scholarly journals In Vivo Addition of Poly(A) Tail and AU-Rich Sequences to the 3′ Terminus of the Sindbis Virus RNA Genome: a Novel 3′-End Repair Pathway

1999 ◽  
Vol 73 (3) ◽  
pp. 2410-2419 ◽  
Author(s):  
Ramaswamy Raju ◽  
Mustapha Hajjou ◽  
Kristie R. Hill ◽  
Vandana Botta ◽  
Sisir Botta
Keyword(s):  
Sindbis Virus ◽  
De Novo ◽  
Genome Replication ◽  
Sequence Motifs ◽  
Rna Sequences ◽  
Cytoplasmic Polyadenylation ◽  
Nontranslated Region ◽  
Rna Genome ◽  
Negative Sense

ABSTRACT Alphaviruses are mosquito-transmitted RNA viruses that cause important diseases in both humans and livestock. Sindbis virus (SIN), the type species of the alphavirus genus, carries a 11.7-kb positive-sense RNA genome which is capped at its 5′ end and polyadenylated at its 3′ end. The 3′ nontranslated region (3′NTR) of the SIN genome carries many AU-rich motifs, including a 19-nucleotide (nt) conserved element (3′CSE) and a poly(A) tail. This 3′CSE and the adjoining poly(A) tail are believed to regulate the synthesis of negative-sense RNA and genome replication in vivo. We have recently demonstrated that the SIN genome lacking the poly(A) tail was infectious and that de novo polyadenylation could occur in vivo (K. R. Hill, M. Hajjou, J. Hu, and R. Raju, J. Virol. 71:2693–2704, 1997). Here, we demonstrate that the 3′-terminal 29-nt region of the SIN genome carries a signal for possible cytoplasmic polyadenylation. To further investigate the polyadenylation signals within the 3′NTR, we generated a battery of mutant genomes with mutations in the 3′NTR and tested their ability to generate infectious virus and undergo 3′ polyadenylation in vivo. Engineered SIN genomes with terminal deletions within the 19-nt 3′CSE were infectious and regained their poly(A) tail. Also, a SIN genome carrying the poly(A) tail but lacking a part or the entire 19-nt 3′CSE was also infectious. Sequence analysis of viruses generated from these engineered SIN genomes demonstrated the addition of a variety of AU-rich sequence motifs just adjacent to the poly(A) tail. The addition of AU-rich motifs to the mutant SIN genomes appears to require the presence of a significant portion of the 3′NTR. These results indicate the ability of alphavirus RNAs to undergo 3′ repair and the existence of a pathway for the addition of AU-rich sequences and a poly(A) tail to their 3′ end in the infected host cell. Most importantly, these results indicate the ability of alphavirus replication machinery to use a multitude of AU-rich RNA sequences abutted by a poly(A) motif as promoters for negative-sense RNA synthesis and genome replication in vivo. The possible roles of cytoplasmic polyadenylation machinery, terminal transferase-like enzymes, and the viral polymerase in the terminal repair processes are discussed.

scholarly journals Alphavirus RNA Genome Repair and Evolution: Molecular Characterization of Infectious Sindbis Virus Isolates Lacking a Known Conserved Motif at the 3′ End of the Genome

2000 ◽  
Vol 74 (20) ◽  
pp. 9776-9785 ◽  
Author(s):  
Jyothi George ◽  
Ramaswamy Raju
Keyword(s):  
Sindbis Virus ◽  
Repeat Sequence ◽  
Genome Replication ◽  
Sequence Element ◽  
Conserved Sequence ◽  
Long Term Stability ◽  
Nontranslated Region ◽  
Viral Promoters ◽  
Sequence Elements

ABSTRACT The 3′ nontranslated region of the genomes of Sindbis virus (SIN) and other alphaviruses carries several repeat sequence elements (RSEs) as well as a 19-nucleotide (nt) conserved sequence element (3′CSE). The 3′CSE and the adjoining poly(A) tail of the SIN genome are thought to act as viral promoters for negative-sense RNA synthesis and genome replication. Eight different SIN isolates that carry altered 3′CSEs were studied in detail to evaluate the role of the 3′CSE in genome replication. The salient findings of this study as it applies to SIN infection of BHK cells are as follows: i) the classical 19-nt 3′CSE of the SIN genome is not essential for genome replication, long-term stability, or packaging; ii) compensatory amino acid or nucleotide changes within the SIN genomes are not required to counteract base changes in the 3′ terminal motifs of the SIN genome; iii) the 5′ 1-kb regions of all SIN genomes, regardless of the differences in 3′ terminal motifs, do not undergo any base changes even after 18 passages; iv) although extensive addition of AU-rich motifs occurs in the SIN genomes carrying defective 3′CSE, these are not essential for genome viability or function; and v) the newly added AU-rich motifs are composed predominantly of RSEs. These findings are consistent with the idea that the 3′ terminal AU-rich motifs of the SIN genomes do not bind directly to the viral polymerase and that cellular proteins with broad AU-rich binding specificity may mediate this interaction. In addition to the classical 3′CSE, other RNA motifs located elsewhere in the SIN genome must play a major role in template selection by the SIN RNA polymerase.

scholarly journals Selection of Functional 5′ cis-Acting Elements Promoting Efficient Sindbis Virus Genome Replication

2004 ◽  
Vol 78 (1) ◽  
pp. 61-75 ◽  
Author(s):  
Rodion Gorchakov ◽  
Richard Hardy ◽  
Charles M. Rice ◽  
Ilya Frolov
Keyword(s):  
Sindbis Virus ◽  
Semliki Forest Virus ◽  
Virus Genome ◽  
Genome Replication ◽  
Negative Strand ◽  
Cis Acting ◽  
Repeat Units ◽  
New Information ◽  
High Level

ABSTRACT The 5′ portion of the Sindbis virus (SIN) genome RNA is multifunctional. Besides initiating translation of the nonstructural polyprotein, RNA elements in the 5′ 200 bases of the SIN genome RNA, or its complement at the 3′ end of the negative-strand intermediate, play key roles in the synthesis of both negative- and positive-strand RNAs. We used here a combination of genetic and biochemical approaches to further dissect the functions of this sequence. Replacement of the SIN 5′ end in defective-interfering (DI) and genome RNAs with sequences from a distantly related alphavirus, Semliki Forest virus (SFV), resulted in nonviable chimeras. The addition of five nucleotides from the 5′ terminus of SIN restored negative-strand RNA synthesis in DI genomes but not their replication in vivo. Pseudorevertants of various SFV-SIN chimeras were isolated, and suppressor mutations were mapped to AU-rich sequences added to the 5′ end of the original SFV 5′ sequence or its “deleted” versions. Early pseudorevertants had heterogeneous 5′ termini that were inefficient for replication relative to the parental SIN 5′ sequence. In contrast, passaging of these pseudorevertant viral populations in BHK cells under competitive conditions yielded evolved, more homogeneous 5′-terminal sequences that were highly efficient for negative-strand synthesis and replication. These 5′-terminal sequences always began with 5′-AU, followed by one or more AU repeats or short stretches of oligo(A). Further analysis demonstrated a positive correlation between the number of repeat units and replication efficiency. Interestingly, some 5′ modifications restored high-level viral replication in BHK-21 cells, but these viruses were impaired for replication in the cells of mosquito origin. These studies provide new information on sequence determinants required for SIN RNA replication and suggest new strategies for restricting cell tropism and optimizing the packaging of alphavirus vectors.

scholarly journals Efficient Dicer processing of virus-derived double-stranded RNAs and its modulation by RIG-I-like receptor LGP2

2021 ◽  
Vol 17 (8) ◽  
pp. e1009790
Author(s):  
Yuqiang Zhang ◽  
Yan Xu ◽  
Yunpeng Dai ◽  
Zhe Li ◽  
Jiaxing Wang ◽  
...  
Keyword(s):  
Virus Infection ◽  
Sindbis Virus ◽  
De Novo ◽  
Somatic Cells ◽  
Interferon Response ◽  
Small Interfering Rnas ◽  
Rnai Suppressor ◽  
Antiviral Function

The interferon-regulated antiviral responses are essential for the induction of both innate and adaptive immunity in mammals. Production of virus-derived small-interfering RNAs (vsiRNAs) to restrict virus infection by RNA interference (RNAi) is a recently identified mammalian immune response to several RNA viruses, which cause important human diseases such as influenza and Zika virus. However, little is known about Dicer processing of viral double-stranded RNA replicative intermediates (dsRNA-vRIs) in mammalian somatic cells. Here we show that infected somatic cells produced more influenza vsiRNAs than cellular microRNAs when both were produced by human Dicer expressed de novo, indicating that dsRNA-vRIs are not poor Dicer substrates as previously proposed according to in vitro Dicer processing of synthetic long dsRNA. We report the first evidence both for canonical vsiRNA production during wild-type Nodamura virus infection and direct vsiRNA sequestration by its RNAi suppressor protein B2 in two strains of suckling mice. Moreover, Sindbis virus (SINV) accumulation in vivo was decreased by prior production of SINV-targeting vsiRNAs triggered by infection and increased by heterologous expression of B2 in cis from SINV genome, indicating an antiviral function for the induced RNAi response. These findings reveal that unlike artificial long dsRNA, dsRNA-vRIs made during authentic infection of mature somatic cells are efficiently processed by Dicer into vsiRNAs to direct antiviral RNAi. Interestingly, Dicer processing of dsRNA-vRIs into vsiRNAs was inhibited by LGP2 (laboratory of genetics and physiology 2), which was encoded by an interferon-stimulated gene (ISG) shown recently to inhibit Dicer processing of artificial long dsRNA in cell culture. Our work thus further suggests negative modulation of antiviral RNAi by a known ISG from the interferon response.

scholarly journals Parsimonious scenario for the emergence of viroid-like repliconsde novo

2019 ◽  
Author(s):  
Pablo Catalán ◽  
Santiago F. Elena ◽  
José A. Cuesta ◽  
Susanna Manrubia
Keyword(s):  
De Novo ◽  
Rna World ◽  
Circular Rna ◽  
Biochemical Properties ◽  
Circular Rnas ◽  
Sequence Motifs ◽  
Rna Sequences ◽  
Specific Sequence ◽  
Evolutionary Pathway ◽  
Rna Molecules

AbstractViroids are small, non-coding, circular RNA molecules that infect plants. Different hypotheses for their evolutionary origin have been put forward, such as an early emergence in a precellular RNA World or severalde novoindependent evolutionary origins in plants. Here we discuss the plausibility ofde novoemergence of viroid-like replicons by giving theoretical support to the likelihood of different steps along a parsimonious evolutionary pathway. While Avsunviroidae-like structures are relatively easy to obtain through evolution of a population of random RNA sequences of fixed length, rod-like structures typical of Pospiviroidae are difficult to fix. Using different quantitative approaches, we evaluate the likelihood that RNA sequences fold into a rod-like structure and bear specific sequence motifs facilitating interactions with other molecules,e.g.RNA polymerases, RNases and ligases. By means of numerical simulations, we show that circular RNA replicons analogous to Pospiviroidae emerge if evolution is seeded with minimal circular RNAs that grow through the gradual addition of nucleotides. Further, these rod-like replicons often maintain their structure if independent functional modules are acquired that impose selective constraints. The evolutionary scenario we propose here is consistent with the structural and biochemical properties of viroids described to date.

scholarly journals A convolutional neural network for the prediction and forward design of ribozyme-based gene-control elements

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Calvin M Schmidt ◽  
Christina D Smolke
Keyword(s):  
Machine Learning ◽  
Rational Design ◽  
De Novo ◽  
Regulation Of Gene Expression ◽  
Regulatory Activity ◽  
Rna Sequences ◽  
Genetic Switch ◽  
Gene Regulatory ◽  
Functional Rnas

Ribozyme switches are a class of RNA-encoded genetic switch that support conditional regulation of gene expression across diverse organisms. An improved elucidation of the relationships between sequence, structure, and activity can improve our capacity for de novo rational design of ribozyme switches. Here, we generated data on the activity of hundreds of thousands of ribozyme sequences. Using automated structural analysis and machine learning, we leveraged these large datasets to develop predictive models that estimate the in vivo gene-regulatory activity of a ribozyme sequence. These models supported the de novo design of ribozyme libraries with low mean basal gene-regulatory activities and new ribozyme switches that exhibit changes in gene-regulatory activity in the presence of a target ligand, producing functional switches for four out of five aptamers. Our work examines how biases in the model and the dataset that affect prediction accuracy can arise and demonstrates that machine learning can be applied to RNA sequences to predict gene-regulatory activity, providing the basis for design tools for functional RNAs.

scholarly journals Parsimonious Scenario for the Emergence of Viroid-Like Replicons De Novo

Viruses ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 425 ◽  
Author(s):  
Pablo Catalán ◽  
Santiago F. Elena ◽  
José A. Cuesta ◽  
Susanna Manrubia
Keyword(s):  
De Novo ◽  
Rna World ◽  
Circular Rna ◽  
Biochemical Properties ◽  
Circular Rnas ◽  
Sequence Motifs ◽  
Rna Sequences ◽  
Specific Sequence ◽  
Evolutionary Pathway ◽  
Rna Molecules

Viroids are small, non-coding, circular RNA molecules that infect plants. Different hypotheses for their evolutionary origin have been put forward, such as an early emergence in a precellular RNA World or several de novo independent evolutionary origins in plants. Here, we discuss the plausibility of de novo emergence of viroid-like replicons by giving theoretical support to the likelihood of different steps along a parsimonious evolutionary pathway. While Avsunviroidae-like structures are relatively easy to obtain through evolution of a population of random RNA sequences of fixed length, rod-like structures typical of Pospiviroidae are difficult to fix. Using different quantitative approaches, we evaluated the likelihood that RNA sequences fold into a rod-like structure and bear specific sequence motifs facilitating interactions with other molecules, e.g., RNA polymerases, RNases, and ligases. By means of numerical simulations, we show that circular RNA replicons analogous to Pospiviroidae emerge if evolution is seeded with minimal circular RNAs that grow through the gradual addition of nucleotides. Further, these rod-like replicons often maintain their structure if independent functional modules are acquired that impose selective constraints. The evolutionary scenario we propose here is consistent with the structural and biochemical properties of viroids described to date.

scholarly journals Initiation of Hepatitis Delta Virus Genome Replication

1998 ◽  
Vol 72 (6) ◽  
pp. 4783-4788 ◽  
Author(s):  
Kate Dingle ◽  
Vadim Bichko ◽  
Harmon Zuccola ◽  
James Hogle ◽  
John Taylor
Keyword(s):  
Hepatitis Delta Virus ◽  
Rna Synthesis ◽  
De Novo ◽  
Virus Genome ◽  
Northern Analysis ◽  
Genome Replication ◽  
Hepatitis Delta ◽  
Delta Virus

ABSTRACT The small, 195-amino-acid form of the hepatitis delta virus (HDV) antigen (δAg-S) is essential for genome replication, i.e., for the transcription, processing, and accumulation of HDV RNAs. To better understand this requirement, we used purified recombinant δAg-S and HDV RNA synthesized in vitro to assemble high-molecular-weight ribonucleoprotein (RNP) structures. After transfection of these RNPs into human cells, we detected HDV genome replication, as assayed by Northern analysis or immunofluorescence microscopy. Our interpretation is that the input δAg-S is necessary for the RNA to undergo limited amounts of RNA-directed RNA synthesis, RNA processing, and mRNA formation, leading to de novo translation of δAg-S. It is this second source of δAg-S which then goes on to support genome replication. This assay made it possible to manipulate in vitro the composition of the RNP and then test in vivo the ability of the complex to initiate RNA-directed RNA synthesis and go on to achieve genome replication. For example, both genomic and antigenomic linear RNAs were acceptable. Substitution for δAg-S with truncated or modified forms of the δAg, and even with HIV nucleocapsid protein and polylysine, was unacceptable; the exception was a form of δAg-S with six histidines added at the C terminus. We expect that further in vitro modifications of these RNP complexes should help define the in vivo requirements for what we define as the initiation of HDV genome replication.

scholarly journals Replication of the Human Hepatitis Delta Virus Genome Is Initiated in Mouse Hepatocytes following Intravenous Injection of Naked DNA or RNA Sequences

2001 ◽  
Vol 75 (7) ◽  
pp. 3469-3473 ◽  
Author(s):  
Jinhong Chang ◽  
Luis J. Sigal ◽  
Anthony Lerro ◽  
John Taylor
Keyword(s):  
Hepatitis Delta Virus ◽  
Virus Genome ◽  
In Vivo Studies ◽  
Genome Replication ◽  
Rna Sequences ◽  
Hepatitis Delta ◽  
Naked Dna ◽  
Delta Virus

ABSTRACT As early as 5 days after DNA copies of the hepatitis delta virus (HDV) genome or even in vitro-transcribed HDV RNA sequences were injected into the mouse tail vein using the hydrodynamics-based transfection procedure of F. Liu et al. (Gene Ther. 6:1258–1266, 1999), it was possible to detect in the liver by Northern analyses of RNA, immunoblots of protein, and immunostaining of liver sections what were considered typical features of HDV genome replication. This transfection strategy should have valuable applications for in vivo studies of HDV replication and pathogenesis and may also be useful for studies of other hepatotropic viruses.

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