing, in tetracycline-regulated cell lines, a comprehensive panel of HCV proteins individually as well as in the context of the entire HCV polyprotein. As visualized by electron microscopy (EM), expression of the combined structural proteins core-E1-E2-p7, the NS3-4A complex, and protein NS4B induced distinct membrane alterations. By immunogold EM (IEM), the membrane-altering proteins were always found to localize to the respective altered membranes. NS4B, a protein of hitherto unknown function, induced a tight structure, designated membranous web, consisting of vesicles in a membranous matrix. Expression of the entire HCV polyprotein gave rise to membrane budding into rough endoplasmic reticulum vacuoles, to the membranous web, and to tightly associated vesicles often surrounding the membranous web. By IEM, all HCV proteins were found to be associated with the NS4B-induced membranous web, forming a membrane-associated multiprotein complex. A similar web-like structure in livers of HCV-infected chimpanzees was previously described (Pfeifer et al., Virchows Arch. B., 33:233-243, 1980). In view of this finding and the observation that all HCV proteins accumulate on the membranous web, we propose that the membranous web forms the viral replication complex in HCV-infected cells.

scholarly journals Replication Complex of Human Parechovirus 1

2003 ◽  
Vol 77 (15) ◽  
pp. 8512-8523 ◽  
Author(s):  
Camilla Krogerus ◽  
Denise Egger ◽  
Olga Samuilova ◽  
Timo Hyypiä ◽  
Kurt Bienz
Keyword(s):  
Viral Protein ◽  
Structural Changes ◽  
Rna Replication ◽  
Viral Rna ◽  
Human Parechovirus ◽  
Electron Microscopic ◽  
Vitro Assay ◽  
Replication Complex ◽  
Infected Cells

ABSTRACT The parechoviruses differ in many biological properties from other picornaviruses, and their replication strategy is largely unknown. In order to identify the viral RNA replication complex in human parechovirus type 1 (HPEV-1)-infected cells, we located viral protein and RNA in correlation to virus-induced membrane alterations. Structural changes in the infected cells included a disintegrated Golgi apparatus and disorganized, dilated endoplasmic reticulum (ER) which had lost its ribosomes. Viral plus-strand RNA, located by electron microscopic (EM) in situ hybridization, and the viral protein 2C, located by EM immunocytochemistry were found on clusters of small vesicles. Nascent viral RNA, visualized by 5-bromo-UTP incorporation, localized to compartments which were immunocytochemically found to contain the viral protein 2C and the trans-Golgi marker 1,4-galactosyltransferase. Protein 2C was immunodetected additionally on altered ER membranes which displayed a complex network-like structure devoid of cytoskeletal elements and with no apparent involvement in viral RNA replication. This protein also exhibited membrane binding properties in an in vitro assay. Our data suggest that the HPEV-1 replication complex is built up from vesicles carrying a Golgi marker and forming a structure different from that of replication complexes induced by other picornaviruses.

Unique Localization of Bovine Viral Diarrhea Virus Non-Structural NS4B Protein in Infected Cells

2016 ◽  
Vol 6 (1) ◽  
pp. 914-921 ◽  
Author(s):  
Yuto Suda ◽  
Daisuke Yamane ◽  
Muhammad Atif Zahoor ◽  
Yassir Mahgoub Mohamed ◽  
Shin Murakami ◽  
...  
Keyword(s):  
Viral Replication ◽  
Bovine Viral Diarrhea Virus ◽  
Bovine Viral Diarrhea ◽  
Replication Complex ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Infected Cells ◽  
Viral Replication Complex ◽  
Viral Diarrhea Virus ◽  
Ns4b Protein

Bovine viral diarrhea virus (BVDV), an important pathogen infecting ruminants, has 2 biotypes: cytopathic (cp) and noncytopathic (ncp), which are related to the onset of disease. The viral replication complex is composed of viral non-structural (NS) proteins, raising the possibility that NS protein(s) play a role in viral biotypes. To gain insight into this possible role, we analysed the intracellular localization of each NS protein in both cp and ncp virus-infected cells, and found that NS4B protein, a possible anchor protein of the viral replication complex, showed a unique dotted localization pattern that markedly merged with an endoplasmic reticulum marker, unlike other NS proteins, although there was no difference in the localization of NS4B protein between the 2 biotypes. 

scholarly journals BothcisandtransActivities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication

2016 ◽  
Vol 90 (15) ◽  
pp. 6864-6883 ◽  
Author(s):  
Morgan R. Herod ◽  
Cristina Ferrer-Orta ◽  
Eleni-Anna Loundras ◽  
Joseph C. Ward ◽  
Nuria Verdaguer ◽  
...  
Keyword(s):  
Disease Virus ◽  
Viral Genome ◽  
Foot And Mouth Disease ◽  
Rna Replication ◽  
Mouth Disease ◽  
Viral Rna ◽  
Genome Replication ◽  
Replication Complex ◽  
Mouth Disease Virus ◽  
Foot And Mouth

ABSTRACTThePicornaviridaeis a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a coordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided intrans(i.e., via expression from a separate RNA molecule), while others are required incis(i.e., expressed from the template RNA molecule).In vitrostudies have suggested that multiple copies of the RNA-dependent RNA polymerase (RdRp) 3D are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically active 3D molecules and those that build a replication complex. We report a novel nonenzymaticcis-acting function of 3D that is essential for viral-genome replication. Using an FMDV replicon in complementation experiments, our data demonstrate that thiscis-acting role of 3D is distinct from the catalytic activity, which is predominantlytransacting. Immunofluorescence studies suggest that bothcis- andtrans-acting 3D molecules localize to the same cellular compartment. However, our genetic and structural data suggest that 3D interacts inciswith RNA stem-loops that are essential for viral RNA replication. This study identifies a previously undescribed aspect of picornavirus replication complex structure-function and an important methodology for probing such interactions further.IMPORTANCEFoot-and-mouth disease virus (FMDV) is an important animal pathogen responsible for foot-and-mouth disease. The disease is endemic in many parts of the world with outbreaks within livestock resulting in major economic losses. Propagation of the viral genome occurs within replication complexes, and understanding this process can facilitate the development of novel therapeutic strategies. Many of the nonstructural proteins involved in replication possess multiple functions in the viral life cycle, some of which can be supplied to the replication complex from a separate genome (i.e., intrans) while others must originate from the template (i.e., incis). Here, we present an analysis ofcisandtransactivities of the RNA-dependent RNA polymerase 3D. We demonstrate a novelcis-acting role of 3D in replication. Our data suggest that this role is distinct from its enzymatic functions and requires interaction with the viral genome. Our data further the understanding of genome replication of this important pathogen.

scholarly journals Noise-induced bistability in the quasineutral coexistence of viral RNA under different replication modes

2018 ◽  
Author(s):  
Josep Sardanyés ◽  
Andreu Arderiu ◽  
Santiago F. Elena ◽  
Tomás Alarcón
Keyword(s):  
Viral Replication ◽  
Evolutionary Dynamics ◽  
Rna Viruses ◽  
Initial Conditions ◽  
Rna Replication ◽  
Viral Rna ◽  
Stochastic Simulations ◽  
Strong Impact ◽  
Deterministic Models ◽  
The Impact

Evolutionary and dynamical investigations on real viral populations indicate that RNA replication can range between two extremes given by so-called stamping machine replication (SMR) and geometric replication (GR). The impact of asymmetries in replication for single-stranded, (+) sense RNA viruses has been up to now studied with deterministic models. However, viral replication should be better described by including stochasticity, since the cell infection process is typically initiated with a very small number of RNA macromolecules, and thus largely influenced by intrinsic noise. Under appropriate conditions, deterministic theoretical descriptions of viral RNA replication predict a quasineutral coexistence scenario, with a line of fixed points involving different strands’ equilibrium ratios depending on the initial conditions. Recent research on the quasineutral coexistence in two competing populations reveals that stochastic fluctuations fundamentally alters the mean-field scenario, and one of the two species outcompetes the other one. In this manuscript we study this phenomenon for RNA viral replication modes by means of stochastic simulations and a diffusion approximation. Our results reveal that noise has a strong impact on the amplification of viral RNA, also causing the emergence of noise-induced bistability. We provide analytical criteria for the dominance of (+) sense strands depending on the initial populations on the line of equilibria, which are in agreement with direct stochastic simulation results. The biological implications of this noise-driven mechanism are discussed within the framework of the evolutionary dynamics of RNA viruses with different modes of replication.

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