2′-5′-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1

2′-5′-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth.     

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.     

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.     

Hepatitis C Virus Non-Structural Protein 3 Interacts with Cytosolic 5′(3′)-Deoxyribonucleotidase and Partially Inhibits Its Activity

Infection with hepatitis C virus (HCV) is etiologically involved in liver cirrhosis, hepatocellular carcinoma and B-cell lymphomas. It has been demonstrated previously that HCV non-structural protein 3 (NS3) is involved in cell transformation. In this study, a yeast two-hybrid screening experiment was conducted to identify cellular proteins interacting with HCV NS3 protein. Cytosolic 5′(3′)-deoxyribonucleotidase (cdN, dNT-1) was found to interact with HCV NS3 protein. Binding domains of HCV NS3 and cellular cdN proteins were also determined using the yeast two-hybrid system. Interactions between HCV NS3 and cdN proteins were further demonstrated by co-immunoprecipitation and confocal analysis in cultured cells. The cellular cdN activity was partially repressed by NS3 protein in both the transiently-transfected and the stably-transfected systems. Furthermore, HCV partially repressed the cdN activity while had no effect on its protein expression in the systems of HCV sub-genomic replicons and infectious HCV virions. Deoxyribonucleotidases are present in most mammalian cells and involve in the regulation of intracellular deoxyribonucleotides pools by substrate cycles. Control of DNA precursor concentration is essential for the maintenance of genetic stability. Reduction of cdN activity would result in the imbalance of DNA precursor concentrations. Thus, our results suggested that HCV partially reduced the cdN activity via its NS3 protein and this may in turn cause diseases.     

NTPase and 5′ to 3′ RNA Duplex-Unwinding Activities of the Hepatitis E Virus Helicase Domain

Hepatitis E virus (HEV) is a causative agent of acute hepatitis, and it is the sole member of the genus Hepevirus in the family Hepeviridae. The open reading frame 1 (ORF1) protein of HEV encodes nonstructural polyprotein with putative domains for methyltransferase, cysteine protease, helicase and RNA-dependent RNA polymerase. It is not yet known whether ORF1 functions as a single protein with multiple domains or is processed to form separate functional units. On the basis of amino acid conserved motifs, HEV helicase has been grouped into helicase superfamily 1 (SF-1). In order to examine the RNA helicase activity of the NTPase/helicase domain of HEV, the region (amino acids 960 to 1204) was cloned and expressed as histidine-tagged protein in Escherichia coli (HEV Hel) and purified. HEV Hel exhibited NTPase and RNA unwinding activities. Enzyme hydrolyzed all rNTPs efficiently, dATP and dCTP with moderate efficiency, while it showed less hydrolysis of dGTP and dTTP. Enzyme showed unwinding of only RNA duplexes with 5′ overhangs showing 5′-to-3′ polarity. We also expressed and purified two HEV Hel mutants. Helicase mutant I, with substitution in the nucleotide-binding motif I (GKS to GAS), showed 30% ATPase activity. Helicase mutant II, with substitutions in the Mg²⁺ binding motif II (DEAP to AAAP), showed 50% ATPase activity. Both mutants completely lost ability to unwind RNA duplexes with 5′ overhangs. These findings represent the first report demonstrating NTPase/RNA helicase activity of the helicase domain of HEV ORF1.Viruses with single-strand positive-sense RNA genomes represent the largest class of viruses, which includes numerous pathogens of humans, plants, and animals. In these viruses, RNA replication occurs through negative-strand RNA intermediate, which may also act as the template for synthesis of subgenomic RNAs in some viruses. During replication, various nonstructural proteins remain associated with the viral polymerase in a small compartmentalized replisome. Most of the other accessory proteins are obtained from the cellular machinery.Helicase seems to be essential for RNA replication by many positive-sense RNA viruses (19). Many positive-strand RNA viruses encode their own RNA helicases and besides RNA-dependent RNA polymerase, helicase is the most conserved viral sequence in these viruses. It has been shown by direct mutagenesis studies in poliovirus (26, 39), alphaviruses (31), brome mosaic virus (2, 41), nidoviruses (40), and flaviviruses (15) that helicase functions are essential for viral replication. In addition, it may be involved in RNA translocation, genome packaging, protection of RNA at the replication center, modulating RNA-protein interactions, etc.Helicases are classified into six superfamilies, SF-1 to SF-6 (11, 35), and can be classified further into subfamilies, A (3′→5′) or B (5′→3′) depending on their unwinding directionality. Classic helicases (exhibiting both NTPase and unwinding activities) are referred to as subtype α, while translocases (with no unwinding activity) are referred to as subtype β (35). SF-1 and SF-2 constitute largest of these superfamilies with seven signature motifs (I, Ia, II, III, IV, V, and VI), which form core of the enzyme. Although these motifs are not comparable between SF-1 and SF-2, universal features of core domains include (i) conserved residues involved in binding and hydrolysis of the NTP and (ii) an arginine finger that plays a key role in energy coupling.Hepatitis E virus (HEV) is a nonenveloped virus in the genus Hepevirus of the family Hepeviridae. Hepatitis E is an important public health disease in many developing countries and is also endemic in some industrialized countries (8). Infection by HEV has a known association with increased mortality during pregnancy (22, 23). HEV has a positive-sense RNA genome of ∼7.2 kb, consisting of a 5′ noncoding region (5′NCR) of 27 to 35 nucleotides (nt), followed by three open reading frames (ORFs)—ORF1, ORF2, and ORF3—and a 3′NCR of 65 to 74 nt, ending with a poly(A) tail of variable length (37). The 5′ end has m⁷G cap (18). ORF1 is known to encode for the viral nonstructural polyprotein with a proposed molecular mass of ∼186 kDa (3). Based on protein sequence homology, the ORF1 polyprotein is proposed to contain four putative domains indicative of methyltransferase, papain-like cysteine protease, RNA helicase (Hel), and RNA-dependent RNA polymerase (RdRp) (24). ORF2 encodes the major structural protein (capsid protein), which has N-terminal signal peptide and three glycosylation sites and is translocated across the endoplasmic reticulum (ER). ORF2 protein associates with the 5′ end of the viral RNA, suggesting its regulatory role in the virus replication (36, 37, 44, 45). ORF3 encodes a protein which gets phosphorylated by the cellular mitogen activated protein kinase and is associated with cellular membranes and cytoskeleton fractions (43).HEV belongs to an “alpha-like” supergroup of positive-sense single-stranded RNA (+ssRNA) viruses with conserved motifs of replication-related proteins in the ORF1, with typical signature sequences homologous with the other members of the family (11, 12, 13). ORF1 of HEV encodes additional domains such as the Y domain, papainlike protease, “proline-rich hinge,” and the X domain. Methyltransferase (25), RdRp (1), and X domain (binding to poly-ADP-ribose) (9) in ORF1 have been characterized, whereas the functions of the other domains are yet to be identified. Intracellularly expressed RdRp localizes itself in the ER membranes (30), suggesting that HEV replicates probably in ER in the cytosolic compartment of the cells. It is still unknown whether ORF1 polyprotein undergoes cleavages to form separate functional units of the replication machinery or functions as a single protein with multiple functional domains.The putative RNA helicase of HEV contains all of the seven conserved segments typical of the SF-1 helicase (12, 13). Putative SF-1 helicases are extremely widespread among +ssRNA viruses. Based on sequence comparisons, such helicases have been identified in a variety of plant virus families, as well as in animal viruses such as alphavirus, rubivirus, hepatitis E virus, and coronavirus (11). When compared to other +ssRNA viral helicases belonging to SF-1, HEV helicase showed the highest overall similarity with the helicase of beet necrotic yellow vein virus, a plant furovirus. HEV helicase was speculated to have N-terminal NTPase and C-terminal RNA-binding domains (24). A major obstacle in studying HEV replication has been lack of cell culture system. We report here experimental verification of the helicase activity of the recombinant helicase domain protein of HEV.     

Aminoterminal Amphipathic α-Helix AH1 of Hepatitis C Virus Nonstructural Protein 4B Possesses a Dual Role in RNA Replication and Virus Production

Nonstructural protein 4B (NS4B) is a key organizer of hepatitis C virus (HCV) replication complex formation. In concert with other nonstructural proteins, it induces a specific membrane rearrangement, designated as membranous web, which serves as a scaffold for the HCV replicase. The N-terminal part of NS4B comprises a predicted and a structurally resolved amphipathic α-helix, designated as AH1 and AH2, respectively. Here, we report a detailed structure-function analysis of NS4B AH1. Circular dichroism and nuclear magnetic resonance structural analyses revealed that AH1 folds into an amphipathic α-helix extending from NS4B amino acid 4 to 32, with positively charged residues flanking the helix. These residues are conserved among hepaciviruses. Mutagenesis and selection of pseudorevertants revealed an important role of these residues in RNA replication by affecting the biogenesis of double-membrane vesicles making up the membranous web. Moreover, alanine substitution of conserved acidic residues on the hydrophilic side of the helix reduced infectivity without significantly affecting RNA replication, indicating that AH1 is also involved in virus production. Selective membrane permeabilization and immunofluorescence microscopy analyses of a functional replicon harboring an epitope tag between NS4B AH1 and AH2 revealed a dual membrane topology of the N-terminal part of NS4B during HCV RNA replication. Luminal translocation was unaffected by the mutations introduced into AH1, but was abrogated by mutations introduced into AH2. In conclusion, our study reports the three-dimensional structure of AH1 from HCV NS4B, and highlights the importance of positively charged amino acid residues flanking this amphipathic α-helix in membranous web formation and RNA replication. In addition, we demonstrate that AH1 possesses a dual role in RNA replication and virus production, potentially governed by different topologies of the N-terminal part of NS4B.     

Ribavirin Enhances the Action of Interferon-α against Hepatitis C Virus by Promoting the p53 Activity through the ERK1/2 Pathway

Background/Aims

Ribavirin significantly enhances the antiviral response of interferon-α (IFN-α) against Hepatitis C virus (HCV), but the underlying mechanisms remain poorly understood. Recently, p53 has been identified as an important factor involving the suppression of HCV replication in hepatocytes. We, therefore, decided to investigate whether and how ribavirin inhibits the replication of HCV by promoting the activity of p53.

Methods

HepG2 and HCV replicons (JFH1/HepG2) were utilized to study the relationship between ribavirin and p53. The effect of ribavirin on cell cycles was analyzed by flow cytometry. The activation of p53 and the signaling pathways were determined using immunoblotting. By knocking down ERK1/ERK2 and p53 utilizing RNA interference strategy, we further assessed the role of ERK1/2 and p53 in the suppression of HCV replication by ribavirin in a HCV replicon system.

Results

Using HepG2 and HCV replicons, we demonstrated that ribavirin caused the cell cycle arrest at G1 phase and stabilized and activated p53, which was associated with the antiviral activity of ribavirin. Compared to either ribavirin or IFN-α alone, ribavirin plus IFN-α resulted in greater p53 activation and HCV suppression. We further identified ERK1/2 that linked ribavirin signals to p53 activation. More importantly, knockdown of ERK1/2 and p53 partially mitigated the inhibitory effects of ribavirin on the HCV replication, indicating that ERK1/2-p53 pathway was involved in the anti-HCV effects of ribavirin.

Conclusion

Ribavirin stimulates ERK1/2 and subsequently promotes p53 activity which at least partly contributes to the enhanced antiviral response of IFN-α plus ribavirin against HCV.     

Spouse-to-Spouse Transmission and Evolution of Hypervariable Region 1 and 5’ Untranslated Region of Hepatitis C Virus Analyzed by Next-Generation Sequencing

Hepatitis C virus (HCV) transmission between spouses remains poorly characterized, largely due to the limited availability of samples from the early stage of infection, as well as methodological constraints. A fifty-eight year-old male developed acute hepatitis C infection and his 53-year old spouse has been HCV-positive for over 10 years. Serum samples were collected from both at the time of acute hepatitis C diagnosis in male (baseline) and then at 9 and 13 months. Hypervariable region 1 (HVR1) and 5’ untranslated region (5’UTR) sequences were amplified and subjected to next generation sequencing (NGS) using a pyrosequencing platform. Genetic variants were inferred by Shorah reconstruction method and compared by phylogenetic and sequence diversity analysis. As the sequencing error of the procedure was previously determined to be ≤ 1.5%, the analysis was conducted with and without the 1.5% cut-off with regard to the frequency of variants. No identical HVR1 variants were identified in spouses at baseline and follow-up samples regardless whether the cut-off was applied or not. However, there was high similarity (98.3%) between a minor baseline donor variant (1.7% frequency) and the most abundant baseline recipient variant (62.5% frequency). Furthermore, donor and recipient strains clustered together when compared to 10 control subjects from the same area and infected with the same HCV subtype. There was an increase in HVR1 complexity (number of genetic variants) over time in both spouses. In contrast, the 5''UTR region was stable and of low complexity throughout the study. In conclusion, intrafamilial HCV transmission may be established by a very minor variant and investigation of this phenomenon requires high-sensitivity assays, such as NGS.     

Improved Annotation of 3′ Untranslated Regions and Complex Loci by Combination of Strand-Specific Direct RNA Sequencing,RNA-Seq and ESTs

The reference annotations made for a genome sequence provide the framework for all subsequent analyses of the genome. Correct and complete annotation in addition to the underlying genomic sequence is particularly important when interpreting the results of RNA-seq experiments where short sequence reads are mapped against the genome and assigned to genes according to the annotation. Inconsistencies in annotations between the reference and the experimental system can lead to incorrect interpretation of the effect on RNA expression of an experimental treatment or mutation in the system under study. Until recently, the genome-wide annotation of 3′ untranslated regions received less attention than coding regions and the delineation of intron/exon boundaries. In this paper, data produced for samples in Human, Chicken and A. thaliana by the novel single-molecule, strand-specific, Direct RNA Sequencing technology from Helicos Biosciences which locates 3′ polyadenylation sites to within +/− 2 nt, were combined with archival EST and RNA-Seq data. Nine examples are illustrated where this combination of data allowed: (1) gene and 3′ UTR re-annotation (including extension of one 3′ UTR by 5.9 kb); (2) disentangling of gene expression in complex regions; (3) clearer interpretation of small RNA expression and (4) identification of novel genes. While the specific examples displayed here may become obsolete as genome sequences and their annotations are refined, the principles laid out in this paper will be of general use both to those annotating genomes and those seeking to interpret existing publically available annotations in the context of their own experimental data.     

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.     

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.     

Structural and Functional Elements of the Promoter Encoded by the 5′ Untranslated Region of the Venezuelan Equine Encephalitis Virus Genome

Venezuelan equine encephalitis virus (VEEV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. The pathogenesis of this virus depends strongly on the sequences of the structural proteins and on the mutations in the RNA promoter encoded by the 5′ untranslated region (5′UTR) of the viral genome. In this study, we performed a detailed investigation of the structural and functional elements of the 5′-terminal promoter and analyzed the effect of multiple mutations introduced into the VEEV 5′UTR on virus and RNA replication. The results of this study demonstrate that RNA replication is determined by two synergistically functioning RNA elements. One of them is a very 5′-terminal AU dinucleotide, which is not involved in the stable RNA secondary structure, and the second is a short, G-C-rich RNA stem. An increase or decrease in the stem''s stability has deleterious effects on virus and RNA replication. In response to mutations in these RNA elements, VEEV replicative machinery was capable of developing new, compensatory sequences in the 5′UTR either containing 5′-terminal AUG or AU repeats or leading to the formation of new, heterologous stem-loops. Analysis of the numerous compensatory mutations suggested that at least two different mechanisms are involved in their generation. Some of the modifications introduced into the 5′ terminus of the viral genome led to an accumulation of the mutations in the VEEV nsPs, which suggested to us that there is a direct involvement of these proteins in promoter recognition. Furthermore, our data provide new evidence that the 3′ terminus of the negative-strand viral genome in the double-stranded RNA replicative intermediate is represented by a single-stranded RNA. Both the overall folding and the sequence determine its efficient function as a promoter for VEEV positive-strand RNA genome synthesis.Alphaviruses are a group of important human and animal pathogens. They are widely distributed both in the New and the Old Worlds and circulate between mosquito vectors and vertebrate hosts (45). In mosquitoes, they cause a persistent, life-long infection characterized by virus accumulation in salivary glands, which is required for infecting vertebrate hosts during a blood meal (50). In vertebrates, alphaviruses develop high-titer viremia, and their replication induces a variety of diseases with symptoms depending on both the host and the causative virus (11). Venezuelan equine encephalitis virus (VEEV), the New World alphavirus, is one of the most pathogenic members of the genus (16, 45). Representatives of the VEEV serocomplex circulate in Central, South, and North America and cause severe, and sometimes fatal, encephalitis in humans and horses (3, 11, 16, 24). Accordingly, VEEV represents a serious public health threat in the United States (39, 48, 51, 53), and during VEEV epizootics, equine mortality can reach 83%, and in humans, neurological diseases can be detected in up to 14% of all infected individuals, especially children (15). The overall mortality rate for humans is below 1%, but it is usually higher among children, the elderly, and, most likely, immunocompromised individuals (49). In spite of the continuous threat of VEEV epidemics, the biology of this virus, its pathogenesis, and the mechanism of replication are insufficiently understood. To date, no safe and efficient vaccine and therapeutic means have been developed for this pathogen.The VEEV genome is represented by a single-stranded, almost 11.5-kb-long RNA molecule of positive polarity. This RNA mimics the structure of cellular mRNAs by containing a cap at the 5′ ends and a poly(A) tail at the 3′ ends of the genome (18). The genomic RNA encodes two polyproteins: the 5′-terminal open reading frame (ORF) is translated into viral nonstructural proteins (nsP1 to nsP4), forming the replication enzyme complex (RC). The second ORF corresponds to the 3′-terminal one-third of the genome and encodes all of the viral structural proteins, C, E2, and E1. The latter proteins are translated from the subgenomic RNA synthesized during virus replication (45).The replication of the alphavirus genome is a highly regulated, multistep process, which includes the synthesis of three different RNA species (45). The regulation of their synthesis is achieved by differential processing of viral nsPs (22, 23, 43). First, the initially synthesized nonstructural polyprotein is partially processed by the nsP2-associated protease into P123 and nsP4, and this complex is active in negative-strand RNA synthesis (22). The latter RNA is present in the double-stranded RNA (dsRNA) replicative intermediate and is associated with plasma membrane and endosome-like vesicular organelles (8). Further processing of the polyproteins into individual nsP1 to nsP4 makes the RC capable of the synthesis of the positive-strand genome and subgenomic RNA but not of negative-strand RNA (23, 41, 42). Thus, the completely processed nsPs utilize only the promoters located on the negative strand of the viral genome.The defined promoters in the alphavirus genomes include (i) a 3′-terminal 19-nucleotide (nt)-long, conserved sequence element (CSE) adjacent to the poly(A) tail (12, 13, 19); (ii) the subgenomic promoter in the negative-strand copy of the viral genome (25); and (iii) the promoter for the synthesis of the positive-strand viral genome (45). The latter promoter is located at the 3′ end of the negative strand of the viral genome and has a complex structure. The two identified elements include the sequence, encoded by the 5′ untranslated region (5′UTR) (a core promoter) (5, 9, 32), and a 51-nt CSE, found ∼150 nt downstream of the genome''s 5′ terminus in the nsP1-encoding sequence. Our previous results and those of other research groups demonstrated that the 51-nt CSE functions as a replication enhancer in a virus- and cell-dependent mode (4, 33). Clustered mutations in the VEEV 51-nt CSE or its complete deletion either had deleterious effects on RNA replication or completely abolished RNA synthesis (30). However, RNA replication was ultimately recovered due to an accumulation of compensatory, adaptive mutations in either VEEV nsP2 or nsP3 (30). Thus, the 51-nt CSE in the VEEV genome is not absolutely essential for virus replication, but its presence is highly beneficial for achieving the most efficient growth rates in cells of both vertebrate and invertebrate origins. Alphavirus core promoters demonstrate a very low level of sequence conservation and also function in cell- and virus-specific modes (9). Previous studies suggested that the sequence and/or secondary structure of the VEEV core promoter plays a critical role in virus pathogenesis, and the G3→A (A3) mutation, found in an attenuated strain of VEEV TC-83, is one of the determinants of its less pathogenic phenotype (17, 55). However, information about functional elements of the VEEV core promoter remains incomplete, and its structural and functional elements have not yet been dissected.In this study, we applied a combination of molecular approaches to further define the functional components of the VEEV 5′UTR-specific core promoter, which mediates positive-strand genome synthesis. Our results demonstrate the presence of three structural RNA elements, two of which synergistically determine promoter activity. The first element of the promoter is a very short, 5′-terminal sequence, which is not involved in a stable secondary structure. Point mutations in the very 5′-terminal nucleotides have a deleterious effect on genome RNA replication. The second element is the short RNA stem, located in close proximity to the 5′ end of the genome. Mutations changing either the stability or sequence of the stem strongly affect virus replication and cause its rapid evolution, leading to the appearance of heterologous repeating elements in the unpaired 5′ terminus or the generation of other sequences that might potentially fold into stem structures. Surprisingly, the third structural RNA element, the loop, appears to play no important role in RNA replication and can be replaced either by a shorter loop or by the loop having a heterologous sequence without a detectable effect on virus and RNA replication.     

A Human Proteome Microarray Identifies that the Heterogeneous Nuclear Ribonucleoprotein K (hnRNP K) Recognizes the 5′ Terminal Sequence of the Hepatitis C Virus RNA

Stem-loop I (SL1) located in the 5′ untranslated region of the hepatitis C virus (HCV) genome initiates binding to miR-122, a microRNA required for hepatitis HCV replication. However, proteins that bind SL1 remain elusive. In this study, we employed a human proteome microarray, comprised of ∼17,000 individually purified human proteins in full-length, and identified 313 proteins that recognize HCV SL1. Eighty-three of the identified proteins were annotated as liver-expressing proteins, and twelve of which were known to be associated with hepatitis virus. siRNA-induced silencing of eight out of 12 candidate genes led to at least 25% decrease in HCV replication efficiency. In particular, knockdown of heterogeneous nuclear ribonucleoprotein K (hnRNP K) reduced HCV replication in a concentration-dependent manner. Ultra-violet-crosslinking assay also showed that hnRNP K, which functions in pre-mRNA processing and transport, showed the strongest binding to the HCV SL1. We observed that hnRNP K, a nuclear protein, is relocated in the cytoplasm in HCV-expressing cells. Immunoprecipitation of the hnRNP K from Huh7.5 cells stably expressing HCV replicon resulted in the co-immunoprecipitation of SL1. This work identifies a cellular protein that could have an important role in the regulation of HCV RNA gene expression and metabolism.RNA viruses are the cause of numerous human diseases. Because of their relatively simple genomes, successful infection by RNA viruses is intimately linked to host factors that can both contribute to, or antagonize the viral infection process (1–3). Infection by the hepatitis C virus (HCV)¹, a positive-sense RNA virus, can lead to liver cirrhosis and hepatocellular carcinoma. Approximately 2–3% of the world''s population is chronically infected with HCV, with more than 350,000 annual fatalities in recent years (4). As is typical for viruses, a large number of host factors have been reported to facilitate HCV infection including microRNA-122 (miR-122), CD81, claudin-1, cyclophilins, and lipoproteins, to name a few (5–9). These cellular factors interact with viral proteins or RNA, thus promoting HCV entry, genome translation, and replication.The 5′-untranslated region (5′-UTR) of the HCV RNA genome contains complex RNA structures that interact with cellular factors. These structures include the internal ribosomal entry site that regulates cap-independent translation of the viral RNA (10–11). The 5′-most stem-loop (SL) structure, namely SL1, has been reported to interact with miR-122 to increase the stability of the genomic RNA and facilitate HCV RNA replication in cells (12–13). However, host proteins that can bind to SL1 remain largely elusive because of a lack of proper tools. Previously, we have shown that functional protein microarrays, comprised of individually purified yeast proteins, are an ideal tool to identify proteins that directly interact with important RNA structures encoded by an RNA virus (14). Here, we took a similar approach using a human proteome microarray to identify human hnRNP K as a specific HCV SL1-binding protein that is required for efficient HCV RNA replication.