An RNA Activator of Subgenomic mRNA1 Transcription in Tomato Bushy Stunt Virus
2002; Elsevier BV; Volume: 277; Issue: 5 Linguagem: Inglês
10.1074/jbc.m109067200
ISSN1083-351X
AutoresIl‐Ryong Choi, K. Andrew White,
Tópico(s)Animal Virus Infections Studies
ResumoMany (+)-strand RNA viruses transcribe small subgenomic (sg) mRNAs that allow for regulated expression of a subset of their genes. Tomato bushy stunt virus (TBSV) transcribes two such messages and here we report the identification of a long-distance RNA·RNA interaction that is essential for the efficient accumulation of capsid protein-encoding sg mRNA1. The relevant base pairing interaction occurs within the TBSV RNA genome between a 7-nucleotide (nt) long sequence, separated by just 3 nt from the downstream sg mRNA1 initiation site, and a complementary sequence positioned some ∼1000 nt further upstream. Analyses of this interaction indicate that it (i) functions in the (+)-strand, (ii) modulates both (+)- and (−)-strand sg mRNA1 accumulation, (iii) specifically regulates the accumulation of sg mRNA1 (−)-strands, (iv) controls sg mRNA1 expression from an ectopic transcriptional initiation site, (v) may occur in cis and, and (vi) could nucleate the formation of a more complex RNA structure. These data are most consistent with a role for this interaction in regulating sg mRNA1 accumulation at the level of transcription. Many (+)-strand RNA viruses transcribe small subgenomic (sg) mRNAs that allow for regulated expression of a subset of their genes. Tomato bushy stunt virus (TBSV) transcribes two such messages and here we report the identification of a long-distance RNA·RNA interaction that is essential for the efficient accumulation of capsid protein-encoding sg mRNA1. The relevant base pairing interaction occurs within the TBSV RNA genome between a 7-nucleotide (nt) long sequence, separated by just 3 nt from the downstream sg mRNA1 initiation site, and a complementary sequence positioned some ∼1000 nt further upstream. Analyses of this interaction indicate that it (i) functions in the (+)-strand, (ii) modulates both (+)- and (−)-strand sg mRNA1 accumulation, (iii) specifically regulates the accumulation of sg mRNA1 (−)-strands, (iv) controls sg mRNA1 expression from an ectopic transcriptional initiation site, (v) may occur in cis and, and (vi) could nucleate the formation of a more complex RNA structure. These data are most consistent with a role for this interaction in regulating sg mRNA1 accumulation at the level of transcription. subgenomic activator sequence 1 core element cucumber necrosis virus coat protein distal element open reading frame potato virus X receptor sequence 1 red clover necrotic mosaic virus stem-loop tomato bushy stunt virus wild type Viral infections of eukaryotic cells are complex processes that require regulated expression of a variety of viral genes. Depending on the virus, this expression can be regulated at different levels, including transcriptional, post-transcriptional, translational, and post-translational (1Maia I.G. Seron K. Haenni A. Bernardi F. Plant Mol. Biol. 1996; 32: 367-391Crossref PubMed Scopus (41) Google Scholar). For (+)-strand RNA viruses, many utilize RNA-templated transcription of subgenomic (sg)1 mRNAs to allow for regulated expression of specific viral genes (2Miller W.A. Koev G. Virology. 2000; 273: 1-8Crossref PubMed Scopus (155) Google Scholar). The mechanism by which sg mRNAs are synthesized can vary, but the messages produced share the common property of encoding open reading frames (ORFs) that are located 3′-proximally in the viral genomes. Because such 3′-proximal ORFs are generally translationally silent within the context of these genomes, sg mRNA production provides a mode for their efficient translation as well as a mechanism to regulate the timing and amount of viral protein produced (2Miller W.A. Koev G. Virology. 2000; 273: 1-8Crossref PubMed Scopus (155) Google Scholar). Two mechanisms for sg mRNA transcription are well-established: (i) synthesis of sg mRNAs from a full-length (−)-strand genomic template via internal initiation (3Miller W.A. Dreher T.W. Hall T.C. Nature. 1985; 313: 68-70Crossref PubMed Scopus (218) Google Scholar, 4Siegel R.W. Adkins S. Kao C.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11238-11243Crossref PubMed Scopus (102) Google Scholar) and (ii) synthesis of a non-contiguous RNA product during (−)-strand synthesis, which is then used as a template for transcription of sg mRNAs (5Pasternak A.O. Gultyaev A.P. Spaan W.J. Snijder E.J. J. Virol. 2000; 74: 11642-11653Crossref PubMed Scopus (39) Google Scholar, 6van Marle G. Dobbe J.C. Gultyaev A.P. Luytjes W. Spaan W.J. Snijder E.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12056-12061Crossref PubMed Scopus (176) Google Scholar). A third possible mechanism that has been proposed involves premature termination during (−)-strand synthesis of the genome followed by use of the 3′-truncated product as a template to transcribe sg mRNAs (7Zhong W. Rueckert R.R. J. Virol. 1993; 67: 2716-2722Crossref PubMed Google Scholar, 8Miller W.A. Brown C.M. Wang S. Semin. Virol. 1997; 8: 3-13Crossref Scopus (46) Google Scholar, 9Sit T.L. Vaewhongs A.A. Lommel S.A. Science. 1998; 281: 829-832Crossref PubMed Scopus (175) Google Scholar). Although this latter model is consistent with data generated from studies on an assortment of (+)-strand RNA viruses (9Sit T.L. Vaewhongs A.A. Lommel S.A. Science. 1998; 281: 829-832Crossref PubMed Scopus (175) Google Scholar, 10Choi I. Ostrovsky M. Zhang G. White K.A. J. Biol. Chem. 2001; 276: 41761-41768Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 11Price B.D. Roeder M. Ahlquist P. J. Virol. 2000; 74: 11724-11733Crossref PubMed Scopus (65) Google Scholar), overwhelming evidence for this mechanism is still lacking.Tomato bushy stunt virus (TBSV) is the prototype member of both the genus Tombusvirus and the family Tombusviridae. Its (+)-strand RNA genome of ∼4.8 kb encodes five functional ORFs (Fig.1A) (12Hearne P.Q. Knorr D.A. Hillman B.I. Morris T.J. Virology. 1990; 177: 141-151Crossref PubMed Scopus (175) Google Scholar). The viral RNA polymerase (p92) and accessory RNA replication protein (p33) are both translated from the genome, the former via translational readthrough of the amber termination codon of the latter (Fig. 1A) (12Hearne P.Q. Knorr D.A. Hillman B.I. Morris T.J. Virology. 1990; 177: 141-151Crossref PubMed Scopus (175) Google Scholar, 13Oster S.K. Wu B. White K.A. J. Virol. 1998; 72: 5845-5851Crossref PubMed Google Scholar). In contrast, the more 3′-proximal coat protein (CP) ORF (p41) and the overlapping ORFs encoding the movement (p22) and defense (p19) proteins are expressed from two sg mRNAs that are synthesized during TBSV infections (Fig.1A) (14Hillman B.I. Hearne P. Rochon D. Morris T.J. Virology. 1989; 169: 42-52Crossref PubMed Scopus (53) Google Scholar, 15Scholthof H.B. Scholthof K.B. Kikkert M. Jackson A.O. Virology. 1995; 213: 425-438Crossref PubMed Scopus (143) Google Scholar, 16Voinnet O. Pinto Y.M. Baulcombe D.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14147-14152Crossref PubMed Scopus (825) Google Scholar). The transcriptional regulation of the smaller of the two sg mRNAs, sg mRNA2, has been studied previously and RNA sequences important for efficient production of this message have been identified both proximal and distal to its initiation site (17Zhang G. Slowinski V. White K.A. RNA (N. Y.). 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). Efficient accumulation of this sg mRNA requires a long-distance base pairing interaction between a sequence within the core element (CE), located just 5′ to the sg mRNA2 initiation site, and a complementary sequence within the distal element (DE) some ∼1100 nucleotides (nt) upstream (Fig. 1A) (17Zhang G. Slowinski V. White K.A. RNA (N. Y.). 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). This interaction functions in the (+)-strand of the viral genome and is proposed to mediate the proper positioning of other subelements within the DE and CE (10Choi I. Ostrovsky M. Zhang G. White K.A. J. Biol. Chem. 2001; 276: 41761-41768Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Additionally, the production of (−)-strand sg mRNA2 occurs independently of (+)-strand sg mRNA2 accumulation (10Choi I. Ostrovsky M. Zhang G. White K.A. J. Biol. Chem. 2001; 276: 41761-41768Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). This latter finding, along with the observed (+)-strand activity of the CE/DE interaction, is consistent with a premature termination mechanism for sg mRNA2 transcription.Essentially nothing is known about the regulatory RNA elements involved in the synthesis of sg mRNA1. This message is critical for a successful viral infection because it allows for the efficient expression of CP, 180 subunits of which are present in each assembled particle (14Hillman B.I. Hearne P. Rochon D. Morris T.J. Virology. 1989; 169: 42-52Crossref PubMed Scopus (53) Google Scholar). Currently, all that is known about sg mRNA1 transcription is that it can be effectively eliminated by the introduction of substitutions at and immediately 5′ to its site of initiation (17Zhang G. Slowinski V. White K.A. RNA (N. Y.). 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). In the present study, we have sought to define RNA sequences and higher order structures within the TBSV genome that are important for sg mRNA1 transcription. Our results provide evidence for a long-distance RNA·RNA interaction that mediates the accumulation of sg mRNA1. The data also indicate that this interaction occurs in the (+)-strand, likely acts in cis, specifically mediates the accumulation of (−)-strand sg mRNA1, and is responsible for regulating expression from an ectopic transcriptional initiation site. The properties of this long-distance RNA·RNA interaction are compared and contrasted with those of the DE/CE interaction involved in regulating sg mRNA2 accumulation. Collectively, these data provide additional insight into how sg mRNAs are transcribed in TBSV.DISCUSSIONOur analysis of the TBSV genome has allowed us to identify sequences and structures that are important for sg mRNA1 accumulation. Specifically, two sequences, AS1 and RS1, were found to be essential for efficient sg mRNA1 accumulation, and the data support a functional requirement for their base pairing in the (+)-strand. Additionally, the AS1/RS1 interaction was found to be important specifically for (−)-strand sg mRNA1 accumulation and was shown to regulate sg mRNA1 production from an ectopic initiation site. These data support a role for this long-distance RNA·RNA interaction in regulating transcriptional activity.Structural and Functional Features of the AS1/RS1 InteractionThe distant positioning and comparatively small size of AS1 and RS1 prompt the following questions: (i) how do these sequences find each other in the context of the genome? and (ii) could this interaction be facilitated and/or stabilized by other RNA elements? Our MFOLD analysis of tombusvirus sequences suggests that the global folding of the genome likely assists in the formation of this base paired segment in cis via colocalizing the participating sequences. Additionally, local structures could also be involved in mediating this interaction. Therefore, we analyzed AS1 and its flanking sequence by MFOLD for potential structures that could facilitate the AS1/RS1 interaction. Different conformations of a SL structure were predicted depending on the parameters used for the analysis, however, a common feature in each was the presence of all or most of AS1 within a terminal loop (Fig.7). This positioning, in a predicted single-stranded region, could facilitate the presentation of AS1 for base pairing with RS1. Surprisingly, one of the predicted conformers (ΔG = −7.9 at 22 °C) is strikingly similar in general structure to the trans-acting hairpin activator of sg mRNA transcription in the bipartite (+)-strand virus red clover necrotic mosaic virus (RCNMV) (Fig. 7) (9Sit T.L. Vaewhongs A.A. Lommel S.A. Science. 1998; 281: 829-832Crossref PubMed Scopus (175) Google Scholar). In RCNMV, the nucleotides in the terminal loop of the hairpin activator in genomic RNA-2 interactin trans with a sequence just 5′ to the sg mRNA transcriptional initiation site in genomic RNA-1, thereby facilitating sg mRNA accumulation. It was proposed that, as with other bimolecular interactions, the base pairing of these elements would be favored by high concentrations of RNA-1 and RNA-2, which occur late in the infection (9Sit T.L. Vaewhongs A.A. Lommel S.A. Science. 1998; 281: 829-832Crossref PubMed Scopus (175) Google Scholar). This is also the time at which CP, which is translated from the induced RCNMV sg mRNA, is required in large quantities for encapsidation of progeny RNA genomes. The sg mRNA1 of TBSV also encodes CP, therefore, a similar concentration-dependent mechanism for controlling appropriate timing of induction of CP (i.e. late in the infection when progeny genomes are abundant) could also apply. Consistent with this notion are the observed accumulation profiles of sg mRNA1 and sg mRNA2 for TBSV and other tombusviruses during 24-h protoplast infections (17Zhang G. Slowinski V. White K.A. RNA (N. Y.). 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar, 24Johnson J.C. Rochon D.M. Virology. 1995; 214: 100-109Crossref PubMed Scopus (54) Google Scholar, 25Tavazza M. Lucioli A. Calogero A. Pay A. Tavazza R. J. Gen. Virol. 1994; 75: 1515-1524Crossref PubMed Scopus (31) Google Scholar). sg mRNA2 is detectable earliest in the infection, however, its accumulation then levels out at intermediate time points. In contrast, sg mRNA1 accumulation is low early in the infection but increases at later time points. The lack of functional complementation in co-infections with AS1- and RS1-defective mutants suggests that these elements do not function efficientlyin trans. However, because the two mutants could conceivably localize to different replication sites within coinfected cells, these results do not preclude the involvement of intermolecular interactions in WT infections.Figure 7MFOLD analysis of AS1 and its flanking sequences predicts the formation of stem-loop structures. Analysis of AS1 and its immediately adjacent sequences by MFOLD predicts stem-loop structures in which all or most of the AS1 nucleotides (inbold) reside in the terminal loop. Different optimal and suboptimal conformations are predicted at 37° C using MFOLD version 3.1 or at 22° C using MFOLD version 2.3, and the corresponding free energy values are indicated below each structure. The sequence shown is entirely conserved in sequenced tombusviruses, except for an adenylate (denoted by an asterisk) that is a guanylate in CNV and TBSV-S. For comparison, the predicted structure of the RCNMV trans-activator hairpin is shown to theright (9Sit T.L. Vaewhongs A.A. Lommel S.A. Science. 1998; 281: 829-832Crossref PubMed Scopus (175) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Formation of the AS1/RS1 helix is clearly essential for efficient sg mRNA1 accumulation, however, the precise nature of this interaction is unknown. It is possible that the base pairing interaction between AS1 and RS1 nucleates the formation of a larger more complex structure. For the RS1 context, the existence of an adjacent secondary structure, SL1sg1, is supported by MFOLD and comparative sequence analysis (Fig.1B). The formation of such a structure could further stabilize the AS1/RS1 helix through coaxial stacking (Fig.8A). Stabilization of higher order RNA structures by coaxial stacking of helices is quite common (26Batey R.T. Rambo R.P. Doudna J.A. Angew. Chem. Int. Ed. Engl. 1999; 38: 2326-2343Crossref PubMed Scopus (340) Google Scholar) and may be involved in stabilizing other RNA structures within the TBSV genome (27Wu B. Vanti W.B. White K.A. J. Mol. Biol. 2001; 305: 741-756Crossref PubMed Scopus (42) Google Scholar). For the AS1/RS1 interaction, additional types of stabilizing interactions could also exist. For instance, the sequence just 3′ to AS1 is complementary to the sequence just 5′ to SL1sg1, thus base pairing of these sequences could further stabilize the AS1/RS1 helix (Fig. 8A). The above examples illustrate the potential for this RNA interaction to be more complex and these putative structural features are being investigated.Figure 8RNA secondary structure models for the sequences involved in modulating sg mRNA1 and sg mRNA2 transcription in TBSV. The structures presented are based on a combination of compensatory-type mutational analyses, comparative sequence analyses, as well as MFOLD-based structural modeling.A, predicted RNA secondary structure for sg mRNA1 modulating sequences. The AS1/RS1 helix is in boldface, and the predicted adjacent SL1sg1 is delineated by a dotted bracket. A putative helix formed by base pairing of sequences 5′ to SL1sg1 and 3′ to AS1 is indicated by a parenthesis with aquestion sign. The initiation site for sg mRNA1 is indicated by a bent arrow. B, predicted RNA secondary structure for sg mRNA2 modulating sequences. The subelements of the DE and CE are delineated by thick horizontal and vertical lines. The base pairing nucleotides in A and B subelements of both DE and CE are inboldface. The initiation site for sg mRNA2 is indicated by a bent arrow. The 5′ boundary of the DE/CE RNA complex is indicated by a diagonal dashed line that traverses the contiguous sequence connecting the two structures.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Comparison of the AS1/RS1 Interaction with the DE/CE Interaction and Mechanistic InsightsThe AS1/RS1 interaction is the second long-distance interaction shown to be involved in sg mRNA synthesis in TBSV. The previously characterized DE/CE interaction, which is required for efficient sg mRNA2 production, shares both similarities and differences with the AS1/RS1 interaction (Fig. 8, compare A with B). Similarities between the predicted structures include: (i) the key base pairing interactions involve sequences just 5′ to the sites of initiation; (ii) the long-distance interactions span similar distances (∼1000–1100 nt); (iii) the functional interactions occur in the (+)-strand and; (iv) similar sequences surround the initiating nucleotides (underlined, 5′-CUUGA(C/A)CAAGA). There are, however, some distinct differences: (i) the overall RNA structures predicted share no striking similarities, other than those listed above; (ii) the AS1/RS1 base pairing interaction is smaller than the DE/CE base pairing interaction (7 versus 12 bp) and; (iii) the spacing of the base pairing interaction relative to the initiation nucleotides are different (3 versus 11 nt). It is possible that this latter difference reflects unique structural features that contribute to distinct activities for these RNA complexes (e.g. timing and/or amount of sg mRNA synthesized). Alternatively, the structure currently proposed for sg mRNA2 may be incomplete and could be lacking, for example, an additional base pairing interaction involving the CE-C sequence that would correspond to the AS1/RS1 helix. In potato virus X (PVX) long-distance base pairing interactions involving the 5′ terminus of the genome and sequences just 5′ to the initiation sites of its two sg mRNAs act to regulate sg mRNA accumulation (28Kim K.H. Hemenway C.L. Virology. 1997; 232: 187-197Crossref PubMed Scopus (53) Google Scholar, 29Kim K.H. Hemenway C.L. RNA (N. Y.). 1999; 5: 636-645Crossref PubMed Scopus (62) Google Scholar). The spacing between the end of the helix formed and the initiating nucleotides in both of these cases is 11 nt. In contrast, the corresponding spacing in RCNMV is 2 nt. Considering that RCNMV, but not PVX, is closely related to TBSV, and that the spacing for sg mRNA1 in TBSV is 3 nt, the latter of the two possibilities described above seems more plausible and is being explored.We have now defined two working RNA secondary structural models for RNA complexes involved in regulating sg mRNA accumulation in TBSV (Fig.8). Our data indicate that these structures function in the (+)-strand of the genome and likely regulate steps in the transcriptional process. It is interesting to note that, if both long-distance interactions were to occur simultaneously, the RNA complexes would be in close proximity to each another (Fig. 8). Such an association may be relevant to their function and could represent a type of multicomplex center where both structures are conveniently serviced by colocalized cis-and/or trans-acting elements. Some overlap in sequence function is suggested by the dependence of both sg mRNA1 and sg mRNA2 on RS1 (Fig. 3A). However, if sg mRNA2 is directly dependent on the sequence of RS1 (as opposed to its coding capacity, see below), it does not appear to rely on the AS1/RS1 interaction, because there was no recovery of sg mRNA2 levels when this helix was restored (Fig. 3A). Alternatively, it is possible that the minor modification to the extreme C terminus of p92 in RS1m1 is responsible for the observed defect. Indeed, modifications of viral RNA replication proteins have been found to specifically affect sg mRNA levels (30van Dinten L.C. den Boon J.A. Wassenaar A.L. Spaan W.J. Snijder E.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 991-996Crossref PubMed Scopus (166) Google Scholar, 31van Marle G. van Dinten L.C. Spaan W.J. Luytjes W. Snijder E.J. J. Virol. 1999; 73: 5274-5281Crossref PubMed Google Scholar).Viewed collectively, the information gathered on sg mRNA regulatory RNA elements in TBSV are consistent with their involvement in transcriptional regulation via a premature termination mechanism (10Choi I. Ostrovsky M. Zhang G. White K.A. J. Biol. Chem. 2001; 276: 41761-41768Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar,17Zhang G. Slowinski V. White K.A. RNA (N. Y.). 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar): (i) Accumulation of sg mRNA (−)-strands occurs independently of complementary (+)-strand accumulation, as would be expected if (−)-strands are synthesized first and then function as templates for (+)-strand synthesis. (ii) The key secondary structures function in the (+)-strand of the genome, as would be predicted for elements implicated in modulating (−)-strand accumulation. (iii) The AS1/RS1 interaction is required specifically for mediating (−)-strand sg mRNA1 accumulation, in accordance with a putative role for this element in promoting premature termination of (−)-strand synthesis. Although these data are consistent with a premature mechanism, they do not preclude alternative transcriptional models. Therefore, further studies will be necessary to determine conclusively the mechanism(s) utilized by TBSV for sg mRNA transcription. Viral infections of eukaryotic cells are complex processes that require regulated expression of a variety of viral genes. Depending on the virus, this expression can be regulated at different levels, including transcriptional, post-transcriptional, translational, and post-translational (1Maia I.G. Seron K. Haenni A. Bernardi F. Plant Mol. Biol. 1996; 32: 367-391Crossref PubMed Scopus (41) Google Scholar). For (+)-strand RNA viruses, many utilize RNA-templated transcription of subgenomic (sg)1 mRNAs to allow for regulated expression of specific viral genes (2Miller W.A. Koev G. Virology. 2000; 273: 1-8Crossref PubMed Scopus (155) Google Scholar). The mechanism by which sg mRNAs are synthesized can vary, but the messages produced share the common property of encoding open reading frames (ORFs) that are located 3′-proximally in the viral genomes. Because such 3′-proximal ORFs are generally translationally silent within the context of these genomes, sg mRNA production provides a mode for their efficient translation as well as a mechanism to regulate the timing and amount of viral protein produced (2Miller W.A. Koev G. Virology. 2000; 273: 1-8Crossref PubMed Scopus (155) Google Scholar). Two mechanisms for sg mRNA transcription are well-established: (i) synthesis of sg mRNAs from a full-length (−)-strand genomic template via internal initiation (3Miller W.A. Dreher T.W. Hall T.C. Nature. 1985; 313: 68-70Crossref PubMed Scopus (218) Google Scholar, 4Siegel R.W. Adkins S. Kao C.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11238-11243Crossref PubMed Scopus (102) Google Scholar) and (ii) synthesis of a non-contiguous RNA product during (−)-strand synthesis, which is then used as a template for transcription of sg mRNAs (5Pasternak A.O. Gultyaev A.P. Spaan W.J. Snijder E.J. J. Virol. 2000; 74: 11642-11653Crossref PubMed Scopus (39) Google Scholar, 6van Marle G. Dobbe J.C. Gultyaev A.P. Luytjes W. Spaan W.J. Snijder E.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12056-12061Crossref PubMed Scopus (176) Google Scholar). A third possible mechanism that has been proposed involves premature termination during (−)-strand synthesis of the genome followed by use of the 3′-truncated product as a template to transcribe sg mRNAs (7Zhong W. Rueckert R.R. J. Virol. 1993; 67: 2716-2722Crossref PubMed Google Scholar, 8Miller W.A. Brown C.M. Wang S. Semin. Virol. 1997; 8: 3-13Crossref Scopus (46) Google Scholar, 9Sit T.L. Vaewhongs A.A. Lommel S.A. Science. 1998; 281: 829-832Crossref PubMed Scopus (175) Google Scholar). Although this latter model is consistent with data generated from studies on an assortment of (+)-strand RNA viruses (9Sit T.L. Vaewhongs A.A. Lommel S.A. Science. 1998; 281: 829-832Crossref PubMed Scopus (175) Google Scholar, 10Choi I. Ostrovsky M. Zhang G. White K.A. J. Biol. Chem. 2001; 276: 41761-41768Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 11Price B.D. Roeder M. Ahlquist P. J. Virol. 2000; 74: 11724-11733Crossref PubMed Scopus (65) Google Scholar), overwhelming evidence for this mechanism is still lacking. Tomato bushy stunt virus (TBSV) is the prototype member of both the genus Tombusvirus and the family Tombusviridae. Its (+)-strand RNA genome of ∼4.8 kb encodes five functional ORFs (Fig.1A) (12Hearne P.Q. Knorr D.A. Hillman B.I. Morris T.J. Virology. 1990; 177: 141-151Crossref PubMed Scopus (175) Google Scholar). The viral RNA polymerase (p92) and accessory RNA replication protein (p33) are both translated from the genome, the former via translational readthrough of the amber termination codon of the latter (Fig. 1A) (12Hearne P.Q. Knorr D.A. Hillman B.I. Morris T.J. Virology. 1990; 177: 141-151Crossref PubMed Scopus (175) Google Scholar, 13Oster S.K. Wu B. White K.A. J. Virol. 1998; 72: 5845-5851Crossref PubMed Google Scholar). In contrast, the more 3′-proximal coat protein (CP) ORF (p41) and the overlapping ORFs encoding the movement (p22) and defense (p19) proteins are expressed from two sg mRNAs that are synthesized during TBSV infections (Fig.1A) (14Hillman B.I. Hearne P. Rochon D. Morris T.J. Virology. 1989; 169: 42-52Crossref PubMed Scopus (53) Google Scholar, 15Scholthof H.B. Scholthof K.B. Kikkert M. Jackson A.O. Virology. 1995; 213: 425-438Crossref PubMed Scopus (143) Google Scholar, 16Voinnet O. Pinto Y.M. Baulcombe D.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14147-14152Crossref PubMed Scopus (825) Google Scholar). The transcriptional regulation of the smaller of the two sg mRNAs, sg mRNA2, has been studied previously and RNA sequences important for efficient production of this message have been identified both proximal and distal to its initiation site (17Zhang G. Slowinski V. White K.A. RNA (N. Y.). 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). Efficient accumulation of this sg mRNA requires a long-distance base pairing interaction between a sequence within the core element (CE), located just 5′ to the sg mRNA2 initiation site, and a complementary sequence within the distal element (DE) some ∼1100 nucleotides (nt) upstream (Fig. 1A) (17Zhang G. Slowinski V. White K.A. RNA (N. Y.). 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). This interaction functions in the (+)-strand of the viral genome and is proposed to mediate the proper positioning of other subelements within the DE and CE (10Choi I. Ostrovsky M. Zhang G. White K.A. J. Biol. Chem. 2001; 276: 41761-41768Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Additionally, the production of (−)-strand sg mRNA2 occurs independently of (+)-strand sg mRNA2 accumulation (10Choi I. Ostrovsky M. Zhang G. White K.A. J. Biol. Chem. 2001; 276: 41761-41768Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). This latter finding, along with the observed (+)-strand activity of the CE/DE interaction, is consistent with a premature termination mechanism for sg mRNA2 transcription. Essentially nothing is known about the regulatory RNA elements involved in the synthesis of sg mRNA1. This message is critical for a successful viral infection because it allows for the efficient expression of CP, 180 subunits of which are present in each assembled particle (14Hillman B.I. Hearne P. Rochon D. Morris T.J. Virology. 1989; 169: 42-52Crossref PubMed Scopus (53) Google Scholar). Currently, all that is known about sg mRNA1 transcription is that it can be effectively eliminated by the introduction of substitutions at and immediately 5′ to its site of initiation (17Zhang G. Slowinski V. White K.A. RNA (N. Y.). 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). In the present study, we have sought to define RNA sequences and higher order structures within the TBSV genome that are important for sg mRNA1 transcription. Our results provide evidence for a long-distance RNA·RNA interaction that mediates the accumulation of sg mRNA1. The data also indicate that this interaction occurs in the
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