5' and 3' cis-Acting RNA Elements Required for RNA Replication of Porcine Reproductive and Respiratory Syndrome Virus

ABSTRACT Secondary and tertiary RNA structures present in viral RNA genomes play essential regulatory roles during translation, RNA replication, and assembly of new viral particles. In the case of flaviviruses, RNA-RNA interactions between the 5′ and 3′ ends of the genome have been proposed to be required for RNA replication. We found that two RNA elements present at the ends of the dengue virus genome interact in vitro with high affinity. Visualization of individual molecules by atomic force microscopy reveled that physical interaction between these RNA elements results in cyclization of the viral RNA. Using RNA binding assays, we found that the putative cyclization sequences, known as 5′ and 3′ CS, present in all mosquito-borne flaviviruses, were necessary but not sufficient for RNA-RNA interaction. Additional sequences present at the 5′ and 3′ untranslated regions of the viral RNA were also required for RNA-RNA complex formation. We named these sequences 5′ and 3′ UAR (upstream AUG region). In order to investigate the functional role of 5′-3′ UAR complementarity, these sequences were mutated either separately, to destroy base pairing, or simultaneously, to restore complementarity in the context of full-length dengue virus RNA. Nonviable viruses were recovered after transfection of dengue virus RNA carrying mutations either at the 5′ or 3′ UAR, while the RNA containing the compensatory mutations was able to replicate. Since sequence complementarity between the ends of the genome is required for dengue virus viability, we propose that cyclization of the RNA is a required conformation for viral replication.

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Genome-wide prediction of cis-acting RNA elements regulating tissue-specific pre-mRNA alternative splicing

BMC Genomics ◽

10.1186/1471-2164-10-s1-s4 ◽

2009 ◽

Vol 10 (Suppl 1) ◽

pp. S4 ◽

Cited By ~ 16

Author(s):

Xin Wang ◽

Kejun Wang ◽

Milan Radovich ◽

Yue Wang ◽

Guohua Wang ◽

...

Keyword(s):

Alternative Splicing ◽

Tissue Specific ◽

Cis Acting ◽

Genome Wide ◽

Rna Elements

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cis-Acting Sequences Required for Coronavirus Infectious Bronchitis Virus Defective-RNA Replication and Packaging

Journal of Virology ◽

10.1128/jvi.75.1.125-133.2001 ◽

2001 ◽

Vol 75 (1) ◽

pp. 125-133 ◽

Cited By ~ 49

Author(s):

Kevin Dalton ◽

Rosa Casais ◽

Kathy Shaw ◽

Kathleen Stirrups ◽

Sharon Evans ◽

...

Keyword(s):

Infectious Bronchitis Virus ◽

Rna Replication ◽

Replicase Gene ◽

Loop Structure ◽

Stem Loop ◽

Cis Acting ◽

Infectious Bronchitis ◽

Stem Loop Structure ◽

Defective Rna ◽

Region Ii

ABSTRACT The parts of the RNA genome of infectious bronchitis virus (IBV) required for replication and packaging of the RNA were investigated using deletion mutagenesis of a defective RNA (D-RNA) CD-61 (6.1 kb) containing a chloramphenicol acetyltransferase reporter gene. A D-RNA with the first 544, but not as few as 338, nucleotides (nt) of the 5′ terminus was replicated; the 5′ untranslated region (UTR) comprises 528 nt. Region I of the 3′ UTR, adjacent to the nucleocapsid protein gene, comprised 212 nt and could be removed without impairment of replication or packaging of D-RNAs. A D-RNA with the final 338 nt, including the 293 nt in the highly conserved region II of the 3′ UTR, was replicated. Thus, the 5′-terminal 544 nt and 3′-terminal 338 nt contained the necessary signals for RNA replication. Phylogenetic analysis of 19 strains of IBV and 3 strains of turkey coronavirus predicted a conserved stem-loop structure at the 5′ end of region II of the 3′ UTR. Removal of the predicted stem-loop structure abolished replication of the D-RNAs. D-RNAs in which replicase gene 1b-derived sequences had been removed or replaced with all the downstream genes were replicated well but were rescued poorly, suggesting inefficient packaging. However, no specific part of the 1b gene was required for efficient packaging.

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The 5′ Terminal Trailer Region of Vesicular Stomatitis Virus Contains a Position-Dependent cis-Acting Signal for Assembly of RNA into Infectious Particles

Journal of Virology ◽

10.1128/jvi.73.1.307-315.1999 ◽

1999 ◽

Vol 73 (1) ◽

pp. 307-315 ◽

Cited By ~ 33

Author(s):

Sean P. J. Whelan ◽

Gail W. Wertz

Keyword(s):

Vesicular Stomatitis Virus ◽

Rna Synthesis ◽

Rna Replication ◽

Genomic Rna ◽

Wild Type ◽

Rna Sequences ◽

Defective Interfering Particles ◽

Cis Acting ◽

Efficient Replication ◽

Vesicular Stomatitis

ABSTRACT The cis-acting genomic RNA requirements for the assembly of vesicular stomatitis virus (VSV) ribonucleocapsids into infectious particles were investigated. Using a biological assay based on particle infectivity, we demonstrated that subgenomic replicons that contained all four possible combinations of the natural genomic termini, the 3′ leader (Le) and 5′ trailer (Tr) regions, were replication competent; however, a 3′ copyback replicon (3′CB), containing the natural 3′ terminus but having the 5′ Tr replaced by a sequence complementary to the 3′ Le for 46 nucleotides, was unable to assemble infectious particles, despite efficient replication. When a copy of Tr was inserted 51 nucleotides from the 5′ end of 3′CB, infectious particles were produced. However, analysis of the replication products of these particles showed that the 51 nucleotides which corresponded to the Le complement sequences at the 5′ terminus were removed during RNA replication, thus restoring the wild-type 5′ Tr to the exact 5′ terminus. These data showed that acis-acting signal was necessary for assembly of VSV RNAs into infectious particles and that this signal was supplied by Tr when located at the 5′ end. The regions within Tr required for assembly were analyzed by a series of deletions and exchanges for Le complement sequences, which demonstrated that the 5′ terminal 29 nucleotides of Tr allowed assembly of infectious particles but that the 5′ terminal 22 nucleotides functioned poorly. Deletions in Tr also altered the balance between negative- and positive-strand genomic RNA and affected levels of replication. RNAs that retained fewer than 45 but at least 22 nucleotides of the 5′ terminus could replicate but were impaired in RNA replication, and RNAs that retained only 14 nucleotides of the 5′ terminus were severely reduced in ability to replicate. These data define the VSV Tr as a position-dependent, cis-acting element for the assembly of RNAs into infectious particles, and they delineate RNA sequences that are essential for negative-strand RNA synthesis. These observations are consistent with, and offer an explanation for, the absence of 3′ copyback defective interfering particles in nature.

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cis-acting lesions targeted to the hydrophobic domain of a poliovirus membrane protein involved in RNA replication.

Journal of Virology ◽

10.1128/jvi.66.10.6045-6057.1992 ◽

1992 ◽

Vol 66 (10) ◽

pp. 6045-6057 ◽

Cited By ~ 53

Author(s):

C Giachetti ◽

S S Hwang ◽

B L Semler

Keyword(s):

Membrane Protein ◽

Rna Replication ◽

Hydrophobic Domain ◽

Cis Acting

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A Comprehensive Analysis of cis-Acting RNA Elements in the SARS-CoV-2 Genome by a Bioinformatics Approach

Frontiers in Genetics ◽

10.3389/fgene.2020.572702 ◽

2020 ◽

Vol 11 ◽

Author(s):

Firoz Ahmed ◽

Monika Sharma ◽

Abdulsalam Abdullah Al-Ghamdi ◽

Sultan Muhammad Al-Yami ◽

Abdulaziz Musa Al-Salami ◽

...

Keyword(s):

Comparative Analysis ◽

Rna Structure ◽

Viral Genome ◽

Genomic Sequence ◽

Evolutionary Relationship ◽

Cis Acting ◽

Full Genomic Sequence ◽

Rna Elements ◽

Global Threat ◽

Genomic Regions

The emergence of a new coronavirus (CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for severe respiratory disease in humans termed coronavirus disease of 2019 (COVID-19), became a new global threat for health and the economy. The SARS-CoV-2 genome is about a 29,800-nucleotide-long plus-strand RNA that can form functionally important secondary and higher-order structures called cis-acting RNA elements. These elements can interact with viral proteins, host proteins, or other RNAs and be involved in regulating translation and replication processes of the viral genome and encapsidation of the virus. However, the cis-acting RNA elements and their biological roles in SARS-CoV-2 as well as their comparative analysis in the closely related viral genome have not been well explored, which is very important to understand the molecular mechanism of viral infection and pathogenies. In this study, we used a bioinformatics approach to identify the cis-acting RNA elements in the SARS-CoV-2 genome. Initially, we aligned the full genomic sequence of six different CoVs, and a phylogenetic analysis was performed to understand their evolutionary relationship. Next, we predicted the cis-acting RNA elements in the SARS-CoV-2 genome using the structRNAfinder tool. Then, we annotated the location of these cis-acting RNA elements in different genomic regions of SARS-CoV-2. After that, we analyzed the sequence conservation patterns of each cis-acting RNA element among the six CoVs. Finally, the presence of cis-acting RNA elements across different CoV genomes and their comparative analysis was performed. Our study identified 12 important cis-acting RNA elements in the SARS-CoV-2 genome; among them, Corona_FSE, Corona_pk3, and s2m are highly conserved across most of the studied CoVs, and Thr_leader, MAT2A_D, and MS2 are uniquely present in SARS-CoV-2. These RNA structure elements can be involved in viral translation, replication, and encapsidation and, therefore, can be potential targets for better treatment of COVID-19. It is imperative to further characterize these cis-acting RNA elements experimentally for a better mechanistic understanding of SARS-CoV-2 infection and therapeutic intervention.

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