Positive-stranded RNA viruses are classified into three superfamilies based on the evolutionary relatedness of their RNA-dependent RNA polymerases (RdRPs). The Flaviviridae are members of superfamily 2, bearing distant similarity to coliphages and the plant-infecting carmo-, tombus-, diantho-, and subgroup I luteoviruses.⁴¹⁸ Before the era of molecular biology, some members of the family Flaviviridae were classified as Togaviridae.

This chapter is organized around common features of the family Flaviviridae life cycle (Fig. 25.2). The enveloped virions are composed of a lipid bilayer with two or more species of envelope (E) glycoprotein surrounding a nucleocapsid, which consists of a single-stranded, positive-sense RNA genome complexed with multiple copies of a small, basic capsid (C) protein. Binding and uptake are believed to involve receptor-mediated endocytosis. The low pH of the endosome induces fusion of the virion envelope with cellular membranes. Following uncoating of the nucleocapsid, the RNA genome is released into the cytoplasm. The genome serves three discrete roles within the life cycle: as the messenger RNA (mRNA) for translation of all viral proteins, a template during RNA replication, and the genetic material packaged within new virus particles. The organization
of the genome is similar for all genera. Viral proteins are produced as part of a single polyprotein that is cleaved by a combination of host and viral proteases. The structural proteins are located in the N-terminal portion of the polyprotein with the nonstructural (NS) proteins in the remainder. Sequence motifs characteristic of a serine protease, RNA helicase, and an RdRP are found in similar locations in the polyproteins of all three genera.⁵⁸⁵ RNA replication occurs entirely in the cytoplasm in close association with intracellular membranes; the synthesis of a genome-length minus-strand RNA provides the intermediate. Progeny virions assemble by budding into an intracellular membrane compartment, most likely the endoplasmic reticulum (ER), then transit through the host secretory pathway and are released at the cell surface.

The Flavivirus genus consists of more than 50 species, many of which are arthropod-borne human pathogens. Flaviviruses cause a variety of diseases, including fever, encephalitis, and hemorrhagic fevers. Entities of major global concern include dengue virus (DENV)—with its associated dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS)—Japanese encephalitis virus (JEV), West Nile virus (WNV), and YFV (reviewed in⁵³⁷). Other flaviviruses with regional or endemic distribution include Murray Valley encephalitis virus (MVEV) and St. Louis encephalitis virus (SLEV). Tick-borne encephalitis virus (TBEV) is a name commonly applied to either central European encephalitis virus or Far Eastern encephalitis virus, although these are clearly distinct species.²⁰⁸ Decreases in mosquito control efforts during the latter part of the 20th century, coupled with societal factors (e.g., increased transportation and dense urbanization), have contributed to the re-emergence of flaviviruses such as DENV in South and Central America. Following an outbreak in New York City in 1999, WNV has spread throughout much of North America and Central America.

Flavivirus species are further categorized into antigenic complexes and subcomplexes based on serologic criteria or into clusters, clades, and species, according to molecular phylogenetics.¹¹⁷ Mosquito-borne and tick-borne flaviviruses, although
distinct, appear to have evolved via a common ancestral line that diverged from viruses with no known arthropod vector. DENV circulates as four distinct serotypes, which show significant sequence diversity (reviewed in³³⁷). Some reports have documented intertypic recombination among DENV isolates, although the taxonomic status of these isolates is currently unclear.

The development of the first live-attenuated flavivirus vaccine, YFV strain 17D,⁸⁴⁹ led to Max Theiler’s recognition by the Nobel Prize committee in 1951. Only a limited number of flavivirus vaccines are available, including inactivated TBEV and JEV for use in humans and inactivated WNV for use in animals.⁶⁹⁴ Development of effective DENV vaccines that exhibit cross-protection between serotypes is proving to be particularly challenging. The ability to genetically manipulate flaviviruses has led to novel approaches, including live attenuated chimeric vaccines based on the YFV-17D backbone.

Infectious flavivirus particles are roughly spherical, approximately 50 nm in diameter, and surrounded by a lipid envelope⁶¹⁵ (Fig. 25.3). Viruses sediment between 170 and 210S and have buoyant densities of 1.19 to 1.23 g/cm³ depending on the lipid composition, which can vary by host.⁷⁴⁵ The outer shell of the particle is made up of two viral proteins, envelope (E) and membrane (M). The E glycoprotein is the major antigenic determinant of the virion and mediates binding and fusion during virus entry. The M protein is a small proteolytic fragment of the precursor (pr)M protein and is produced during viral maturation within the secretory pathway. Removal of the lipid envelope with nonionic detergents reveals discrete nucleocapsids (120 to 140S; 1.30 to 1.31 g/cm³), which consist of capsid (C) protein and genomic RNA (reviewed in⁷⁴⁵). Isolated nucleocapsids become unstable under high salt conditions, disassembling into C protein dimers.³⁹⁹

Cryoelectron microscopy and image reconstruction have provided a wealth of information on flavivirus structure. Mature infectious particles of DENV⁴³⁰ and WNV⁶¹⁰ display a relatively smooth outer surface. Fitting the E protein crystal structure⁷²² into the electron density maps showed that glycoprotein dimers lie flat across the surface of the virion. Interestingly, the 180 copies of E are tightly packed in an unusual herringbone array that completely covers the lipid bilayer. Beneath the protein shell, the M protein associates closely with the membrane. Notably, the nucleocapsid lacks discernible symmetry, and neither E nor M sequences extend through the membrane to make contacts with the nucleocapsid.⁹⁵⁶

Immature flavivirus particles adopt various appearances as they egress through the secretory pathway.⁶⁶⁷ Soon after they are formed, immature virions are larger (60 nm in diameter) than mature virions and display 60 prominent spikes on their surface.⁹⁵⁷ Each protrusion is composed of three E-prM heterodimers, with the prM molecule capping the E fusion peptide. As immature particles pass through the low pH environment of the trans-Golgi network, a dramatic rearrangement of the glycoproteins occurs. The immature virions now adopt a smooth appearance almost indistinguishable from mature particles, with the exception that prM remains attached.⁹⁴⁶ This conformational change is followed by cleavage of prM by the host cell enzyme furin⁸⁰² and the release of the protective fragment upon exit from the cell, revealing the mature virions.⁹⁴⁶ This process is not always efficient, and immature or partially mature particles can be released in significant quantities. Although immature particles are deemed noninfectious because they cannot undergo fusion,³⁰³ they have recently been shown to initiate infection when internalized in complex with anti-prM antibodies by cells bearing the Fc receptor.⁷³⁵ The mechanism of this is not well understood, but it may invoke prM cleavage in the endosome and could be especially relevant during secondary infection when antibody levels are high. In addition, partially matured particles that retain only some prM have been visualized in flavivirus populations and can undergo attachment and fusion to initiate infection similar to fully mature particles.¹³⁷

Small, noninfectious subviral particles (SVPs) are the final class of particles released from flavivirus-infected cells. SVPs
contain E and M proteins but lack C and RNA.⁷⁹⁷ They complete the same maturation process as whole virions and can undergo fusion with a target cell⁷⁶⁰; due to lack of a genome, however, they are not infectious. Recombinant subviral particles (RSPs) form in cells experimentally transfected with only prM and E, indicating that interactions between these envelope proteins are sufficient to drive budding.¹⁴,⁵¹²,⁷⁶⁰ RSPs are generally about 30 nm in diameter and slightly less dense than infectious virus (1.14 g/cm³),⁷⁶⁰ although virion-sized particles have also been observed in these expression systems.¹⁶,⁵¹² Cryoelectron microscopy and image reconstruction of TBEV RSPs suggests a markedly different arrangement of the E proteins compared to infectious virions. Thirty E dimers lie flat against the surface in a T = 1 icosahedral shell²³² rather than a herringbone array.⁴³⁰ It is hypothesized that this arrangement may resemble a fusion intermediate that is adopted as E dimers rearrange to trimers upon virus entry.⁶¹¹

Flaviviruses infect a variety of target cells through receptor-mediated endocytosis, followed by intracellular membrane fusion. Flavivirus receptors are not well characterized, perhaps because these viruses use a range of entry factors for different cell types and employ more than one host molecule to enter a target cell. Highly sulphated glycosaminoglycans, such as heparin sulphate, are ubiquitously expressed molecules used as initial attachment factors by many viruses. These factors have also been shown to play a role in binding and entry of flaviviruses, such as DENV,¹³² YFV,²⁶⁹ TBE,⁴²⁶ and JEV.¹³⁰ However, high affinity for glycosaminoglycans in tissue culture–adapted strains is associated with in vivo attenuation.⁴⁵⁸ C-type lectins are cellular proteins that bind mannose-rich glycans and are involved in flavivirus infection of dendritic cells (DCs). Intradermal DCs are often the primary targets encountered by the arthropod-delivered pathogens and can transport the virus to draining lymph nodes where a second round of replication initiates viremia. C-type lectin Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is thought to function as an attachment receptor for DENV infection of DCs.⁵¹⁶,⁶²⁴,⁸³³ WNV preferentially uses DC-SIGN-related (DC-SIGNR),¹⁸² while YFV-17D, which lacks glycan modifications on E, can infect DC cells in a lectin-independent manner.³⁹ The mannose receptor is a C-type lectin that is constitutively internalized by clathrin-mediated endocytosis and has been suggested to play a role in endocytosis of DENV, JEV, and TBEV.⁵⁸⁴ Interestingly, C-type lectin domain family 5, member A (CLEC5) interacts with DENV but does not mediate its entry; instead, CLEC5 binding triggers the release of inflammatory cytokines, leading to DHF/DSS-like symptoms in mice.¹³¹

Additional receptors that have been implicated in flavivirus entry include the glycosphingolipid neolactotetraosylceramide,²⁶ low-density lipoprotein receptor (LDL-R),¹³⁸ laminin receptor 1,⁸⁵⁰,⁸⁵⁷ αvβ3 integrins,¹⁴⁴ and a CD14-containing complex.¹³⁴ Heat shock proteins have also been suggested as entry factors. GRP78 (BIP) plays a role in liver cell uptake of DENV,¹¹²,³⁶⁸ and Hsp90/Hsp70 acts in DENV and JEV entry into human monocytes/macrophages, neuroblastoma cell lines, and mosquito cells.¹⁸⁰,⁷²⁰,⁷²³ Finally, virus particles opsonized with subneutralizing concentrations of immunoglobulins show enhanced binding and infection of cells expressing Fc receptors.⁶⁶¹,⁷⁶⁴ It is widely speculated that antibody-enhanced infection is relevant to the pathogenesis of DSS and DHF, which occur more frequently in people previously exposed to other DENV serotypes.

After capture by the appropriate receptor(s), flaviviruses are internalized by endocytosis. Single particle tracking of fluorescently labeled DENV particles has shown that virions diffuse across the surface of the cell until they encounter a preformed clathrin-coated pit.⁸⁷¹ Following internalization, DENV particles are delivered to early or intermediate endosomes, which then mature into late endosomes.⁸⁷¹ Fusion of the viral and host membranes occurs during endosomal trafficking, although the exact compartment that triggers this event seems to differ between strains and flavivirus species, perhaps indicating an optimal pH.⁸⁷¹ In the acidic environment, E protein dimers dissociate and undergo an irreversible conformational change to become fusogenic trimers.¹³,⁸¹⁰ The fusion peptide, previously buried at the E homodimer interface, is exposed and inserts into the endosomal membrane (Video 25.1). The efficiency of fusion is influenced by the lipid composition of target membranes: cholesterol, oleic acid, and anionic lipids such as bis(monoacylglycero)phosphate and phosphatidylserine enhance fusion, whereas lyophosphatidylcholine inhibits the process.⁸¹²,⁸¹³,⁹⁵⁰ Lipid composition can also influence the pH threshold of fusion.⁴²⁰ Following fusion, viral genomes are immediately accessible for translation.⁴²⁰

As for other positive-strand RNA viruses, the genomes of flaviviruses are infectious.⁶⁶² Full-length infectious complementary DNA (cDNA) clones have been constructed for several species, allowing flavivirus biology to be dissected by reverse genetics.⁴⁹¹,⁷²⁵,⁷⁴² Flavivirus genomes consist of a single, positive-strand RNA of approximately 11 kilobases (kb) in length (sedimentation, 42S) with a 5′ type 1 cap, m⁷GpppAmN¹⁵¹,⁹⁰¹ (Fig. 25.4). The cap structure serves to stabilize the viral RNA, initiate translation, and subvert innate antiviral defenses.¹⁷⁸,²⁵³ Unlike cellular mRNAs, flavivirus genomes lack a 3′ polyadenylate tail.⁹⁰¹ Genomes encode a single long open reading frame (ORF, ∼3,400 codons) flanked by 5′ and 3′ noncoding regions (NCRs) of ∼100 nucleotides (nt) and 400 to 700 nt, respectively⁵⁴⁴ (Fig. 25.4).

The sequence of the 5′ NCR is not well conserved between flaviviruses, although common secondary structural elements have been identified, including a bifurcating 5′ stem-loop (5′ SL). These structures influence viral genome translation, as antisense oligonucleotides complementary to the 5′ SL abolish DENV translation and replication, and second-site mutations in this region compensate for replication defects caused by reduced viral cap methylation. In addition, 5′ SL likely acts as a promoter to initiate RNA replication by binding the viral NS5 polymerase/methyltransferase protein.¹⁹⁶,²³⁶,²³⁷,⁵⁰⁶ Consistent with this, deletions within the 5′ NCR cause severe defects in DENV-4 replication, but not viral translation.¹¹³ Interestingly, one of the viable mutants exhibited a limited host-range growth phenotype, suggesting that host-specific factors interact with either the 5′ NCR or the complementary 3′ end of the negative strand. Indeed, several human proteins, including La and TIAR, can bind to the 3′ end of negative-strand RNA.⁴⁷²,⁷⁷⁸,⁹³⁸ WNV replication is inhibited in a TIAR-knockout cell line,⁴⁷² and mutagenesis of the TIA-1/TIAR binding sites suggests a role in initiating positive-strand RNA synthesis.²¹⁷

The organization of the 3′ NCR differs between mosquito-borne viruses, tick-borne viruses, and viruses with no known vector. Nevertheless, conserved regions, sequence duplications, and predicted RNA secondary structures are shared among the groups (Fig. 25.4). The greatest structural similarity is a long (90 to 120 nt) 3′ stem-loop (3′ SL) that differs in primary sequence between mosquito-borne and tick-borne flaviviruses.²⁹⁶ Mutational analysis of DENV-2 and WNV revealed essential virus-specific and host-specific functional regions within the 3′ SL.²¹⁶,⁸⁵⁶,⁹⁴⁷,⁹⁵³ The 3′ SL enhances translation of reporter mRNAs containing the DENV 3′ NCR,¹⁴⁰,³³⁵ while DENV-2 translation and replication were inhibited by a corresponding antisense oligonucleotide.³³⁶ The 3′ SL also interacts with several important proteins, including the viral NS2A, NS3, and NS5 proteins¹²⁹,¹⁷⁵,⁵³⁵ and translation elongation factor 1A (EF1A).⁷⁵,¹⁸³,¹⁸⁵ These results are intriguing, because EF1A, and its prokaryotic homolog EF-Tu, contribute to the replication of other positive-strand RNA viruses.⁸¹,³¹⁴,³⁷⁹,⁹⁵¹ In addition, the human La autoantigen,¹⁸⁵,²⁶²,⁸⁷⁵ polypyrimidine tract binding (PTB) protein,¹⁸⁵ and murine Mov34 protein⁸²² were found to bind 3′ SL of DENV-4 and JEV, although the functional relevance of these interactions is presently unknown.

Upstream of the 3′ SL lie conserved sequence repeats (CS1, CS2, CS3, RCS2, and RCS3), secondary structures, and putative pseudoknots.²⁹⁶ Some of these structures confer resistance to the cellular 5′-3′ exoribonuclease Xrn-1,²⁵²,⁷⁸⁵ suggesting that this region of the genome forms a compact structure.

Flavivirus genomes can be circularized through long-distance base pairing between elements located near the 5′ and 3′ ends. In mosquito-borne flaviviruses, these interactions are mediated by the 5′ UAR (upstream of AUG region), 5′ DAR (downstream of AUG region), and 5′ CS (conserved sequence), which base-pair with the 3′ UAR, 3′ DAR, and 3′ CS1 regions, respectively, located more than 10 kb downstream at the base of the 3′ SL.³⁵⁴ A distinct set of long-distance interactions circularizes the genomes of tick-borne flaviviruses.³⁹⁴,⁴¹⁰ These long-range base pairs are important for RNA replication, presumably by bringing the 5′ SL–bound NS5 protein in proximity to the 3′ site of minus-strand initiation.¹⁸,⁹⁶,¹⁶⁷,¹⁹⁶,²³⁶,²³⁷,²⁴⁸,³⁵⁴,³⁹⁴,⁴¹⁰,⁹⁴⁰ It should be noted that genome circularization requires the melting of local secondary structures within the 5′ and 3′ ends and leads to the occlusion of the translation start site. Thus, large-scale conformation changes within the flavivirus genome may regulate the switch from translation to RNA replication.

The efficiency of genome translation is a primary determinant of flavivirus infectivity.²⁰⁹ The viruses therefore use several mechanisms to ensure translational competence, including specialized structures within the 5′ and 3′ NCRs. Translation is cap dependent, and 2′-O methylation of the 5′ cap helps to overcome innate antiviral defenses that down-regulate translation in infected cells.¹⁷⁸ While translation initiates via ribosomal scanning, many mosquito-borne flaviviruses lack a canonical Kozak initiation motif and contain several AUG codons near the correct start site. To help ensure proper AUG selection, DENV uses a small RNA stem-loop embedded within the C gene to induce ribosomal pausing over the authentic initiation codon.¹⁵⁵

Translation of the single, long ORF produces a large polyprotein that is co- and posttranslationally cleaved into at least 10 proteins (Fig. 25.4B). The N-terminal region of the polyprotein encodes the structural proteins (C-prM-E), which are followed by the NS proteins (NS1-NS2A-NS2B-NS3-NS4A-2K-NS4B-NS5).¹¹⁹,¹²⁰,⁷²⁶ Host signal peptidase is responsible for cleavages between C/prM, prM/E, E/NS1, and 2K-NS4B. A virus-encoded serine protease, NS2B-3, processes at the NS2A/NS2B, NS2B/NS3, NS3/NS4A, NS4A/2K, and NS4B/NS5 junctions. The enzyme responsible for NS1–2A cleavage is presently unknown. The expected topology of the flavivirus polyprotein is depicted in Figure 25.4C.

The flavivirus NS proteins presumably recruit the viral genome out of translation and into a replication complex. Replication begins with the synthesis of a genome-length minus-strand RNA, which then serves as a template for new plus-strand genomes. Minus-strand RNA has been detected as early as 3 hours after infection.⁴⁹² Viral RNA synthesis is asymmetric, with approximately 10-fold more positive strands accumulating compared to minus strands.¹⁵²,⁶²⁰ Flavivirus replication can be followed by metabolic labeling of virus-specific RNA in the presence of actinomycin D, an inhibitor of DNA-dependent RNA polymerases. Three major species of labeled flavivirus RNA have been described, including the plus-strand genome, a double-stranded replicative form (RF), and a heterogeneous population of replicative intermediates (RIs) that most likely represent duplex regions and recently synthesized RNAs displaced by nascent strands undergoing elongation.¹⁴⁶,¹⁵² Pulse-chase analyses indicate that RF and RI are precursors to genome RNA,¹⁴⁶,¹⁵² indicating semiconservative and asymmetric replication.¹⁴⁶

In addition to genome-length plus- and minus-strand products of RNA replication, 0.2- to 0.6-kb subgenomic flavivirus RNAs (sfRNAs) are produced in infected cells.⁴⁸⁵,⁷⁶²,⁸⁶⁹ sfRNAs are co-linear with the 3′ end of the genome and are produced through incomplete degradation of the genome by the cellular 5′-3′ exoribonuclease Xrn1.⁶⁷⁸,⁷⁸⁵ The Xrn1 resistance of this region is due to conserved secondary structures and pseudoknots located within the 3′ NCR.²⁵²,⁷⁸⁵ While controversy exists over whether sfRNAs are needed for efficient RNA replication, a WNV mutant that does not produce sfRNAs is less cytopathic in cell culture and less pathogenic in mice.²⁵²,⁶⁷⁸,⁷⁸⁵ Interestingly, supplying sfRNA in trans restored WNV cytopathic effect in cell culture, suggesting that these small RNAs may have a specific but as yet unknown target.⁶⁷⁸

Similar to replication, early ultrastructural studies indicated that flavivirus morphogenesis occurs in close association with intracellular membranes.⁶¹⁵ The assembly process is thought to commence by association of C protein dimers with genomic RNA, followed by budding into ER membranes containing the E–prM glycoprotein complex. Recent electron tomography studies of DENV-infected cells have shown that replicase-containing vesicle packets and ER-associated sites of virus budding are part of one continuous network. Pores within the replication vesicles appear to release newly synthesized viral RNA directly adjacent to budding DENV particles.⁸⁹⁸ These findings suggest a potential mechanism for coupling of flavivirus replication and assembly.³⁹⁷ Interdependence of these processes is a common theme among RNA viruses and might serve to reduce the propagation of defective genomes. Coupling flavivirus replication and assembly may also explain the roles of NS2A and NS3 proteins in infectious virus assembly.⁴³⁴,⁴⁶⁵,⁴⁹⁷,⁶⁵⁵

Following assembly, nascent virions are transported through the secretory pathway and released at the cell surface.⁵³⁶ It is during secretion that immature particles undergo acid-induced E–prM rearrangement and prM cleavage (Fig. 25.10). Additional virion maturation steps occur during egress, including glycan modification of prM and E (for some viruses) by trimming and terminal addition.¹²³,¹⁶⁹,³⁰⁹,⁵⁴⁷ This implies that virions move through an exocytosis pathway similar to that used for host cell surface glycoproteins. In addition to the secretory pathway machinery, other host factors have been implicated in flavivirus morphogenesis. Inhibitor and RNA interference studies implicate the Src family kinase c-Yes in WNV egress from the ER.³³³ A serine/threonine protein phosphatase inhibitor, I(2)(PP2A), has also been found to bind the WNV C protein, although its importance for infectivity was minimal.³⁵⁰

HCV was identified in 1989 through expression cloning of immunoreactive cDNA from the plasma of a chimpanzee infected with an etiologic agent known to cause non-A non-B viral hepatitis.¹⁴³ The virus currently infects more than 130 million people worldwide and remains a significant public health concern. HCV typically causes chronic hepatotropic infections, which can progress over decades to fibrosis, cirrhosis, and liver cancer. Based on difficulties in detecting HCV in vivo, the virus was originally thought to replicate poorly. Yet mathematical models of HCV dynamics indicate that a chronically infected patient produces approximately 10¹² virions per day, with a virion half-life of only a few hours.⁶⁶⁶

Based on its evolutionary history, HCV is classified into seven genotypes, which differ from each other by more than 30% at the nucleotide level.⁶¹⁴,⁷⁸⁸ Each genotype is further divided into numerous subtypes. HCV genotypes show differences in worldwide distribution, disease progression, and susceptibility to
treatment. In addition, intergenotypic and intersubtype recombinant HCV genomes have been described.⁷⁸⁸

While no HCV vaccine exists, advances in treatment have been made. Pegylated IFN-α and ribavirin—the standard of care for over a decade—have burdensome side effects and do not cure many of the patients treated. The most common HCV genotype, genotype 1, is also the most difficult to treat with this therapy.³³⁴ Recently, however, HCV-specific antiviral drugs—such as protease inhibitors—have begun to reach the clinic. While the new drugs must still be used in combination with IFN and ribavirin, first-generation protease inhibitors have almost doubled the chances of treatment success.

In addition to HCV, the genus Hepacivirus may contain a few related viruses that are awaiting taxonomic classification. An HCV-like virus was recently identified in domestic dogs with respiratory illness.³⁸⁸ This virus appears to be closely related to HCV and has been tentatively designated canine hepacivirus (CHV), although it has not been formally classified within this genus. In addition, GB virus B (see later) appears to be closely related to the hepaciviruses.

HCV particles are enveloped and contain the viral proteins core (C), E1, and E2, as well as the genomic RNA. Virions are between 30 and 80 nm in diameter.⁸⁸,³¹⁷,⁹⁴⁹ For a virus of this size, HCV exhibits an unusually low and heterogeneous buoyant density. In highly infectious acute-phase chimpanzee serum, HCV-specific RNA is detected in fractions with densities ranging from 1.03 to 1.10 g/mL.⁸⁷,³³¹ The particle density is inversely correlated with infectivity, with low-density virions being more infectious than high-density particles. Similar to serum-derived virus, the peak of HCVcc RNA has a buoyant density of approximately 1.15 g/mL,¹¹⁶,⁴⁸⁷,⁸⁸³,⁹³⁷,⁹⁵⁹ whereas the peak infectivity is near 1.10 g/mL.¹¹⁶,⁴⁸⁷,⁹⁵⁹ Passage of cell culture–grown virus in animals⁴⁸⁸ or primary hepatocyte cultures⁶⁸² increases the proportion of low-density, high-infectivity virus, highlighting the impact of host cell environment on virion composition.

The high density observed for noninfectious particles may come from immunoglobulin binding to the virus and/or represent nonenveloped nucleocapsids.²³ The low buoyant density of infectious HCV appears to reflect the ability of the virus to interact with serum lipoproteins.²²,⁶³¹,⁶⁹²,⁸⁵⁵ The lipidome of HCV particles is very similar to low-density lipoproteins (LDLs) and very-low-density lipoproteins (VLDLs), including an enrichment of cholesteryl esters.⁵⁶⁹ Immunoprecipitation and immunogold-electron microscopy (EM) studies have detected apolipoprotein (Apo) AI,⁴¹⁶ ApoB,⁴¹⁶,⁶³¹,⁸⁵⁵ ApoC1,⁵⁷² and ApoE⁶³¹ in association with serum-derived HCV. Apolipoprotein E and ApoC1 are also associated with HCVcc,¹²⁶,⁵⁶⁹,⁵⁷² but ApoB is generally lacking, leading to controversy about
its functional importance (reviewed in⁴⁴). The propensity of HCV to interact closely with lipids has led to the hypothesis that at least a portion of circulating virus is internalized in host lipoproteins to form a “lipoviral” particle,²² which may provide camouflage for the virus to escape neutralizing antibodies.

Although unambiguous visualization of HCV from patient samples remains a challenge, negative stain and cryoelectron microscopy studies have started to reveal structural features of HCV grown in cell culture²⁶⁴,⁵⁶⁹,⁸⁸³ (Fig. 25.11). One recent study detected two predominant classes of HCV particles, each making up approximately half of the preparation. The first were spherical, pleomorphic particles, ∼55 nm in diameter, containing a lipid bilayer and an electron-dense core (Fig. 25.11C). These particles, which are presumably infectious HCV, were enriched in low-density fractions and had a relatively smooth, featureless surface that reacted with E2- and ApoE-specific antibodies. The second class of particles, electron-dense structures ∼45 nm in diameter, is presumed to be naked nucleocapsids. This class was enriched in high-density, low-infectivity fractions and lacked a membrane or visible surface projections. Similar particles were seen after detergent treatment of infectious virus, which is expected to remove the viral envelope.²⁶⁴ Unfortunately, the reconstruction of high-resolution models of the virus particle is currently limited by particle heterogeneity, relatively low yields, and lack of symmetry in both classes of HCV particles.

HCV infects target hepatocytes through a multistep process that involves several cell surface molecules. Virus particles initially attach to glycosaminoglycans⁴⁵,²⁶⁸ and possibly the LDL receptor (LDL-R),⁴,⁵⁹⁸ which may interact with virion-associated ApoB and ApoE. Following attachment, virus particles employ four specific entry factors to mediate uptake: scavenger receptor class B type I (SR-BI, aka SCARBI),⁷⁵⁸ CD81,⁶⁷⁹ claudin 1 (CLDN1),²²⁴ and occludin (OCLN).⁶⁸¹

SR-BI, which is highly expressed on hepatocytes, plays important physiologic roles in the selective uptake of cholesterol and cholesteryl esters from lipoproteins. SR-BI can interact with the E2 hypervariable region 1 (HVR1),⁷⁵⁸ and its overexpression enhances virion binding to CHO cells,²²⁴ suggesting a role for SR-BI in primary attachment. Blocking antibody studies have both supported a role for SR-BI in binding,¹²¹ as well as shown the importance of SR-B1 at a postbinding step that is closely linked to CD81.³⁸⁶,⁹⁵² Antibodies directed at E2 HVR1 similarly block a postattachment step of HCV entry.⁸⁷⁸ The physiologic SR-BI binding partner, PDZK1, is also important for entry.²²⁵ Perhaps because the lipoviral particle shares commonalities with the natural ligands of SR-BI, various lipoproteins modulate HCV infectivity. Specifically, high-density lipoproteins (HDLs) can enhance HCV fusion in a manner dependent on SR-BI lipid transfer, ApoCI, and the E2 HVR1.⁴⁷,¹⁹⁹,⁵⁷¹,⁸⁸⁰ In contrast, oxidized LDL,⁸⁸² VLDL,⁵⁴² and serum amyloid A¹¹⁴,⁴⁵³ inhibit HCV entry.

The tetraspanin CD81 binds tightly to the glycoprotein E2 via residues in the CD81 large extracellular loop.³²⁸,⁶⁷⁰,⁶⁷⁹ Antibodies targeting CD81 do not affect HCVpp binding to hepatocytes¹⁶⁶ but inhibit HCV infection after virus attachment.⁴²¹ Because HVR1 masks the CD81 binding site, a conformational change may be required to prime E2 for binding to this co-receptor.³⁷ CD81 interacts with another HCV entry factor, CLDN1,³¹³,⁹²⁹ forming a complex that is essential for HCV infection.³¹² Receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR), appear to regulate CD81–CLDN1 interactions and to promote HCV membrane fusion.⁵²⁷ Interaction of CD81 with another host factor, EWI-2wint, inhibits virus uptake.⁷³⁴ In addition to its role in entry, interaction of HCV with lymphocyte-expressed CD81 may contribute to the immune dysregulation seen in some patients with chronic infection.¹⁷³,⁷³⁹,⁸⁶⁵

Two tight junction proteins, CLDN1²²⁴ and OCLN,⁵⁹,⁴⁹⁶,⁶⁸¹ are essential for postbinding steps of HCV uptake. In addition, CLDN family members-6 and −9 can also mediate entry.⁵⁶³ The involvement of tight junction proteins suggests that HCV could traffic across the surface of the hepatocyte in order to engage all of its entry factors. Indeed, binding to CD81 activates a Rho guanosine triphosphatase (GTPase), which may mediate cytoskeleton rearrangements. Furthermore, recombinant E2 promotes relocalization of CD81 to tight junctions.⁹⁴ In conflict with this hypothesis, however, is the finding that CLDN1
can form complexes with CD81 on the basolateral surface of hepatocytes.³¹² In addition, single particle tracking has visualized HCV internalization outside tight junctions—although this may represent the nonpolarized nature of the cultured hepatoma cells.¹⁵⁹

Following attachment, HCV is taken up via clathrin-mediated endocytosis.⁷⁶,⁵⁶⁴ Internalized virions co-localize with clathrin light chain and the E3 ubiquitin ligase c-Cbl prior to uncoating.¹⁵⁹ Intracellular HCV is delivered to Rab5a-positive early endosomes,¹⁵⁹ which likely provide the acidic environment necessary to induce rearrangement of the HCV glycoproteins into their fusogenic form.³⁴³,⁸⁶⁴ Surprisingly, extracellular HCVcc particles are resistant to inactivation by low pH, suggesting that interactions at or near the cell surface prime the virus for fusion.⁸⁶⁴ This is functionally similar to pestiviruses⁴²⁵ but in contrast to flavivirus particles, which are primed during maturation,⁹⁴⁶ and to HCVpp,³⁴³,⁶⁴⁴ which have structural and compositional differences from authentic HCV particles.

The HCV genome is an uncapped, 9.6-kb RNA containing highly structured 5′ and 3′ ends (Fig. 25.12A). The 5′ NCR is a well-conserved, 341-nt sequence element that folds into a complex structure consisting of four major domains and a pseudoknot.³²⁴ The first 120 nt serves as a minimal replication element, although compelling genetic evidence shows that the entire 5′ NCR (or its complement in the negative strand) plays an important role in efficient positive-strand RNA synthesis.⁷⁰,²⁴⁷,²⁸²,²⁹⁰,⁴⁰⁴,⁵²⁵,⁷²¹ The 5′ NCR also contains an IRES, which directs the cap-independent translation of the single large ORF.

Interestingly, the liver-specific micro-RNA (miR)-122 binds to two sites within the HCV 5′ NCR, and these interactions are required for efficient viral replication.³⁷⁷,⁴⁶⁹ While miR-122 can stimulate IRES-mediated translation in an Argonaute 2-dependent manner,³²⁵,³⁶⁶,⁷³²,⁹⁰⁷ elegant genetic experiments revealed that a major function of miR-122 is to sequester the 5′ end of the genome, perhaps protecting it from RNA degradation or from activation of innate antiviral responses.⁵³² Nuclease-resistant oligonucleotides that antagonize miR-122 function in vivo profoundly decrease HCV replication in experimentally infected chimpanzees and show promise for future therapeutics,⁴⁴⁷ although viral escape can occur in cell culture.⁴⁷⁴

A number of functionally important RNA structures have been identified within the HCV ORF.⁸⁶⁷ Genetic analysis indicated that an RNA stem-loop within the C gene is important for viral fitness.⁵⁶¹,⁸⁷⁶ An RNA structure in NS5B, termed 5BSL3.2, forms a long-distance base-pairing interaction with upstream RNA sequences with the 3′ NCR.²⁴⁶ This “kissing” interaction is essential for replication, although its function remains to be determined. In addition, the HCV ORF contains fewer UA and UU dinucleotides than expected
by chance.³⁰⁸ This is significant because these dinucleotides are the preferred cleavage sites of the IFN-stimulated RNase L. Based on the large-scale computerized folding of numerous positive-strand RNA viral genomes, the HCV ORF is also predicted to contain an unusually high rate of internal base pairing.⁷⁸⁹ Intriguingly, this feature is common among viruses that cause persistent infections. Thus, the observed bias in nucleotide composition and propensity for internal base pairing within the HCV ORF may reflect adaptation to evolutionary pressure exerted by innate antiviral pathways.

The 3′ NCR was originally thought to terminate in polyadenosine or polyuridine. Improved methods for producing 3′ terminal cDNAs later revealed, however, that the HCV 3′ NCR actually consists of a short (∼40 nt) variable domain and a polyuridine/polypyrimidine (polyU/UC) tract, followed by a highly conserved 98-nt 3′ X domain.⁴¹³,⁸²⁷ Mutagenesis studies revealed that the 3′ X domain is essential for RNA replication and that the polyU/UC tract must be at least 26 nt in length.²⁴⁵,⁹²⁶,⁹³⁵,⁹⁴¹ A number of cellular factors have been found to bind to the HCV 3′ NCR, including RNA-binding proteins polypyrimidine-tract binding (PTB) protein,²⁷⁸,³⁶³,⁸⁶⁶ heterogeneous ribonuclear protein C,¹⁵⁰,²⁷⁷ glyceraldehyde-3-phosphate dehydrogenase,⁶⁷¹ HuR,⁸⁰¹ and La autoantigen.⁷⁹⁹

In addition to its roles in replication, the HCV 5′ NCR acts as an IRES to direct cap-independent translation of the viral genome. The IRES is encoded by 5′ NCR domains II through IV (Fig. 25.12A), although flanking sequences can influence activity. In the current model of HCV IRES function, free 40S ribosomal subunits directly bind to the 5′ NCR domains IIId through f and adopt an mRNA-bound conformation.⁶⁴⁵,⁶⁶⁹,⁷⁹⁸ The IRES–40S complex then binds eIF3 and the ternary complex eIF2–GTP–Met-tRNA_i to form a 48S intermediate complex, in which the initiating AUG codon at nt 342 is positioned within the ribosomal P site.³⁶⁷,⁶⁴⁵ The HCV IRES directly interacts with eIF3 via determinants in domain IIIb¹⁶⁴ and thereby functionally mimics the 5′ cap-binding complex eIF4F.⁷⁹³ Following GTP hydrolysis and release of eIF2, the 60S ribosomal subunit is recruited to form a translationally active 80S complex.³⁶⁷,⁶⁴⁵,⁶⁶⁹ Under some conditions the HCV IRES does not require eIF3 or ternary complex and is also capable of directing translation from non-AUG initiation sites.⁴⁴⁴,⁸⁴⁶ This mechanism, which utilizes eIF5B to recruit Met-tRNA_i, may allow the virus to initiate translation despite cellular eIF2 phosphorylation by the viral RNA sensor protein kinase R (PKR).

A number of cellular factors have been reported to contribute to HCV IRES activity. The human La protein stimulates translation by binding near the initiator AUG and recruiting the 40S ribosomal subunit¹⁶⁸,⁶⁹³; inhibiting La strongly decreases HCV gene expression and replication.¹⁶⁸,¹⁹⁴ Other host factors that stimulate HCV IRES activity include proteosome α-subunit PSMA7⁴²⁸ and nucleolin.³⁶⁵ In addition, polycytidine binding proteins 1 and 2 and PTB were shown to bind the 5′ NCR, although their functions in the virus life cycle remain to be defined.⁴³ IRES-mediated translation can be inhibited by long-distance base pairing of 5′ NCR nt 24 to 38 to a stem-loop region (nt 428 to 442) within the C gene region.⁴⁰⁵ Interestingly, this part of the 5′ NCR overlaps with one of the miR-122 binding sites.³⁷⁷

Translation of the HCV genome produces a large polyprotein (∼3,000 amino acids) that is proteolytically cleaved to produce 10 viral proteins (Fig. 25.12B): the virion structural components core (C) and glycoproteins E1 and E2, and the presumed NS proteins p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. Mature forms of the HCV proteins arise via co- and posttranslational cleavage mediated by cellular and host-encoded proteases. During structural region processing, cleavages between C/E1, E1/E2, and E2/p7 are mediated by signal peptidase; mature C protein is released from the E1 signal peptide via signal peptide peptidase (SPP). Within the NS region, signal peptidase mediates cleavage of p7/NS2. The remainder of the NS region is processed by two virus-encoded proteases: the NS2 cysteine autoprotease mediates cleavage at the NS2/3 junction, whereas the NS3-4A serine protease cleaves at all downstream junctions (Fig. 25.12C).

In addition to proteins encoded by the large ORF, it appears that small protein products can be produced from the +1 reading frame of the C gene. At least three different forms of alternative reading frame protein (ARFP) have been described: ARFP/F (frameshift), ARFP/DF (double frameshift), or ARFP/S (short form). Although it is thought that these products are produced via ribosomal frameshifting, alternate translational initiation sites may also be involved.⁸⁹ It is equally unclear what role, if any, ARFP expression plays in the virus life cycle. Mutant genomes with a disrupted +1 reading frame are highly attenuated in vivo,⁵⁶¹,⁸⁷⁶ although this may be due to disruption of conserved RNA secondary structures within the C gene.