Interplay of RNA Elements in the Dengue Virus 5′ and 3′ Ends Required for Viral RNA Replication

Dengue virus (DENV) is a member of the Flavivirus genus of positive-sense RNA viruses. DENV RNA replication requires cyclization of the viral genome mediated by two pairs of complementary sequences in the 5′ and 3′ ends, designated 5′ and 3′ cyclization sequences (5′-3′ CS) and the 5′ and 3′ upstream of AUG region (5′-3′ UAR). Here, we demonstrate that another stretch of six nucleotides in the 5′ end is involved in DENV replication and possibly genome cyclization. This new sequence is located downstream of the AUG, designated the 5′ downstream AUG region (5′ DAR); the motif predicted to be complementary in the 3′ end is termed the 3′ DAR. In addition to the UAR, CS and DAR motifs, two other RNA elements are located at the 5′ end of the viral RNA: the 5′ stem-loop A (5′ SLA) interacts with the viral RNA-dependent RNA polymerase and promotes RNA synthesis, and a stem-loop in the coding region named cHP is involved in translation start site selection as well as RNA replication. We analyzed the interplay of these 5′ RNA elements in relation to RNA replication, and our data indicate that two separate functional units are formed; one consists of the SLA, and the other includes the UAR, DAR, cHP, and CS elements. The SLA must be located at the 5′ end of the genome, whereas the position of the second unit is more flexible. We also show that the UAR, DAR, cHP, and CS must act in concert and therefore likely function together to form the tertiary RNA structure of the circularized DENV genome.Dengue virus (DENV), a member of the Flaviviridae family, is a human pathogen causing dengue fever, the most common mosquito-borne viral disease in humans. The virus has become a major international public health concern, with 3 billion people at risk for infection and an estimated 50 million dengue cases worldwide every year (28). Neither specific antiviral therapies nor licensed vaccines are available, and the biology of the virus is poorly understood.DENV is a small enveloped virus containing a positive-stranded RNA genome with a length of approximately 10.7 kb. The virus encodes one large polyprotein that is co- and posttranslationally cleaved into 10 viral proteins. The structural proteins C, prM/M, and E are located in the N terminus, followed by the nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (6, 10). NS5, the largest of the viral proteins, functions as an RNA-dependent RNA polymerase (RdRP) (29). The coding region is flanked at both ends by untranslated regions (UTR). The 5′ end has a type I cap structure (m⁷GpppAmp) mediating cap-dependent translation, but the virus can switch to a noncanonical translation mechanism under conditions in which translation factors are limiting (13). Cellular mRNAs are known to circularize via a protein-protein bridge between eIF4G and eIF4E (the cap binding complex) at the 5′ end and the poly(A) binding protein (PABP) at the 3′ end, enhancing translation efficiency. Despite the fact that the DENV 3′ UTR lacks a poly(A) tail, recent findings demonstrated binding of PABP to the 3′ UTR and an effect on RNA translation, suggesting a similar mechanism (12, 26).In addition to a presumed protein-mediated genome circularization regulating viral translation, an RNA-RNA-based 5′ and 3′ (5′-3′) end interaction, which can occur in the absence of proteins, leads to circularization of the viral genome (1, 3, 4, 18, 20, 30, 33, 34). This cyclization of the genome is necessary for viral RNA replication, and thus far, two complementary sequences at the 5′ and 3′ ends have been identified (3). The first are the cyclization sequences (CS) present in the capsid-coding region at the 5′ end (5′ CS) and upstream of the 3′ stem-loop (3′ SL) in the 3′ UTR (3′ CS) (2, 4, 18, 20, 30). A second sequence, known as the 5′ upstream AUG region (5′ UAR) element in the 5′ UTR, base pairs with its complementary 3′ UAR counterpart, which is located at the bottom part of 3′ SL (1, 4, 30). Recently, the structure of the 5′ end of the DENV genome hybridized to the 3′ end was determined in solution (25), confirming previous computer-predicted structures for genome cyclization (4, 20, 30). Besides the base pairing between 5′-3′ UAR and 5′-3′ CS sequences, a third stretch of nucleotides was identified to form a double-stranded (ds) region between the 5′ and 3′ ends.In addition to RNA sequences involved in 5′-3′-end interactions that are necessary for cyclization, the 5′ end of the viral genome harbors at least two more functional RNA elements, the stem-loop A (SLA) and capsid-coding region hairpin (cHP). The SLA consists of the first 70 nucleotides (nt) of the genome, forming a stable stem-loop structure. This structure has been confirmed in several studies and identified as a promoter element for RNA synthesis that recruits the viral RdRp NS5 (16, 22). Once NS5 is bound to the SLA at the 5′ end, it is believed to be delivered to the initiation site of minus-strand RNA synthesis at the 3′ end via 5′-3′ RNA-RNA circularization. In addition, a short poly(U) tract located immediately downstream of SLA has been shown to be necessary for RNA synthesis, although it is not involved in genome circularization (22). Finally, the cHP element resides within the capsid-coding region; it directs start codon selection and is essential for RNA replication (8, 9). The cHP structure is more important than its primary sequence. For start codon selection, it is believed that the cHP stalls the scanning initiation complex over the first AUG, favoring its recognition (9). In the case of RNA replication, the cHP likely stabilizes the overall 5′-3′ panhandle structure or participates in recruitment of factors associated with the replicase machinery (8).In this study, we demonstrate that in addition to the 5′ CS and 5′ UAR sequences, a third stretch of nucleotides in the 5′ end is required for RNA replication and appears to be involved in genome circularization. This new motif is located downstream of the AUG and was therefore designated the downstream AUG region (5′ DAR) element, with the predicted counterpart in the 3′ end designated the 3′ DAR. Our results indicate that the 5′ DAR modulates RNA-RNA interaction and RNA replication, and restoring complementarity between the 5′ DAR and 3′ DAR rescues detrimental effects caused by mutations in the 5′ DAR on genome circularization and RNA replication. Although the role of the predicted 3′ DAR counterpart is less conclusive, it may serve to make other structures and sequences in the 3′ end available for 5′-3′ RNA-RNA interaction to facilitate the replication-competent conformation of the DENV genome.Furthermore, we analyzed the functional interplay of RNA elements in the viral 5′ end, showing that two separate units are formed during replication. The first consists of the SLA, and it must be located at the very 5′ end of the genome. The second unit includes UAR, DAR, cHP, and CS elements, and the positional requirements are more flexible within the DENV RNA 5′ terminus. However, all four elements in the second unit must act in concert, forming a functional tertiary RNA structure of the circularized viral genome.     

5′-O-Fluorosulfonylbenzoyl Esters of Purine Nucleosides as Potential Inhibitors of NTPase/Helicase and Polymerase of Flaviviridae Viruses

Abstract

Synthesis and interactions of guanosine, inosine and ribavirin 5′-fluorosulfonyl-benzoyl esters with hepatitis C virus (HCV) and Flaviviruses NTPase/helicase and polymerase are described.     

Dimerization of Hepatitis E Virus Capsid Protein E2s Domain Is Essential for Virus–Host Interaction

Hepatitis E virus (HEV), a non-enveloped, positive-stranded RNA virus, is transmitted in a faecal-oral manner, and causes acute liver diseases in humans. The HEV capsid is made up of capsomeres consisting of homodimers of a single structural capsid protein forming the virus shell. These dimers are believed to protrude from the viral surface and to interact with host cells to initiate infection. To date, no structural information is available for any of the HEV proteins. Here, we report for the first time the crystal structure of the HEV capsid protein domain E2s, a protruding domain, together with functional studies to illustrate that this domain forms a tight homodimer and that this dimerization is essential for HEV–host interactions. In addition, we also show that the neutralizing antibody recognition site of HEV is located on the E2s domain. Our study will aid in the development of vaccines and, subsequently, specific inhibitors for HEV.     

Crystal Structure of the Dengue Virus Methyltransferase Bound to a 5′-Capped Octameric RNA

The N-terminal domain of the flavivirus NS5 protein functions as a methyltransferase (MTase). It sequentially methylates the N7 and 2′-O positions of the viral RNA cap structure (GpppA→^7meGpppA→^7meGpppA_2′-O-me). The same NS5 domain could also have a guanylyltransferase activity (GTP+ppA-RNA→GpppA). The mechanism by which this protein domain catalyzes these three distinct functions is currently unknown. Here we report the crystallographic structure of DENV-3 MTase in complex with a 5′-capped RNA octamer (G_pppAGAACCUG) at a resolution of 2.9 Å. Two RNA octamers arranged as kissing loops are encircled by four MTase monomers around a 2-fold non-crystallography symmetry axis. Only two of the four monomers make direct contact with the 5′ end of RNA. The RNA structure is stabilised by the formation of several intra and intermolecular base stacking and non-canonical base pairs. The structure may represent the product of guanylylation of the viral genome prior to the subsequent methylation events that require repositioning of the RNA substrate to reach to the methyl-donor sites. The crystal structure provides a structural explanation for the observed trans-complementation of MTases with different methylation defects.     

A long-range RNA–RNA interaction between the 5′ and 3′ ends of the HCV genome

The RNA genome of the hepatitis C virus (HCV) contains multiple conserved structural cis domains that direct protein synthesis, replication, and infectivity. The untranslatable regions (UTRs) play essential roles in the HCV cycle. Uncapped viral RNAs are translated via an internal ribosome entry site (IRES) located at the 5′ UTR, which acts as a scaffold for recruiting multiple protein factors. Replication of the viral genome is initiated at the 3′ UTR. Bioinformatics methods have identified other structural RNA elements thought to be involved in the HCV cycle. The 5BSL3.2 motif, which is embedded in a cruciform structure at the 3′ end of the NS5B coding sequence, contributes to the three-dimensional folding of the entire 3′ end of the genome. It is essential in the initiation of replication. This paper reports the identification of a novel, strand-specific, long-range RNA–RNA interaction between the 5′ and 3′ ends of the genome, which involves 5BSL3.2 and IRES motifs. Mutants harboring substitutions in the apical loop of domain IIId or in the internal loop of 5BSL3.2 disrupt the complex, indicating these regions are essential in initiating the kissing interaction. No complex was formed when the UTRs of the related foot and mouth disease virus were used in binding assays, suggesting this interaction is specific for HCV sequences. The present data firmly suggest the existence of a higher-order structure that may mediate a protein-independent circularization of the HCV genome. The 5′–3′ end bridge may have a role in viral translation modulation and in the switch from protein synthesis to RNA replication.     

The C-Terminal α-Helix Domain of Apolipoprotein E Is Required for Interaction with Nonstructural Protein 5A and Assembly of Hepatitis C Virus

We have recently demonstrated that human apolipoprotein E (apoE) is required for the infectivity and assembly of hepatitis C virus (HCV) (K. S. Chang, J. Jiang, Z. Cai, and G. Luo, J. Virol. 81:13783-13793, 2007; J. Jiang and G. Luo, J. Virol. 83:12680-12691, 2009). In the present study, we have determined the molecular basis underlying the importance of apoE in HCV assembly. Results derived from mammalian two-hybrid studies demonstrate a specific interaction between apoE and HCV nonstructural protein 5A (NS5A). The C-terminal third of apoE per se is sufficient for interaction with NS5A. Progressive deletion mutagenesis analysis identified that the C-terminal α-helix domain of apoE is important for NS5A binding. The N-terminal receptor-binding domain and the C-terminal 20 amino acids of apoE are dispensable for the apoE-NS5A interaction. The NS5A-binding domain of apoE was mapped to the middle of the C-terminal α-helix domain between amino acids 205 and 280. Likewise, deletion mutations disrupting the apoE-NS5A interaction resulted in blockade of HCV production. These findings demonstrate that the specific apoE-NS5A interaction is required for assembly of infectious HCV. Additionally, we have determined that using different major isoforms of apoE (E2, E3, and E4) made no significant difference in the apoE-NS5A interaction. Likewise, these three major isoforms of apoE are equally compatible with infectivity and assembly of infectious HCV, suggesting that apoE isoforms do not differentially modulate the infectivity and/or assembly of HCV in cell culture.Hepatitis C virus (HCV) remains a major global health problem, chronically infecting approximately 170 million people worldwide, with severe consequences such as hepatitis, fibrosis/cirrhosis, and hepatocellular carcinoma (HCC) (2, 57). The current standard therapy for hepatitis C is pegylated alpha interferon in combination with ribavirin. However, this anti-HCV regimen has limited efficacy (<50% sustained antiviral response for the dominant genotype 1 HCV) and causes severe side effects (17, 39). Recent clinical studies on the HCV protease- and polymerase-specific inhibitors showed promising results but also found that drug-resistant HCV mutants emerged rapidly (3, 27), undermining the efficacy of specific antiviral therapy for hepatitis C. Therefore, future antiviral therapies for hepatitis C likely require a combination of several safer and more efficacious antiviral drugs that target different steps of the HCV life cycle. The lack of knowledge about the molecular details of the HCV life cycle has significantly impeded the discovery of antiviral drugs and development of HCV vaccines.HCV is a small enveloped RNA virus classified as a member of the Hepacivirus genus in the family Flaviviridae (46, 47). It contains a single positive-sense RNA genome that encodes a large viral polypeptide, which is proteolytically processed by cellular peptidases and viral proteases into different structural and nonstructural proteins in the order of C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (30, 31). Other novel viral proteins derived from the C-coding region have also been discovered (11, 13, 55, 59). The nucleotides at both the 5′ and 3′ untranslated regions (UTR) are highly conserved and contain cis-acting RNA elements important for internal ribosome entry site (IRES)-mediated initiation of protein translation and viral RNA replication (15, 16, 33, 56, 60).The success in the development of HCV replicon replication systems has made enormous contributions to the determination of the roles of the conserved RNA sequences/structures and viral NS proteins in HCV RNA replication (4, 5, 7, 32). However, the molecular mechanisms of HCV assembly, morphogenesis, and egression have not been well understood. A breakthrough advance has been the development of robust cell culture systems for HCV infection and propagation, which allow us to determine the roles of viral and cellular proteins in the HCV infectious cycle (9, 29, 54, 63). We have recently demonstrated that infectious HCV particles are enriched in apolipoprotein E (apoE) and that apoE is required for HCV infection and assembly (10, 23). apoE-specific monoclonal antibodies efficiently neutralized HCV infectivity. The knockdown of endogenous apoE expression by a specific small interfering RNA (siRNA) and the blockade of apoE secretion by microsomal triglyceride transfer protein (MTP) inhibitors remarkably suppressed HCV assembly (10, 23). More importantly, apoE was found to interact with the HCV NS5A in the cell and purified HCV particles, as determined by yeast two-hybrid and coimmunoprecipitation (co-IP) studies (6, 23). These findings suggest that apoE has dual functions in HCV infection and assembly via distinct interactions with cell surface receptors and HCV NS5A. To further understand the molecular mechanism of apoE in HCV assembly, we carried out a mutagenesis analysis of apoE and determined the importance of the apoE-NS5A interaction in HCV assembly. Progressive deletion mutagenesis analysis has mapped the NS5A-binding domain of apoE to the C-terminal α-helix region between amino acid residues 205 and 280. Mutations disrupting the apoE-NS5A interaction also blocked HCV production. Additionally, we have determined the effects of three major isoforms of apoE on HCV infection and assembly. Our results demonstrate that apoE isoforms do not determine the infectivity and assembly of infectious HCV in cell culture.     

Nucleotide Variability and Translation Efficiency of the 5′ Untranslated Region of Hepatitis A Virus: Update from Clinical Isolates Associated with Mild and Severe Hepatitis

Mutations in the internal ribosome entry site (IRES) of hepatitis A virus (HAV) have been associated with enhanced in vitro replication and viral attenuation in animal models. To address the possible role of IRES variability in clinical presentation, IRES sequences were obtained from HAV isolates associated with benign (n = 8) or severe (n = 4) hepatitis. IRES activity was assessed using a bicistronic dual-luciferase expression system in adenocarcinoma (HeLa) and hepatoma (HuH7) cell lines. Activity was higher in HuH7 than in HeLa cells, except for an infrequently isolated genotype IIA strain. Though globally low, significant variation in IRES-dependent translation efficiency was observed between field isolates, reflecting the low but significant genetic variability of this region (94.2% ± 0.5% nucleotide identity). No mutation was exclusive of benign or severe hepatitis, and variations in IRES activity were not associated with a clinical phenotype, indirectly supporting the preponderance of host factors in determining the clinical presentation.Hepatitis A virus (HAV) is a nonenveloped RNA virus of the Picornaviridae family. The viral genome consists of an approximately 7,500-nucleotide (nt)-long, positive-stranded RNA divided in three parts: a 5′ untranslated region (5′ UTR), a single open reading frame that encodes both structural and nonstructural proteins, and a 3′ UTR with a short poly(A) tail. By sequencing of the VP1-2A junction and the VP1 gene, 3 genotypes (I, II, and III) divided into A and B subtypes have been described in humans (7, 27). HAV is the main cause of acute viral hepatitis worldwide. The majority of cases follow a benign course, but some may be present with fulminant forms, characterized by acute liver failure (factor V levels of <50% and encephalopathy). HAV-induced liver disease appears to result primarily from immunologic mechanisms, chiefly on the basis of in vitro studies. Most HAV strains have no detectable cytopathic effect in cell culture and no apparent effect on cell growth or metabolism (16), and HAV-infected cells are lysed by cytotoxic T cells isolated from the liver of acutely infected patients (30, 31). Clinical studies have suggested that host factors such as age and underlying liver disease were involved in the severity of liver diseases (32, 33) and that the host immune response also played a role in the fulminant forms of hepatitis A, as evidenced by markedly low viral loads (26).Nevertheless, the existence of viral determinants of hepatitis A severity is suggested by both experimental and clinical studies. Indeed, mutations within the VP1-2A and 2C genes have been shown to enhance virulence in tamarins (9). It has also been suggested that 5′ UTR mutations associated with viral adaptation to cell culture were also responsible for viral attenuation in vivo (15). The 5′ UTR of HAV is about 735 nucleotides long and is considered the most conserved region of the genome. The 5′ UTR is involved in genome replication and translation initiation. Folding predictions and biochemical probing showed that this region forms a highly ordered secondary structure containing a pyrimidine-rich tract (PRT) and an internal ribosomal entry site (IRES) with 10 to 12 AUG triplets upstream of the initiator codon (18). The IRES allows the initiation of the cap-independent translation of the viral genome. Most knowledge of HAV IRES activity is derived from studies of the HM-175 reference strain and its cell culture-adapted variants (4, 5, 36). These experiments have shown that HAV presents the lowest IRES-dependent translation initiation activity among picornaviruses both in reticulocyte lysates and in a variety of cell lines, including the human hepatoma cell line HepG2 (type III IRES) (3, 6). These features have been attributed to a lower affinity of the HAV 5′ UTR for translation factors (6). The hypothesis that the slow growth of HAV in cell culture could be related to this inefficient translation is supported by the emergence of 5′ UTR mutations in cell culture-adapted variants with enhanced viral replication (8). The finding that these mutations were associated with viral attenuation in vivo supports the hypothesis of viral determinants of virulence in the 5′ UTR (15). Among the few clinical studies which have addressed this question, Fujiwara et al., by comparing full-length HAV genomes obtained from Japanese patients with benign or fulminant hepatitis, found less nucleotide variation in the 5′ UTRs from patients with fulminant hepatitis (12, 13) and suggested that two IRES mutations (G324A and C372G/T) might influence the course of HAV infection (14).The aim of the present study was to further examine the genetic variability of 5′ UTR sequences from field isolates, to assess the potential impact of nucleotide variations on IRES activity by using validated techniques, and to search for a relationship with disease severity by comparing isolates obtained from patients with benign or fulminant forms of hepatitis A.     

Contribution of charged and polar residues for the formation of the E1–E2 heterodimer from Hepatitis C Virus

The transmembrane domains of the envelope glycoprotein E1 and E2 have crucial multifunctional roles in the biogenesis of hepatitis C virus. We have performed molecular dynamics simulations to investigate a structural model of the transmembrane segments of the E1–E2 heterodimer. The simulations support the key role of the Lys370–Asp728 ion pair for mediating the E1–E2 heterodimerization. In comparison to these two residues, the simulation results also reveal the differential effect of the conserved Arg730 residue that has been observed in experimental studies. Furthermore, we discovered the formation of inter-helical hydrogen bonds via Asn367 that stabilize dimer formation. Simulations of single and double mutants further demonstrate the importance of the ion-pair and polar interactions between the interacting helix monomers. The conformation of the E1 fragment in the simulation of the E1–E2 heterodimer is in close agreement with an NMR structure of the E1 transmembrane segment. The proposed model of the E1–E2 heterodimer supports the postulated cooperative insertion of both helices by the translocon complex into the bilayer.     

Synthesis of 6-Arylthio Analogs of 2′,3′-Dideoxy-3′-Fluoroguanosine and Their Effect against Hepatitis B Virus Replication

A key compound, 2-amino-6-chloro-9-(2,3-dideoxy-3-fluoro-β-D-erythro-pentofuranosyl)purine, was prepared from 2-amino-6-chloropurine riboside in 5 steps, then subjected to the nucleophilic displacement with benzenethiols to afford 6-arylthio congeners. These compounds showed a similar anti-HBV effect to that of 2′,3′-dideoxy-3′-fluoroguanosine.