Monitoring reversion of hepatitis C virus-induced cellular alterations by direct-acting antivirals using cryo soft X-ray tomography and infrared microscopy.

Ana J Perez-Berna,María José Rodríguez,Eva Pereiro,Nuria Benseny-Cases,José L Carrascosa,Pablo Gastaminza,Ricardo Valcarcel

doi:10.1107/s2059798321009955

Ana J Perez-Berna, María José Rodríguez + Show 5 more

Open Access

https://doi.org/10.1107/s2059798321009955

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Abstract

Hepatitis C virus (HCV) is an enveloped RNA virus. One of the hallmarks of HCV infection is a rearrangement of the host cell membranes, known as the `membranous web'. Full-field cryo soft X-ray tomography (cryo-SXT) in the water-window energy range (284-543 eV) was performed on the MISTRAL beamline to investigate, in whole unstained cells, the morphology of the membranous rearrangements induced in HCV replicon-harbouring cells in conditions close to the living physiological state. All morphological alterations could be reverted by a combination of sofosbuvir/daclatasvir, which are clinically approved antivirals (direct-acting antivirals; DAAs) for HCV infection. Correlatively combining cryo-SXT and 2D synchrotron-based infrared microscopy provides critical information on the chemical nature of specific infection-related structures, which allows specific patterns of the infection process or the DAA-mediated healing process to be distinguished.

Highlights

The prevalence of chronic hepatitis C virus (HCV) infection has been estimated at 1.2–1.7% of the adult global population
HCVinfected cells show vesicular alterations that can be classified as single- and double-membrane vesicles, and multiplemembrane vesicles. 3D reconstruction of HCV-infected cell sections revealed that the membranous web, which is likely to be derived from modified endoplasmic reticulum cisternae, consists of enlarged endoplasmic reticulum tubules with double-membrane vesicles that are either connected or tightly apposed to the endoplasmic reticulum, with a minor proportion of double-membrane vesicles separated by the endoplasmic reticulum network (Romero-Brey et al, 2012; Romero-Brey & Bartenschlager, 2014)
We have analysed the ultrastructural and chemical alterations that liver cells undergo after HCV infection by cryo-SXT and SR-FTIR

Summary

Introduction

The prevalence of chronic hepatitis C virus (HCV) infection has been estimated at 1.2–1.7% of the adult global population. Once the polyprotein has been translated and processed into individual proteins, viral replicase complexes are assembled, generating progeny genomes using a full-length, negative-strand intermediate as a template These progeny genomes are either translated or encapsidated into infectious virions that are assembled and secreted by mechanisms involving elements of the very lowdensity lipoprotein biosynthetic machinery (Asselah et al, 2010). One of the hallmarks of HCV infection is a rearrangement of the host cell membranes, known as the membranous web This membranous web is likely to host the viral replicase, as viral RNA and nonstructural proteins have been localized within these vesicular structures (Romero-Brey et al, 2012; Romero-Brey & Bartenschlager, 2014). We studied the regression of the pathological membranous web proliferation by the use of the experimental inhibitor 2-C-methyladenosine (2MAd) and clinically relevant DAAs (Pawlotsky, 2013) such as daclatasvir, telaprevir and sofosbuvir, demonstrating that the observed alterations were induced by HCV replication and reverted after seven and 20 days of treatment (Perez-Bernaet al., 2016)

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