sgRNA Promoter Research Articles

Efficient gene editing in the slow-growing, non-sporulating, melanized, endophytic fungus Berkleasmium sp. Dzf12 using a CRISPR/Cas9 system.

The endophytic fungus Berkleasmium sp. Dzf12 that was isolated from Dioscorea zingiberensis, is a proficient producer of palmarumycins, which are intriguing polyketides of the spirobisnaphthalene class. These compounds displayed a wide range of bioactivities, including antibacterial, antifungal, and cytotoxic activities. However, conventional genetic manipulation of Berkleasmium sp. Dzf12 is difficult and inefficient, partially due to the slow-growing, non-sporulating, and highly pigmented behavior of this fungus. Herein, we developed a CRISPR/Cas9 system suitable for gene editing in Berkleasmium sp. Dzf12. The protoplast preparation was optimized, and the expression of Cas9 in Berkleasmium sp. Dzf12 was validated. To assess the gene disruption efficiency, a putative 1, 3, 6, 8-tetrahydroxynaphthalene synthase encoding gene, bdpks, involved in 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis, was selected as the target for gene disruption. Various endogenous sgRNA promoters were tested, and different strategies to express sgRNA were compared, resulting in the construction of an optimal system using the U6 snRNA-1 promoter as the sgRNA promoter. Successful disruption of bdpks led to a complete abolishment of the production of spirobisnaphthalenes and melanin. This work establishes a useful gene targeting disruption system for exploration of gene functions in Berkleasmium sp. Dzf12, and also provides an example for developing an efficient CRISPR/Cas9 system to the fungi that are difficult to manipulate using conventional genetic tools.

Adaptation of high-efficiency CRISPR/Cas9-based multiplex genome editing system in white lupin by using endogenous promoters.

White lupin (Lupinus albus L.) is an important crop with high phosphorus (P) use efficiency; however, technologies for its functional genomic and molecular analyses are limited. Cluster regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system has been applied to gene editing and function genomics in many crops, but its application in white lupin has not been well documented. Here, we adapted the CRISPR/Cas9-based multiplex genome editing system by using the native U3/U6 and ubiquitin (UBQ) promoters to drive sgRNAs and Cas9. Two target sites (T1 and T2) within the Lalb_Chr05g0223881 gene, encoding a putative trehalase, were selected to validate its efficacy in white lupin based on the Agrobacterium rhizogenes-mediated transformation. We found that the T0 hairy roots were efficiently mutated at T1 and T2 with a frequency of 6.25%-35% and 50%-92.31%, respectively. The mutation types include nucleotide insertion, deletion, substitution, and complicated variant. Simultaneous mutations of the two targets were also observed with a range of 6.25%-35%. The combination of LaU6.6 promoter for sgRNA and LaUBQ12 promoter for Cas9 generated the highest frequency of homozygous/biallelic mutations at 38.46%. In addition, the target-sgRNA sequence also contributes to the editing efficiency of the CRISPR/Cas9 system in white lupin. In conclusion, our results expand the toolbox of the CRISPR/Cas9 system and benefit the basic research in white lupin.

CRISPR/Cas9 technology and its application in horticultural crops

Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated 9 (CRISPR/Cas9) system has recently become one popular technology due to its efficiency, precision, and simplicity compared with other genome editing tools such as Zinc Finger Nucleases (ZFNs) and Transcription Activator Like Effector Nucleases (TALENs). Horticultural crops provide energy and health-keeping nutrients to humankind. Genome-editing technology has become widely adopted in horticultural breeding with the increasing demand for high yield and better-quality horticultural crops. Here, we describe the CRISPR/Cas9 system construction, its optimization, including sgRNA promoter, sgRNA design, Cas9 protein promoter, SpCas9 variants and orthologs, and vector delivery methods. We also summarized the application of this technology in horticultural plants for stress responses enhancement, fruit quality improvement, and cultivation traits modification. This detailed review was compiled to help establish comprehensive understanding of the CRISPR/Cas9 systems and provide a reference for further developing this technology to manipulate horticultural plant traits effectively.

Optimization of heat shock temperature and time on the transformation of pRGEB32 into Escherichia coli DH5α

Genome editing technique is one of the methods for studying the expression of gene, eliminating unfavorable traits or phenotypes and generating the new characters of species. The pRGEB32 plasmid is one of the vectors that used in genome editing with carrying the Cas9 gene, restriction site of sgRNA (single guide RNA) and specific promoters that can be expressed in plants. The first step in the genome editing process is inserting pRGEB32 into Escherichia coli for propagation. The large size of the plasmid molecule becomes a challenge to determine the right method in the transformation process. This study aims to determine the temperature and time of heat shock transformation of plasmid pRGEB32 into E. coli. The transformation of pRGEB32 into plasmids was carried out with variations in temperature and time, 42℃ (30 seconds and 60 seconds) and 55℃ (30 seconds and 60 seconds). The results showed that a heat shock temperature of 55℃ with a time of 60 seconds was the best temperature for the transformation of pRGEB32 into E. coli. This optimization of heat shock condition will increase the transformation efficiency, which is in the range of 3322-10.989 cfu/µg.ÂÂ

NbPsbO1 Interacts Specifically with the Bamboo Mosaic Virus (BaMV) Subgenomic RNA (sgRNA) Promoter and Is Required for Efficient BaMV sgRNA Transcription.

ABSTRACTMany positive-strand (+) RNA viruses produce subgenomic RNAs (sgRNAs) in the infection cycle through the combined activities of viral replicase and host proteins. However, knowledge about host proteins involved in direct sgRNA promoter recognition is limited. Here, in the partially purified replicase complexes from Bamboo mosaic virus (BaMV)-infected tissue, we have identified the Nicotiana benthamiana photosystem II oxygen-evolving complex protein, NbPsbO1, which specifically interacted with the promoter of sgRNA but not that of genomic RNA (gRNA). Silencing of NbPsbO1 expression suppressed BaMV accumulation in N. benthamiana protoplasts without affecting viral gRNA replication. Overexpression of wild-type NbPsbO1 stimulated BaMV sgRNA accumulation. Fluorescent microscopy examination revealed that the fluorescence associated with NbPsbO1 was redistributed from chloroplast granal thylakoids to stroma in BaMV-infected cells. Overexpression of a mislocalized mutant of NbPsbO1, dTPPsbO1-T7, inhibited BaMV RNA accumulation in N. benthamiana, whereas overexpression of an NbPsbO1 derivative, sPsbO1-T7, designed to be targeted to chloroplast stroma, upregulated the sgRNA level. Furthermore, depletion of NbPsbO1 in BaMV RdRp preparation significantly inhibited sgRNA synthesis in vitro but exerted no effect on (+) or (−) gRNA synthesis, which indicates that NbPsbO1 is required for efficient sgRNA synthesis. These results reveal a novel role for NbPsbO1 in the selective enhancement of BaMV sgRNA transcription, most likely via direct interaction with the sgRNA promoter.IMPORTANCE Production of subgenomic RNAs (sgRNAs) for efficient translation of downstream viral proteins is one of the major strategies adapted for viruses that contain a multicistronic RNA genome. Both viral genomic RNA (gRNA) replication and sgRNA transcription rely on the combined activities of viral replicase and host proteins, which recognize promoter regions for the initiation of RNA synthesis. However, compared to the cis-acting elements involved in the regulation of sgRNA synthesis, the host factors involved in sgRNA promoter recognition mostly remain to be elucidated. Here, we found a chloroplast protein, NbPsbO1, which specifically interacts with Bamboo mosaic virus (BaMV) sgRNA promoter. We showed that NbPsbO1 is relocated to the BaMV replication site in BaMV-infected cells and demonstrated that NbPsbO1 is required for efficient BaMV sgRNA transcription but exerts no effect on gRNA replication. This study provides a new insight into the regulating mechanism of viral gRNA and sgRNA synthesis.

Single-plasmid systems based on CRISPR-Cas9 for gene editing in Lactococcus lactis

Lactococcus lactis is a food-grade lactic acid bacterial species that is widely used in food and medical industries. Due to its relatively small genome and simple metabolism, L. lactis is commonly engineered to produce large quantities of recombinant proteins. The most common single-gene knockout strategy in L. lactis involves RecA-dependent homologous double-crossover recombination, which is relatively time-consuming and laborious. In this study, a precise and efficient genome-editing plasmid for L. lactis NZ9000 genome engineering, pLL, was established based on clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology. By studying the effects of different single guide RNA (sgRNA) promoters, the efficiency of gene deletion was optimized. For LLNZ_02045 (ldh), gene deletion efficiency of up to 50% was achieved. Effective sequential gene deletion of LLNZ_11240 (upp) and LLNZ_04580 (upp1) was also demonstrated using this tool. Additionally, the gene that encodes for uracil phosphoribosyltransferase was identified using this system. Similar robust gene deletion efficiencies of sgRNA that targeted different regions of a single gene suggested that gene deletion was not affected by the location of sgRNA binding. Thus, our study established a new gene-editing tool that may allow further investigation and understanding of the L. lactis NZ9000 genome.

Prediction of putative regulatory elements in the subgenomic promoters of cucumber green mottle mosaic virus and their interactions with the RNA dependent RNA polymerase domain.

Characterization of the subgenomic RNA (sgRNA) promoter of many plant viruses is important to understand the expression of downstream genes and also to configure their genome into a suitable virus gene-vector system. Cucumber green mottle mosaic virus (CGMMV, genus Tobamovirus) is one of the RNA viruses, which is extensively being exploited as the suitable gene silencing and protein expression vector. Even though, characters of the sgRNA promoters (SGPs) of CGMMV are yet to be addressed. In the present study, we predicted the SGP for the movement protein (MP) and coat protein (CP) of CGMMV. Further, we identified the key regulatory elements in the SGP regions of MP and CP, and their interactions with the core RNA dependent RNA polymerase (RdRp) domain of CGMMVwas deciphered. The modeled structure of core RdRp contains two palm (1-41 aa, and 63-109 aa), one finger (42-62 aa) subdomains with three conserved RdRp motifs that played important role in binding to the SGP nucleic acids. RdRp strongly preferred the double helix form of the stem region in the stem and loop (SL) structures, and the internal bulge elements. In MP-SGP, a total of six elements was identified; of them, the affinity of binding to - 26nt to - 17nt site (CGCGGAAAAG) was higher through the formation of strong hydrogen bonds with LYS16, TYR17, LYS19, SER20, etc. of the motif A in the palm subdomain of RdRp. Similar strong interactions were noticed in the internal bulge (CAACUUU) located at + 33 to + 39 nt adjacent to the translation start site (TLSS) (+ 1). These could be proposed as the putative core promoter elements in MP-SGP. Likewise, total five elements were predicted within - 114nt to + 144 nt region of CP-SGPwith respect to CP-TLSS. Of them, RdRp preferred to bind at the small hairpin located at - 60 nt to - 43 nt (UUGGAGGUUUAGCCUCCA) in the upstream region, and at the complex duplex structure spanning between + 99 and + 114 nt in the downstream region, thus indicating the distribution of core promoter within - 60 nt to + 114 nt region of CP-SGPwith respect to TLSS (+ 1)of the CP; whereas, the - 114 nt to + 144 nt region of CP-SGP might be necessary for the full activity of the CP-SGP. Ourinsilicoprediction certifies the gravity of these nucleotide stretches as the RNA regulatory elements and identifies their potentiality for binding with of palm and finger sub-domain of RdRp. Identification of such elements will be helpful to anticipate the critical length of the SGPs. Our finding will not only be helpful to delineate the SGPs of CGMMV but also their subsequent application in the efficient construction of virus gene-vector for the expression of foreign protein in plant.

Exploration of expression activity of subgenomic RNA promoter of coat protein gene from Odontoglossum ringspot virus

The subgenomic mRNA (sgRNA) promoter for coat protein (CP) was isolated from the cDNA infectious clone of Odontoglossum ringspot virus Taiwan strain (ORSV-TW), which is a positive single-stranded RNA virus of the Tobamovirus family. The 5′-end deletion analysis of the 290 nucleotides ORSV CP sgRNA promoter was performed to investigate the promoter activity. A series of different ORSV CP sgRNA promoter deletions were characterized by driving the expression of a fused green fluorescent protein (GFP)-β-glucuronidase (GUS) gene using Agrobacterium-mediated infiltration in Phalaenopsis petals and Nicotiana benthamiana leaves. The results of deletion analysis indicated that the region between 24 nucleotides upstream and 18 nucleotides downstream of translation start codon was sufficient for promoter activity. The activity of this minimal promoter was relatively stronger than other promoters with longer length. A minimal ORSV CP sgRNA promoter was found to accumulate high levels of GFP and GUS, suggesting that this sequence might drive the foreign protein expression in heterologous systems.

An in vitro assay to study chikungunya virus RNA synthesis and the mode of action of inhibitors.

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that causes severe persistent arthralgia. To better understand the molecular details of CHIKV RNA synthesis and the mode of action of inhibitors, we have developed an in vitro assay to study CHIKV replication/transcription complexes isolated from infected cells. In this assay (32)P-CTP was incorporated into the CHIKV genome, subgenomic (sg) RNA and into a ~7.5 kb positive-stranded RNA, termed RNA II. We mapped RNA II, which was also found in CHIKV-infected cells, to the 5' end of the genome up to the start of the sgRNA promoter region. Most of the RNA-synthesizing activity, negative-stranded RNA and a relatively large proportion of nsP1 and nsP4 were recovered from a crude membrane fraction obtained by pelleting at 15,000 G: . Positive-stranded RNA was mainly found in the cytosolic S15 fraction, suggesting it was released from the membrane-associated replication/transcription complexes (RTCs). The newly synthesized RNA was relatively stable and remained protected from cellular nucleases, possibly by encapsidation. A set of compounds that inhibit CHIKV replication in cell culture was tested in the in vitro RTC assay. In contrast to 3'dNTPs, chain terminators that acted as potent inhibitors of RTC activity, ribavirin triphosphate and 6-aza-UTP did not affect the RNA-synthesizing activity in vitro. In conclusion, this in vitro assay for CHIKV RNA synthesis is a useful tool for mechanistic studies on the RTC and mode of action studies on compounds with anti-CHIKV activity.

Infection of capilloviruses requires subgenomic RNAs whose transcription is controlled by promoter-like sequences conserved among flexiviruses

The first open-reading frame (ORF) of apple stem grooving virus (ASGV), of the genus Capillovirus, encodes an apparently chimeric polyprotein containing conserved regions for replicase (Rep) and coat protein (CP). However, our previous study revealed that ASGV mutants with distinct and discontinuous Rep- and CP-coding regions successfully infect plants, indicating that CP expressed via a subgenomic RNA (sgRNA) is sufficient for viability of the virus. Here we identified a transcription start site of the CP sgRNA and revealed that CP translated from the sgRNA is essential for ASGV infection. We mapped the transcription start sites of both the CP and the movement protein (MP) sgRNAs of ASGV and found a hexanucleotide motif, UUAGGU, conserved upstream from both sgRNA transcription start sites. Mutational analysis of the putative CP initiation codon and of the UUAGGU sequence upstream from the transcription start site of CP sgRNA demonstrated their importance for ASGV accumulation. Our results also demonstrated that potato virus T (PVT), an unassigned species closely related to ASGV, produces two sgRNAs putatively deployed for the CP and MP expression and that the same hexanucleotide motif as found in ASGV is located upstream from the transcription start sites of both sgRNAs. This motif, which constituted putative core elements of the sgRNA promoter, is broadly conserved among viruses in the families Alphaflexiviridae and Betaflexiviridae, suggesting that the gene expression strategy of the viruses in both families has been conserved throughout evolution.

Characterization of the subgenomic RNAs produced by Pelargonium flower break virus: Identification of two novel RNAs species

Pelargonium flower break virus (PFBV), a member of the genus Carmovirus, has a single-stranded positive-sense genomic RNA (gRNA) of 3.9 kb. The 5′ half of the gRNA encodes two proteins involved in replication, the p27 and its readthrough product, p86 (the viral RNA dependent-RNA polymerase, RdRp), and the 3′ half encodes two small movement proteins, p7 and p12, and the coat protein (CP). As other members of the family Tombusviridae, carmoviruses express ORFs that are not 5′-proximal from subgenomic RNAs (sgRNAs). Analysis of double-stranded RNAs extracted from PFBV-infected leaves and Northern blot hybridizations of total RNA from infected plants or protoplasts revealed than PFBV produces four 3′-coterminal sgRNAs of 3.2, 2.9, 1.7 and 1.4 kb, respectively. The 5′ termini of the 1.7 and 1.4 kb sgRNAs mapped 26 and 143 nt upstream of the initiation codons of the p7 and CP genes, respectively, whereas the 5′-ends of the 3.2 and 2.9 kb sgRNAs were located within the readthrough portion of the RdRp gene. The PFBV sgRNAs begin with a motif which is also present at the 5′ terminus of the gRNA and the minus polarity of the regions preceding their corresponding start sites (in the gRNA) may be folded into hairpin structures resembling those found for the sgRNA promoters of other carmoviruses. The results indicate that, besides the sgRNAs involved in the translation of the movement proteins and the CP identified in most carmoviral infections, PFBV produces two additional sgRNAs whose biological significance is currently unknown. The possible participation of the 3.2 and 2.9 kb PFBV sgRNAs in the expression of readthrough portions of the RdRp is discussed.

Analyses of Subgenomic Promoters of Hibiscus Chlorotic Ringspot Virus and Demonstration of 5′ Untranslated Region and 3′-Terminal Sequences Functioning as Subgenomic Promoters

Hibiscus chlorotic ringspot virus (HCRSV), which belongs to the genus Carmovirus, generates two 3'-coterminal subgenomic RNAs (sgRNAs) of 1.4 kb and 1.7 kb. Transcription start sites of the two sgRNAs were identified at nucleotides (nt) 2178 and 2438, respectively. The full promoter of sgRNA1, a 118-base sequence, is localized between positions +6 and -112 relative to its transcription start site (+1). Similarly, a 132-base sequence, from +6 to -126, defines the sgRNA2 promoter. Computer analysis revealed that both sgRNA promoters share a similar two-stem-loop (SL1 + SL2) structure, immediately upstream of the transcription start site. Mutational analysis of the primary sequence and secondary structures showed further similarities between the two subgenomic promoters. The basal portion of SL2, encompassing the transcription start site, was essential for transcription activity in each promoter, while SL1 and the upper portion of SL2 played a role in transcription enhancement. Both the 5' untranslated region (UTR) and the last 87 nt at the 3' UTR of HCRSV genomic RNA are likely to be the putative genomic plus-strand and minus-strand promoters, respectively. They function well as individual sgRNA promoters to produce ectopic subgenomic RNAs in vivo but not to the same levels of the actual sgRNA promoters. This suggests that HCRSV sgRNA promoters share common features with the promoters for genomic plus-strand and minus-strand RNA synthesis. To our knowledge, this is the first demonstration that both the 5' UTR and part of the 3' UTR can be duplicated and function as sgRNA promoters within a single viral genome.

Characterization of regulatory elements within the coat protein (CP) coding region of Tobacco mosaic virus affecting subgenomic transcription and green fluorescent protein expression from the CP subgenomic RNA promoter

A replicon based on Tobacco mosaic virus that was engineered to express the open reading frame (ORF) of the green fluorescent protein (GFP) gene in place of the native coat protein (CP) gene from a minimal CP subgenomic (sg) RNA promoter was found to accumulate very low levels of GFP. Regulatory regions within the CP ORF were identified that, when presented as untranslated regions flanking the GFP ORF, enhanced or inhibited sg transcription and GFP expression. Full GFP expression from the CP sgRNA promoter required more than the first 20 nt of the CP ORF but not beyond the first 56 nt. Further analysis indicated the presence of an enhancer element between nt +25 and +55 with respect to the CP translation start site. The inclusion of this enhancer sequence upstream of the GFP ORF led to elevated sg transcription and to a 50-fold increase in GFP accumulation in comparison with a minimal CP promoter in which the entire CP ORF was displaced by the GFP ORF. Inclusion of the 3'-terminal 22 nt had a minor positive effect on GFP accumulation, but the addition of extended untranslated sequences from the 3' terminus of the CP ORF downstream of the GFP ORF was basically found to inhibit sg transcription. Secondary structure analysis programs predicted the CP sgRNA promoter to reside within two stable stem-loop structures, which are followed by an enhancer region.

Transcription strategy in a Closterovirus: a novel 5'-proximal controller element of Citrus Tristeza Virus produces 5'- and 3'-terminal subgenomic RNAs and differs from 3' open reading frame controller elements.

Citrus tristeza virus (CTV) produces more than thirty 3'- or 5'-terminal subgenomic RNAs (sgRNAs) that accumulate to various extents during replication in protoplasts and plants. Among the most unusual species are two abundant populations of small 5'-terminal sgRNAs of approximately 800 nucleotides (nt) termed low-molecular-weight tristeza (LMT1 and LMT2) RNAs. Remarkably, CTV replicons with all 10 3' genes deleted produce only the larger LMT1 RNAs. These 5'-terminal positive-sense sgRNAs do not have corresponding negative strands and were hypothesized to be produced by premature termination during plus-strand genomic RNA synthesis. We characterized a cis-acting element that controls the production of the LMT1 RNAs. Since manipulation of this cis-acting element in its native position (the L-ProI region of replicase) was not possible because the mutations negatively affect replication, a region (5'TR) surrounding the putative termination sites (nt approximately 550 to 1000) was duplicated in the 3' end of a CTV replicon to allow characterization. The duplicated sequence continued to produce a 5'-terminal plus-strand sgRNA, here much larger ( approximately 11 kb), apparently by termination. Surprisingly, a new 3'-terminal sgRNA was observed from the duplicated 5'TR. A large 3'-terminal sgRNA resulting from the putative promoter activity of the native 5'TR was not observed, possibly because of the down-regulation of a promoter approximately 19 kb from the 3' terminus. However, we were able to observe a sgRNA produced from the native 5'TR of a small defective RNA, which placed the native 5'TR closer to the 3' terminus, demonstrating sgRNA promoter activity of the native 5'TR. Deletion mutagenesis mapped the promoter and the terminator activities of the 5'TR (in the 3' position in the CTV replicon) to a 57-nt region, which was folded by the MFOLD computer program into two stem-loops. Mutations in the putative stem-loop structures equally reduced or prevented production of both the 3'- and 5'-terminal sgRNAs. These mutations, when introduced in frame in the native 5'TR, similarly abolished the synthesis of the LMT1 RNAs and presumably the large 3'-terminal sgRNA while having no impact on replication, demonstrating that neither 5'- nor 3'-terminal sgRNA is necessary for replication of the replicon or full-length CTV in protoplasts. Differences between the 5'TR, which produced two plus-strand sgRNAs, and the cis-acting elements controlling the 3' open reading frames, which produced additional minus-strand sgRNAs corresponding to the 3'-terminal mRNAs, suggest that the different sgRNA controller elements had different origins in the modular evolution of closteroviruses.

Mapping of the Tobacco Mosaic Virus Movement Protein and Coat Protein Subgenomic RNA Promoters in Vivo

The Tobacco mosaic virus movement protein (MP) and coat protein (CP) are expressed from 3′-coterminal subgenomic RNAs (sgRNAs). The transcription start site of the MP sgRNA, previously mapped to positions 4838 (Y. Watanabe, T. Meshi, and Y. Okada (1984), FEBS Lett. 173, 247–250) and 4828 (K. Lehto, G. L. Grantham, and W. O. Dawson (1990), Virology 174, 145–157) for the TMV OM and U1 strains, respectively, has been reexamined and mapped to position 4838 for strain U1. Sequences of the MP and CP sgRNA promoters were delineated by deletion analysis. The boundaries for minimal and full MP sgRNA promoter activity were localized between −35 and +10 and −95 and +40, respectively, relative to the transcription start site. The minimal CP sgRNA promoter was mapped between −69 and +12, whereas the boundaries of the fully active promoter were between −157 and +54. Computer analysis predicted two stem–loop structures (SL1 and SL2) upstream of the MP sgRNA transcription start site. Deletion analysis and site-directed mutagenesis suggested that SL1 secondary structure, but not its sequence, was required for MP sgRNA promoter activity, whereas a 39-nt deletion removing most of the SL2 region increased MP sgRNA accumulation fourfold. Computer-predicted folding of the fully active CP sgRNA promoter revealed one long stem–loop structure. Deletion analysis suggested that the upper part of this stem–loop, located upstream of the transcription start site, was essential for transcription and that the lower part of the stem had an enhancing role.

A positive-strand RNA virus with three very different subgenomic RNA promoters.

Numerous RNA viruses generate subgenomic mRNAs (sgRNAs) for expression of their 3'-proximal genes. A major step in control of viral gene expression is the regulation of sgRNA synthesis by specific promoter elements. We used barley yellow dwarf virus (BYDV) as a model system to study transcriptional control in a virus with multiple sgRNAs. BYDV generates three sgRNAs during infection. The sgRNA1 promoter has been mapped previously to a 98-nucleotide (nt) region which forms two stem-loop structures. It was determined that sgRNA1 is not required for BYDV RNA replication in oat protoplasts. In this study, we show that neither sgRNA2 nor sgRNA3 is required for BYDV RNA replication. The promoters for sgRNA2 and sgRNA3 synthesis were mapped by using deletion mutagenesis. The minimal sgRNA2 promoter is approximately 143 nt long (nt 4810 to 4952) and is located immediately downstream of the putative sgRNA2 start site (nt 4809). The minimal sgRNA3 core promoter is 44 nt long (nt 5345 to 5388), with most of the sequence located downstream of sgRNA3 start site (nt 5348). For both promoters, additional sequences upstream of the start site enhanced sgRNA promoter activity. These promoters contrast to the sgRNA1 promoter, in which almost all of the promoter is located upstream of the transcription initiation site. Comparison of RNA sequences and computer-predicted secondary structures revealed little or no homology between the three sgRNA promoter elements. Thus, a small RNA virus with multiple sgRNAs can have very different subgenomic promoters, which implies a complex system for promoter recognition and regulation of subgenomic RNA synthesis.

Sequence Analysis and Genome Organisation of Poinsettia Mosaic Virus (PnMV) Reveal Closer Relationship to Marafiviruses Than to Tymoviruses

Sequence comparison and genome organisation of poinsettia mosaic virus (PnMV), a putative member of the tymoviruses, revealed a closer relationship to marafiviruses. The complete nucleotide sequence of PnMV was determined. The 6099-nt RNA genome encodes a putative 221-kDa polyprotein that lacks a stop codon between the replicase and the coat protein genes, as in most tymovirus RNAs. The genomic RNA has a poly(A) tail at its 3′-terminus in contrast to the tRNA-like structure found in the RNA of most tymoviruses, and no homology was observed to the conserved noncoding region of the tymoviral 3′-termini. The tymobox of PnMV, a 16-nt region of the subgenomic RNA (sgRNA) promoter shared by most tymoviruses, differs in 3 nt from the RNA sequence of tymoviruses but is identical to the sequence of marafiviruses. At least three sgRNAs were found in PnMV-infected Euphorbia pulcherrima and in isolated PnMV particles; one that is 650 nt long encodes the 21.4-kDa coat protein, and the others are about 3.5 and 1.7 kb and contain the 5′- and the 3′-terminal parts of genomic RNA, respectively. Like tymoviruses, PnMV particles sediment as top and bottom components. The particles of the top component contain the sgRNA (650 nt) encoding the coat protein, and those of bottom component contain both genomic and sgRNAs.

Primary and secondary structural elements required for synthesis of barley yellow dwarf virus subgenomic RNA1.

Barley yellow dwarf luteovirus (BYDV) generates three 3'-coterminal subgenomic RNAs (sgRNAs) in infected cells. The promoter of sgRNA1 is a putative hot spot for RNA recombination in luteovirus evolution. The sgRNA1 transcription start site was mapped previously to either nucleotide 2670 or nucleotide 2769 of BYDV genomic RNA (gRNA) in two independent studies. Our data support the former initiation site. The boundaries of the sgRNA1 promoter map between nucleotides 2595 and 2692 on genomic RNA. Computer prediction, phylogenetic comparison, and structural probing revealed two stem-loops (SL1 and SL2) in the sgRNA1 promoter region on the negative strand. Promoter function was analyzed by inoculating protoplasts with a full-length infectious clone of the BYDV genome containing mutations in the sgRNA promoter. Because the promoter is located in an essential coding region of the replicase gene, we duplicated it in a nonessential part of the genome from which a new sgRNA was expressed. Mutational analysis revealed that secondary structure, but not the nucleotide sequence, was important at the base of SL1. Regions with both RNA primary and secondary structural features that contributed to transcription initiation were found at the top of SL1. Primary sequence, but not the secondary structure, was required in SL2, which includes the initiation site. Disruption of base pairing near the sgRNA1 start site increased the level of transcription three- to fourfold. We propose that both primary and secondary structures of the sgRNA1 promoter of BYDV play unique roles in sgRNA1 promoter recognition and transcription initiation.

Minimal Sequence and Structural Requirements of a Subgenomic RNA Promoter for Turnip Crinkle Virus

Infection of plants or protoplasts with turnip crinkle virus (TCV) results in the synthesis of the genomic RNA and two subgenomic (sg) RNAs of 1.7 kb and 1.45 kb, respectively. Both of the sgRNA promoters were characterized previously and their secondary structures predicted by computer analysis [J. Wang and A. E. Simon (1997).Virology232, 174–186]. Secondary structure-sensitive chemical and enzymatic probes have now been used to determine the structure of the promoter directing synthesis of the 1.45-kb sgRNA, namely the 1.45-kb sgRNA promoter, in solution. The newly obtained structure conforms with the previously predicted hairpin structure except for the hairpin base: four CG base pairs and a CA bulge are present instead of an A bulge. Studies of deletions within the 96-nucleotide (nt) 1.45-kb sgRNA promoter defined a minimal 30-nt core sequence as essential for promoter activity: a 21-nt hairpin and a 9-nt flanking single-stranded sequence. Mutational analysis in the stem section of the core promoter supported a role for the primary sequence and secondary structure in promoter activity. Sequence alterations in the flanking single-stranded region further suggest that the sequence CCCAUUA, encompassing the transcription start site, is required for efficient transcription of the 1.45-kb sgRNA by the TCV RNA-dependent RNA polymerasein vivo.

Analysis of the Two Subgenomic RNA Promoters for Turnip Crinkle Virusin Vivoandin Vitro

Infection of plants or protoplasts with turnip crinkle virus (TCV), a monopartite RNA virus, results in the synthesis of the genomic RNA and two subgenomic (sg) RNAs. The transcription start site for the 1.45-kb sgRNA was previously mapped to position 2606 (J. C. Carrington, T.J. Morris, P.G. Stockley, and S.C. Harrison, (1987).J. Mol. Biol.194, 265–276) corresponding to position 2607 in the TCVms isolate and the start site for the 1.7-kb sgRNA has now been mapped to position 2333 in TCVms. A 96-base sequence (90 bases upstream and 6 bases downstream) encompassing the transcription start site for the 1.45-kb sgRNA was sufficient for full promoter activity. Similarly, a 94-base sequence (90 bases upstream and 4 bases downstream) encompassing the start site was required for full activity of the 1.7-kb sgRNA promoter. The 1.45-kb sgRNA promoter, but not the 1.7-kb sgRNA promoter, was able to direct synthesis of a nontemplate RNAin vitrousing partially purified TCV RNA-dependent RNA polymerase. Computer generated secondary structures for the two sgRNA promoters revealed an extensive hairpin just upstream from the transcription start site. Comparisons of corresponding sequences from related viruses indicates higher sequence conservation for the 1.45-kb sgRNA promoter compared with the 1.7-kb sgRNA promoter, despite the latter's location within the RNA-dependent RNA polymerase open reading frame.