Continuous de novo generation of spatially segregated hepatitis C virus replication organelles revealed by pulse-chase imaging

Wang, Hongliang; Tai, Andrew W.

doi:10.1016/j.jhep.2016.08.018

Cited by 18 publications

(24 citation statements)

References 38 publications

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“…While goodness of fit based on cumulative AIC values for the three viruses (Table S4) demonstrates a marginal advantage in favour of our main model, measured CM formation dynamics [32,33] support our choice of the model. Moreover, independent experimental corroborations like recovery of steady state levels of replication intermediate [55,17,56] and steady state positive/negative RNA ratios [44,43], (Table S4) further supports our model. Due to the lack of molecular details for virus assembly, our model only qualitatively captures the virus assembly and release dynamics and we cannot discriminate between alternate sites (CMs vs cytoplasm) for virus assembly.…”

Section: Comparative Analysis Of Monopartite (+)Rna Virusessupporting

confidence: 76%

Life cycle process dependencies of positive-sense RNA viruses suggest strategies for inhibiting productive cellular infection

Chhajer¹,

Rizvi

Roy

2020

Preprint

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Positive strand (+)RNA viruses are the most common and clinically important human pathogens. Their life cycle processes are broadly conserved across many virus families but they employ different life cycle strategies for their growth in the cell. Upon RNA genome release into the cytoplasm post cellular entry, viral translation generates structural and non-structural proteins that induce intracellular remodelling, forming membrane compartments that foster viral replication leading to virus particle formation. We present a generalized dynamical model for intracellular (+)ssRNA virus growth that accounts for these critical steps. Our model can capture experimental growth dynamics for several RNA viruses as well as parse the effect of viral mutations and host cell permissivity. We show that Poliovirus (PV) employs rapid replication and virus assembly whereas Japanese Encephalitis virus leverages its higher rate of translation and efficient host membrane reorganization for enhanced viral dynamics compared to Hepatitis C virus. Since the slow membrane reorganization represents a crucial bottleneck for replication, stochastic simulations demonstrate that an infection event, even with multiple viral genomes, can go to extinction if all seeding viral RNA degrade before establishing robust viral replication. We estimate this probability of productive cellular infection, termed ‘Cellular Infectivity (Φ)’ using stochastic simulations. Φ varies for a virus-host pair with initial virus seeding and life cycle perturbations like increase in cytoplasmic RNA degradation and delay in compartment formation can reduce infectivity. Extent of synergy among these parameters while seemingly diverse for viruses is defined by Φ. Therefore, our model suggests new avenues for inhibition of viral infections by targeting early life cycle bottlenecks.

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Section: Comparative Analysis Of Monopartite (+)Rna Virusessupporting

confidence: 76%

Life cycle process dependencies of positive-sense RNA viruses suggest strategies for inhibiting productive cellular infection

Chhajer¹,

Rizvi

Roy

2020

Preprint

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“…In agreement with others (42), we found that exogenously added Topfluor-cholesterol that mimics the partitioning and trafficking of cellular unesterified cholesterol (34) is recruited to NS5A-positive sites. Wang and Tai argued that TFC was mainly recruited to old NS5A sites, representing DMVs and multimembrane vesicles (MMVs), where cholesterol may be important for association of the ROs with LDs, and thus for viral assembly (42). Indeed, we found that NS5A and TFC were present at DMVs in close proximity to LDs, but an even larger fraction of both protein and lipid colocalized to DMVs that were remote from LDs or other cellular organelles.…”

Section: Discussionmentioning

confidence: 68%

Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis

Stoeck

Lee

Tabata

et al. 2018

J Virol

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Similar to other positive-strand RNA viruses, hepatitis C virus (HCV) causes massive rearrangements of intracellular membranes, resulting in a membranous web (MW) composed of predominantly double-membrane vesicles (DMVs), the presumed sites of RNA replication. DMVs are enriched for cholesterol, but mechanistic details on the source and recruitment of cholesterol to the viral replication organelle are only partially known. Here we focused on selected lipid transfer proteins implicated in direct lipid transfer at various endoplasmic reticulum (ER)-membrane contact sites. RNA interference (RNAi)-mediated knockdown identified several hitherto unknown HCV dependency factors, such as steroidogenic acute regulatory protein-related lipid transfer domain protein 3 (STARD3), oxysterol-binding protein-related protein 1A and -B (OSBPL1A and -B), and Niemann-Pick-type C1 (NPC1), all residing at late endosome and lysosome membranes and required for efficient HCV RNA replication but not for replication of the closely related dengue virus. Focusing on NPC1, we found that knockdown or pharmacological inhibition caused cholesterol entrapment in lysosomal vesicles concomitant with decreased cholesterol abundance at sites containing the viral replicase factor NS5A. In untreated HCV-infected cells, unesterified cholesterol accumulated at the perinuclear region, partially colocalizing with NS5A at DMVs, arguing for NPC1-mediated endosomal cholesterol transport to the viral replication organelle. Consistent with cholesterol being an important structural component of DMVs, reducing NPC1-dependent endosomal cholesterol transport impaired MW integrity. This suggests that HCV usurps lipid transfer proteins, such as NPC1, at ER-late endosome/lysosome membrane contact sites to recruit cholesterol to the viral replication organelle, where it contributes to MW functionality. A key feature of the replication of positive-strand RNA viruses is the rearrangement of the host cell endomembrane system to produce a membranous replication organelle (RO). The underlying mechanisms are far from being elucidated fully. In this report, we provide evidence that HCV RNA replication depends on functional lipid transport along the endosomal-lysosomal pathway that is mediated by several lipid transfer proteins, such as the Niemann-Pick type C1 (NPC1) protein. Pharmacological inhibition of NPC1 function reduced viral replication, impaired the transport of cholesterol to the viral replication organelle, and altered organelle morphology. Besides NPC1, our study reports the importance of additional endosomal and lysosomal lipid transfer proteins required for viral replication, thus contributing to our understanding of how HCV manipulates their function in order to generate a membranous replication organelle. These results might have implications for the biogenesis of replication organelles of other positive-strand RNA viruses.

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“…1D). We also transfected a subgenomic replicon containing a Renilla luciferase reporter (17) into the knockout cells to assess whether the specific step of viral replication requires VAPA or VAPB. We found significant decreases in luciferase activity in all knockout cell pools compared to levels in the control at both 48 and 72 h posttransfection (Fig.…”

Section: Resultsmentioning

confidence: 99%

Nir2 Is an Effector of VAPs Necessary for Efficient Hepatitis C Virus Replication and Phosphatidylinositol 4-Phosphate Enrichment at the Viral Replication Organelle

Wang

Tai

2019

J Virol

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The endoplasmic reticulum (ER)-resident proteins vesicle-associated membrane protein (VAMP)-associated protein A and B (VAPA and VAPB) have been reported to be necessary for efficient hepatitis C virus (HCV) replication, but the specific mechanisms are not well understood. VAPs are known to recruit lipid transfer proteins to the ER, including oxysterol binding protein (OSBP), which has been previously shown to be necessary for cholesterol delivery to the HCV replication organelle in exchange for phosphatidylinositol 4-phosphate [PI(4)P]. Here, we show that VAPA and VAPB are redundant for HCV infection and that dimerization is not required for their function. In addition, we identify the phosphatidylinositol transfer protein Nir2 as an effector of VAPs to support HCV replication. We propose that Nir2 functions to replenish phosphoinositides at the HCV replication organelle to maintain elevated steady-state levels of PI(4)P, which is removed by OSBP. Thus, Nir2, along with VAPs, OSBP, and the phosphatidylinositol 4-kinase, completes a cycle of phosphoinositide flow between the ER and viral replication organelles to drive ongoing viral replication. IMPORTANCE Hepatitis C virus (HCV) is known for its ability to modulate phosphoinositide signaling pathways for its replication. Elevated levels of phosphatidylinositol 4-phosphate [PI(4)P] in HCV replication organelles (ROs) recruits lipid transfer proteins (LTPs), like oxysterol-binding protein (OSBP). OSBP exchanges PI(4)P with cholesterol, thus removing PI(4)P from the HCV RO. Here, we found that the phosphatidylinositol transfer protein Nir2 acts as an LTP and may replenish PI at the HCV RO by interacting with VAMP-associated proteins (VAPs), enabling continuous viral replication during chronic infection. Therefore, the coordination of OSBP, Nir2, and VAPs completes our understanding of the phosphoinositide cycle between the ER and HCV ROs.

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Continuous de novo generation of spatially segregated hepatitis C virus replication organelles revealed by pulse-chase imaging

Cited by 18 publications

References 38 publications

Life cycle process dependencies of positive-sense RNA viruses suggest strategies for inhibiting productive cellular infection

Life cycle process dependencies of positive-sense RNA viruses suggest strategies for inhibiting productive cellular infection

Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis

Nir2 Is an Effector of VAPs Necessary for Efficient Hepatitis C Virus Replication and Phosphatidylinositol 4-Phosphate Enrichment at the Viral Replication Organelle

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