The rate of hepatitis C virus infection initiation in vitro is directly related to particle density

Sabahi, Ali; Marsh, Katherine A.; Dahari, Harel; Corcoran, Patricia A.; Lamora, Jennifer M.; Yu, Xiaobing; Garry, Robert F.; Uprichard, Susan L.

doi:10.1016/j.virol.2010.07.026

Cited by 17 publications

(16 citation statements)

References 74 publications

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“…Specifically, as depicted in Fig. 3A, confluent Huh7 cell cultures were inoculated with 50 FFU of HCVcc for 17 h to allow time for ϳ80% viral entry into ϳ40 individual single cells (45). Cells were then washed, and cell-free virus spread was blocked by the addition of anti-E2 at a concentration that neutralizes extracellular virus infection of cells (6,13,16).…”

Section: Resultsmentioning

confidence: 99%

Quantification of Hepatitis C Virus Cell-to-Cell Spread Using a Stochastic Modeling Approach

Graw

Martin

Perelson

et al. 2015

J Virol

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It has been proposed that viral cell-to-cell transmission plays a role in establishing and maintaining chronic infections. Thus, understanding the mechanisms and kinetics of cell-to-cell spread is fundamental to elucidating the dynamics of infection and may provide insight into factors that determine chronicity. Because hepatitis C virus (HCV) spreads from cell to cell and has a chronicity rate of up to 80% in exposed individuals, we examined the dynamics of HCV cell-to-cell spread in vitro and quantified the effect of inhibiting individual host factors. Using a multidisciplinary approach, we performed HCV spread assays and assessed the appropriateness of different stochastic models for describing HCV focus expansion. To evaluate the effect of blocking specific host cell factors on HCV cell-to-cell transmission, assays were performed in the presence of blocking antibodies and/or small-molecule inhibitors targeting different cellular HCV entry factors. In all experiments, HCV-positive cells were identified by immunohistochemical staining and the number of HCV-positive cells per focus was assessed to determine focus size. We found that HCV focus expansion can best be explained by mathematical models assuming focus size-dependent growth. Consistent with previous reports suggesting that some factors impact HCV cell-to-cell spread to different extents, modeling results estimate a hierarchy of efficacies for blocking HCV cell-to-cell spread when targeting different host factors (e.g., CLDN1 > NPC1L1 > TfR1). This approach can be adapted to describe focus expansion dynamics under a variety of experimental conditions as a means to quantify cell-to-cell transmission and assess the impact of cellular factors, viral factors, and antivirals. IMPORTANCEThe ability of viruses to efficiently spread by direct cell-to-cell transmission is thought to play an important role in the establishment and maintenance of viral persistence. As such, elucidating the dynamics of cell-to-cell spread and quantifying the effect of blocking the factors involved has important implications for the design of potent antiviral strategies and controlling viral escape. Mathematical modeling has been widely used to understand HCV infection dynamics and treatment response; however, these models typically assume only cell-free virus infection mechanisms. Here, we used stochastic models describing focus expansion as a means to understand and quantify the dynamics of HCV cell-to-cell spread in vitro and determined the degree to which cellto-cell spread is reduced when individual HCV entry factors are blocked. The results demonstrate the ability of this approach to recapitulate and quantify cell-to-cell transmission, as well as the impact of specific factors and potential antivirals. V iral entry into permissive cells is the first step in establishing infection and is thus a common and often effective target for antiviral therapy. However, after replication and assembly of viral particles in an infected cell, many viruses, including hepatitis C virus...

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Section: Resultsmentioning

confidence: 99%

Quantification of Hepatitis C Virus Cell-to-Cell Spread Using a Stochastic Modeling Approach

Graw

Martin

Perelson

et al. 2015

J Virol

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“…The number of total foci observed was slightly reduced when factors involved in initial cellfree infection were blocked (Fig. 1B), likely because some HCV infection initiation can continue up to 24 h after inoculation in vitro (48). However, differences in HCV cell-to-cell spread were assessed by counting the number of cells per foci (Fig.…”

Section: Antibody Blocking Of Npc1l1 Prevents Hcv Cell-to-cell Spreadmentioning

confidence: 99%

“…We used a previously established antibody-blocking approach (22), in which a neutralizing antibody to the HCV E2 glycoprotein was used to block cell-free virus spread while the role of various host cell receptors in cell-to-cell spread was determined using specific blocking antibodies. Specifically, confluent Huh7 cells were inoculated with 100 FFU JFH-1 HCVcc per well for 16 h to allow time for sufficient amounts of initial viral entry (48). Cells were then washed, and cell-free virus spread was blocked by the addition of anti-E2 at a concentration which neutralizes extracellular virus infection of cells (22).…”

Section: Antibody Blocking Of Npc1l1 Prevents Hcv Cell-to-cell Spreadmentioning

confidence: 99%

Determining the Involvement and Therapeutic Implications of Host Cellular Factors in Hepatitis C Virus Cell-to-Cell Spread

Barretto

Sáinz

Hussain

et al. 2014

J Virol

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Hepatitis C virus (HCV) infects 180 million people worldwide and is a leading cause of liver diseases such as fibrosis, cirrhosis, and hepatocellular carcinoma. It has been shown that HCV can spread to naive cells using two distinct entry mechanisms, "cellfree" entry of infectious extracellular virions that have been released by infected cells and direct "cell-to-cell" transmission. Here, we examined host cell requirements for HCV spread and found that the cholesterol uptake receptor NPC1L1, which we recently identified as being an antiviral target involved in HCV cell-free entry/spread, is also required for the cell-to-cell spread. In contrast, the very low density lipoprotein (VLDL) pathway, which is required for the secretion of cell-free infectious virus and thus has been identified as an antiviral target for blocking cell-free virus secretion/spread, is not required for cell-to-cell spread. Noting that HCV cell-free and cell-to-cell spread share some common factors but not others, we tested the therapeutic implications of these observations and demonstrate that inhibitors that target cell factors required for both forms of HCV spread exhibit synergy when used in combination with interferon (a representative inhibitor of intracellular HCV production), while inhibitors that block only cell-free spread do not. This provides insight into the mechanistic basis of synergy between interferon and HCV entry inhibitors and highlights the broader, previously unappreciated impact blocking HCV cell-to-cell spread can have on the efficacy of HCV combination therapies. IMPORTANCEHCV can spread to naive cells using distinct mechanisms: "cell-free" entry of extracellular virus and direct "cell-to-cell" transmission. Herein, we identify the host cell HCV entry factor NPC1L1 as also being required for HCV cell-to-cell spread, while showing that the VLDL pathway, which is required for the secretion of cell-free infectious virus, is not required for cell-to-cell spread. While both these host factors are considered viable antiviral targets, we demonstrate that only inhibitors that block factors required for both forms of HCV entry/spread (i.e., NPC1L1) exhibit synergy when used in combination with interferon, while inhibitors that block factors required only for cell-free spread (i.e., VLDL pathway components) do not. Thus, this study advances our understanding of HCV cell-to-cell spread, provides mechanistic insight into the basis of drug synergy, and highlights inhibition of HCV spread as a previously unappreciated consideration in HCV therapy design.

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“…, those not associated with TRL [125]. Indeed, the virus particles that are higher in density in cell-culture are marked by a more rapidly-infecting phenotype than the low-density viruses [126]. One of the first identified HCV entry factors was LDL receptor (LDLr), by detecting HCV RNA taken up by the hepatoma line HepG2 (Figure 2) [127].…”

Section: Lipoprotein and Cholesterol’s Role In Viral Entrymentioning

confidence: 99%

Hepatitis C Virus, Cholesterol and Lipoproteins — Impact for the Viral Life Cycle and Pathogenesis of Liver Disease

Felmlee

Hafirassou

Lefèvre

et al. 2013

Viruses

132

119

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Hepatitis C virus (HCV) is a leading cause of chronic liver disease, including chronic hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. Hepatitis C infection associates with lipid and lipoprotein metabolism disorders such as hepatic steatosis, hypobetalipoproteinemia, and hypocholesterolemia. Furthermore, virus production is dependent on hepatic very-low-density lipoprotein (VLDL) assembly, and circulating virions are physically associated with lipoproteins in complexes termed lipoviral particles. Evidence has indicated several functional roles for the formation of these complexes, including co-opting of lipoprotein receptors for attachment and entry, concealing epitopes to facilitate immune escape, and hijacking host factors for HCV maturation and secretion. Here, we review the evidence surrounding pathogenesis of the hepatitis C infection regarding lipoprotein engagement, cholesterol and triglyceride regulation, and the molecular mechanisms underlying these effects.

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The rate of hepatitis C virus infection initiation in vitro is directly related to particle density

Cited by 17 publications

References 74 publications

Quantification of Hepatitis C Virus Cell-to-Cell Spread Using a Stochastic Modeling Approach

Quantification of Hepatitis C Virus Cell-to-Cell Spread Using a Stochastic Modeling Approach

Determining the Involvement and Therapeutic Implications of Host Cellular Factors in Hepatitis C Virus Cell-to-Cell Spread

Hepatitis C Virus, Cholesterol and Lipoproteins — Impact for the Viral Life Cycle and Pathogenesis of Liver Disease

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