Assembly of hepatitis delta virus particles.

Hepatitis delta virus (HDV) is a subviral satellite of hepatitis B virus (HBV). Since the RNA genome of HDV can replicate in cultured cells in the absence of HBV, it has been suggested that the only helper function of HBV is to supply HBV coat proteins in the assembly process of HDV particles. To examine the factors involved in such virion assembly, we transiently cotransfected cells with various hepadnavirus constructs and cDNAs of HDV and analyzed the particles released into the medium. We report that the HDV genomic RNA and the delta antigen can be packaged by coat proteins of either HBV or the related hepadnavirus woodchuck hepatitis virus (WHV). Among the three co-carboxy-terminal coat proteins of WHV, the smallest form was sufficient to package the HDV genome; even in the absence of HDV RNA, the delta antigen could be packaged by this WHV coat protein. Also, of the two co-amino-terminal forms of the delta antigen, only the larger form was essential for packaging.     

Assembly of infectious hepatitis C virus particles

A hallmark of the hepatitis C virus (HCV) replication cycle is its tight link with host cell lipid synthesis. This is best illustrated by the peculiar pathway used for the assembly of infectious HCV particles. Research in the past few years has shown that formation of HC-virions is closely connected to lipid droplets that could serve as an assembly platform. Moreover, HCV particle production appears to be strictly linked to very-low-density lipoproteins. In this review, we focus on new insights into the molecular aspects of the architecture and assembly of this unique type of virus particle.     

Interaction of hepatitis C virus nonstructural protein 5A with core protein is critical for the production of infectious virus particles

Nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) possesses multiple and diverse functions in RNA replication, interferon resistance, and viral pathogenesis. Recent studies suggest that NS5A is involved in the assembly and maturation of infectious viral particles; however, precisely how NS5A participates in virus production has not been fully elucidated. In the present study, we demonstrate that NS5A is a prerequisite for HCV particle production as a result of its interaction with the viral capsid protein (core protein). The efficiency of virus production correlated well with the levels of interaction between NS5A and the core protein. Alanine substitutions for the C-terminal serine cluster in domain III of NS5A (amino acids 2428, 2430, and 2433) impaired NS5A basal phosphorylation, leading to a marked decrease in NS5A-core interaction, disturbance of the subcellular localization of NS5A, and disruption of virion production. Replacing the same serine cluster with glutamic acid, which mimics the presence of phosphoserines, partially preserved the NS5A-core interaction and virion production, suggesting that phosphorylation of these serine residues is important for virion production. In addition, we found that the alanine substitutions in the serine cluster suppressed the association of the core protein with viral genome RNA, possibly resulting in the inhibition of nucleocapsid assembly. These results suggest that NS5A plays a key role in regulating the early phase of HCV particle formation by interacting with core protein and that its C-terminal serine cluster is a determinant of the NS5A-core interaction.     

Cloned hepatitis delta virus cDNA is infectious in the chimpanzee.

A head-to-tail trimer of a full-length cDNA clone of the hepatitis delta virus (HDV) genome was examined for infectivity by direct inoculation into the liver of a chimpanzee that was already infected with hepatitis B virus. Five weeks after inoculation, a marked elevation of serum alanine aminotransferase activity was observed, followed by the appearance of high levels of HDV RNA and antigen in both liver and serum and a high level of viral particles in the serum. A transient suppression of hepatitis B virus replication was evident during the acute phase of HDV infection. Seroconversion for antibodies to delta antigen occurred 3 weeks after the onset of the disease. These results demonstrate that a typical HDV infection can be initiated by inoculation of a susceptible animal with recombinant HDV cDNA.     

Engineered serine protease inhibitor prevents furin-catalyzed activation of the fusion glycoprotein and production of infectious measles virus.

We have identified the major cellular endoprotease that activates the fusion (F) glycoprotein of measles virus (MV) and have engineered a serine protease inhibitor (serpin) to target the endoprotease and inhibit the production of infectious MV. The F-protein precursor of MV was not cleaved efficiently into the mature F protein in human colon carcinoma cells lacking functional furin, indicating that furin is the major enzyme responsible for activation of the MV F protein. A human serpin alpha 1-antitrypsin variant was engineered to specifically inhibit furin. When expressed from a recombinant vaccinia virus in primate cells infected by MV, the engineered serpin (alpha 1-PDX) specifically inhibited furin-catalyzed cleavage of the F-protein precursor without affecting synthesis of other MV proteins. We generated human glioma cells stably expressing alpha 1-PDX. MV infection in these cells did not result in syncytia. The infected cells produced all the MV proteins, but the F-protein precursor remained largely uncleaved. This did not prevent virus assembly. However, the released virions contained inactive F-protein precursor rather than mature F protein, and infectious-virus titers were reduced by 3 to 4 orders of magnitude. These results show that a mature F protein is not required for the assembly of MV but is crucial for virus infectivity. The engineered serpin may offer a novel molecular antiviral approach against MV.     

Efficient trans-encapsidation of hepatitis C virus RNAs into infectious virus-like particles

Recently, complete replication of hepatitis C virus (HCV) in tissue culture was established using the JFH1 isolate. To analyze determinants of HCV genome packaging and virion assembly, we developed a system that supports particle production based on trans-packaging of subgenomic viral RNAs. Using JFH1 helper viruses, we show that subgenomic JFH1 replicons lacking the entire core to NS2 coding region are efficiently encapsidated into infectious virus-like particles. Similarly, chimeric helper viruses with heterologous structural proteins trans-package subgenomic JFH1 replicons. Like authentic cell culture-produced HCV (HCVcc) particles, these trans-complemented HCV particles (HCV_TCP) penetrate target cells in a CD81 receptor-dependent fashion. Since HCV_TCP production was limited by competition between the helper and subgenomic RNA and to avoid contamination of HCV_TCP stocks with helper viruses, we created HCV packaging cells. These cells encapsidate various HCV replicons with high efficiency, reaching infectivity titers up to 10⁶ tissue culture infectious doses 50 per milliliter. The produced particles display a buoyant density comparable to HCVcc particles and can be propagated in the packaging cell line but support only a single-round infection in naïve cells. Together, this work demonstrates that subgenomic HCV replicons are assembly competent, thus excluding cis-acting RNA elements in the core-to-NS2 genomic region essential for RNA packaging. The experimental system described here should be helpful to decipher the mechanisms of HCV assembly and to identify RNA elements and viral proteins involved in particle formation. Similar to other vector systems of plus-strand RNA viruses, HCV_TCP may prove valuable for gene delivery or vaccination approaches.Hepatitis C virus (HCV) is an enveloped plus-strand RNA virus of the genus Hepacivirus within the family Flaviviridae (34). The HCV genome is approximately 9.6 kb in length and consists of a single open reading frame encoding a polyprotein of ca. 3,000 amino acids and nontranslated regions (NTRs) located at the 5′ and 3′ termini. These NTRs are highly structured RNA segments encompassing critical cis-active RNA elements essential for genome replication and RNA translation (31). Viral proteins are expressed in a cap-independent manner by means of an internal ribosome entry site (IRES) located in the 5′ NTR. Co- and posttranslational cleavages liberate 10 viral proteins: core; envelope protein 1 (E1) and E2, representing the structural proteins that constitute the virion; a small membrane-associated ion channel protein designated p7 that is essential for virus assembly (16, 22, 43, 57); and six nonstructural (NS) proteins (NSs 2, 3, 4A, 4B, 5A, and 5B). HCV proteins NS3 to NS5B are both necessary and sufficient to establish membrane-bound replication complexes catalyzing RNA replication (13, 36). More recent data indicate that the NS2 protease that catalyzes cleavage at the NS2-NS3 site in addition participates in assembly and release of infectious viruses (22). Finally, ribosomal frame-shifting and internal translation initiation yield a group of additional proteins designated ARFP (alternative reading frame protein) or core+1 proteins. However, their function for the HCV replication cycle is currently not known.One hallmark of HCV is its high propensity to establish a persistent infection, which frequently causes progressive morbidity ranging from hepatic fibrosis to cirrhosis and hepatocellular carcinoma (20). Despite considerable progress in the treatment of HCV infection, the currently available therapy (a combination of pegylated interferon alpha with ribavirin) is not well tolerated and is efficacious in only ca. 50% of patients infected with the most prevalent genotype 1 (38). Therapeutic or prophylactic vaccines are not available. For these reasons and with currently ca. 170 million persistently infected individuals, HCV infection represents a considerable global health problem necessitating pertinent basic and applied research efforts.In recent years three major advances enabled analysis of the HCV replication cycle in tissue culture. First, Lohmann and colleagues developed subgenomic HCV replicons (36). These autonomously replicating RNA molecules carry all the genetic elements necessary for self-replication (the NTRs and NS3 to NS5B), including a selectable marker or a reporter gene in place of the viral structural proteins, and an internal IRES for expression of the HCV replicase genes (reviewed in reference 45). Second, HCV pseudotype particles, i.e., retroviral particles surrounded by an envelope carrying HCV E1-E2 complexes in place of their cognate envelope proteins, were established (3, 21). As these particles carry functional HCV glycoprotein complexes on their surface, HCV pseudotype particles have been instrumental for the analysis of E1-E2 receptor interactions and the early events of HCV infection (reviewed in reference 2). Finally, in 2005 fully permissive cell culture systems which are based on the JFH1 clone were described (33, 66, 72). This isolate replicates with unprecedented efficiency in transfected Huh7 human hepatoma cells and produces particles infectious both in vitro and in vivo, thus providing a model system reproducing the complete HCV replication cycle.Use of these novel models has considerably expanded our knowledge of viral and host cell factors involved in HCV replication (for a recent review, see reference 59). It is now known that similar to virtually all other plus-strand RNA viruses, HCV induces intracellular membrane alterations and replicates its genome in conjunction with vesicular membrane structures, the so-called “membranous web” (10, 13). Presumably as a consequence of this specific, rather secluded architecture of the membrane-associated replication machinery, all viral proteins involved in RNA replication, with the exception of NS5A function in cis, cannot be complemented in trans (1). Restricted trans-complementation of viral replicase proteins has been observed for other plus-strand RNA viruses as well, thus indicating that a rather “closed” replication machinery is a shared feature of these viruses (15, 27, 60). In contrast, for a number of plus-strand RNA viruses from diverse virus families like Picornaviridae (poliovirus), Alphaviridae (Sindbis virus, Semliki Forest virus, and Venezuelan equine encephalitis virus), Coronaviridae (human coronavirus E229), and Flaviviridae (tick-borne encephalitis virus, Kunjin virus, West Nile virus, and yellow fever virus), assembly of progeny viruses can be achieved when structural proteins are expressed in trans and independent from the RNA molecule that encodes the replicase proteins. Similarly, Miyanari recently reported that HCV genomes with lethal mutations in core protein can be rescued by ectopic expression of functional core protein (39). This flexibility has been extensively used to create viral vectors for gene delivery as well as viral vector-based immunization approaches (32, 48, 49, 61, 68) (for a recent review on alphaviral vectors, the most frequently used among plus strand RNA vectors, see reference 37). In these systems the viral genome region encoding the structural proteins is replaced by a transgene. The resulting defective vector genomes are capable of RNA replication but due to the lack of structural proteins are unable to produce progeny virus particles. This defect is rescued by expression of the structural proteins in trans via helper viruses (28, 55) or, in some cases, by DNA constructs stably expressed in packaging cell lines (17). The resulting virus-like particles are infectious but support only single-round infection and are unable to spread, thus improving the safety of the viral transduction system.Given the success of plus-strand RNA vector technology for basic and applied clinical research, in this study we developed a trans-complementation system for HCV that provided new insights into the basic principles of HCV particle assembly.     

Characterization of infectious retroviral pseudotype particles bearing hepatitis C virus glycoproteins

The recent development of infectious retroviral pseudotypes bearing hepatitis C virus (HCV) glycoproteins represents an opportunity to study the functionally active form of the HCV E1 and E2 glycoproteins. In the culture supernatant of cells producing HCV retroviral pseudotypes, the majority of E2 was not associated with infectious particles and failed to sediment on sucrose gradients. The E2 that was incorporated into infectious particles appeared as a triplet of diffuse bands at 60, 70, and 90 kDa. Similarly, three major forms of E1 were incorporated into the pseudotype particles, migrating at 33, 31, and 25 kDa. Endoglycosidase H (endo-H) treatment of particles demonstrated that the incorporated E1 was partially or completely sensitive to digestion. In contrast, the majority of the incorporated E2 was endo-H resistant. Purified pseudotype particles were found to contain both disulfide-linked aggregates and nonaggregated E1 and E2. HCV pseudotypes generated from cells expressing E1E2p7 showed similar heterogeneity in the incorporated glycoproteins and were of comparable infectivity to those generated by expression of E1E2. Our results demonstrate the heterogenous nature of E1 and E2 incorporated into retroviral pseudotypes and highlight the difficulty in identifying forms of the HCV glycoproteins that initiate infection.     

Differential biophysical properties of infectious intracellular and secreted hepatitis C virus particles

The recent development of a cell culture infection model for hepatitis C virus (HCV) permits the production of infectious particles in vitro. In this report, we demonstrate that infectious particles are present both within the infected cells and in the supernatant. Kinetic analysis indicates that intracellular particles constitute precursors of the secreted infectious virus. Ultracentrifugation analyses indicate that intracellular infectious viral particles are similar in size (approximately 65 to 70 nm) but different in buoyant density (approximately 1.15 to 1.20 g/ml) from extracellular particles (approximately 1.03 to 1.16 g/ml). These results indicate that infectious HCV particles are assembled intracellularly and that their biochemical composition is altered during viral egress.     

Production of infectious hepatitis delta virus in vitro and neutralization with antibodies directed against hepatitis B virus pre-S antigens.

Hepatitis delta virus (HDV) particles were produced in Huh7 human hepatoma cells by transfection with cloned hepatitis B virus (HBV) DNA and HDV cDNA. The particles were characterized by their buoyant density, the presence of encapsidated viral RNA, and their ability to infect primary cultures of chimpanzee hepatocytes. Successful infection was evidenced by the appearance of increasing amounts of intracellular HDV RNA after exposure to particles. Infection was prevented when particles were incubated with antibodies directed against synthetic peptides specific for epitopes of the pre-S1 or pre-S2 domains of the HBV envelope proteins before exposure to hepatocytes. These data demonstrate that HDV particles produced in vitro are infectious and indicate (i) that infectious particles are coated with HBV envelope proteins that contain the pre-S1 and pre-S2 regions, (ii) that epitopes of the pre-S1 and pre-S2 domains of HBV envelope proteins are exposed at the surface of HDV particles, and (iii) that antibodies directed against those epitopes have neutralizing activity against HDV.     

A circular trans-acting hepatitis delta virus ribozyme.

A circular trans-acting ribozyme designed to adopt the motif of the hepatitis delta virus (HDV) trans-acting ribozyme was produced. The circular form was generated in vitro by splicing a modified group I intron precursor RNA in which the relative order of the 5' and 3' splice sites, flanking the single HDV-like ribozyme sequence-containing exon, is reversed. Trans-cleavage activity of the circular HDV-like ribozyme was comparable to linear permutations of HDV ribozymes containing the same core sequence, and was shown not to be due to linear contaminants in the circular ribozyme preparation. In nuclear and cytoplasmic extracts from HeLa cells, the circular ribozyme had enhanced resistance to nuclease degradation relative to a linear form of the ribozyme, suggesting that circularization may be a viable alternative to chemical modification as a means of stabilizing ribozymes against nuclease degradation.     

Mutations in the carboxyl-terminal domain of the small hepatitis B virus envelope protein impair the assembly of hepatitis delta virus particles

The carboxyl-terminal domain of the small (S) envelope protein of hepatitis B virus was subjected to mutagenesis to identify sequences important for the envelopment of the nucleocapsid during morphogenesis of hepatitis delta virus (HDV) virions. The mutations consisted of carboxyl-terminal truncations of 4 to 64 amino acid residues and small combined deletions and insertions spanning the entire hydrophobic domain between residues 163 and 224. Truncation of as few as 14 residues partially inhibited glycosylation and secretion of S and prevented assembly or stability of HDV virions. Short internal combined deletions and insertions were tolerated for secretion of subviral particles with the exceptions of those affecting residues 164 to 173 and 219 to 223. However, mutants competent for subviral particle secretion had a reduced capacity for HDV assembly compared to that of the wild type. One exception was a mutant carrying a deletion of residues 214 to 218, which exhibited a twofold increase in HDV assembly (or stability), whereas deletions of residues 179 to 183, 194 to 198, and 199 to 203 were the most inhibitory. Substitutions of single amino acids between residues 194 and 198 demonstrated that HDV assembly deficiency could be assigned to the replacement of the tryptophan residue at position 196. We concluded that assembly of stable HDV particles requires a specific function of the carboxyl terminus of S which is mediated at least in part by Trp-196.     

Knockdown of autophagy-related gene decreases the production of infectious Hepatitis C virus particles

Hepatitis C virus (HCV) is a positive strand RNA virus, and classified within the Flaviridae family. It has been reported that Atg7-knockdown decreases the amount of HCV replicon RNA, when HCV JFH1 RNA and HCV subgenomic replicon were transfected into Huh7.5 cells. However, when infectious naïve HCV particles are directly infected into Huh7.5.1 cells, it is still unclear whether Atg7-knockdown decreases the production of intracellular HCV-related proteins, HCV mRNA and infectious HCV particles. When Atg7 protein in HCV-infected Huh7.5.1 cells was knocked down by RNA-interference, the levels of intracellular HCV core, NS3, NS5A proteins, HCV mRNA, and secreted albumin remained unchanged compared with those in the control HCV-infected cells. However, the level of infectious HCV particles released in the medium was decreased by the Atg7-knockdown. The similar results were obtained, when beclin1 was knocked down by RNA-interference. The colocalization of endogenous LC3-puncta with HCV core, HS5A proteins, and lipid droplets was also investigated. However, little endogenous LC3-puncta colocalized with HCV core, NS5A proteins or lipid droplets. These results suggested that autophagy contributed to the effective production of HCV particles, but little to the intracellular production of HCV-related proteins, HCV mRNA, and secretory pathway, in naïve HCV particles-infection system.     

High-throughput screening and rapid inhibitor triage using an infectious chimeric hepatitis C virus

The recent development of a Hepatitis C virus (HCV) infectious virus cell culture model system has facilitated the development of whole-virus screening assays which can be used to interrogate the entire virus life cycle. Here, we describe the development of an HCV growth assay capable of identifying inhibitors against all stages of the virus life cycle with assay throughput suitable for rapid screening of large-scale chemical libraries. Novel features include, 1) the use of an efficiently-spreading, full-length, intergenotypic chimeric reporter virus with genotype 1 structural proteins, 2) a homogenous assay format compatible with miniaturization and automated liquid-handling, and 3) flexible assay end-points using either chemiluminescence (high-throughput screening) or Cellomics ArrayScan™ technology (high-content screening). The assay was validated using known HCV antivirals and through a large-scale, high-throughput screening campaign that identified novel and selective entry, replication and late-stage inhibitors. Selection and characterization of resistant viruses provided information regarding inhibitor target and mechanism. Leveraging results from this robust whole-virus assay represents a critical first step towards identifying inhibitors of novel targets to broaden the spectrum of antivirals for the treatment of HCV.     

Interactions between hepatitis delta virus proteins

The 195- and 214-amino-acid (aa) forms of the delta protein (deltaAg-S and deltaAg-L, respectively) of hepatitis delta virus (HDV) differ only in the 19-aa C-terminal extension unique to deltaAg-L. deltaAg-S is needed for genome replication, while deltaAg-L is needed for particle assembly. These proteins share a region at aa 12 to 60, which mediates protein-protein interactions essential for HDV replication. H. Zuccola et al. (Structure 6:821-830, 1998) reported a crystal structure for a peptide spanning this region which demonstrates an antiparallel coiled-coil dimer interaction with the potential to form tetramers of dimers. Our studies tested whether predictions based on this structure could be extrapolated to conditions where the peptide was replaced by full-length deltaAg-S or deltaAg-L, and when the assays were not in vitro but in vivo. Nine amino acids that are conserved between several isolates of HDV and predicted to be important in multimerization were mutated to alanine on both deltaAg-S and deltaAg-L. We found that the predicted hierarchy of importance of these nine mutations correlated to a significant extent with the observed in vivo effects on the ability of these proteins to (i) support in trans the replication of the HDV genome when expressed on deltaAg-S and (ii) act as dominant-negative inhibitors of replication when expressed on deltaAg-L. We thus infer that these biological activities of deltaAg depend on ordered protein-protein interactions.     

A tryptophan-rich motif in the carboxyl terminus of the small envelope protein of hepatitis B virus is central to the assembly of hepatitis delta virus particles

The small hepatitis B virus surface antigen (S-HBsAg) is capable of driving the assembly and secretion of hepatitis delta virus (HDV) particles by interacting with the HDV ribonucleoprotein (RNP). Previously, a specific domain of the S-HBsAg protein carboxyl terminus, including a tryptophan residue at position 196 (W196), was proven essential for HDV maturation (S. Jenna and C. Sureau, J. Virol. 73: 3351-3358, 1999). Mutation of W196 to phenylalanine (W196F) was permissive for HBV subviral particle (SVP) secretion but deleterious to HDV virion assembly. Here, the W196F S-HBsAg deficiency was assigned to a loss of its ability for interaction with the large HDV antigen (L-HDAg), a major component of the RNP. Because the overall S-HBsAg carboxyl terminus is particularly rich in tryptophan, an amino acid frequently involved in protein-protein interactions, site-directed mutagenesis was conducted to investigate the function of the S-HBsAg Trp-rich domain in HDV assembly. Single substitutions of tryptophan between positions 163 and 201 with alanine or phenylalanine were tolerated for SVP secretion, but those affecting W196, W199, and W201 were detrimental for HDV assembly. This was proven to result from a reduced capacity of the mutants for interaction with L-HDAg. In addition, a W196S S-HBsAg mutant, which has been described in HBV strains that arose in a few cases of lamivudine-treated HBV-infected patients, was deficient for HDV assembly as a consequence of its impaired capacity for interacting with L-HDAg. Interestingly, the fact that even the most conservative substitution of phenylalanine for tryptophan at positions 196, 199, or 201 was sufficient to ablate interaction of S-HBsAg with L-HDAg suggests that W196, W199, and W201 are located at a binding interface that is central to HDV maturation.     

A novel structural rearrangement of hepatitis delta virus antigenomic ribozyme

A bioinformatic covariation analysis of a collection of 119 novel variants of the antigenomic, self-cleaving hepatitis delta virus (HDV) RNA motif supported the formation of all of the Watson–Crick base pairs (bp) of the catalytic centre except the C19–G81 pair located at the bottom of the P2 stem. In fact, a novel Watson–Crick bp between C19 and G80 is suggested by the data. Both chemical and enzymatic probing demonstrated that initially the C19–G81 pair is formed in the ribozyme (Rz), but upon substrate (S) binding and the formation of the P1.1 pseudoknot C19 switches its base-pairing partner from G81 to G80. As a result of this finding, the secondary structure of this ribozyme has been redrawn. The formation of the C19–G80 bp results in a J4/2 junction composed of four nucleotides, similar to that seen in the genomic counterpart, thereby increasing the similarities between these two catalytic RNAs. Additional mutagenesis, cleavage activity and probing experiments yield an original characterization of the structural features involving the residues of the J4/2 junction.     

Substitution rates in hepatitis delta virus

Substitution rates were estimated for the coding and noncoding regions of the hepatitis delta virus (HDV). The estimated rates of synonymous substitution in HDV were lower than the rates of substitution at nonsynonymous sites and in the noncoding region. HDV has lower synonymous substitution rates than the hepatitis C virus, though both are RNA viruses. The relatively low rate of synonymous substitution in HDV may be due to a strong preference of G and C nucleotides at third codon positions. Variation in substitution rate among HDV lineages may be correlated with the clinical development of the HDV-induced hepatitis. The phylogenetic tree inferred for 24 HDV strains reveals similarities between lineages isolated from the same geographic region. Correspondence to: W.-H. Li     

Characterization of hepatitis delta antigen: specific binding to hepatitis delta virus RNA.

It has previously been shown that human hepatitis virus delta antigen has an RNA-binding activity (Chang et al., J. Virol. 62:2403-2410, 1988). In the present study, the specificity of such an RNA-protein interaction was demonstrated by expressing various domains of the delta antigen in Escherichia coli as TrpE fusion proteins and testing their RNA-binding activities in a Northwestern protein-RNA immunoblot assay and RNA gel mobility shift assay. Hepatitis delta virus (HDV) RNA bound specifically to the delta antigen in the presence of an excess amount of unrelated RNAs and a relatively high salt concentration. Both genome- and antigenome-sense HDV RNAs and at least two different regions of HDV genomic RNA bound to the delta antigen. Surprisingly, these two different regions of HDV genomic RNA could compete with each other for delta antigen binding, although they do not have common nucleotide sequences. In contrast, this binding could not be competed with by other viral or cellular RNA. Since both the genomic and antigenomic HDV RNAs had strong intramolecular complementary sequences, these results suggest that the binding of delta antigen is probably specific for a secondary structure unique to the HDV RNA. By expressing different subdomains of the delta antigen, we found that the middle one-third of delta antigen was responsible for binding HDV RNA. Neither the N-terminal nor the C-terminal domain bound HDV RNA. Binding between the delta antigen and HDV RNA was also demonstrated within the HDV particles isolated from the plasma of a human delta hepatitis patient. This in vivo binding resisted treatment with 0.1% sodium dodecyl sulfate and 0.5% Nonidet P-40. In addition, we showed that the antiserum from a human patient with delta hepatitis reacted with all three subdomains of the delta antigen, indicating that all of the domains are immunogenic in vivo. These studies demonstrated the specific interaction between delta antigen and HDV RNA.