Analysis of hepatitis C virus RNA dimerization and core–RNA interactions

The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3′-untranslated region (3′-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623–2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3′-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus

A Cooperative Interaction between Nontranslated RNA Sequences and NS5A Protein Promotes In Vivo Fitness of a Chimeric Hepatitis C/GB Virus B

GB virus B (GBV-B) is closely related to hepatitis C virus (HCV), infects small non-human primates, and is thus a valuable surrogate for studying HCV. Despite significant differences, the 5′ nontranslated RNAs (NTRs) of these viruses fold into four similar structured domains (I-IV), with domains II-III-IV comprising the viral internal ribosomal entry site (IRES). We previously reported the in vivo rescue of a chimeric GBV-B (vGB/IIIHC) containing HCV sequence in domain III, an essential segment of the IRES. We show here that three mutations identified within the vGB/IIIHC genome (within the 3′NTR, upstream of the poly(U) tract, and NS5A coding sequence) are necessary and sufficient for production of this chimeric virus following intrahepatic inoculation of synthetic RNA in tamarins, and thus apparently compensate for the presence of HCV sequence in domain III. To assess the mechanism(s) underlying these compensatory mutations, and to determine whether 5′NTR subdomains participating in genome replication do so in a virus-specific fashion, we constructed and evaluated a series of chimeric subgenomic GBV-B replicons in which various 5′NTR subdomains were substituted with their HCV homologs. Domains I and II of the GBV-B 5′NTR could not be replaced with HCV sequence, indicating that they contain essential, virus-specific RNA replication elements. In contrast, domain III could be swapped with minimal loss of genome replication capacity in cell culture. The 3′NTR and NS5A mutations required for rescue of the related chimeric virus in vivo had no effect on replication of the subgenomic GBneoD/IIIHC RNA in vitro. The data suggest that in vivo fitness of the domain III chimeric virus is dependent on a cooperative interaction between the 5′NTR, 3′NTR and NS5A at a step in the viral life cycle subsequent to genome replication, most likely during particle assembly. Such a mechanism may be common to all hepaciviruses

The Core Protein of Classical Swine Fever Virus Is Dispensable for Virus Propagation In Vitro

Core protein of Flaviviridae is regarded as essential factor for nucleocapsid formation. Yet, core protein is not encoded by all isolates (GBV- A and GBV- C). Pestiviruses are a genus within the family Flaviviridae that affect cloven-hoofed animals, causing economically important diseases like classical swine fever (CSF) and bovine viral diarrhea (BVD). Recent findings describe the ability of NS3 of classical swine fever virus (CSFV) to compensate for disabling size increase of core protein (Riedel et al., 2010). NS3 is a nonstructural protein possessing protease, helicase and NTPase activity and a key player in virus replication. A role of NS3 in particle morphogenesis has also been described for other members of the Flaviviridae (Patkar et al., 2008; Ma et al., 2008). These findings raise questions about the necessity and function of core protein and the role of NS3 in particle assembly. A reverse genetic system for CSFV was employed to generate poorly growing CSFVs by modification of the core gene. After passaging, rescued viruses had acquired single amino acid substitutions (SAAS) within NS3 helicase subdomain 3. Upon introduction of these SAAS in a nonviable CSFV with deletion of almost the entire core gene (Vp447Δc), virus could be rescued. Further characterization of this virus with regard to its physical properties, morphology and behavior in cell culture did not reveal major differences between wildtype (Vp447) and Vp447Δc. Upon infection of the natural host, Vp447Δc was attenuated. Hence we conclude that core protein is not essential for particle assembly of a core-encoding member of the Flaviviridae, but important for its virulence. This raises questions about capsid structure and necessity, the role of NS3 in particle assembly and the function of core protein in general

Activation of 2′ 5′-oligoadenylate synthetase by stem loops at the 5′-end of the West Nile virus genome

West Nile virus (WNV) has a positive sense RNA genome with conserved structural elements in the 5′ and 3′ -untranslated regions required for polyprotein production. Antiviral immunity to WNV is partially mediated through the production of a cluster of proteins known as the interferon stimulated genes (ISGs). The 2′ 5′-oligoadenylate synthetases (OAS) are key ISGs that help to amplify the innate immune response. Upon interaction with viral double stranded RNA, OAS enzymes become activated and enable the host cell to restrict viral propagation. Studies have linked mutations in the OAS1 gene to increased susceptibility to WNV infection, highlighting the importance of OAS1 enzyme. Here we report that the region at the 5′-end of the WNV genome comprising both the 5′-UTR and initial coding region is capable of OAS1 activation in vitro. This region contains three RNA stem loops (SLI, SLII, and SLIII) whose relative contribution to OAS1 binding affinity and activation were investigated using electrophoretic mobility shift assays and enzyme kinetics experiments. Stem loop I, comprising nucleotides 1-73, is dispensable for maximum OAS1 activation, as a construct containing only SLII and SLIII was capable of enzymatic activation. Mutations to the RNA binding site of OAS1 confirmed the specificity of the interaction. The purity, monodispersity and homogeneity of the 5′-end (SLI/II/III) and OAS1 were evaluated using dynamic light scattering and analytical ultra-centrifugation. Solution conformations of both the 5′-end RNA of WNV and OAS1 were then elucidated using small-angle x-ray scattering. In the context of purified components in vitro, these data demonstrate the recognition of conserved secondary structural elements of the WNV genome by a member of the interferon-mediated innate immune response

Dengue Virus Capsid Protein Usurps Lipid Droplets for Viral Particle Formation

Dengue virus is responsible for the highest rates of disease and mortality among the members of the Flavivirus genus. Dengue epidemics are still occurring around the world, indicating an urgent need of prophylactic vaccines and antivirals. In recent years, a great deal has been learned about the mechanisms of dengue virus genome amplification. However, little is known about the process by which the capsid protein recruits the viral genome during encapsidation. Here, we found that the mature capsid protein in the cytoplasm of dengue virus infected cells accumulates on the surface of ER-derived organelles named lipid droplets. Mutagenesis analysis using infectious dengue virus clones has identified specific hydrophobic amino acids, located in the center of the capsid protein, as key elements for lipid droplet association. Substitutions of amino acid L50 or L54 in the capsid protein disrupted lipid droplet targeting and impaired viral particle formation. We also report that dengue virus infection increases the number of lipid droplets per cell, suggesting a link between lipid droplet metabolism and viral replication. In this regard, we found that pharmacological manipulation of the amount of lipid droplets in the cell can be a means to control dengue virus replication. In addition, we developed a novel genetic system to dissociate cis-acting RNA replication elements from the capsid coding sequence. Using this system, we found that mislocalization of a mutated capsid protein decreased viral RNA amplification. We propose that lipid droplets play multiple roles during the viral life cycle; they could sequester the viral capsid protein early during infection and provide a scaffold for genome encapsidation

Identification of novel RNA secondary structures within the hepatitis C virus genome reveals a cooperative involvement in genome packaging

The specific packaging of the hepatitis C virus (HCV) genome is hypothesised to be driven by Core- RNA interactions. To identify the regions of the viral genome involved in this process, we used SELEX (systematic evolution of ligands by exponential enrichment) to identify RNA aptamers which bind specifically to Core in vitro. Comparison of these aptamers to multiple HCV genomes revealed the presence of a conserved terminal loop motif within short RNA stem-loop structures. We postulated that interactions of these motifs, as well as sub-motifs which were present in HCV genomes at statistically significant levels, with the Core protein may drive virion assembly. We mutated 8 of these predicted motifs within the HCV infectious molecular clone JFH-1, thereby producing a range of mutant viruses predicted to possess altered RNA secondary structures. RNA replication and viral titre were unaltered in viruses possessing only one mutated structure. However, infectivity titres were decreased in viruses possessing a higher number of mutated regions. This work thus identified multiple novel RNA motifs which appear to contribute to genome packaging. We suggest that these structures act as cooperative packaging signals to drive specific RNA encapsidation during HCV assembly

The chromatin structuring protein HMGA2 influences human subtelomere stability and cancer chemosensitivity

10.1371/journal.pone.0215696PLoS ONE145e021569