Regulatory Activity of Distal and Core RNA Elements in Tombusvirus Subgenomic mRNA2 Transcription

Positive-strand RNA viruses that encode multiple cistrons often mediate expression of 3′-encoded open reading frames via RNA-templated transcription of subgenomic (sg) mRNAs. Tomato bushy stunt virus (TBSV) is a positive-strand RNA virus that transcribes two such sg mRNAs during infections. We have previously identified a distal element (DE), located ∼1100 nucleotides upstream from the initiation site of sg mRNA2 transcription, part of which must base pair with a portion of a core element (CE), located just 5′ to the initiation site, for efficient transcription to occur (Zhang, G., Slowinski, V., and White, K. A. (1999) RNA5, 550–561). Here we have analyzed further this long distance RNA-RNA interaction and have investigated the regulatory roles of other subelements within the DE and CE. Our results indicate that (i) the functional base-pairing interaction between these elements occurs in the positive strand and that the interaction likely acts to properly position other subelements, (ii) two previously undefined subelements within the DE and CE are important and essential, respectively, for efficient sg mRNA2 accumulation, and (iii) the production of (−)-strand sg mRNA2 can be uncoupled from the synthesis of its (+)-strand complement. These data provide important insight into the mechanism of sg mRNA2 transcription. Positive-strand RNA viruses that encode multiple cistrons often mediate expression of 3′-encoded open reading frames via RNA-templated transcription of subgenomic (sg) mRNAs. Tomato bushy stunt virus (TBSV) is a positive-strand RNA virus that transcribes two such sg mRNAs during infections. We have previously identified a distal element (DE), located ∼1100 nucleotides upstream from the initiation site of sg mRNA2 transcription, part of which must base pair with a portion of a core element (CE), located just 5′ to the initiation site, for efficient transcription to occur (Zhang, G., Slowinski, V., and White, K. A. (1999) RNA5, 550–561). Here we have analyzed further this long distance RNA-RNA interaction and have investigated the regulatory roles of other subelements within the DE and CE. Our results indicate that (i) the functional base-pairing interaction between these elements occurs in the positive strand and that the interaction likely acts to properly position other subelements, (ii) two previously undefined subelements within the DE and CE are important and essential, respectively, for efficient sg mRNA2 accumulation, and (iii) the production of (−)-strand sg mRNA2 can be uncoupled from the synthesis of its (+)-strand complement. These data provide important insight into the mechanism of sg mRNA2 transcription. open reading frame core element distal element polymerase chain reaction Red clover necrotic mosaic virus subgenomic Tomato bushy stunt virus wild type nucleotide(s) Many (+)-strand (i.e. messenger-sensed) RNA viruses possess polycistronic coding organizations (1Maia I.G. Seron K. Haenni A. Bernardi F. Plant Mol. Biol. 1996; 32: 367-391Crossref PubMed Scopus (41) Google Scholar). However, this coding strategy poses a problem for translation of sequentially encoded open reading frames (ORFs)1 within these genomes. The difficulty arises due to the 5′ to 3′ and linear nature of conventional ribosome scanning, which generally allows for only the first ORF encoded in a message to be translated efficiently (2Kozak M. Cell. 1978; 15: 1109-1123Abstract Full Text PDF PubMed Scopus (508) Google Scholar). A coping strategy used commonly by a wide variety of (+)-strand RNA viruses is to synthesize smaller viral mRNAs via RNA-templated transcription (3Miller W.A. Koev G. Virology. 2000; 273: 1-8Crossref PubMed Scopus (159) Google Scholar). These so-called subgenomic (sg) mRNAs represent 5′-truncated 3′-coterminal copies of the genome that permit efficient translation of their most 5′-proximal ORFs (3Miller W.A. Koev G. Virology. 2000; 273: 1-8Crossref PubMed Scopus (159) Google Scholar). There is compelling evidence for two distinct mechanisms for the transcription of sg mRNAs. The first mechanism, which has been studied most extensively in Brome mosaic virus, involves the initiation of transcription at a localized internal promoter within the full-length (−)-strand of the genome (4Miller W.A. Dreher T.W. Hall T.C. Nature. 1985; 313: 68-70Crossref PubMed Scopus (218) Google Scholar, 5Siegel R.W. Adkins S. Kao C.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11238-11243Crossref PubMed Scopus (102) Google Scholar). The second defined mechanism, which occurs in the arteriviruses, involves the discontinuous synthesis of (−)-strands that are then used as templates in the production of sg mRNAs, which include a portion of the 5′-untranslated region of the genome (6Pasternak A.O. Gultyaev A.P. Spaan W.J. Snijder E.J. J. Virol. 2000; 74: 11642-11653Crossref PubMed Scopus (39) Google Scholar, 7van 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 (179) Google Scholar). A third mechanism that has been proposed is that of premature termination of (−)-strand synthesis during the copying of the genome and subsequent use of the 3′-truncated product as a template for the transcription of sg mRNAs (8Zhong W. Rueckert R.R. J. Virol. 1993; 67: 2716-2722Crossref PubMed Google Scholar, 9Miller W.A. Brown C.M. Wang S. Semin. Virol. 1997; 8: 3-13Crossref Scopus (46) Google Scholar, 10Sit T.L. Vaewhongs A.A. Lommel S.A. Science. 1998; 281: 829-832Crossref PubMed Scopus (178) Google Scholar). Although there is no compelling experimental evidence validating this latter model, it is supported indirectly by the observation that (−)-strand sg mRNAs do accumulate in various (+)-strand RNA viral infections (8Zhong W. Rueckert R.R. J. Virol. 1993; 67: 2716-2722Crossref PubMed Google Scholar, 11Chapman S. Hills G. Watts J. Baulcombe D. Virology. 1992; 191: 223-230Crossref PubMed Scopus (173) Google Scholar, 12Tavazza M. Lucioli A. Calogero A. Pay A. Tavazza R. J. Gen. Virol. 1994; 75: 1515-1524Crossref PubMed Scopus (31) Google Scholar, 13Price B.D. Roeder M. Ahlquist P. J. Virol. 2000; 74: 11724-11733Crossref PubMed Scopus (66) Google Scholar). However, the origin and function of these (−)-strand sg mRNAs remain to be determined. Tomato bushy stunt virus (TBSV) is the prototype member of the family Tombusviridae (14Hearne P.Q. Knorr D.A. Hillman B.I. Morris T.J. Virology. 1990; 177: 141-151Crossref PubMed Scopus (176) Google Scholar). Its (+)-strand RNA genome is 4.8 kilobases in length and encodes five functional ORFs (see Fig.1 A). The 5′-terminally encoded p33 and its readthrough product p92 are the only viral proteins required for viral RNA synthesis, and both are translated directly from the viral genome (15Oster S.K. Wu B. White K.A. J. Virol. 1998; 72: 5845-5851Crossref PubMed Google Scholar). The three ORFs encoded more 3′ in the genome are involved in viral assembly, movement, and suppression of host defense mechanisms and are translated from two sg mRNAs that are synthesized during infections (Fig. 1 A) (16Scholthof H.B. Scholthof K.B. Kikkert M. Jackson A.O. Virology. 1995; 213: 425-438Crossref PubMed Scopus (145) Google Scholar, 17Voinnet O. Pinto Y.M. Baulcombe D.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14147-14152Crossref PubMed Scopus (844) Google Scholar). RNA sequence elements within the genome that are required for transcription of the smaller sg mRNA2 have been identified previously (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar, 19Johnson J.C. Rochon D.M. Virology. 1995; 214: 100-109Crossref PubMed Scopus (54) Google Scholar). Efficient transcription of this message requires sequences both at the site of initiation, termed the core element (CE), as well as sequences some ∼1100 nucleotides (nt) upstream from the start site, termed the distal element (DE) (Fig.1 A). The DE and CE can each be divided further into subelements A, B, and C based on their relative positions and/or structural properties. The sequence of the CE-C subelement is highly conserved within tombusvirus genomes and is located just 5′ to the sg mRNA2 initiation site (Fig. 1 C) (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). The DE-C element is not well conserved within the genus Tombusvirus and displays no significant complementarity to CE-C. In contrast, the DE-A and CE-A subelements and the DE-B and CE-B subelements are complementary to each other, and previous studies have provided compelling evidence that base pairing of the former pair is required for efficient sg mRNA2 synthesis (Fig. 1, B and C) (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). A functional role for the DE-A/CE-A base-pairing interaction was supported by comparative sequence analysis of tombusvirus genomes as well as deletion and compensatory-type mutational analyses (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). Interestingly, long distance base-pairing interactions have also been found to be important for sg mRNA transcription in the unrelated virus Potato virus X. (20Kim K.H. Hemenway C.L. Virology. 1997; 232: 187-197Crossref PubMed Scopus (54) Google Scholar,21Kim K.H. Hemenway C.L. RNA. 1999; 5: 636-645Crossref PubMed Scopus (63) Google Scholar). This suggests that long range RNA interactions involved in regulating sg mRNAs may be a common feature in diverse groups of (+)-strand RNA viruses. However, the functions of these far-spanning interactions may be quite different within the unique contexts of these distinct viral genomes. In a previous study we confirmed the functional requirement for the DE-A/CE-A base-pairing interaction (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar); however, these analyses did not determine whether this interaction occurred in the (+)- or (−)-strand of the genome nor did they investigate the specific role of the interaction in sg mRNA2 transcription. To address these questions and to determine the possible roles of other yet uncharacterized subelements, we have carried out additional analyses of the DE and CE. Our results indicate that the DE-A/CE-A base-pairing interaction is functional in the (+)-strand and that the DE-C and CE-C subelements represent newly defined components that are important and essential, respectively, for efficient sg mRNA transcription. Additionally, by localizing both the DE and CE at different positions within the genome, we have deduced that the likely function of the DE-A/CE-A interaction is to position other subelements optimally. Furthermore, mutational analysis of the initiating nucleotide for sg mRNA2 transcription has provided clues to the origin of (−)-strand sg mRNAs and, together with other results, provided important new insight into the mechanism of sg mRNA2 transcription. The plasmid T-100, containing a full-length cDNA copy of the wild type (WT) TBSV genome, has been described previously (14Hearne P.Q. Knorr D.A. Hillman B.I. Morris T.J. Virology. 1990; 177: 141-151Crossref PubMed Scopus (176) Google Scholar). Construction of mutant derivatives of T-100 containing modifications to the DE and/or CE (i.e. Psg20/26 and ΔPsg1) have also been described (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). All mutant viral constructs used in this study by and polymerase chain reaction J. A and are derivatives of the previously described constructs or Additionally, new mutant constructs to that only the modifications as well as and derivatives of Psg20/26 (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). and or respectively, used to containing the modifications (see A). The with and and used to the in and or respectively, used to containing the modifications (see A). The with and and used to the in The constructs and by the in with in This was by the in with the from All described in from Psg20/26 (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google only by the in A. The and in which the DE was to the CE derivatives of (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). The strategy used was to and or The containing the localized sequences (see A) then used to the and in To constructs with the localized sequences the position of the constructs and used to constructs and This was by of the former with and and of the by of the of the with polymerase and subsequent The and as derivatives of Psg20/26 with and or respectively, and the to the in These the CE-C sequence or a CE-C that the nucleotide respectively, and are to Psg20/26 (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google for the modifications described The mutant in which the DE-C was from its is a of Psg20/26 and was the as described for The and are derivatives of T-100 (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google in which the initiation nucleotide for sg mRNA2 transcription was or with or and was with pair and by of the product with and and of the in and in that the product used for was pair and the All of these constructs are to T-100 for the nucleotide and a deletion directly 5′ to the site in templates for in transcription by containing viral with RNA synthesized the K.A. Morris T.J. J. Virol. 1994; PubMed Google Scholar). from to and with of each viral RNA and in a at for K.A. Morris T.J. J. Virol. 1994; PubMed Google Scholar). from as described previously K.A. Morris T.J. J. Virol. 1994; PubMed Google that for the of (−)-strand viral in with (+)-strand viral RNA of the in and to analysis complementary to the of the TBSV genome K.A. Morris T.J. J. Virol. 1994; PubMed Google Scholar). by of J. A Scholar). viral with in RNA to the of the TBSV and of the RNA in as by the both (+)- and (−)-strand of was for J. M. J. Mol. Biol. 1999; PubMed Scopus Google Scholar, M. J. RNA and The Scholar). results a requirement for a base-pairing interaction between the DE-A and CE-A for efficient sg mRNA2 transcription (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). However, the (i.e. of of the base involved are of the this interaction occur in the (+)- or (−)-strand (Fig.1 In the study we to this by base into DE-A or CE-A that and the DE-A/CE-A base-pairing interaction in the (+)- or (−)-strand A). These into the previously described mutant genome Psg20/26 that is to due to the of the DE-A and CE-A subelements (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). Psg20/26 both the DE-B and CE-B subelements (Fig. A) sg mRNA2 at that of the genome (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). (−)-strand was by or three base into DE-A/CE-A nucleotide within the (+)-strand of mutant derivatives and (Fig. A). These base to be to the (+)-strand interaction to the (−)-strand interaction that complementary (Fig. A). or three into DE-A/CE-A in the (+)-strand of Psg20/26 to the (+)-strand and (Fig. A). In to the genome (Fig. 1 T-100, and mutant genomes synthesized in and into analysis was then on to determine whether the modifications the of sg mRNA2 (Fig. The relative of sg mRNA2 by analysis of the and the represent of sg mRNA2 to their RNA to that for Psg20/26 (Fig. which to be in the (−)-strand DE-A/CE-A a in sg mRNA2 for to a that of the Psg20/26 In contrast, which to be in the (+)-strand a more in sg mRNA2 to that of the Psg20/26 These data indicate that the (+)-strand DE-A/CE-A interaction is more to its (−)-strand and a functional role for this interaction in the the more for be to the in nucleotide in DE-C to their base-pairing To address this we base-pairing for and by additional into DE-A that base pairing the in and (Fig. A). of these that sg mRNA2 Psg20/26 in the to C in these the functional is likely (+)-strand base pairing nucleotide In the TBSV genome the DE and CE are by ∼1100 The for the long distance of these two elements is not may be to the by which they In to insight into the function of these we a of in which the two elements directly to in the In these mutant the sequence the DE and CE was to a site A). of the first of the previously mutant genome was used as the base (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). In sg synthesis is by five nucleotide in and its site of initiation (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). Additionally, and more to this the DE is from its upstream position (Fig. in only of sg mRNA2 that of T-100, To determine whether sg mRNA2 synthesis be by the DE to CE in the DE sequence was just 5′ to the CE The (Fig. and was then into to the on sg mRNA2 analysis of viral a in sg mRNA2 to that of the T-100 also in which various subelements In and the subelements or both the and DE-A/CE-A subelements, respectively, from their localized positions A). These modifications in relative sg mRNA of and (Fig. These results that the nor the DE-A/CE-A subelement interactions are required for the in sg mRNA2 and DE-C as a possible subelement for To further this latter the DE-C subelement was from the and contexts to and A). of these genomes of sg mRNA2 to that of important role for DE-C within this the of localized (Fig. A) was the DE The of the localized in this second of was to in the however, the Although these also derivatives of their genomes ∼1100 due to the of the sequence between the DE and CE in these constructs the genomes of these (Fig. This was in which the CE be from its and just 3′ to the DE the ∼1100 5′ to the the sg mRNA from such a mutant be ∼1100 the sg This highly sg mRNA be to have that with sg The analysis of which the CE element the DE sg mRNA2 that of T-100 (Fig. of the or and DE-A/CE-A subelements in and respectively, in in sg mRNA2 to and (Fig. This of on these subelements is to that for the localized at the CE (Fig. C) and suggests that the and DE-A/CE-A subelements are for the (i.e. that of ΔPsg1) for these localized Interestingly, the deletion of the DE-C subelement in and did not to in sg mRNA2 These latter results are in to for and deletion of DE-C in of sg mRNA2 to that of (Fig. This that the requirement for DE-C is To the role of the newly defined and previously uncharacterized CE-C two mutant genomes within a Psg20/26 In the first the CE-C subelement was however, the initiating located just 3′ to CE-C (see 1 C) was A). the second the sequence of CE-C was to to the nucleotide not the sequence (Fig. A). the initiating just 3′ to CE-C was for their to sg mRNA2 transcription, both of sg mRNA2 (Fig. This CE-C as a new subelement that is for efficient sg mRNA2 The requirement for the DE-C subelement in the more Psg20/26 was also by mutant in which the DE-C was (Fig. A). of this subelement to a significant in the relative of sg mRNA2 that of This DE-C as important for sg mRNA2 it is in a To investigate the role of the initiating nucleotide located just 3′ to the in the genome was to A, or the and A). of the three to in sg mRNA2 (Fig. In contrast, (−)-strand viral analyzed for the infections there was no in the relative (−)-strand sg mRNA2 and, in two relative (Fig. These indicate that the initiating nucleotide is required for efficient of sg mRNA2 and suggests that it can also to a the relative of sg mRNA2 In this study we have carried out a more analysis of the RNA subelements in the of sg mRNA2 transcription in results from have confirmed the of the DE-A/CE-A base-pairing interaction for efficient sg mRNA2 transcription (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). Our results that this interaction is functional in the (+)-strand (Fig. of this interaction by the of with base did not sg mRNA2 accumulation, three base (Fig. This suggests that this interaction may be at its required for The (+)-strand defined for the DE-A/CE-A interaction also likely to the interaction that was proposed to role in and/or the DE-A/CE-A interaction (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). that these two interactions occur in the (+)-strand from previous comparative sequence analyses of elements in other of the genus Tombusvirus (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). of the a of and base within the analyzed base are common in and are to be more their (−)-strand S. Mol. Biol. 1997; PubMed Scopus Google Scholar). these data the functional interaction of and by in the (+)-strand of the viral is the function of the DE-A/CE-A interaction in the A is to in Red clover necrotic mosaic virus it occurs just upstream from the initiation site of sg mRNA synthesis T.L. Vaewhongs A.A. Lommel S.A. Science. 1998; 281: 829-832Crossref PubMed Scopus (178) Google Scholar). in the interaction is that it occurs in as it involves base pairing between the two RNA the the it was proposed that of this acts to the viral polymerase during (−)-strand synthesis and that in it to and (−)-strand sg mRNAs T.L. Vaewhongs A.A. Lommel S.A. Science. 1998; 281: 829-832Crossref PubMed Scopus (178) Google Scholar). These (−)-strand sg mRNAs then be used as templates for (+)-strand sg mRNA transcription. Our previous studies that the DE-A/CE-A interaction is for sg mRNA2 synthesis the DE and CE are by ∼1100 within the genome (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). However, results indicate that the DE and CE are directly to each other, there is no requirement for the DE-A/CE-A elements containing only the subelements to sg mRNA2 transcription (Fig. C and This observation suggests that the role of the DE-A/CE-A interaction within the of the genome may be in the relative of other subelements, such as DE-C and/or their (see additional roles for this interaction be The the DE-A and is not to have significant complementarity to its in the CE-C (Fig. 1 Our results indicate that the of the DE-C subelement for sg mRNA2 is the DE was the CE deletion of DE-C to a in sg mRNA2 (Fig. However, the CE was localized the DE no such was deletion of CE-C (Fig. In the more of the DE-C was also found to be important for sg mRNA2 (Fig. on its structural a possible role for this element may be to not base pair with CE-C. This in CE-C to with other sequences and/or This is with the observation that there is no significant sequence DE-C elements from different of the genus yet a common feature is that they complementarity to their CE-C subelements (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). In contrast, the sequence the CE-C is highly conserved for of this genus and is located just 5′ to the site of initiation of sg mRNA2 (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar). This subelement was found to be for efficient sg mRNA2 (Fig. In the sequence just 5′ to the initiation site of its sg mRNA with a complementary viral sequence to mediate sg mRNA transcription T.L. Vaewhongs A.A. Lommel S.A. Science. 1998; 281: 829-832Crossref PubMed Scopus (178) Google Scholar). to the of these viruses (i.e. they are both of the family it is possible that sg mRNA2 transcription in TBSV also requires base pairing of its CE-C. The of complementarity between CE-C and DE-C the latter from in this However, possible base-pairing for the CE-C sequence have been identified within the TBSV sequence and are analyzed to determine whether they in regulating sg mRNA2 transcription. The initiating nucleotide for sg mRNA2 synthesis has been by to a just 3′ to the CE-C B.I. P. Rochon D. Morris T.J. Virology. PubMed Scopus Google Scholar). Our results have that for efficient transcription of sg mRNA2 there is a requirement for the of the initiating nucleotide to be a However, the requirement was not for the production of (−)-strands to sg mRNA2 These (−)-strands have been previously as possible templates for the synthesis of (+)-strand sg mRNA2 (18Zhang G. Slowinski V. White K.A. RNA. 1999; 5: 550-561Crossref PubMed Scopus (62) Google Scholar); however, they are the sg mRNA2 (−)-strands accumulate as synthesized from sg mRNAs from the full-length (−)-strand of the genome represent synthesized during (−)-strand synthesis of the (+)-strand In the latter the sg mRNA2 (−)-strands as by as templates for the transcription of (+)-strand sg Our that (−)-strand not with (+)-strand the (−)-strands from (+)-strand sg This of on (+)-strand sg mRNA has also been for virus B.D. Roeder M. Ahlquist P. J. Virol. 2000; 74: 11724-11733Crossref PubMed Scopus (66) Google Scholar). in some the between the two is with (+)-strand to (−)-strand accumulation, as for and (Fig. B and These results the that the (−)-strand sg mRNAs are not from (+)-strand sg they represent that during (−)-strand synthesis of the viral genome in as templates for sg mRNA2 The of the (−)-strands from the and to transcription of be to the requirement of tombusvirus to efficiently only to C within in viral templates J. Virology. 2000; PubMed Scopus Google Scholar). results are with the that sg mRNA2 transcription occur via a premature termination However, other such as the the of be out at the are to further investigate the by which sg mRNA2 is

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