In this study, we identified an activity of the hepatitis delta antigen that both modulates the cis-cleaving activities of hepatitis delta virus (HDV) genomic RNA fragments and facilitates the trans-cleavage reactions between hammerhead ribozymes and the cognate substrates of various lengths in vitro. Hepatitis delta antigen peptides exert their effect by accelerating the unfolding and refolding of RNA molecules and by promoting strand annealing and strand dissociation. In addition, the stimulatory effect of hepatitis delta antigen peptide on hammerhead catalysis is observed whether the peptide is removed or not by phenol/chloroform extraction prior to the initiation of trans-cleavage reaction. Therefore, hepatitis delta antigen peptide acts as an RNA chaperone. The RNA chaperone domain of hepatitis delta antigen overlaps with the coiled-coil domain that is rich in lysine residues. The RNA binding domains of hepatitis delta antigen previously identified are not required for the RNA chaperone activity identified herein. The RNA chaperone activity of hepatitis delta antigen may be important for the regulation of HDV replication in vivo.Hepatitis delta virus (HDV) 1 is a subviral pathogen that requires hepatitis B virus (HBV) to supply envelope protein for completion of package, secretion, and infection (1-3). The genome of HDV is a single-stranded circular RNA of ϳ1700 nt and HDV RNA and is replicated through a rolling circle mechanism (4). HDV codes one protein of two forms during infection: the small delta antigen (SdAg) contains 195 aa and the large delta antigen (LdAg) has an extra 19 aa at the C terminus (5). Transfection studies with HDV cDNA elucidated that the two protein forms have distinct functions. SdAg initiates genome replication (6) and LdAg promotes package (7). There are two RNA binding domains in each protein form. The first is the arginine-rich sequence near the N terminus, and the second is the arginine-rich motifs (ARMs) located at the middle one-third of the protein (8, 9). The RNA binding activity is important for the function of the two protein forms: the second RNA binding domain of SdAg is required to initiate genome replication (9 -12), and the first RNA binding domain of LdAg is responsible for potent inhibition of replication (13). The specific interactions between the hepatitis delta antigen and HDV RNA appear to be involved in the regulation of virus replication although a molecular mechanism has not yet been elucidated.HDV RNAs of genomic and antigenomic senses cis-cleaved in the absence of protein factors in vitro (14). The ribozyme activity of HDV RNA, which requires a pseudoknot-like structure of the RNA molecule (15, 16) and the catalysis of divalent cations (17), is essential for generating monomeric size RNA molecules during replication (18). Recently, Jeng et al. (19) illustrated that hepatitis delta antigen may enhance, though is not required for, the processing of multiple length HDV RNA in vivo. Conceivably, hepatitis delta antigen per se or together with some other facto...

The virion of dengue virus (DENV) is composed of a viral envelope covering a nucleocapsid formed by a complex of viral genomic RNA and core protein (CP). DENV CP forms a dimer via the internal ␣2 and ␣4 helices of each monomer. Pairing of ␣2-␣2= creates a continuous hydrophobic surface, while the ␣4-␣4= helix pair joins the homodimer via side-chain interactions of the inner-edge residues. However, the importance of dimer conformation and the ␣4 helix of DENV CP in relation to its function are poorly understood. Loss of association between CP and lipid droplets (LDs) due to mutation suggests that the CP hydrophobic surface was not exposed, offering a possible explanation for the absence of dimers. Further assays suggest the connection between CP folding and protein stability. Attenuation of full-length RNA-derived virus production is associated with CP mutation, since no significant defects were detected in virus translation and replication. The in vitro characterization assays further highlighted that the ␣4-␣4= helix pair conformation is critical in preserving the overall ␣-helical content, thermostability, and dimer formation ability of CP, features correlated with the efficiency of nucleocapsid formation. Addition of Tween 20 improves in vitro nucleocapsid-like particle formation, suggesting the role of the LD in nucleocapsid formation in vivo. This study provides the first direct link between the ␣4-␣4= helix pair interaction and the CP dimer conformation that is the basis of CP function, particularly in nucleocapsid formation during virion production. IMPORTANCEStructure-based mutagenesis study of the dengue virus core protein (CP) reveals that the ␣4-␣4= helix pair is the key to maintaining its dimer conformation, which is the basis of CP function in nucleocapsid formation and virus production. Attenuation of full-length RNA-derived virus production is associated with CP mutation, since no significant defects in virus translation and replication were detected. In vitro inefficiency and size of nucleocapsid-like particle (NLP) formation offer a possible explanation for in vivo virus production inefficiency upon CP mutation. Further, the transition of NLP morphology from an incomplete state to an intact particle shown by ␣4-␣4= helix pair mutants in the presence of a nonionic detergent suggests the regulatory role of the intracellular lipid droplet (LD) in CP-LD interaction and in promoting nucleocapsid formation. This study provides the first direct link between the ␣4-␣4= helix pair interaction and CP dimer conformation that is the fundamental requirement of CP function, particularly in nucleocapsid formation during virion production. V iruses of the Flaviviridae family are enveloped and have a monopartite, linear, and positive-polarity single-stranded RNA genome. A member of this family in the Flavivirus genus, Dengue virus (DENV), is the most important human arthropodborne viral pathogen, putting 3 billion people at risk and resulting in 50 million to 100 million infections and around 22,000 deat...

To determine the sequence requirements and structural features of the self-cleavage domain of hepatitis delta virus (HDV) antigenomic RNA, we constructed a series of mutants and measured the rate constant of the cleavage reaction for each. The self-cleavage activity of HDV RNA of antigenomic sense was found to reside in a region of less than 90 nucleotides in length. The catalytic domain contained a long complementary sequence which could be deleted to half of its original size. Moreover, this region could be replaced by other sequences as long as they could fold into a stem-and-loop structure. The catalytic domain also required a 6-basepair helix adjacent to the cleaving point for activity. The structural features of these two base-pairing regions are quite similar to those of the HDV genomic self-cleavage domain. The cleavage site as well as the the hinge region (the sequence between the two stems) requires specific sequences for activity.

bDengue virus (DENV) causes disease globally, resulting in an estimated 25 to 100 million new infections per year. No effective DENV vaccine is available, and the current treatment is only supportive. Thus, there is an urgent need to develop therapeutic agents to cure this epidemic disease. In the present study, we identified a potential small-molecule inhibitor, BP13944, via highthroughput screening (HTS) of 60,000 compounds using a stable cell line harboring an efficient luciferase replicon of DENV serotype 2 (DENV-2). BP13944 reduced the expression of the DENV replicon reporter in cells, showing a 50% effective concentration (EC 50 ) of 1.03 ؎ 0.09 M. Without detectable cytotoxicity, the compound inhibited replication or viral RNA synthesis in all four serotypes of DENV but not in Japanese encephalitis virus (JEV). Sequencing analyses of several individual clones derived from BP13944-resistant RNAs purified from cells harboring the DENV-2 replicon revealed a consensus amino acid substitution (E66G) in the region of the NS3 protease domain. Introduction of E66G into the DENV replicon, an infectious DENV cDNA clone, and recombinant NS2B/NS3 protease constructs conferred 15.2-, 17.2-, and 3.1-fold resistance to BP13944, respectively. Our results identify an effective small-molecule inhibitor, BP13944, which likely targets the DENV NS3 protease. BP13944 could be considered part of a more effective treatment regime for inhibiting DENV in the future. D engue virus (DENV) (serotypes 1 to 4) belongs to the familyFlaviviridae, a group of enveloped RNA viruses that includes the genera Hepacivirus, Flavivirus, and Pestivirus. The genus Flavivirus consists of arthropod-borne disease agents such as yellow fever virus (YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV), tick-borne encephalitis virus (TBEV), and DENV (1). More than 70 members of the Flavivirus genus are important human pathogens that cause significant morbidity and mortality (2). DENV is a public health threat to an estimated 2.5 billion people living in areas where dengue is epidemic, leading to 50 to 100 million human infections each year (3, 4). DENV infection frequently leads to dengue fever, life-threatening dengue hemorrhagic fever (DHF), or dengue shock syndrome (DSS) (5-7). Approximately 500,000 cases of DHF and DSS have been reported among more than 100 countries, causing approximately 12,500 deaths per year (3). Despite the tremendous efforts invested in anti-DENV research, no clinically approved vaccine or antiviral therapeutic agents are available for humans, and disease treatment is limited to supportive care (4,8,9). Considering the spread of this epidemic and the severity of DENV, an effective anti-DENV drug is urgently needed.DENV is an enveloped RNA virus consisting of a 10.7-kb single-stranded, positive-polarity RNA genome associated with multiple copies of capsid proteins. DENV RNA is translated as a single polyprotein upon entering the host cell and is cleaved by host proteases and the virus-encoded two-component protease (NS...

Edited by Michael IbbaKeywords: DENV Core protein NS5 NS3 helicase RNA chaperone Strand annealing RNA binding protein a b s t r a c tIn this study we showed that the dengue virus (DENV) core protein forms a dimer with an a-helixrich structure, binds RNA and facilitates the strand annealing process. To assess the RNA chaperone activity of this core protein and other dengue viral RNA-interacting proteins, such as NS3 helicase and NS5 proteins, we engineered cis-and trans-cleavage hammerhead ribozyme constructs carrying DENV genomic RNA elements. Our results indicate that DENV core protein facilitates typical hammerhead structure formation by acting as an RNA chaperone and DENV NS5 has a weak RNA chaperone activity, while DENV NS3 helicase failed to refold RNA with a complex secondary structure.

We have previously shown that the N-terminal domain of hepatitis delta virus (NdAg) has an RNA chaperone activity in vitro (Huang, Z. S., and Wu, H. N. (1998) J. Biol. Chem. 273, 26455-26461). Here we investigate further the basis of the stimulatory effect of NdAg on RNA structural rearrangement: mainly the formation and breakage of base pairs. Duplex dissociation, strand annealing, and exchange of complementary RNA oligonucleotides; the hybridization of yeast U4 and U6 small nuclear RNAs and of hammerhead ribozymes and cognate substrates; and the cis-cleavage reaction of hepatitis delta ribozymes were used to determine directly the role of NdAg in RNA-mediated processes. The results showed that NdAg could accelerate the annealing of complementary sequences in a selective fashion and promote strand exchange for the formation of a more extended duplex. These activities would prohibit NdAg from modifying the structure of a stable RNA, but allow NdAg to facilitate a trans-acting hammerhead ribozyme to find a more extensively matched target in cognate substrate. These and other results suggest that hepatitis delta antigen may have a biological role as an RNA chaperone, modulating the folding of viral RNA for replication and transcription. Hepatitis delta virus (HDV)1 is a satellite virus of hepatitis B virus. The genome of HDV comprises single-stranded circular RNA of ϳ1700 nucleotides, and HDV RNA replicates through a symmetrical rolling circle mechanism (1). Hepatitis delta antigen is the only protein coded by HDV that is critical for viral replication (2) and virion assembly (3), although the molecular mechanisms have not yet been elucidated. HDV RNA, of both genomic and antigenomic senses, contains a ribozyme domain that can adopt a pseudoknot-like structure and undergo ciscleavage in vitro (4 -7). The cis-cleaving activity is essential for generating monomeric size RNA molecules during viral replication (8, 9). Hepatitis delta antigen may enhance, although it is not required for, the processing of multimeric size HDV RNA in vivo (10). Conceivably, hepatitis delta antigen by itself, or together with unidentified cellular factors, acts as an RNA chaperone that modulates the ribozyme activity of HDV RNA.RNA chaperones are defined as proteins that aid in the folding of RNA by preventing misfolding or by resolving misfolded structures (11). The RNA chaperone activities in vitro of several proteins that interact with RNA with broad specificity have been explored through their effect on ribozyme reactions. These proteins, including several Escherichia coli ribosomal proteins (12), the C-terminal domain of heterogeneous nuclear ribonucleoprotein A1 protein (13), and the nucleocapsid protein of human immunodeficiency virus (14, 15), can overcome the general limitations of ribozyme reactions and facilitate ribozyme catalysis. Proteins with RNA chaperone activity are thought to lower the activation energy necessary for breaking and reforming of base pairs, although the molecular mechanism underlying RNA chaperone activity...

We used synthetic DNA oligos to investigate the nucleic acid chaperone properties of the N terminal domain of hepatitis delta antigen (NdAg). We found that NdAg possessed a bona fide chaperone activity. NdAg could distinguish subtle differences in the thermal stability of the base pairing region, and enabled DNA oligos to form a more stable duplex among competing sequences through facilitating strand annealing selectively, stimulating duplex conversion selectively, and stabilizing the more stable duplex. The property of NdAg identified in this study could be applied to improve the efficiency and specificity of dot blot hybridization under conditions of low stringency.

Edited by Hans-Dieter KlenkKeywords: HCV NS3 helicase RNA structure conversion Strand annealing dsRNA unwinding RNA chaperone DExH/D-box protein a b s t r a c t NS3H, the helicase domain of HCV NS3, possesses RNA-stimulated ATPase and ATP hydrolysis-dependent dsRNA unwinding activities. Here, the ability of NS3H to facilitate RNA structural rearrangement is studied using relatively long RNA strands as the model substrates. NS3H promotes intermolecular annealing, resolves three-stranded RNA duplexes, and assists dsRNA and ssRNA inter-conversions to establish a steady state among RNA structures. NS3H facilitates RNA structure conversions in a mode distinct from an ATP-independent RNA chaperone. These findings expand the known function of HCV NS3 helicase and reveal a role for viral helicase in assisting RNA structure conversions during virus life cycle.