118 resultados para virus RNA


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A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3′2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5′ end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo.

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Many examples of extreme virus resistance and posttranscriptional gene silencing of endogenous or reporter genes have been described in transgenic plants containing sense or antisense transgenes. In these cases of either cosuppression or antisense suppression, there appears to be induction of a surveillance system within the plant that specifically degrades both the transgene and target RNAs. We show that transforming plants with virus or reporter gene constructs that produce RNAs capable of duplex formation confer virus immunity or gene silencing on the plants. This was accomplished by using transcripts from one sense gene and one antisense gene colocated in the plant genome, a single transcript that has self-complementarity, or sense and antisense transcripts from genes brought together by crossing. A model is presented that is consistent with our data and those of other workers, describing the processes of induction and execution of posttranscriptional gene silencing.

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The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus conserved all of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified. This demonstration opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses.

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We provide the first report, to our knowledge, of a helper-independent system for rescuing a segmented, negative-strand RNA genome virus entirely from cloned cDNAs. Plasmids were constructed containing full-length cDNA copies of the three Bunyamwera bunyavirus RNA genome segments flanked by bacteriophage T7 promoter and hepatitis delta virus ribozyme sequences. When cells expressing both bacteriophage T7 RNA polymerase and recombinant Bunyamwera bunyavirus proteins were transfected with these plasmids, full-length antigenome RNAs were transcribed intracellularly, and these in turn were replicated and packaged into infectious bunyavirus particles. The resulting progeny virus contained specific genetic tags characteristic of the parental cDNA clones. Reassortant viruses containing two genome segments of Bunyamwera bunyavirus and one segment of Maguari bunyavirus were also produced following transfection of appropriate plasmids. This accomplishment will allow the full application of recombinant DNA technology to manipulate the bunyavirus genome.

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Protein synthesis is believed to be initiated with the amino acid methionine because the AUG translation initiation codon of mRNAs is recognized by the anticodon of initiator methionine transfer RNA. A group of positive-stranded RNA viruses of insects, however, lacks an AUG translation initiation codon for their capsid protein gene, which is located at the downstream part of the genome. The capsid protein of one of these viruses, Plautia stali intestine virus, is synthesized by internal ribosome entry site-mediated translation. Here we report that methionine is not the initiating amino acid in the translation of the capsid protein in this virus. Its translation is initiated with glutamine encoded by a CAA codon that is the first codon of the capsid-coding region. The nucleotide sequence immediately upstream of the capsid-coding region interacts with a loop segment in the stem–loop structure located 15–43 nt upstream of the 5′ end of the capsid-coding region. The pseudoknot structure formed by this base pair interaction is essential for translation of the capsid protein. This mechanism for translation initiation differs from the conventional one in that the initiation step controlled by the initiator methionine transfer RNA is not necessary.

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Cucumber mosaic virus (CMV) and tomato aspermy virus (TAV) belong to the Cucumovirus genus. They have a tripartite genome consisting of single-stranded RNAs, designated 1, 2, and 3. Previous studies have shown that viable pseudorecombinants could be created in vitro by reciprocal exchanges between CMV and TAV RNA 3, but exchanges of RNAs 1 and 2 were replication deficient. When we coinoculated CMV RNAs 2 and 3 along with TAV RNAs 1 and 2 onto Nicotiana benthamiana, a hybrid quadripartite virus appeared that consisted of TAV RNA 1, CMV RNAs 2 and 3, and a distinctive chimeric RNA originating from a recombination between CMV RNA 2 and the 3′-terminal 320 nucleotides of TAV RNA 2. This hybrid arose by means of segment reassortment and RNA recombination to produce an interspecific hybrid with the TAV helicase subunit and the CMV polymerase subunit. To our knowledge, this is the first report demonstrating the evolution of a new plant or animal virus strain containing an interspecific hybrid replicase complex.

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The genome expression of positive-stranded RNA viruses starts with translation rather than transcription. For some viruses, the genome is the only viral mRNA and expression is regulated primarily at the translational level and by limited proteolysis of polyproteins. Other virus groups also generate subgenomic mRNAs later in the reproductive cycle. For nidoviruses, subgenomic mRNA synthesis (transcription) is discontinuous and yields a 5′ and 3′ coterminal nested set of mRNAs. Nidovirus transcription is not essential for genome replication, which relies on the autoprocessing products of two replicase polyproteins that are translated from the genome. We now show that the N-terminal replicase subunit, nonstructural protein 1 (nsp1), of the nidovirus equine arteritis virus is in fact dispensable for replication but crucial for transcription, thereby coupling replicase expression and subgenomic mRNA synthesis in an unprecedented manner. Nsp1 is composed of two papain-like protease domains and a predicted N-terminal zinc finger, which was implicated in transcription by site-directed mutagenesis. The structural integrity of nsp1 is essential, suggesting that the protease domains form a platform for the zinc finger to operate in transcription.

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Flock house virus (FHV), a single-stranded RNA insect virus, has previously been reported to cross the kingdom barrier and replicate in barley protoplasts and in inoculated leaves of several plant species [Selling, B. H., Allison, R. F. & Kaesberg, P. (1990) Proc. Natl. Acad. Sci. USA 87, 434–438]. There was no systemic movement of FHV in plants. We tested the ability of movement proteins (MPs) of plant viruses to provide movement functions and cause systemic spread of FHV in plants. We compared the growth of FHV in leaves of nontransgenic and transgenic plants expressing the MP of tobacco mosaic virus or red clover necrotic mosaic virus (RCNMV). Both MPs mobilized cell-to-cell and systemic movement of FHV in Nicotiana benthamiana plants. The yield of FHV was more than 100-fold higher in the inoculated leaves of transgenic plants than in the inoculated leaves of nontransgenic plants. In addition, FHV accumulated in the noninoculated upper leaves of both MP-transgenic plants. RCNMV MP was more efficient in mobilizing FHV to noninoculated upper leaves. We also report here that FHV replicates in inoculated leaves of six additional plant species: alfalfa, Arabidopsis, Brassica, cucumber, maize, and rice. Our results demonstrate that plant viral MPs cause cell-to-cell and long-distance movement of an animal virus in plants and offer approaches to the study of the evolution of viruses and mechanisms governing mRNA trafficking in plants as well as to the development of promising vectors for transient expression of foreign genes in plants.

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RNA viruses evolve rapidly. One source of this ability to rapidly change is the apparently high mutation frequency in RNA virus populations. A high mutation frequency is a central tenet of the quasispecies theory. A corollary of the quasispecies theory postulates that, given their high mutation frequency, animal RNA viruses may be susceptible to error catastrophe, where they undergo a sharp drop in viability after a modest increase in mutation frequency. We recently showed that the important broad-spectrum antiviral drug ribavirin (currently used to treat hepatitis C virus infections, among others) is an RNA virus mutagen, and we proposed that ribavirin's antiviral effect is by forcing RNA viruses into error catastrophe. However, a direct demonstration of error catastrophe has not been made for ribavirin or any RNA virus mutagen. Here we describe a direct demonstration of error catastrophe by using ribavirin as the mutagen and poliovirus as a model RNA virus. We demonstrate that ribavirin's antiviral activity is exerted directly through lethal mutagenesis of the viral genetic material. A 99.3% loss in viral genome infectivity is observed after a single round of virus infection in ribavirin concentrations sufficient to cause a 9.7-fold increase in mutagenesis. Compiling data on both the mutation levels and the specific infectivities of poliovirus genomes produced in the presence of ribavirin, we have constructed a graph of error catastrophe showing that normal poliovirus indeed exists at the edge of viability. These data suggest that RNA virus mutagens may represent a promising new class of antiviral drugs.

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The intercistronic region between the maturation and coat-protein genes of RNA phage MS2 contains important regulatory and structural information. The sequence participates in two adjacent stem-loop structures, one of which, the coat-initiator hairpin, controls coat-gene translation and is thus under strong selection pressure. We have removed 19 out of the 23 nucleotides constituting the intercistronic region, thereby destroying the capacity of the phage to build the two hairpins. The deletion lowered coat-protein yield more than 1000-fold, and the titer of the infectious clone carrying the deletion dropped 10 orders of magnitude as compared with the wild type. Two types of revertants were recovered. One had, in two steps, recruited 18 new nucleotides that served to rebuild the two hairpins and the lost Shine-Dalgarno sequence. The other type had deleted an additional six nucleotides, which allowed the reconstruction of the Shine-Dalgarno sequence and the initiator hairpin, albeit by sacrificing the remnants of the other stem-loop. The results visualize the immense genetic repertoire created by, what appears as, random RNA recombination. It would seem that in this genetic ensemble every possible new RNA combination is represented.

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The complete nucleotide sequence, 5178 bp, of the totivirus Helminthosporium vicotoriae 190S virus (Hv190SV) double-stranded RNA, was determined. Computer-assisted sequence analysis revealed the presence of two large overlapping ORFs; the 5'-proximal large ORF (ORF1) codes for the coat protein (CP) with a predicted molecular mass of 81 kDa, and the 3'-proximal ORF (ORF2), which is in the -1 frame relative to ORF1, codes for an RNA-dependent RNA polymerase (RDRP). Unlike many other totiviruses, the overlap region between ORF1 and ORF2 lacks known structural information required for translational frameshifting. Using an antiserum to a C-terminal fragment of the RDRP, the product of ORF2 was identified as a minor virion-associated polypeptide of estimated molecular mass of 92 kDa. No CP-RDRP fusion protein with calculated molecular mass of 165 kDa was detected. The predicted start codon of the RDRP ORF (2605-AUG-2607) overlaps with the stop codon (2606-UGA-2608) of the CP ORF, suggesting RDRP is expressed by an internal initiation mechanism. Hv190SV is associated with a debilitating disease of its phytopathogenic fungal host. Knowledge of its genome organization and expression will be valuable for understanding its role in pathogenesis and for potential exploitation in the development of biocontrol measures.