RNA-Based Regulation of Transcription and Translation of Aureusvirus Subgenomic mRNA1

Abstract

Cucumber leaf spot virus (CLSV) is an aureusvirus (family Tombusviridae) that has a positive-sense RNA genome encoding five proteins. During infections, CLSV transcribes two subgenomic (sg) mRNAs and the larger of the two, sg mRNA1, encodes coat protein. Here, the viral RNA sequences and structures that regulate transcription and translation of CLSV sg mRNA1 were investigated. A medium-range RNA-RNA interaction in the CLSV genome, spanning 148 nucleotides, was found to be required for the efficient transcription of sg mRNA1. Further analysis indicated that the structure formed by this interaction acted as an attenuation signal required for transcription of sg mRNA1 via a premature termination mechanism. Translation of coat protein from sg mRNA1 was determined to be facilitated by a 5-terminal stem-loop structure in the message that resembled a tRNA anticodon stem-loop. The results from mutational analysis indicated that the 5-terminal stem-loop mediated efficient base pairing with a 3-cap-independent translational enhancer at the 3 end of the message, leading to efficient translation of coat protein from sg mRNA1. Comparison of the regulatory RNA structures for sg mRNA1 of CLSV to those used by the closely related tombusviruses and certain cellular RNAs revealed interesting differences and similarities that provide evolutionary and mechanistic insights into RNA-based regulatory strategies.Positive-strand RNA viruses use a variety of mechanisms for expressing their genes. For viruses in the family Tombusviridae, subgenomic (sg) mRNA transcription is utilized to mediate the expression of viral proteins encoded 3Ј proximally in their genomes (28, 50). These smaller, 3Ј-coterminal messages are efficient templates for translation and allow for expression of a subset of viral proteins. Interestingly, the plus-strand RNA genomes of the members of Tombusviridae are not 5Ј capped or 3Ј polyadenylated, and several have been shown to contain 3Ј-cap independent translational enhancers (3ЈCITEs) in their 3Ј-untranslated regions (3ЈUTRs) (1,6,9,10,19,30,31,33,38,39,41,43,45,46,51). Some of these 3ЈCITEs are known to function by recruiting translational machinery to the message (13, 41, 44), and several have been shown to communicate with their cognate 5ЈUTRs via RNA-RNA interactions (9,10,14,16,17). In addition, since sg mRNAs are 3Ј-coterminal with their genomes, they also contain 3ЈCITEs and can utilize them for efficient translation of their encoded proteins (18,29).Cucumber leaf spot virus (CLSV) is a member of the genus Aureusvirus (family Tombusviridae) (27) and it is very closely related to Tomato bushy stunt virus (TBSV; genus Tombusvirus, family Tombusviridae) (15). These viruses share a similar coding strategy in which RNA replication-related proteins are encoded 5Ј proximally in the viral genome, followed by coat protein (CP) and, finally, overlapping cell-to-cell movement and gene silencing suppressor proteins that are coded in different reading frames (Fig. 1) (26,27,35,37,50). In both cases, the CP and the 3Ј-pr...

Subgenomic mRNA transcription in Tombusviridae

“…28,29,36 As noted before, the fact that these heterologous structures function in transcription indicates that thermodynamic stability is the key property required, however these results also provide some insight into the relative effectiveness of the higher-order structural elements. Due to their compact and stable nature, the RNA hairpin structures used are predicted to dominate at equilibrium.…”

“…3C). 13,36 Such diverse intervening distances both between and within viruses are probably consequences of "random" spatial proximity of RS elements to potential AS elements during genesis of the attenuator structures. That is, for an AS-RS element to originate, the RS element would have to encounter a compatible upstream partner sequence (i.e., pre-AS element) to base pair with and form an effective attenuation structure.…”

“…28 The same is also true for CLSV, where substituted heterologous AS-RS-like elements function well. 36 For these cases, the formation of a stable RNA secondary structure seems to be what is vital. The situation is more complicated for TNV-D, where both the identity within the attenuation structure and the intervening sequence influence transcriptional activity (Jiwan SD and White KA, unpublished data).…”

“…The transcriptional promoters characterized are about 8 to 10 nts long and AU-rich. 22,25,36,37 In TBSV, CLSV and TCV, transcription initiates with a guanylate, while RCNMV initiates with an adenylate. For all cases, the transcriptional promoters tend to be linear and similar in sequence and length to the genomic promoters for plus-strand synthesis (Fig.…”

“…The fact that an RNA hairpin can act as an attenuation structure served as the starting point to develop an RNA-based molecular switch to allow for exogenous regulation of viral sg mRNA transcription in vivo. 29,36 To accomplish this, an RNA hairpin structure was integrated with an RNA aptamer. RNA aptamers are small RNA structures that are able to specifically bind to small metabolites.…”