Generation of Subgenomic RNA Directed by a Satellite RNA Associated with Bamboo Mosaic Potexvirus: Analyses of Potexvirus Subgenomic RNA Promoter

Abstract

Satellite RNA of bamboo mosaic potexvirus (satBaMV), a single-stranded positive-sense RNA encoding a nonstructural protein of 20 kDa (P20), depends on bamboo mosaic potexvirus (BaMV) for replication and encapsidation. A full-length cDNA clone of satBaMV was used to examine the sequences required for the synthesis of potexvirus subgenomic RNAs (sgRNAs). Subgenomic promoter-like sequences (SGPs), 107 nucleotides (nt) upstream from the capsid protein (CP) gene of BaMV-V, were inserted upstream of the start codon of the P20 gene of satBaMV. Insertion of SGPs gave rise to the synthesis of sgRNA of satBaMV in protoplasts of Nicotiana benthamiana and leaves of Chenopodium quinoa when coinoculated with BaMV-V genomic RNA. Moreover, both the satBaMV cassette and its sgRNA were encapsidated. From analysis of the SGPs by deletion mutation, we concluded that an SGP contains one core promoterlike sequence (nt ؊30 through ؉16), two upstream enhancers (nt ؊59 through ؊31 and ؊91 through ؊60), and one downstream enhancer (nt ؉17 through ؉52), when the transcription initiation site is taken as ؉1. Site-directed mutagenesis and compensatory mutation to disrupt and restore potential base pairing in the core promoter-like sequence suggest that the stem-loop structure is important for the function of SGP in vivo. Likewise, the insertion of a putative SGP of the BaMV open reading frame 2 gene or a heterologous SGP of potato virus X resulted in generation of an sgRNA. The satBaMV cassette should be a useful tool to gain insight into sequences required for the synthesis of potexvirus sgRNAs.The production of subgenomic RNAs (sgRNAs) is one of the strategies by which internally located open reading frames (ORFs) of multicistronic RNA virus may be expressed and regulated during replication. The capsid proteins (CPs) of alpha-like superfamily viruses are expressed by sgRNA transcription (24). The proteins encoded by the triple gene block of potex-, carla-, furo-, and hordeiviruses and the movement proteins of tobamo-and tombusviruses are also expressed by the same strategy (24). Several mechanisms are proposed for the transcription of sgRNAs. The first is leader-primed transcription, which occurs in the production of coronavirus sgRNAs. This process involves fusion of noncontinuous sequences from the 5Ј leader sequences to the sgRNA by a discontinuous process (14). Recently, a new model for coronavirus transcription was proposed. This model predicts that subgenome-length negative strands containing the common 5Ј leader sequence were directly derived from genome RNA and served as a template for the production of sgRNA (27). The second mechanism proposed for transcription of sgRNAs is exemplified by transcription of RNA-mediated trans-activation. A 34-nucleotide (nt) trans-activator on red clover necrotic mosaic virus RNA2 was proposed to bind to the element upstream of the sgRNA initiation site on RNA1 and to prevent the synthesis of full-length minus-strand RNA1. This truncated minus-strand RNA1 may serve as a template for viral RNA-depen...

Molecular biology of potexviruses: recent advances

“…2a, c) . Potexviruses have three subgenomic RNAs for expression of TGB and CP (Huisman et al, 1988;Lee et al, 2000). An octanucleotide sequence (AACUAAAC) in the PVX 59 NTR was identified that can base pair with sequences Fig.…”

A dual gene‐silencing vector system for monocot and dicot plants

“…P20 is an RNA-binding protein that can be replaced with other proteins such as chloramphenicol acetyltransferase (CAT). Thus, satBaMV has been developed as a useful plant expression vector (Lee et al, 2000;Lin et al, 1996). The helper virus BaMV, a member of the Potexvirus genus, contains a single-stranded positive-sense RNA genome with 5 conserved ORFs Yang et al, 1997).…”

Production of Human IFNγ Protein in Nicotiana benthamiana Plant through an Enhanced Expression System Based on Bamboo mosaic Virus

“…Since CP is required for cell-to-cell movement of potexviruses [33,34], we have constructed potexvirus-based viral vectors by replacing the CP open reading frame (ORF) with that of the TP in order to prevent the undesired spread of the viral vectors. It has been previously shown that nucleotides (nts) +1 to +15 at the 5′-terminal of BaMV-S strain CP ORF are essential for CP subgenomic RNA promoter activity (SGP) [35]. Thus, a pKB-derived vector, pKB△C His , was generated, containing multiple cloning sites ( Mlu I- Stu I- Not I -Spe I) and 6X His tag to facilitate the purification of TP and nts +1 to 15 at the 5’ terminus of BaMV CP gene, of which initiation codon was mutated.…”