Negative Sense RNA

6 Key excerpts on "Negative Sense RNA"

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  eBook - ePub

  Reverse Genetics of RNA Viruses

  Applications and Perspectives

  • Anne Bridgen(Author)
  • 2012(Publication Date)
  • Wiley

    (Publisher)

  cis -acting signals must be maintained in modified genomes.

  Early attempts to rescue Negative Sense RNA viruses relied on the reconstitution of viral RNP complexes. The first virus for which this approach was successfully used was the segmented virus influenza A (Luytjes et al ., 1989). RNA transcripts, generated in vitro and containing the authentic terminal sequences from an influenza A genome segment flanking the gene for chloramphenicol acetyl transferase (CAT), were encapsidated in vitro by purified influenza A virus N protein, and the viral polymerase proteins (PA, PB1 and PB2) to produce a synthetic RNP complex. After transfection of this RNP complex into influenza A virus-infected cells, the synthetic genome segment was successfully replicated and transcribed to generate CAT protein. Interestingly, this approach failed to rescue non-segmented, Negative Sense RNA viruses probably due to the tighter RNP complex structure in these viruses (Baudin et al ., 1994). The first successful demonstration of rescue of a foreign gene for MV involved the use of a negative-sensed minigenome; the MV 3' and 5' non-coding termini (NCT) consisting of the Le and Tr (containing the GP and AGP promoters) and the N 5' UTR and L 3' UTR (containing the N GS and the L GE signals), flanking the ORF for the CAT reporter gene (Sidhu et al ., 1995). These sequences were cloned into a plasmid designed to produce an in vitro minigenome RNA transcript of equivalent polarity to the genome but lacking all of the viral genes. The RNA was expressed using a T7 promoter (Plate 3A ). Generation of a precise 3' terminus is important for the subsequent replication of the minigenome inside cells. This was achieved by incorporating a hepatitis δ ribozyme sequence into the minigenome clone. The hepatitis δ ribozyme sequence is ideal for this purpose, in that only sequences downstream of the cleavage site are required for the autocatalytic activity (Perrotta and Been, 1991). Therefore upon in vitro transcription and ribozyme folding there is an autocatalytic, intramolecular cleavage event within the nascent RNA transcript producing RNA molecules with the exact 3' end of the virus minigenome (Rosenstein and Been, 1991). The difference between this and the influenza rescue described above was that the RNP complex was not formed outside of the cell. Instead the synthetic minigenomic RNA was transcribed in vitro

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  Negative Strand Rna Virus

  • Ming Luo(Author)
  • 2011(Publication Date)
  • World Scientific

    (Publisher)

  Chapter 1 Overview of Negative-Strand RNA Viruses Biao He ∗ 1. Non-Segmented Negative-Strand RNA Viruses There are four families of non-segmented negative-strand RNA viruses (NNSV): Paramyxoviridae , Filoviridae , Rhabdoviridae , and Bornaviridae . The single-stranded RNA genomes of the Mononegavirales range from approximately 11,000 to 19,000 nucleotides in length and contain a series of tandemly-linked genes sep-arated by non-transcribed sequences. For paramyxoviruses, the gene order is 3 -NP-P(V/W/C)-M-F-(SH)-HN-L-5 where genes in parenthesis are not found in all species (reviewed in Ref. 1). The viral RNA-dependent RNA polymerase (vRdRp), which transcribes the nucleocap-sid protein (NP)-encapsidated RNA into 5 capped and 3 polyadenylated mRNAs, minimally consists of two proteins, phosphoprotein (P) and the large (L) poly-merase protein. 2 The vRdRp is thought to bind the genomic RNA at a single 3 entry site and to transcribe the genome by a sequential and polar process. The vRdRp also replicates the viral RNA genome. 3–6 The functional template for tran-scription of non-segmented negative-sense RNA viruses is the helical nucleocapsid (NC) (reviewed in Ref. 1). Although the exact details of mRNA production are unknown, the process is currently believed to involve termination and reinitiation (stop and start) at each gene junction and these junctions consist of three nucleotide sequence elements. At the gene end (GE) sequence, polyadenylation occurs through ∗ Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA. E-mail: [email protected] 1 2 B. He reiterative copying of a 4–7 uridyl (U) residue tract, and transcription terminates releasing a polyadenylated RNA. At this juncture, the vRdRp either leaves the template (attenuation) or passes over an intergenic sequence (IGS) region not found in mRNAs, and the vRdRp reinitiates mRNA synthesis at a downstream transcriptional gene start (GS) sequence.

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  Viral Diagnostics

  Advances and Applications

  • Robert S. Marks, Leslie Lobel, Amadou Sall, Robert S. Marks, Leslie Lobel, Amadou Sall(Authors)
  • 2014(Publication Date)
  • Jenny Stanford Publishing

    (Publisher)

  8.2 Negative-Stranded RNA Viruses NS RNA viruses are classified into eight families. 1 Five of these ( Bornaviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae , and an unassigned family represented by Deltavirus) have single-RNA-molecule genomes, while the remaining three families ( Orthomyx-oviridae, Bunyaviridae, and Arenaviridae ) are characterized by segmented genomes. The NS RNA viruses are a broad group of mammalian viruses that comprise several classic pathogens, including influenza, measles, mumps, and rabies, and many newly emergent and reemerging pathogens, such as Ebola, Hantaviruses, and Rift Valley fever virus (see Table 8.1). Most of these pathogens are capable of causing severe morbidity and significant mortality, being responsible for thousands of deaths every year. Some of the viruses are on the National Institute of Allergy and Infectious Diseases (NIAID) and Centers for Disease Control and Prevention (CDC) priority A list of potential bioterror pathogens. 8.2.1 Genome Structure NS RNA viruses are divided into two major groups, nonsegmented negative stranded (NNS) and segmented negative stranded (SNS) (see Table 8.1). All the NNS and SNS RNA viruses have several features in common: by definition, the polarity of the genomic RNA of NS RNA viruses is opposite (negative sense) to mRNA (positive sense); all of them are enveloped viruses, and the majority replicate in the cell cytoplasm. Two exceptions to this rule are orthomyxo-and bornaviruses, which transcribe and replicate in the nucleus. The major difference between NNS and SNS RNA viruses is the structure of their genome. The genome of SNS RNA viruses consists of several segments, each segment usually encoding one protein, Negative-Stranded RNA Viruses 151 Table 8.1 NS RNA viruses can be divided into two major groups.

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  New Generation Vaccines

  • Myrone M. Levine, Myron M. Levine, Gordon Dougan, Michael F. Good, Margaret A. Liu, Gary J. Nabel, James P. Nataro, Rino Rappuoli, Myrone M. Levine, Myron M. Levine, Gordon Dougan, Michael F. Good, Gary J. Nabel, James P. Nataro, Rino Rappuoli, Myrone M. Levine, Myron M. Levine, Gordon Dougan, Michael F. Good, Gary J. Nabel, James P. Nataro, Rino Rappuoli(Authors)
  • 2016(Publication Date)
  • CRC Press

    (Publisher)

  Negative stranded RNA genomes carry their genetic information in the complementary sense relative to mRNA. Therefore, successful virus propagation requires that the viral replicase/transcriptase enzymes required in the earliest stages of intracellular replication be introduced into the cell along with the genome. In systems designed for reverse genetics of negative stranded viruses, in vitro transcripts from cDNA clones (usually transcripts that are the positive sense comple-ment of the genome) are introduced into cells where the replicase proteins are being expressed from a second expres-sion system (often the vaccinia T7 system). With either positive or Negative Sense RNA genomes, the systems used to regenerate infectious viruses from cDNA clones have also been employed to add immunizing genes from target pathogens to the viral genomes. This results in a fully infectious and propagation competent virus vector, which expresses an exogenous immunogen in addition to a full complement of viral proteins in the successive cells it infects (Fig. 1A.1). A second iteration on this theme is the substitution of an immunogen gene for one or more of the structural protein genes of the vector virus. The resulting RNA genome is self-replicating inside an appropriate cell, thus the term ‘‘replicon.’’ The replicon expresses the immunizing gene and can be packaged into ‘‘replicon particles’’ when the structural proteins are produced in trans within the same cell (Fig. 1B). When inoculated into an animal or human, the replicon particles target to those cells normally infected first by the complete virus, the replicon genome expresses the immunizing gene, but the infection cannot be propagated to additional cells due to the absence of the structural protein genes from the replicon genome. Alternatively, a positive sense replicon can be tran-scribed, or launched, from a DNA vaccine in vivo.

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  Nucleic Acid Sensors and Antiviral Immunity

  • Dr. Prakash Sambhara(Author)
  • 2012(Publication Date)
  • CRC Press

    (Publisher)

  ©2013 Landes Bioscience. Antiviral Actions ofDouble-Stranded RNA 219 Figure 1. DsRNA-mediated antiviral effects. DsRNA, produced by virus infection is recognized by various dsRNA sensors to initiate the host defense processes. Some of the cellular dsRNA sensors and their mode of action are shown. Sources of dsRNA The most well characterized source of dsRNA in infected cells is viral dsRNA. For dsRNA viruses, the genome of infecting viruses can direcdy generate dsRNA inside the cells. The ssRNA viruses also generate dsRNA as replication intermediates. In addition, ssRNAs with extensive secondary structures such as hairpin loop formations are also effectively recognized by the receptors. The ssRNA virus populations, containing defective particles with ds defective genomes also serve as a source of dsRNA. For DNA viruses, complementary mRNAs are often produced; they are encoded by partially overlapping genes, located on the opposite strands of the viral genome. Recendy, cytosolic RNA polymerase III has been shown to generate dsRNA from dsDNA, which are produced by DNA viruses and intracellular bacteria.5 In addition to viral infections, extracellular RNA, generated by tissue damage or necrotic cells, has enough ds structures to serve as potential sources ofdsRNA. Extracellular dsRNA is endocytosed and transported to endosomal lumen for presentation to dsRNA recognizing protein TLR3, whereas cytosolic dsRNA generated during viral replication is direcdy recognized by cytosolic RNA helicases such as RIG-I or MDA-5. A synthetic dsRNA, polyl.polyC, is often used as an experimental mimic to trigger host response to virus infection.

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  Biotechnology Annual Review

  • M.R. El-Gewely(Author)
  • 1995(Publication Date)
  • Elsevier Science

    (Publisher)

  Besides a detailed analysis of the cellular mechanisms that underly the effects obtained with antisense-, sense- and triple helix-forming oligonucleotides, continu- ing efforts have been applied to the search for additional “tools” in order to improve delivery, specificity and efficiency. A brief introduction to antisense technology and catalytic RNAs (ribozymes) is given in the first sections. In the second part, we discuss another approach for the sequence-specific inactivation of RNA which is based on a universal cellular en- zyme, the structure-specific ribonuclease P (RNase P). Targets for Antisense Strategies Gene expression can be subdivided into several steps leading from genomic DNA to the functional protein (Fig. l), which depend on specific interactions of nucleic acids with other nucleic acids and proteins. The main targets of antisense strategies are cellular or viral mRNAs. Antisense nucleic acids can interfere with splicing, nuclear export, mRNA stability and accessibility for components of the translational machinery. triple helix antisense DNARNA (interacts with RNA) / I / transcription post transcript. export translation 1 DNA - -mRNA 7 7 protein modification nucleus cytoplasm sense DNARNA Fig. I. Sites of interference of gene expression inside a eukaryotic cell. Transcription, processing and modification occurs in the nuclear compartment (shaded area). Subsequently, mRNAs are exported into the cytoplasm, where translation takes place. Triple helix forming oligonucleotides are thought to inter- fere with gene expression mainly by binding to the DNA, while sense nucleic acids act by competing for essential cellular factors. Antisense nucleic acids are designed to interfere with splicing, export into the cytoplasm, translation or RNA stability by virtue of their capability to hybridize with the target (pre-) mRNA.

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