Activation of Heterologous Gene Expression by the Large Isoform of Hepatitis Delta Antigen

Hepatitis delta virus (HDV) encodes two isoforms of its principal gene product, hepatitis delta antigen (HDAg). These two forms play distinctive and complementary roles in viral replication. Here we report that the large (LHDAg), but not the small (SHDAg), isoform of HDAg has the capacity to activate the expression of cotransfected genes driven by a variety of promoters, including the pre-S, S, and C promoters of hepatitis B virus. Mutational analysis of the C-terminal 19 amino acids unique to LHDAg shows that changing prolines to alanines in the two PXXP motifs in this region specifically ablates the activation function without abolishing another activity of LHDAg, namely, its ability to inhibit HDV RNA synthesis. However, C-terminal truncations that also disrupt these PXXP motifs only slightly diminished the activation function, indicating that the proline mutations were not acting by inactivating potential SH3 interactions that could be mediated by these motifs. Mutation of the isoprenylated cysteine to serine decreases but does not abolish the activation activity, and overexpression of SHDAg does not interfere with the transactivation function of LHDAg. Although the mechanism and biological significance of this activity of LHDAg remain unknown, the presence of this activity serves as yet another marker that functionally distinguishes this protein from the closely related isoform SHDAg.Hepatitis delta virus (HDV) is an RNA virus that requires coinfection with hepatitis B virus (HBV) to complete its life cycle. The helper function supplied by HBV is limited to the provision of envelope proteins (hepatitis B surface antigens) for the completion of HDV assembly (28, 29, 31). HDV RNA replication is independent of its HBV helper (19). In fact, the presence of HDV suppresses HBV replication in vivo (30, 39). Nonetheless, clinical studies have shown that HDV infection can be associated with more severe hepatitis than HBV alone and is often implicated in cases of fulminant hepatitis (4, 32).The genome of HDV is a circular, single-stranded RNA of about 1,700 nucleotides (nt), of which approximately 70% are self-complementary (for a review, see references 20 and 21). This self-complemetarity allows the genome to form an unbranched rod-like structure. A unique functional protein, hepatitis delta antigen (HDAg), is encoded by the genome (3, 38), and two isoforms of this protein are produced during infection. The canonical small form of HDAg (SHDAg) is 195 amino acids (aa) long; it harbors an N-terminal coiled-coil domain responsible for oligomerization (37), a central domain responsible for binding to the RNA genome (7, 23), a nuclear localization signal (2, 7), and a C-terminal glycine- and proline-rich region with an uncertain function. This form of HDAg is essential for viral RNA replication, although it is not itself a polymerase. Host RNA polymerase II is thought to supply the polymerase function for replication (15, 26). During viral replication, an RNA editing event occurs at the UAG termination codon of SHDAg, allowing readthrough of another 19 aa (Fig. ​(Fig.1)1) to generate the large isoform of the protein, LHDAg (25). Since LHDAg contains all of the domains of SHDAg, it too can form multimers with itself and with the SHDAg isoform, bind HDV RNA (as a homo- or heteromultimer), and be localized to the nucleus. Open in a separate windowFIG. 1Sequence of the 19 aa unique to the C terminus of LHDAg. The PXXP motifs are underlined. Below are shown the amino acid changes present in the mutants employed in this study. The positions of the termination codons introduced into the truncation mutants are indicated by asterisks.Despite these similarities, the two HDAgs have very distinct functions (22) and play complementary roles in HDV replication, which takes place largely in the nuclei of infected cells (34). While SHDAg activates HDV RNA replication, LHDAg is a trans-dominant inhibitor of this process (8). By contrast, LHDAg, but not SHDAg, is capable of interacting with the HBV envelope proteins to mediate envelopment of the HDV ribonucleoprotein in viral assembly (6). This interaction has been shown to require farnesylation of a cysteine residue found in the C-terminal 19 aa unique to LHDAg (27, 16). Furthermore, it has been shown recently that only LHDAg is phosphorylated in cells (1).In this report, we describe yet another activity of LHDAg that further differentiates it from the related isoform SHDAg, i.e., the ability to activate gene expression in trans.     

Multimerization of Hepatitis Delta Antigen Is a Critical Determinant of RNA Binding Specificity

Hepatitis delta virus (HDV) RNA forms an unbranched rod structure that is associated with hepatitis delta antigen (HDAg) in cells replicating HDV. Previous in vitro binding experiments using bacterially expressed HDAg showed that the formation of a minimal ribonucleoprotein complex requires an HDV unbranched rod RNA of at least about 300 nucleotides (nt) and suggested that HDAg binds the RNA as a multimer of fixed size. The present study specifically examines the role of HDAg multimerization in the formation of the HDV ribonucleoprotein complex (RNP). Disruption of HDAg multimerization by site-directed mutagenesis was found to profoundly alter the nature of RNP formation. Mutant HDAg proteins defective for multimerization exhibited neither the 300-nt RNA size requirement for binding nor specificity for the unbranched rod structure. The results unambiguously demonstrate that HDAg binds HDV RNA as a multimer and that the HDAg multimer is formed prior to binding the RNA. RNP formation was found to be temperature dependent, which is consistent with conformational changes occurring on binding. Finally, analysis of RNPs constructed with unbranched rod RNAs successively longer than the minimum length indicated that multimeric binding is not limited to the first HDAg bound and that a minimum RNA length of between 604 and 714 nt is required for binding of a second multimer. The results confirm the previous proposal that HDAg binds as a large multimer and demonstrate that the multimer is a critical determinant of the structure of the HDV RNP.Human hepatitis delta virus (HDV) is an unusual subviral agent that increases the severity of acute and chronic liver disease in those infected with its helper, hepatitis B virus (23). The HDV genome is a 1,680-nucleotide (nt) single-stranded circular RNA that is replicated by a double-rolling-circle mechanism (reviewed in references 15 and 28). Both the genome and antigenome RNAs form a characteristic unbranched rod structure due to 70% sequence complementarity between the noncoding and coding regions of the RNA (10, 11, 31). HDV encodes just one protein, hepatitis delta antigen (HDAg), which forms ribonucleoprotein (RNP) complexes with both the genome and the antigenome in cells replicating HDV (3, 5, 30). These complexes play fundamental roles in viral RNA replication and packaging and their characterization is essential for understanding these processes, which are not well characterized.HDAg has been shown to form dimers and higher order multimers, even in the absence of HDV RNA (25, 30, 32). The multimerization activity has been localized to the amino-terminal third of the 195-amino-acid (aa) protein (12, 24, 30, 32). X-ray crystallographic analysis of a peptide comprised of aa 12 to 60 indicated that antiparallel dimers are stabilized by a coiled coil (aa 16 to 48), as well as a hydrophobic core region (aa 50 to 60) that also stabilizes interactions between dimers such that an octameric structure may form (35). Zuccola et al. found that bacterially expressed HDAg could be cross-linked in an octameric structure, and Cornillez-Ty et al. obtained evidence supporting such a structure in cells replicating HDV (7, 35). Site-directed mutations of HDAg amino acids critical for dimerization and/or multimerization abolish the ability of HDAg to support RNA replication (18, 32), indicating that the formation of HDAg multimers is essential for this process.We recently showed that bacterially expressed, C-terminally truncated HDAg forms stable RNP complexes in vitro with segments of HDV RNA that form unbranched rod structures (8). No particular sequences or structures in the RNA, other than the HDV unbranched rod, were essential for complex formation, but, remarkably, binding required that the RNA have a minimum length of at least about 300 nt. Overall, the results were consistent with the formation of a large RNP containing multiple copies of the 19-kDa protein that bound to the RNA either in a highly cooperative manner or as a preformed multimer. On the other hand, based on indirect measures of the RNA-binding activity of site-directed HDAg mutations in cells, others have found that HDAg multimerization might not be required for RNA-binding activity (18).Here, we directly analyze the role of HDAg multimerization in the formation of the HDV RNP complex. We find that HDAg binds to HDV unbranched rod RNA as a preformed multimer. Site-directed mutations that disrupted protein multimerization did not abolish binding but profoundly altered the nature of the RNA-protein complex. In particular, we found that multimerization is associated with RNA-binding specificity, including the RNA length requirement for binding. For the wild-type protein, RNP formation was found to be strongly temperature dependent, suggesting that conformational changes occur on binding, and providing a plausible explanation of the RNA length requirement for binding. Furthermore, we show that the protein binds as multiple multimeric units on longer RNAs, provided the length of the RNA is sufficient. We conclude that the HDAg multimer plays a critical role in the formation of properly structured HDV RNPs.     

The Isomerase Active Site of Cyclophilin A Is Critical for Hepatitis C Virus Replication

Cyclosporine A and nonimmunosuppressive cyclophilin (Cyp) inhibitors such as Debio 025, NIM811, and SCY-635 block hepatitis C virus (HCV) replication in vitro. This effect was recently confirmed in HCV-infected patients where Debio 025 treatment dramatically decreased HCV viral load, suggesting that Cyps inhibitors represent a novel class of anti-HCV agents. However, it remains unclear how these compounds control HCV replication. Recent studies suggest that Cyps are important for HCV replication. However, a profound disagreement currently exists as to the respective roles of Cyp members in HCV replication. In this study, we analyzed the respective contribution of Cyp members to HCV replication by specifically knocking down their expression by both transient and stable small RNA interference. Only the CypA knockdown drastically decreased HCV replication. The re-expression of an exogenous CypA escape protein, which contains escape mutations at the small RNA interference recognition site, restored HCV replication, demonstrating the specificity for the CypA requirement. We then mutated residues that reside in the hydrophobic pocket of CypA where proline-containing peptide substrates and cyclosporine A bind and that are vital for the enzymatic or the hydrophobic pocket binding activity of CypA. Remarkably, these CypA mutants fail to restore HCV replication, suggesting for the first time that HCV exploits either the isomerase or the chaperone activity of CypA to replicate in hepatocytes and that CypA is the principal mediator of the Cyp inhibitor anti-HCV activity. Moreover, we demonstrated that the HCV NS5B polymerase associates with CypA via its enzymatic pocket. The study of the roles of Cyps in HCV replication should lead to the identification of new targets for the development of alternate anti-HCV therapies.Hepatitis C virus (HCV)² is the main contributing agent of acute and chronic liver diseases worldwide (1). Primary infection is often asymptomatic or associated with mild symptoms. However, persistently infected individuals develop high risks for chronic liver diseases such as hepatocellular carcinoma and liver cirrhosis (1). The combination of IFNα and ribavirin that serves as current therapy for chronically HCV-infected patients not only has a low success rate (about 50%) (2) but is often associated with serious side effects (2). There is thus an urgent need for the development of novel anti-HCV treatments (2).The immunosuppressive drug cyclosporine A (CsA) was reported to be clinically effective against HCV (3). Controlled trials showed that a combination of CsA with IFNα is more effective than IFNα alone, especially in patients with a high viral load (4, 5). Moreover, recent in vitro studies provided evidence that CsA prevents both HCV RNA replication and HCV protein production in an IFNα-independent manner (6–10). CsA exerts this anti-HCV activity independently of its immunosuppressive activity because the nonimmunosuppressive Cyp inhibitors such as Debio 025, NIM811, and SCY-635 also block HCV RNA and protein production (9, 11–14). Unlike CsA, these molecules do not display calcineurin affinity and specifically inhibit the peptidyl-prolyl cis-trans-isomerase (PPIase) Cyps. Most importantly, recent clinical data demonstrated that Debio 025 dramatically decreased HCV viral load (3.6 log decrease) in patients coinfected with HCV and HIV (15). This 14-day Debio 025 treatment (1200 mg orally administered twice daily) was effective against the three genotypes (genotypes 1, 3, and 4) represented in the study. More recently, the anti HCV effect of Debio 025 in combination with peginterferon α 2a (peg-IFNα2a) was investigated in treatment-inexperienced patients with chronic hepatitis C. Debio 025 (600 mg administered once daily) in combination with peg-IFNα2a (180 μg/week) for 4 weeks induced a continuous decay in viral load that reached −4.61 ± 1.88 IU/ml in patients with genotypes 1 and 4 and −5.91 ± 1.11 IU/ml in patients with genotypes 2 and 3 at week 4 (16). The Debio 025 findings are critical because they suggest that Cyp inhibitors represent a novel class of anti-HCV agents. However, it remains unclear how these compounds control HCV replication. The fact that several recent studies using small RNA interference knockdown approaches suggest that Cyps are critical for the HCV life cycle (9, 17, 18) strongly implies that there is a direct or indirect link between the CsA- and CsA derivative-mediated inhibitory effect on HCV replication and host Cyps.The discovery 20 years ago of the first cellular protein showing PPIase activity (19) was entirely unrelated to the discovery of CypA as an intracellular protein possessing a high affinity for CsA (20). It is only a few years later that Fischer et al. (21) demonstrated that the 18-kDa protein with PPIase activity and CypA represent a single unique protein. All Cyps contain a common domain of 109 amino acids, called the Cyp-like domain, which is surrounded by domains specific to each Cyp members and which dictates their cellular compartmentalization and function (22). Bacteria, fungi, insects, plants, and mammals contain Cyps, which all have PPIase activity and are structurally conserved (22). To date, 16 Cyp members have been identified, and 7 of them are found in humans: CypA, CypB, CypC, CypD, CypE, Cyp40, and CypNK (22).Although there is a growing body of evidence that Cyps control HCV replication in human hepatocytes, a major disagreement currently exists on the respective roles of Cyp members in HCV replication. One study suggests that CypB, but not CypA, is critical for HCV replication (17), another suggests that CypA, but not CypB and CypC, is critical for HCV replication (18), and a third study suggests that three Cyps, CypA, B, and C, are all required for HCV replication (9). Thus, although it becomes evident that Cyps serve as HCV co-factors, their respective contributions and roles in the HCV life cycle remain to be determined. An understanding of the mechanisms that control the Cyp inhibitor-mediated anti-HCV effect is imperative because it will provide new alternate anti-HCV therapies and shed light on the still poorly understood early and late steps of the HCV life cycle.     

Heterogeneous Ribonucleoprotein K (hnRNP K) Binds miR-122, a Mature Liver-Specific MicroRNA Required for Hepatitis C Virus Replication

Heterogeneous ribonucleoprotein K (hnRNP K) binds to the 5′ untranslated region of the hepatitis C virus (HCV) and is required for HCV RNA replication. The hnRNP K binding site on HCV RNA overlaps with the sequence recognized by the liver-specific microRNA, miR-122. A proteome chip containing ∼17,000 unique human proteins probed with miR-122 identified hnRNP K as one of the strong binding proteins. In vitro kinetic study showed hnRNP K binds miR-122 with a nanomolar dissociation constant, in which the short pyrimidine-rich residues in the central and 3′ portion of the miR-122 were required for hnRNP K binding. In liver hepatocytes, miR-122 formed a coprecipitable complex with hnRNP K. High throughput Illumina DNA sequencing of the RNAs precipitated with hnRNP K was enriched for mature miR-122. SiRNA knockdown of hnRNP K in human hepatocytes reduced the levels of miR-122. These results show that hnRNP K is a cellular protein that binds and affects the accumulation of miR-122. Its ability to also bind HCV RNA near the miR-122 binding site suggests a role for miR-122 recognition of HCV RNA.MicroRNAs (miRNAs) are a class of noncoding RNA of ∼22-nucleotides in length that can regulate gene expression by either targeting RNA for degradation or suppressing their translation through base pairing to the RNAs (1). Since their discovery in 1993 in Caenorhabditis elegans, miRNAs have been found in many species and are involved in the regulation of proliferation, differentiation, apoptosis, and development (1, 2). Moreover, miRNAs are also critical factors in the development of cancers, neurodegenerative diseases, and infectious diseases (3).MiR-122 is a highly abundant RNA in hepatocytes that regulates lipid metabolism, regeneration, and neoplastic transformation (4–6). In addition, miR-122 is required for the replication of the hepatitis C virus (HCV), a positive-strand RNA virus that infects over 170 million people worldwide (7–9). MiR-122 binds to a conserved sequence in the 5′ untranslated region (UTR) of the HCV RNA to increase the stability of the HCV RNA (10). Silencing of miR-122 can abolish HCV RNA accumulation in non-human primates (11). The expression of human miR-122 in non-hepatic cells can confer the ability to replicate HCV RNA (12). MiR-122 is one of the most critical host factors for HCV replication.We previously reported that the HCV RNA sequence that anneals to miR-122 is recognized by the heterogeneous ribonucleoprotein K (hnRNP K), a multifunctional RNA-binding protein known to be involved in RNA processing, translation, and the replication of several RNA viruses (13–15). In an unbiased screen for proteins from human proteome chips containing over 17,000 proteins, we identified 40 proteins that bind mature miR-122, including hnRNP K. Recombinant hnRNP K recognizes short pyrimidine sequences in miR-122 in vitro and a similar sequence in the HCV 5′ UTR. In hepatocytes endogenous hnRNP K can form a coprecipitable complex with miR-122, whether or not the cells contain replicating HCV. HnRNP K is thus a protein that binds a mature microRNA.