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The chaperone-like activity of the hepatitis C virus IRES and CRE elements regulates genome dimerization.

ABSTRACT: The RNA genome of the hepatitis C virus (HCV) establishes a network of long-distance RNA-RNA interactions that direct the progression of the infective cycle. This work shows that the dimerization of the viral genome, which is initiated at the dimer linkage sequence (DLS) within the 3'UTR, is promoted by the CRE region, while the IRES is a negative regulatory partner. Using differential 2'-acylation probing (SHAPE-dif) and molecular interference (HMX) technologies, the CRE activity was found to mainly lie in the critical 5BSL3.2 domain, while the IRES-mediated effect is dependent upon conserved residues within the essential structural elements JIIIabc, JIIIef and PK2. These findings support the idea that, along with the DLS motif, the IRES and CRE are needed to control HCV genome dimerization. They also provide evidences of a novel function for these elements as chaperone-like partners that fine-tune the architecture of distant RNA domains within the HCV genome.

SUBMITTER: Romero-Lopez C

PROVIDER: S-EPMC5324077 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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The chaperone-like activity of the hepatitis C virus IRES and CRE elements regulates genome dimerization.

Romero-López Cristina C Barroso-delJesus Alicia A Berzal-Herranz Alfredo A

Scientific reports 20170224

The RNA genome of the hepatitis C virus (HCV) establishes a network of long-distance RNA-RNA interactions that direct the progression of the infective cycle. This work shows that the dimerization of the viral genome, which is initiated at the dimer linkage sequence (DLS) within the 3'UTR, is promoted by the CRE region, while the IRES is a negative regulatory partner. Using differential 2'-acylation probing (SHAPE-dif) and molecular interference (HMX) technologies, the CRE activity was found to m ...[more]

PMID: 28233845

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Project description:Paramyxoviruses rely on the matrix (M) protein to orchestrate viral assembly and budding at the plasma membrane. Although the mechanistic details remain largely unknown, structural data suggested that M dimers and/or higher-order oligomers may facilitate membrane budding. To gain functional insights, we employed a structure-guided mutagenesis approach to investigate the role of canine distemper virus (CDV) M protein self-assembly in membrane-budding activity. Three six-alanine-block (6A-block) mutants with mutations located at strategic oligomeric positions were initially designed. While the first one includes residues potentially residing at the protomer-protomer interface, the other two display amino acids located within two distal surface-exposed α-helices proposed to be involved in dimer-dimer contacts. We further focused on the core of the dimeric interface by mutating asparagine 138 (N138) to several nonconservative amino acids. Cellular localization combined with dimerization and coimmunopurification assays, performed under various denaturing conditions, revealed that all 6A-block mutants were impaired in self-assembly and cell periphery accumulation. These phenotypes correlated with deficiencies in relocating CDV nucleocapsid proteins to the cell periphery and in virus-like particle (VLP) production. Conversely, all M-N138 mutants remained capable of self-assembly, though to various extents, which correlated with proper accumulation and redistribution of nucleocapsid proteins at the plasma membrane. However, membrane deformation and VLP assays indicated that the M-N138 variants exhibiting the most reduced dimerization propensity were also defective in triggering membrane remodeling and budding, despite proper plasma membrane accumulation. Overall, our data provide mechanistic evidence that the efficiency of CDV M dimerization/oligomerization governs both cell periphery localization and membrane-budding activity.IMPORTANCE Despite the availability of effective vaccines, both measles virus (MeV) and canine distemper virus (CDV) still lead to significant human and animal mortality worldwide. It is assumed that postexposure prophylaxis with specific antiviral compounds may synergize with vaccination campaigns to better control ongoing epidemics. Targeting the matrix (M) protein of MeV/CDV is attractive, because M coordinates viral assembly and egress through interaction with multiple cellular and viral components. However, the lack of basic molecular knowledge of how M orchestrates these functions precludes the rational design of antivirals. Here we combined structure-guided mutagenesis with cellular, biochemical, and functional assays to investigate a potential correlation between CDV M self-assembly and virus-like particle (VLP) formation. Altogether, our findings provide evidence that stable M dimers at the cell periphery are required to productively trigger VLPs. Such stabilized M dimeric units may facilitate further assembly into robust higher-order oligomers necessary to promote plasma membrane-budding activity.

Project description:Hepatitis C virus (HCV) is a single-stranded positive-sense RNA virus from the genus Hepacivirus. The viral genomic +RNA is 9.6 kb long and contains highly structured 5' and 3' untranslated regions (UTRs) and codes for a single large polyprotein, which is co- and post-translationally processed by viral and cellular proteases into at least 11 different polypeptides. Most of the 5' UTR and an initial part of the polyprotein gene are occupied by an internal ribosome entry site (IRES), which mediates cap-independent translation of the viral proteins and allows the virus to overcome cellular antiviral defense based on the overall reduction of the cap-dependent translation initiation. We reconsidered published results concerning a search for possible correlation between patient response to interferon-based antiviral therapy and accumulation of nucleotide changes within the HCV IRES. However, we were unable to identify any such correlation. Rather than searching for individual mutations, we suggest to focus on determination of individual and collective activities of the HCV IRESs found in patient specimens. We developed a combined, fast, and undemanding approach based on high-throughput cloning of the HCV IRES species to a bicistronic plasmid followed by determination of the HCV IRES activity by flow cytometry. This approach can be adjusted for measurement of the individual HCV IRES activity and for estimation of the aggregate ability of the whole HCV population present in the specimen to synthesize viral proteins. To detect nucleotide variations in the individual IRESs, we used denaturing gradient gel electrophoresis (DGGE) analysis that greatly improved identification and classification of HCV IRES variants in the sample. We suggest that determination of the collective activity of the majority of HCV IRES variants present in one patient specimen in a given time represents possible functional relations among variant sequences within the complex population of viral quasispecies better than bare information about their nucleotide sequences. A similar approach might be used for monitoring of sequence variations in quasispecies populations of other RNA viruses in all cases when changes in primary sequence represent changes in measurable and easily quantifiable phenotypes.

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The chaperone-like activity of the hepatitis C virus IRES and CRE elements regulates genome dimerization.

Publications

The chaperone-like activity of the hepatitis C virus IRES and CRE elements regulates genome dimerization.

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