ABSTRACT
Subgenomic RNAs are produced by several RNA viruses through incomplete degradation of their genomic RNA by the exoribonuclease Xrn1, and have been shown to be essential for viral growth and pathogenicity. Within the flavivirus genus of the Flaviviridae family, two distinct classes of Xrn1-resistant RNA motifs have been proposed; one for mosquito-borne and insect-specific flaviviruses, and one for tick-borne flaviviruses and no-known-vector flaviviruses. We investigated tick-borne and no-known-vector flavivirus Xrn1-resistant RNA motifs through systematic in vitro mutational analysis and showed that both classes actually possess very similar structural configurations, including a double pseudoknot and a base-triple at identical, conserved locations. For the no-known-vector flavivirus Modoc virus, we show that in vivo generation of subgenomic flaviviral RNA was affected by mutations targeted at nucleotides involved in the structural features of flaviviral Xrn1-resistant RNA motifs that were defined in this work. Our results suggest that throughout the genus flavivirus Xrn1-resistant RNA motifs adopt the same topologically conserved structure.
Subject(s)
Flavivirus , RNA Stability/genetics , RNA, Viral/chemistry , 3' Untranslated Regions , Animals , Base Sequence , Cells, Cultured , Conserved Sequence , Cricetinae , Culicidae/virology , Exoribonucleases/metabolism , Flavivirus/classification , Flavivirus/genetics , Genome, Viral , Nucleic Acid Conformation , RNA, Viral/metabolism , Sequence Analysis, RNAABSTRACT
RNA structures are increasingly recognized to be of importance during influenza A virus replication. Here, we investigated a predicted conserved hairpin in the M gene segment (nt 967-994) within the region of the vRNA 5' packaging signal. The existence of this RNA structure and its possible role in virus replication was investigated using a compensatory mutagenesis approach. Mutations were introduced in the hairpin stem, based on natural variation. Virus replication properties were studied for the mutant viruses with disrupted and restored RNA structures. Viruses with structure-disrupting mutations had lower virus titers and a significantly reduced median plaque size when compared with the wild-type (WT) virus, while viruses with structure restoring-mutations replicated comparable to WT. Moreover, virus replication was also reduced when mutations were introduced in the hairpin loop, suggesting its involvement in RNA interactions. Northern blot and FACS experiments were performed to study differences in RNA levels as well as production of M1 and M2 proteins, expressed via alternative splicing. Stem-disruptive mutants caused lower vRNA and M2 mRNA levels and reduced M2 protein production at early time-points. When the RNA structure was restored, vRNA, M2 mRNA and M2 protein levels were increased, demonstrating a compensatory effect. Thus, this study provides evidence for functional importance of the predicted M RNA structure and suggests its role in splicing regulation.
Subject(s)
Influenza A virus/genetics , RNA, Messenger/chemistry , RNA, Viral/chemistry , Viral Matrix Proteins/chemistry , Virus Replication , Alternative Splicing , Animals , Base Pairing , Conserved Sequence , Dogs , HEK293 Cells , Humans , Influenza A virus/growth & development , Influenza A virus/metabolism , Inverted Repeat Sequences , Madin Darby Canine Kidney Cells , Mutagenesis , Nucleic Acid Conformation , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Structure-Activity Relationship , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolism , Virus AssemblyABSTRACT
Small RNAs play an important role in regulation of gene expression in eukaryotic and eubacterial cells by modulating gene expression both at the level of transcription and translation. Here, we show that short complementary RNAs can also affect gene expression by stimulating ribosomal frameshifting in vitro. This finding has important implications for understanding the process of ribosomal frameshifting and for the potential application of small RNAs in the treatment of diseases that are due to frameshift mutations.
Subject(s)
Frameshifting, Ribosomal/genetics , RNA, Small Interfering/metabolism , Ribosomes/metabolism , Base Pairing , Codon , Codon, Terminator , Enhancer Elements, Genetic , Gene Expression Regulation , Oligonucleotides/metabolism , Open Reading Frames , Protein Biosynthesis , RNA Interference , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Fusion Proteins/metabolism , Thermodynamics , Transcription, GeneticABSTRACT
Double-stranded RNA preparations produced from potato plants graft-inoculated with a Peruvian isolate of Potato yellow vein virus (PYVV; genus Crinivirus, family Closteroviridae) contain five RNA species denoted RNA 1, RNA 2, RNA 3, x and y of approximately 8, 5.3, 3.8, 2.0 and 1.8 kbp, respectively. The complete nucleotide sequences of PYVV RNAs 1, 2 and 3 and Northern hybridization analysis showed that PYVV RNA 1 contained the replication module and an additional open reading frame (p7), while two distinct species, RNAs 2 and 3, contain the Closteroviridae hallmark gene array. Pairwise comparisons and phylogeny of genome-encoded proteins showed that PYVV shares significant homology with other criniviruses but is most closely related to the Trialeurodes vaporariorum-vectored Cucumber yellows virus. Secondary structure prediction of the 3'-untranslated regions of all three PYVV RNAs revealed four conserved stem-loop structures and a 3'-terminal pseudoknot structure, also predicted for all fully characterized members of the genus Crinivirus and some members of the genera Closterovirus and Ampelovirus.
Subject(s)
Crinivirus/genetics , Genome, Viral , Potyvirus/genetics , RNA, Viral/genetics , Solanum tuberosum/virology , 3' Untranslated Regions/genetics , Amino Acid Sequence , Base Sequence , Conserved Sequence , Crinivirus/classification , Crinivirus/isolation & purification , Molecular Sequence Data , Open Reading Frames , Phylogeny , Potyvirus/classification , Potyvirus/isolation & purification , RNA, Viral/chemistryABSTRACT
A model system of a single-stranded trisegment Brome mosaic bromovirus (BMV) was used to analyze the mechanism of homologous RNA recombination. Elements capable of forming strand-specific stem-loop structures were inserted at the modified 3' noncoding regions of BMV RNA3 and RNA2 in either positive or negative orientations, and various combinations of parental RNAs were tested for patterns of the accumulating recombinant RNA3 components. The structured negative-strand stem-loops that were inserted in both RNA3 and RNA2 reduced the accumulation of RNA3-RNA2 recombinants to a much higher extent than those in positive strands or the unstructured stem-loop inserts in either positive or negative strands. The use of only one parental RNA carrying the stem-loop insert reduced the accumulation of RNA3-RNA2 recombinants even further, but only when the stem-loops were in negative strands of RNA2. We assume that the presence of a stable stem-loop downstream of the landing site on the acceptor strand (negative RNA2) hampers the reattachment and reinitiation processes. Besides RNA3-RNA2 recombinants, the accumulation of nontargeted RNA3-RNA1 and RNA3-RNA3 recombinants were observed. Our results provide experimental evidence that homologous recombination between BMV RNAs more likely occurs during positive- rather than negative-strand synthesis.
Subject(s)
Bromovirus/genetics , RNA, Viral/genetics , Recombination, Genetic , Base Sequence , Molecular Sequence Data , RNA, Viral/chemistryABSTRACT
We used an infectious cDNA clone of porcine reproductive and respiratory syndrome virus (PRRSV) to investigate the presence of essential replication elements in the region of the genome encoding the structural proteins. Deletion analysis showed that a stretch of 34 nucleotides (14653 to 14686) within ORF7, which encodes the nucleocapsid protein, is essential for RNA replication. Strand-specific reverse transcription-PCR analysis of viral RNA isolated from transfected BHK-21 cells revealed that this region is required for negative-strand genomic RNA synthesis. The 34-nucleotide stretch is highly conserved among PRRSV isolates and folds into a putative hairpin. A 7-base sequence within the loop of this structure was suggested to base-pair with a sequence present in the loop of a hairpin located in the 3' noncoding region, resulting in a kissing interaction. Mutational analyses confirmed that this kissing interaction is required for RNA replication.
Subject(s)
3' Untranslated Regions/physiology , Porcine respiratory and reproductive syndrome virus/genetics , RNA, Viral/physiology , Virus Replication/genetics , Animals , Cell Line , Cricetinae , Nucleic Acid Conformation , Nucleocapsid/genetics , Open Reading Frames , Porcine respiratory and reproductive syndrome virus/physiology , Swine , Viral Matrix Proteins/genetics , Viral Nonstructural Proteins/geneticsABSTRACT
Alfalfa mosaic virus (AMV) and Prunus necrotic ringspot virus (PNRSV) belong to the genera ALFAMOVIRUS: and ILARVIRUS:, respectively, of the family BROMOVIRIDAE: Initiation of infection by AMV and PNRSV requires binding of a few molecules of coat protein (CP) to the 3' termini of the inoculum RNAs and the CPs of the two viruses are interchangeable in this early step of the replication cycle. CIS:-acting sequences in PNRSV RNA 3 that are recognized by the AMV replicase were studied in in vitro replicase assays and by inoculation of AMV-PNRSV RNA 3 chimeras to tobacco plants and protoplasts transformed with the AMV replicase genes (P12 plants). The results showed that the AMV replicase recognized the promoter for minus-strand RNA synthesis in PNRSV RNA 3 but not the promoter for plus-strand RNA synthesis. A chimeric RNA with PNRSV movement protein and CP genes accumulated in tobacco, which is a non-host for PNRSV.
