The genomes of RNA viruses often contain RNA structures that are crucial for translation and RNA replication and may play additional, uncharacterized roles during the viral replication cycle. For the picornavirus family member poliovirus, a number of functional RNA structures have been identified, but much of its genome, especially the open reading frame, has remained uncharacterized. We have now generated a global RNA structure map of the poliovirus genome using a chemical probing approach that interrogates RNA structure with single-nucleotide resolution. In combination with orthogonal evolutionary analyses, we uncover several conserved RNA structures in the open reading frame of the viral genome. To validate the ability of our global analyses to identify functionally important RNA structures, we further characterized one of the newly identified structures, located in the region encoding the RNA-dependent RNA polymerase, 3D pol , by site-directed mutagenesis. Our results reveal that the structure is required for viral replication and infectivity, since synonymous mutants are defective in these processes. Furthermore, these defects can be partially suppressed by mutations in the viral protein 3C pro , which suggests the existence of a novel functional interaction between an RNA structure in the 3D pol -coding region and the viral protein(s) 3C pro and/or its precursor 3CD pro .T he genomes of RNA viruses, such as poliovirus (PV), often contain complex RNA secondary and tertiary structures. These structures are crucial for translation and replication of the viral genome and may play additional roles in other processes, such as genome packaging and modulation of the host antiviral response.Poliovirus, the prototypical picornavirus and causative agent of poliomyelitis, is a nonenveloped virus with a single-stranded RNA genome of positive polarity. The virion consists of an icosahedral protein shell, composed of four capsid proteins (VP1, VP2, VP3, and VP4), which encapsidates the RNA genome (1). Poliovirus has a rapid replication cycle, with approximately 8 h elapsing between infection and release of progeny virions upon host cell lysis. During an infection, high yields of both viral proteins and genomes are produced. These yields ensure synthesis of up to 10,000 virions per cell (2), which can be widely disseminated to neighboring cells and/or new hosts. The compact nature of the viral genome (less than 7,500 nucleotides [nt] long) facilitates this rapid exponential growth. The viral genome acts as an mRNA and can be divided into a highly conserved 742-nt 5= untranslated region (5= UTR), a single long open reading frame encoding the viral polyprotein, a 68-nt 3= untranslated region (3= UTR), and a polyadenosine tract of a variable length (see Fig. 2A). A small viral protein of 22 amino acids, VPg (3B), is covalently attached to the 5= end of the RNA. The 5= UTR contains a structure critical for viral translation (the internal ribosome entry site or IRES) (3, 4), as well the 5=-cloverleaf (5=-CL) structure, which i...
Prior reports have suggested that variants in the genes for maturity-onset diabetes of the young (MODY) may confer susceptibility to type 2 diabetes, but results have been conflicting and coverage of the MODY genes has been incomplete. To complement our previous studies of HNF4A, we examined the other five known MODY genes for association with type 2 diabetes in Finnish individuals. For each of the five genes, we selected 1) nonredundant single nucleotide polymorphisms (SNPs) (r 2 < 0.8 with other SNPs) from the HapMap database or another linkage disequilibrium map, 2) SNPs with previously reported type 2 diabetes association, and 3) nonsynonymous coding SNPs. We tested 128 SNPs for association with type 2 diabetes in 786 index cases from type 2 diabetic families and 619 normal glucose-tolerant control subjects. We followed up 35 of the most significant SNPs by genotyping them on another 384 case subjects and 366 control subjects from Finland. We also supplemented our previous HNF4A results by genotyping 12 SNPs on additional Finnish samples. After correcting for testing multiple correlated SNPs within a gene, we find evidence of type 2 diabetes association with SNPs in five of the six known MODY genes: GCK, HNF1A, HNF1B, NEUROD1, and HNF4A. Our data suggest that common variants in several MODY genes play a modest role in type 2 diabetes susceptibility. Diabetes 55:
Poliovirus (PV) is the prototypical picornavirus. It is a non‐enveloped RNA virus with a small (∼7.5‐kb) genome of positive polarity. It has long served as a model to study RNA virus biology, pathogenesis, and evolution. cDNA clones of several strains are available, and infectious virus can be produced by the transfection of in vitro transcribed viral genomes into an appropriate host cell. PV infects many human and non‐human primate cell lines including HeLa and HeLa S3 cells, and can grow to high titer in culture. Protocols for the production, propagation, quantification, and purification of PV are presented. Curr. Protoc. Microbiol. 29:15H.1.1‐15H.1.27. © 2013 by John Wiley & Sons, Inc.
Poliovirus (PV) is the prototypical picornavirus. It is a non‐enveloped RNA virus with a small (∼7.5‐kb) genome of positive polarity. cDNA clones of several strains are available, and infectious virus can be produced by the transfection of in vitro–transcribed viral genomes into an appropriate host cell. The ease of genetic studies in poliovirus is a primary reason that it has long served as a model to study RNA virus biology, pathogenesis, and evolution. Protocols for the generation and characterization of PV mutants are presented. Curr. Protoc. Microbiol. 29:15H.2.1‐15H.2.32. © 2013 by John Wiley & Sons, Inc.
Selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE) provides a means to investigate RNA structure with better resolution and higher throughput than has been possible with traditional methods. We present several protocols, which are based on a variety of previously published methods and were adapted and optimized for the analysis of poliovirus RNA in the Andino laboratory. These include methods for nondenaturing RNA extraction, RNA modification and primer extension, and data processing in ShapeFinder. Curr. Protoc. Microbiol. 30:15H.3.1‐15H.3.12. © 2013 by John Wiley & Sons, Inc.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.