Subgenomic mRNA

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Eric J. Snijder - One of the best experts on this subject based on the ideXlab platform.

  • nucleocapsid protein of equine arteritis virus
    2016
    Co-Authors: Marieke A. Tijms, Yvonne Van Der Meer, Eric J. Snijder
    Abstract:

    RNA synthesis (genome replication and Subgenomic mRNA transcription) directed by equine arteritis virus (EAV; family Arteriviridae, order Nidovirales) occurs on modified cytoplasmic membranes to which most viral replicase subunits localize. Re-markably, a fraction of non-structural protein 1 (nsp1), a protein essential for transcription but dis-pensable for genome replication, is present in the host cell nucleus, in particular during the earlier stagesof infection.ExpressionofGFP-tagged fusion proteins revealed that nsp1 is actively imported into the nucleus. Although the signals responsible for nsp1 transport could not be identified, our studies revealed that another EAV protein with a partially nuclear localization, the nucleocapsid (N

  • Arterivirus Subgenomic mRNA Synthesis and Virion Biogenesis Depend on the Multifunctional nsp1 Autoprotease
    Journal of virology, 2007
    Co-Authors: Marieke A. Tijms, Alexander E. Gorbalenya, Danny D. Nedialkova, Jessika C. Zevenhoven-dobbe, Eric J. Snijder
    Abstract:

    Many groups of plus-stranded RNA viruses produce additional, Subgenomic mRNAs to regulate the expression of part of their genome. Arteriviruses and coronaviruses (order Nidovirales) are unique among plusstranded RNA viruses for using a mechanism of discontinuous RNA synthesis to produce a nested set of 5- and 3-coterminal Subgenomic mRNAs, which serve to express the viral structural protein genes. The discontinuous step presumably occurs during minus-strand synthesis and joins noncontiguous sequences copied from the 3and 5-proximal domains of the genomic template. Nidovirus genome amplification (“replication”) and Subgenomic mRNA synthesis (“transcription”) are driven by 13 to 16 nonstructural proteins (nsp’s), generated by autocatalytic processing of two large “replicase” polyproteins. Previously, using a replicon system, the Nterminal nsp1 replicase subunit of the arterivirus equine arteritis virus (EAV) was found to be dispensable for replication but crucial for transcription. Using reverse genetics, we have now addressed the role of nsp1 against the background of the complete EAV life cycle. Mutagenesis revealed that nsp1 is in fact a multifunctional regulatory protein. Its papain-like autoprotease domain releases nsp1 from the replicase polyproteins, a cleavage essential for viral RNA synthesis. Several mutations in the putative N-terminal zinc finger domain of nsp1 selectively abolished transcription, while replication was either not affected or even increased. Other nsp1 mutations did not significantly affect either replication or transcription but still dramatically reduced the production of infectious progeny. Thus, nsp1 is involved in at least three consecutive key processes in the EAV life cycle: replicase polyprotein processing, transcription, and virion biogenesis.

  • equine arteritis virus non structural protein 1 an essential factor for viral Subgenomic mRNA synthesis interacts with the cellular transcription co factor p100
    Journal of General Virology, 2003
    Co-Authors: Marieke A. Tijms, Eric J. Snijder
    Abstract:

    Non-structural protein 1 (nsp1), the N-terminal subunit of the replicase polyprotein of the arterivirus Equine arteritis virus (EAV), is essential for viral Subgenomic mRNA synthesis, but fully dispensable for genome replication. However, at the molecular level, the role of nsp1 in EAV Subgenomic mRNA synthesis is poorly understood. A yeast two-hybrid screen did not reveal interactions between EAV nsp1 and other viral non-structural proteins or the nucleocapsid protein, although both nsp1 and the nucleocapsid protein were found to form homomers. Subsequently, a yeast two-hybrid screen of a HeLa cell cDNA library was performed using nsp1 as bait. Remarkably, this analysis revealed (potential) interactions between EAV nsp1 and factors that are involved in host cell transcriptional regulation. The interaction of nsp1 with one of these proteins, p100, a transcription co-activator that also interacts with regulatory proteins of other viruses, was confirmed by mutual co-immunoprecipitation from lysates of EAV-susceptible mammalian cells.

  • The stability of the duplex between sense and antisense transcription-regulating sequences is a crucial factor in arterivirus Subgenomic mRNA synthesis.
    Journal of virology, 2003
    Co-Authors: Alexander O. Pasternak, Willy J. M. Spaan, Erwin Van Den Born, Eric J. Snijder
    Abstract:

    Subgenomic mRNAs of nidoviruses (arteriviruses and coronaviruses) are composed of a common leader sequence and a "body" part of variable size, which are derived from the 5'- and 3'-proximal part of the genome, respectively. Leader-to-body joining has been proposed to occur during minus-strand RNA synthesis and to involve transfer of the nascent RNA strand from one site in the template to another. This discontinuous step in Subgenomic RNA synthesis is guided by short transcription-regulating sequences (TRSs) that are present at both these template sites (leader TRS and body TRS). Sense-antisense base pairing between the leader TRS in the plus strand and the body TRS complement in the minus strand is crucial for strand transfer. Here we show that extending the leader TRS-body TRS duplex beyond its wild-type length dramatically enhanced the Subgenomic mRNA synthesis of the arterivirus Equine arteritis virus (EAV). Generally, the relative amount of a Subgenomic mRNA correlated with the calculated stability of the corresponding leader TRS-body TRS duplex. In addition, various leader TRS mutations induced the generation of minor Subgenomic RNA species that were not detected upon infection with wild-type EAV. The synthesis of these RNA species involved leader-body junction events at sites that bear only limited resemblance to the canonical TRS. However, with the mutant leader TRS, but not with the wild-type leader TRS, these sequences could form a duplex that was stable enough to direct Subgenomic RNA synthesis, again demonstrating that the stability of the leader TRS-body TRS duplex is a crucial factor in arterivirus Subgenomic mRNA synthesis.

  • A zinc finger-containing papain-like protease couples Subgenomic mRNA synthesis to genome translation in a positive-stranded RNA virus.
    Proceedings of the National Academy of Sciences of the United States of America, 2001
    Co-Authors: Marieke A. Tijms, Leonie C. Van Dinten, Alexander E. Gorbalenya, Eric J. Snijder
    Abstract:

    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.

Henry V. Huang - One of the best experts on this subject based on the ideXlab platform.

  • Sequence Requirements for Sindbis Virus Subgenomic mRNA Promoter Function in Cultured Cells
    Journal of virology, 2001
    Co-Authors: Matthew M. Wielgosz, Ramaswamy Raju, Henry V. Huang
    Abstract:

    The Sindbis virus minimal Subgenomic mRNA promoter (spanning positions 219 to 15 relative to the Subgenomic mRNA start site) is approximately three- to sixfold less active than the fully active 298 to 114 promoter region. We identified two elements flanking the 219 to 15 region which increase its transcription to levels comparable to the 298 to 114 region. These elements span positions 240 to 220 and 1 6t o114 and act synergistically to enhance transcription. Nine different virus libraries were constructed containing blocks of five randomized nucleotides at various positions in the 240 to 114 region. On passaging these libraries in mosquito cells, a small subset of the viruses came to dominate the population. Sequence analysis at the population level and for individual clones revealed that in general, wild-type bases were preferred for positions 215 to 15 of the minimal promoter. Base mutagenesis experiments indicated that the selection of wild-type bases in this region was primarily due to requirements for Subgenomic mRNA transcription. Outside of the minimal promoter, the 235 to 229 region contained four positions which also preferred wildtype bases. However, the remaining positions generally preferred non-wild-type bases. On passaging of the virus libraries on hamster cells, the 215 to 15 region again preferred the wild-type base but most of the remaining positions exhibited almost no base preference. The promoter thus consists of an essential central region from 215 to 15 and discrete flanking sites that render it fully active, depending on the host environment.

  • A novel viral RNA species in Sindbis virus-infected cells.
    Journal of virology, 1997
    Co-Authors: Matthew M. Wielgosz, Henry V. Huang
    Abstract:

    Sindbis virus (SIN), the type alphavirus, has been studied extensively to identify the viral cis-acting sequences and proteins involved in RNA transcription and replication. However, very little is known about how these processes are coordinated. For example, synthesis of the genomic RNA and the Subgenomic mRNA depends on the minus strand. Do these activities occur independently on different templates, or can replication and transcription take place simultaneously on the same template? We describe the appearance of a SIN-specific, plus-sense RNA that is intermediate in size between the genomic and Subgenomic RNA species. This RNA, designated RNA II, is observed in a number of different cell lines, both early and late in infection. The number of RNA II species, their sizes, and their abundances are influenced by the Subgenomic promoter. We have mapped the 3' end of RNA II to a site within the Subgenomic promoter, four nucleotides before the initiation site of the Subgenomic mRNA. Our results indicate that the appearance of RNA II is correlated with Subgenomic mRNA transcription, such that strong or active promoters tend to increase the abundance of RNA II, relative to weak or less active promoters. RNA II is most abundantly detected with the full promoter and is at much lower abundance with the minimal promoter. The possible origins of RNA II are discussed.

  • Evolution of the Sindbis virus Subgenomic mRNA promoter in cultured cells.
    Journal of virology, 1995
    Co-Authors: J M Hertz, Henry V. Huang
    Abstract:

    Transcription of the Subgenomic mRNA of alphaviruses initiates at an internal site, called the promoter, which is highly conserved. To determine the functional significance of this conservation, we used an approach that randomizes positions -13 to -9 of the promoter to generate a library containing all possible sequences within this region, including the wild-type sequence. Viruses in the mixed population with more-efficient promoters were selected for during passaging in mammalian (BHK-21) cells. Results from early passage populations indicate that a large number of different promoters are functionally active. Analysis of eight individual viruses found that although each contained a promoter with different degrees of sequence identity to the wild-type sequence, all eight viruses produced progeny. This suggests that the mechanism for transcription allows for a diversity of sequences to serve as promoters. Further passaging of the viral library led to a population consensus sequence that increasingly resembled the wild-type sequence, despite the fact that these promoters are not constrained by the need to encode the carboxyl terminus of the nsP4 protein. Thus, conservation of the region of the promoter from -13 to -9 is in large part due to selection for promoter function, and the wild-type sequence and sequences closely similar to it seem to be optimal for promoter function in BHK-21 cells.

  • Host-dependent evolution of the Sindbis virus promoter for Subgenomic mRNA synthesis.
    Journal of virology, 1995
    Co-Authors: J M Hertz, Henry V. Huang
    Abstract:

    Alphaviruses are alternately transmitted between arthropod and vertebrate hosts. In each host, the virus transcribes a Subgenomic mRNA that encodes the viral structural proteins which encapsidate the genome to form progeny virions. Transcription initiates at an internal site called the promoter. To determine if promoter utilization varies in mammalian versus mosquito cells, we used these cells as hosts to select for active promoters among a library of different mutant promoters. Compared with that in BHK-21 cells, selection was more rapid in mosquito (C7-10) cells, with much less diversity of promoters remaining after fewer passages. Thus, promoter selection is host dependent. With further passaging, both BHK-21 and C7-10 cells selected for similar sequences that closely resemble the wild-type promoter sequence. The difference in the rates of selection is not because BHK-21-derived promoters cannot function in mosquito cells. Instead, part of the host dependence is probably due to posttranscriptional differences between BHK-21 and C7-10 cells that may require more active promoters in mosquito cells. Part of the host dependence may also be attributed to the decreased rate of transcription versus that of replication in mosquito cells. This change in regulation of Subgenomic to genomic RNA synthesis appears to correlate with the extent of cleavage or pausing of the genomic RNA synthesis at or close to the promoter.

  • Analysis of Sindbis virus promoter recognition in vivo, using novel vectors with two Subgenomic mRNA promoters.
    Journal of virology, 1991
    Co-Authors: Ramaswamy Raju, Henry V. Huang
    Abstract:

    Four types of Sindbis virus vectors, each carrying two promoters for Subgenomic mRNA synthesis, were designed to measure relative promoter strengths and to survey potential contextual effects on promoter strengths. One of the promoters in each vector was used as the reference promoter, while the other was the one being tested. We used these vectors to measure the relative strengths of four promoters: the minimal promoter, an extended sequence believed to have full promoter activity, and two mutant promoters, one with an inactivating 3-nucleotide insertion called CR4.1 and the other with a 4-nucleotide deletion called delta 4. The strengths of the promoters were measured by quantitating the RNA transcribed from each promoter in vivo and also by assaying for chloramphenicol acetyltransferase activity encoded by one of the two transcripts. We found that the relative strengths of the promoters were similar in different contexts. The complete promoter was 6-fold more active, the delta 4 promoter was (surprisingly) about twice as active, and the CR4.1 promoter was 100-fold less active than the minimal promoter. At least two contextual effects were identified that can alter the activity of one or both promoters in the vectors. One effect is that given identical promoters, the 3'-proximal promoter on the minus-strand template can be more active than the 5'-proximal promoter. This may be due to preferential association of one or more components of the transcription complex for the 3' end of the minus-strand template. A second effect is promoter competition, particularly when the promoters are closely spaced.

Michael M C Lai - One of the best experts on this subject based on the ideXlab platform.

  • Transcription of Subgenomic mRNA of Hepatitis Delta Virus Requires a Modified Hepatitis Delta Antigen That Is Distinct from Antigenomic RNA Synthesis
    Journal of virology, 2008
    Co-Authors: Chung-hsin Tseng, King-song Jeng, Michael M C Lai
    Abstract:

    Hepatitis delta virus (HDV) contains a viroid-like, 1.7-kb circular RNA genome, which replicates via a double-rolling-circle model. However, the exact mechanism involved in HDV genome RNA replication and Subgenomic mRNA transcription is still unclear. Our previous studies have shown that the replications of genomic and antigenomic HDV RNA strands have different sensitivities to alpha-amanitin and are associated with different nuclear bodies, suggesting that these two strands are synthesized in different transcription machineries in the cells. In this study, we developed a unique quantitative reverse transcription-PCR (qRT-PCR) procedure for detection of various HDV RNA species from an RNA transfection system. Using this qRT-PCR procedure and a series of HDV mutants, we demonstrated that Arg-13 methylation, Lys-72 acetylation, and Ser-177 phosphorylation of small hepatitis delta antigen (S-HDAg) are important for HDV mRNA transcription. In addition, these three S-HDAg modifications are dispensable for antigenomic RNA synthesis but are required for genomic RNA synthesis. Furthermore, the three RNA species had different sensitivities to acetylation and deacetylation inhibitors, showing that the metabolic requirements for the synthesis of HDV antigenomic RNA are different from those for the synthesis of genomic RNA and mRNA. In sum, our data support the hypothesis that the cellular machinery involved in the synthesis of HDV antigenomic RNA is different from that of genomic RNA synthesis and mRNA transcription, even though the antigenomic RNA and the mRNA are made from the same RNA template. We propose that acetylation and deacetylation of HDAg may provide a molecular switch for the synthesis of the different HDV RNA species.

  • Polypyrimidine Tract-Binding Protein Binds to the Complementary Strand of the Mouse Hepatitis Virus 3′ Untranslated Region, Thereby Altering RNA Conformation
    Journal of virology, 1999
    Co-Authors: Peiyong Huang, Michael M C Lai
    Abstract:

    Mouse hepatitis virus (MHV) RNA transcription is regulated mainly by the leader and intergenic (IG) sequences. However, a previous study has shown that the 3′ untranslated region (3′-UTR) of the viral genome is also required for Subgenomic mRNA transcription; deletion of nucleotides (nt) 270 to 305 from the 3′-UTR completely abolished Subgenomic mRNA transcription without affecting minus-strand RNA synthesis (Y.-J. Lin, X. Zhang, R.-C. Wu, and M. M. C. Lai, J. Virol. 70:7236–7240, 1996), suggesting that the 3′-UTR affects positive-strand RNA synthesis. In this study, by UV-cross-linking experiments, we found that several cellular proteins bind specifically to the minus-strand 350 nucleotides complementary to the 3′-UTR of the viral genome. The major protein species, p55, was identified as the polypyrimidine tract-binding protein (PTB, also known as heterogeneous nuclear RNP I) by immunoprecipitation of the UV-cross-linked protein and binding of the recombinant PTB. A strong PTB-binding site was mapped to nt 53 to 149, and another weak binding site was mapped to nt 270 to 307 on the complementary strand of the 3′-UTR (c3′-UTR). Partial substitutions of the PTB-binding nucleotides reduced PTB binding in vitro. Furthermore, defective interfering (DI) RNAs harboring these mutations showed a substantially reduced ability to synthesize Subgenomic mRNA. By enzymatic and chemical probing, we found that PTB binding to nt 53 to 149 caused a conformational change in the neighboring RNA region. Partial deletions within the PTB-binding sequence completely abolished the PTB-induced conformational change in the mutant RNA even when the RNA retained partial PTB-binding activity. Correspondingly, the MHV DI RNAs containing these deletions completely lost their ability to transcribe mRNAs. Thus, the conformational change in the c3′-UTR caused by PTB binding may play a role in mRNA transcription.

  • The 3' untranslated region of coronavirus RNA is required for Subgenomic mRNA transcription from a defective interfering RNA.
    Journal of virology, 1996
    Co-Authors: Yi Jyun Lin, Xuming Zhang, Michael M C Lai
    Abstract:

    The 3'-end of mouse hepatitis virus (MHV) genomic RNA contains a recognition sequence (55 nucleotides [nt]) required for minus-strand RNA synthesis. To determine whether the 3'-end sequence is also involved in Subgenomic mRNA transcription, we have constructed MHV defective interfering (DI) RNAs which contain a chloramphenicol acetyltransferase (CAT) gene placed behind an intergenic sequence and a 3'-end sequence with various degrees of internal deletions. The DI RNAs were transfected into MHV-infected cells, and CAT activities, which represent Subgenomic mRNA transcription from the intergenic site, were determined. The results demonstrated that the deletions of sequence upstream of the 350 nt at the 3'-end, which include the 3'-untranslated region (3'-UTR), of MHV genomic RNA did not affect Subgenomic mRNA transcription. However, deletions that reduced the 3'-end sequences to 270 nt or less completely abolished the mRNA transcription despite the fact that all of these clones synthesized minus-strand RNAs. These results indicated that mRNA transcription from an intergenic site in the MHV DI RNA requires most of the 3'-UTR as a cis-acting signal, which likely exerts its effects during plus-strand RNA synthesis. A substitution of the corresponding bovine coronavirus sequence for the MHV sequence within nt 270 to 305 from the 3'-end abrogated the CAT gene expression, suggesting a very rigid sequence requirement in this region. The deletion of a putative pseudoknot structure within the 3'-UTR also abolished the CAT gene expression. These findings suggest that the 3'-UTR may interact with the other RNA regulatory elements to regulate mRNA transcription.

  • Requirement of the 5'-end genomic sequence as an upstream cis-acting element for coronavirus Subgenomic mRNA transcription.
    Journal of virology, 1994
    Co-Authors: Ching-len Liao, Michael M C Lai
    Abstract:

    We have developed a defective interfering (DI) RNA containing a chloramphenicol acetyltransferase reporter gene, placed behind an intergenic sequence, for studying Subgenomic mRNA transcription of mouse hepatitis virus (MHV), a prototype coronavirus. Using this system, we have identified the sequence requirement for MHV Subgenomic mRNA transcription. We show that this sequence requirement differs from that for RNA replication. In addition to the previously identified requirement for an intergenic (promoter) sequence, additional sequences from the 5' end of genomic RNA are required for Subgenomic mRNA transcription. These upstream sequences include the leader RNA and a spacer sequence between the leader and intergenic sequence, which is derived from the 5' untranslated region and part of gene 1. The spacer sequence requirement is specific, since only the sequence derived from the 5' end of RNA genome, but not from other MHV genomic regions or heterologous sequences, could initiate Subgenomic transcription from the intergenic sequence. These results strongly suggest that the wild-type viral Subgenomic mRNAs (mRNA2 to mRNA7) and probably their counterpart Subgenomic negative-sense RNAs cannot be utilized for mRNA amplification. Furthermore, we have demonstrated that a partial leader sequence present at the 5' end of genome, which lacks the leader-mRNA fusion sequence, could still support Subgenomic mRNA transcription. In this case, the leader sequences of the Subgenomic transcripts were derived exclusively from the wild-type helper virus, indicating that the MHV leader RNA initiates in trans Subgenomic mRNA transcription. Thus, the leader sequence can enhance Subgenomic transcription even when it cannot serve as a primer for mRNA synthesis. These results taken together suggest that the 5'-end leader sequence of MHV not only provides a trans-acting primer for mRNA initiation but also serves as a cis-acting element required for the transcription of Subgenomic mRNAs. The identification of an upstream cis-acting element for MHV Subgenomic mRNA synthesis defines a novel sequence requirement for regulating mRNA synthesis in RNA viruses.

  • Coronavirus leader RNA regulates and initiates Subgenomic mRNA transcription both in trans and in cis.
    Journal of virology, 1994
    Co-Authors: Xuming Zhang, Ching-len Liao, Michael M C Lai
    Abstract:

    Mouse hepatitis virus (MHV), a coronavirus, utilizes a discontinuous transcription mechanism for Subgenomic mRNA synthesis. Previous studies (C.-L. Liao and M. C. C. Lai, J. Virol. 68:4727-4737, 1994) have demonstrated that an upstream cis-acting leader sequence serves as a transcriptional enhancer, but the mechanism of transcriptional regulation is not clear. In this study, we constructed a series of defective interfering (DI) RNAs containing the chloramphenicol acetyltransferase (CAT) gene behind a differentially expressed transcription initiation (intergenic) sequence (for mRNA2-1). These DI RNAs had different copy numbers of the UCUAA pentanucleotide sequence at the 3' end of the leader. Transfection of these DI RNA constructs into cells infected with a helper MHV, which contains either two or three UCUAA copies at the 3' end of the leader, resulted in differential expression of CAT activities. We demonstrated that the copy number of UCUAA repeats in the leaders of both helper viral and DI RNAs affected the level of CAT activity, suggesting that MHV leader RNA could regulate both in trans and in cis the transcription of Subgenomic mRNAs. The leader RNA of Subgenomic mRNAs was derived from either the trans- or the cis-acting leader. Furthermore, insertion of a UA-rich sequence (UUUAUAAAC) immediately downstream of the leader in DI RNA, to match the sequence of helper viral RNA, enhanced the CAT activity by threefold, suggesting that this nine-nucleotide sequence is a cis-acting element. Interestingly, when the nine-nucleotide sequence was absent in DI RNA, the leaders of Subgenomic mRNAs were exclusively derived from the helper virus. In contrast, when the nine-nucleotide sequence was present in DI RNA, the leaders were derived from both helper viral and DI RNAs. These results suggest that the nine-nucleotide sequence either is required for the leader RNA to initiate mRNA synthesis or, alternatively, serves as a transcription terminator for the leader RNA synthesis. However, when a constitutively expressed intergenic sequence (for mRNA7) was used, no difference in transcription efficiency was noted, regardless of the copy number of UCUAA in the DI RNA and helper virus. This study thus indicates that MHV Subgenomic RNA transcription requires the interaction among the intergenic (promoter) sequence, a trans-acting leader, and a cis-acting leader sequence. A novel model of transcriptional regulation of coronavirus Subgenomic mRNAs is presented.

K A White - One of the best experts on this subject based on the ideXlab platform.

  • Activation of viral transcription by stepwise largescale folding of an RNA virus genome.
    Nucleic acids research, 2020
    Co-Authors: Tamari Chkuaseli, K A White
    Abstract:

    The genomes of RNA viruses contain regulatory elements of varying complexity. Many plus-strand RNA viruses employ largescale intra-genomic RNA-RNA interactions as a means to control viral processes. Here, we describe an elaborate RNA structure formed by multiple distant regions in a tombusvirus genome that activates transcription of a viral Subgenomic mRNA. The initial step in assembly of this intramolecular RNA complex involves the folding of a large viral RNA domain, which generates a discontinuous binding pocket. Next, a distally-located protracted stem-loop RNA structure docks, via base-pairing, into the binding site and acts as a linchpin that stabilizes the RNA complex and activates transcription. A multi-step RNA folding pathway is proposed in which rate-limiting steps contribute to a delay in transcription of the capsid protein-encoding viral Subgenomic mRNA. This study provides an exceptional example of the complexity of genome-scale viral regulation and offers new insights into the assembly schemes utilized by large intra-genomic RNA structures.

  • Intragenomic Long-Distance RNA-RNA Interactions in Plus-Strand RNA Plant Viruses.
    Frontiers in Microbiology, 2018
    Co-Authors: T Chkuaseli, K A White
    Abstract:

    Plant viruses that contain positive-strand RNA genomes represent an important class of pathogen. The genomes of these viruses harbor RNA sequences and higher-order RNA structures that are essential for the regulation of viral processes during infections. In recent years, it has become increasingly evident that, in addition to locally positioned RNA structures, long-distance intragenomic interactions, involving nucleotide base pairing over large distances, also contribute significantly to the control of various viral events. Viral processes that are modulated by such interactions include genome replication, translation initiation, translational recoding, and Subgenomic mRNA transcription. Here, we review the structure and function of different types of long-distance RNA-RNA interactions, herein termed LDRIs, present in members of the family Tombusviridae and other plus-strand RNA plant viruses.

  • Subgenomic mRNA transcription in tobacco necrosis virus.
    Virology, 2011
    Co-Authors: S D Jiwan, Baodong Wu, K A White
    Abstract:

    Abstract Tobacco necrosis virus-D (TNV-D), a positive-strand RNA Necrovirus in the family Tombusviridae , transcribes two Subgenomic (sg) mRNAs during infections. We have investigated the strategy used by TNV-D in this process and uncovered evidence that it employs a premature termination (PT) mechanism for the transcription of its sg mRNAs. Structural and mutational analysis of the TNV-D genome identified local RNA structures upstream from transcriptional initiation sites that functioned in the plus-strand as attenuation structures and mediated the production of sg mRNA-sized minus-strands. Other evidence in support of a PT mechanism included the ability to uncouple minus-strand sg RNA production from plus-strand sg mRNA synthesis and the sequence similarities observed between the sg mRNA promoter and that for the viral genome. Accordingly, our results indicate that the necrovirus TNV-D, like several other genera in the family Tombusviridae , uses a PT mechanism for transcription of its sg mRNAs.

  • Subgenomic mRNA transcription in tombusviridae
    RNA Biology, 2011
    Co-Authors: S D Jiwan, K A White
    Abstract:

    Members of the large virus family Tombusviridae are plus-strand RNA viruses that infect a wide variety of plant hosts. As part of their gene expression strategy, these viruses transcribe viral Subgenomic (sg) mRNAs during infections. These short viral messages are synthesized so as to allow for efficient translation of a subset of viral genes. Over the past decade, evidence has been mounting that different members of this virus family utilize a premature termination mechanism for transcription of their sg mRNAs. Here, we describe what is currently known about this unique transcriptional process as gleaned from studies on members of Tombusviridae.

  • Subgenomic mRNA transcription in an aureusvirus: down-regulation of transcription and evolution of regulatory RNA elements.
    Virology, 2007
    Co-Authors: Wei Xu, K A White
    Abstract:

    Abstract The genus Aureusvirus is composed of a group of positive-strand RNA plant viruses that belong to the family Tombusviridae . Expression of certain aureusvirus genes requires the transcription of two Subgenomic (sg) mRNAs. Interestingly, the level of sg mRNA2 accumulation in aureusvirus infections is considerably lower than that of sg mRNA1. The nature of this difference was investigated using the aureusvirus Cucumber leaf spot virus (CLSV). Analysis of sg mRNA2 transcription indicated that it is synthesized by a premature termination mechanism. The results also implicated the transcriptional promoter, the attenuation signal, and global RNA folding of the viral genome as mediators of sg mRNA2 suppression. Additionally, evaluation of the transcriptional regulatory RNA elements in aureusviruses and related tombusviruses revealed alternative strategies for building functionally-equivalent stem-loop structures and showed that sequences encoding a critical and invariant amino acid can be successfully incorporated into essential long-distance tertiary RNA–RNA interactions.

David A Brian - One of the best experts on this subject based on the ideXlab platform.

  • 5′-Proximal Hot Spot for an Inducible Positive-to-Negative-Strand Template Switch by Coronavirus RNA-Dependent RNA Polymerase
    Journal of virology, 2007
    Co-Authors: David A Brian
    Abstract:

    Coronaviruses have a positive-strand RNA genome and replicate through the use of a 3' nested set of Subgenomic mRNAs each possessing a leader (65 to 90 nucleotides [nt] in length, depending on the viral species) identical to and derived from the genomic leader. One widely supported model for leader acquisition states that a template switch takes place during the generation of negative-strand antileader-containing templates used subsequently for Subgenomic mRNA synthesis. In this process, the switch is largely driven by canonical heptameric donor sequences at intergenic sites on the genome that match an acceptor sequence at the 3' end of the genomic leader. With experimentally placed 22-nt-long donor sequences within a bovine coronavirus defective interfering (DI) RNA we have shown that matching sites occurring anywhere within a 65-nt-wide 5'-proximal genomic acceptor hot spot (nt 33 through 97) can be used for production of templates for Subgenomic mRNA synthesis from the DI RNA. Here we report that with the same experimental approach, template switches can be induced in trans from an internal site in the DI RNA to the negative-strand antigenome of the helper virus. For these, a 3'-proximal 89-nt acceptor hot spot on the viral antigenome (nt 35 through 123), largely complementary to that described above, was found. Molecules resulting from these switches were not templates for Subgenomic mRNA synthesis but, rather, ambisense chimeras potentially exceeding the viral genome in length. The results suggest the existence of a coronavirus 5'-proximal partially double-stranded template switch-facilitating structure of discrete width that contains both the viral genome and antigenome.

  • downstream sequences influence the choice between a naturally occurring noncanonical and closely positioned upstream canonical heptameric fusion motif during bovine coronavirus Subgenomic mRNA synthesis
    Journal of Virology, 2001
    Co-Authors: Aykut Ozdarendeli, Sylvie Rochat, Gwyn D Williams, Savithra D Senanayake, David A Brian
    Abstract:

    Mechanisms leading to Subgenomic mRNA (sgmRNA) synthesis in coronaviruses are poorly understood but are known to involve a heptameric signaling motif, originally called the intergenic sequence. The intergenic sequence is the presumed crossover region (fusion site) for RNA-dependent RNA polymerase (RdRp) during discontinuous transcription, a process leading to sgmRNAs that are both 5′ and 3′ coterminal. In the bovine coronavirus, the major fusion site for synthesis of mRNA 5 (GGUAGAC) does not conform to the canonical motif (UC[U,C]AAAC) at three positions (underlined), yet it lies just 14 nucleotides downstream from such a sequence (UCCAAAC). The infrequently used canonical sequence, by computer prediction, is buried within the stem of a stable hairpin (−17.2 kcal/mol). Here we document the existence of this stem by enzyme probing and examine its influence and that of neighboring sequences on the unusual choice of fusion sites by analyzing transcripts made in vivo from mutated defective interfering RNA constructs. We learned that (i) mutations that were predicted to unfold the stem-loop in various ways did not switch RdRp crossover to the upstream canonical site, (ii) a totally nonconforming downstream motif resulted in no measurable transcription from either site, (iii) the canonical upstream site does not function ectopically to lend competence to the downstream noncanonical site, and (iv) altering flanking sequences downstream of the downstream noncanonical motif in ways that diminish sequence similarity with the virus genome 5′ end caused a dramatic switch to the upstream canonical site. These results show that sequence elements downstream of the noncanonical site can dramatically influence the choice of fusion sites for synthesis of mRNA 5 and are interpreted as being most consistent with a mechanism of similarity-assisted RdRp strand switching during minus-strand synthesis.

  • nidovirus genome replication and Subgenomic mRNA synthesis pathways followed and cis acting elements required
    Advances in Experimental Medicine and Biology, 2001
    Co-Authors: David A Brian
    Abstract:

    Nidoviruses, specifically coronaviruses and arteriviruses, are recognized as unique among RNA viruses for utilizating a discontinuous transcription step during sgmRNA synthesis. Details of this pathway and of genome replication remain enigmatic, and identification of cis-acting elements and trans-acting factors involved is far from complete. The apparent absence of a common leader in toroviruses and differences in genome size among the viruses hint that fundamental differences in pathways of RNA synthesis might still be revealed. What are the current views?

  • Tandem Placement of a Coronavirus Promoter Results in Enhanced mRNA Synthesis from the Downstream-Most Initiation Site
    Virology, 1996
    Co-Authors: Rajesh Krishnan, Rueyyi Chang, David A Brian
    Abstract:

    Insertion of the 17-nucleotide promoter region for the bovine coronavirus N gene as part of a 27-nucleotide cassette into the open reading frame of a cloned synthetic defective-interfering (DI) RNA resulted in synthesis of subDI RNA transcripts from the replicating DI RNA genome. Duplicating and triplicating the promoter sequence in tandem caused a progressive increase in the efficiency of Subgenomic mRNA synthesis despite a concurrent decrease in the rate of DI RNA accumulation that was not specific to the promoter sequences being added. Although initiation of transcription (leader fusion) occurred at each of the three promoter sites in the tandem construct, almost all of the transcripts were found as a product of the most downstream (3'-most on the genome) promoter. These results show that enhancement of Subgenomic mRNA synthesis is a property that can reside within sequence situated near the promoter. A possible role for the plus strand in the downstream promoter choice is suggested.

  • leader mRNA junction sequences are unique for each Subgenomic mRNA species in the bovine coronavirus and remain so throughout persistent infection
    Virology, 1993
    Co-Authors: Martin A Hofmann, Rueyyi Chang, David A Brian
    Abstract:

    Abstract The common leader sequence on bovine coronavirus Subgenomic mRNAs and genome was determined. To examine leader-mRNA junction sequences on Subgenomic mRNAs, specific oligodeoxynucleotide sets were used in a polymerase chain reaction to amplify junction sequences from either the positive-strand mRNA (eight of nine total identified species) or the negative-strand anti-mRNA (six of the nine species), and sequenced. The mRNA species studied were those for the N, M, S, and HE structural proteins and the 9.5-, 12.7-, 4.8-, and 4.9-kDa putative nonstructural proteins. By defining the leader-mRNA junction sequence as the sequence between (i) the point of mismatch between the leader and genome and (ii) the 3′ end of the consensus heptameric intergenic sequence [(U/A)C(U/C)AAAC)], or its variant, a unique junction sequence was found for each Subgenomic mRNA species studied. In one instance (mRNA for the 12.7-kDa protein) the predicted intergenic sequence UCCAAAC was not part of the junction region, and in its place was the nonconforming sequence GGTAGAC that occurs just 15 nt downstream in the genome. Leader-mRNA junction sequences found after 296 days of persistent infection were the same as those found during acute infection (