Hepatitis C Virus Replication

Abstract

Replication and amplification of the viral genome is a key process for all viruses. For hepatitis C virus (HCV), a positive-strand RNA virus, amplification of the viral genome requires the synthesis of a negative-sense RNA template, which is in turn used for the production of new genomic RNA. This process is governed by numerous proteins, both host and viral, as well as distinct lipids and specific RNA elements within the positive- and negative-strand RNAs. Moreover, this process requires specific changes to host cell ultrastructure to create microenvironments conducive to viral replication. This review will focus on describing the processes and factors involved in facilitating or regulating HCV genome replication.

Figure 1.

Hepatitis C virus (HCV) genome organization and membrane topology of viral proteins. The open reading frame (ORF) encoding the HCV polyprotein and the predicted secondary structures of the flanking 5′ and 3′ untranslated region (UTR) are depicted at the top. Membrane topology of mature viral proteins and their function are shown at the bottom. Co- and posttranslational cleavage of the viral polyprotein are indicated as follows: (dashed vertical arrows) signal peptidase, (star) signal peptide peptidase removing the E1 signal sequence from the carboxyl terminus of core, (dashed curved arrow) NS2-3 protease, (solid arrows) NS3-4A protease. Note that only NS5A is shown as a dimer, but other viral proteins also may form homo- and heterodimers or oligomeric complexes. (D) domain, (ER) endoplasmic reticulum. (Figure adapted from data in Bartenschlager et al. 2013, with permission, from the authors.)

Figure 2.

HCV replication organelle. After entering the cell, the HCV genome is released into the cytosol and translated at the rough endoplasmic reticulum (ER). Viral proteins, in cooperation with host factors, induce intracellular membrane alterations consisting of double-membrane vesicles (DMVs), single-membrane vesicles (SMVs), and multimembrane vesicles (MMVs). DMVs, usually found in close association with lipid droplets (LDs), are protrusions of the ER that contain nonstructural proteins required for genome amplification (inset A). These vesicles are open toward the cytosol or are closed (represented as gray shaded vesicles), possibly reflecting different stages of DMV “maturation” (early and late, respectively). Viral RNA amplification may occur inside DMVs, which would allow the exit of newly synthetized viral genomes as long as the DMV is open. RNA molecules might be delivered by NS5A and NS3 to nearby assembly sites enriched in core protein and E1-E2 envelope glycoprotein complexes that are associated with p7 and NS2. Alternatively, replication might occur on the outer surface of DMVs (not represented). Particles are formed by budding into the lumen of the ER. (Inset B) DMVs emanate from ER membranes that are tightly wrapped around LDs as revealed by a combination of live cell imaging and electron tomography. (Left) Single tomographic slice of an HCV-infected cell revealing two classes of LDs. First, an LD (LD*) that is tightly wrapped by the ER and that stains positive for E2 and NS5A as revealed by fluorescence microscopy (not shown) and, second, several LDs that are not wrapped by the ER and that do not stain for E2 and NS5A (LD) suggesting that HCV proteins trigger LD wrapping by ER membranes. (Right) 3D reconstruction of the membranes surrounding LD*. ER membrane and DMVs are shown in yellow-gray; the LD monolayer membrane is shown in violet. Note the DMVs originating from the wrapping ER membrane. In some cases, a stalk-like connection between DMVs and the ER is visible. Assuming that RNA replication occurs in these DMVs, only short-distance trafficking of viral RNA would be required to the ER lumen to allow virus budding (as indicated in the schematic above). (Images in inset B are adapted from images in Lee et al. 2019 under the terms of the Creative Commons Attribution License [CC BY].)

Figure 3.

Exploitation of lipid pathways and miR-122 by hepatitis C virus (HCV). (A) HCV alters the lipid composition of rearranged membranes. NS5A and NS5B recruit and activate phosphatidylinositol 4-kinase-α (PI4KA) to produce a local accumulation of phosphatidylinositol 4-phosphate (PI4P). This may determine the directionality of cholesterol transfer by lipid transfer proteins (LTPs), such as oxysterol-binding protein (OSBP), which is recruited by NS5A via VAP-A/B and releases cholesterol in exchange for PIP₄ at these membrane contact sites. VAP proteins might serve as anchors for additional host proteins promoting the formation of endoplasmic reticulum (ER)–late endosome (LE) membrane contacts. Here NPC1, possibly in coordination with NPC2, mediates the export of unesterified cholesterol that might be accepted by lipid transfer proteins recruited by HCV (indicated with a question mark). (B,C) HCV infection activates the transcription of lipogenic genes by two distinct pathways. (B) The inactive SREBP precursor traffics from the ER to the Golgi on HCV infection or expression of core or NS4B. There, the transcriptionally active amino-terminal segment is released after two-step proteolytic processing by the site 1 protease (S1P) and S2P. Upon dimerization, the active SREBP enters the nucleus and activates the transcription of lipogenic genes. (C) The HCV 3′UTR interacts with DEAD box polypeptide 3X-linked (DDX3X). This RNA-binding protein activates IKK-α, which stimulates CBP-p300 to promote SREBP-mediated transcription. (D) miR-122, in association with Agonaute-2 (Ago2), binds to the HCV 5′UTR and protects the viral genome from 5′ triphosphate removal and nucleolytic degradation by 5′-3′ exoribonucleases 1 (XRN1) and XRN2.

Figure 4.

RNA elements within the positive-strand hepatitis C virus (HCV) genome and its negative-strand replication intermediate. The HCV genome organization is represented on the top as in Figure 1. A magnification of three regions within the positive-strand (+) RNA are illustrated below, each representing predicted RNA structures. Long-range RNA–RNA interactions are indicated with dashed arrows, whereas predicted binding sites of miR-122 are shown as gray rectangles. Structures within the negative-strand (−) RNA are shown on the bottom. Alternative nomenclatures of the structures are given in parentheses. (RNA stem-loop (SL) structures are adapted from data in Niepmann et al. 2018 and Bartenschlager et al. 2013.)