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Journal of Virology, February 2004, p. 1129-1138, Vol. 78, No. 3
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.3.1129-1138.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Bunyamwera Bunyavirus RNA Synthesis Requires Cooperation of 3'- and 5'-Terminal Sequences

John N. Barr* and Gail W. Wertz

Department of Microbiology, University of Alabama School of Medicine, Birmingham, Alabama 35294

Received 27 August 2003/ Accepted 22 October 2003


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ABSTRACT
 
Bunyamwera virus (BUNV) is the prototype of both the Orthobunyavirus genus and the Bunyaviridae family of segmented negative-sense RNA viruses. The tripartite BUNV genome consists of small (S), medium (M), and large (L) segments that are each transcribed to yield a single mRNA and are replicated to generate an antigenome that acts as a template for synthesis of further genomic strands. As for all negative-sense RNA viruses, the 3'- and 5'-terminal nontranslated regions (NTRs) of the BUNV S, M, and L segments exhibit nucleotide complementarity and, except for one conserved U-G pairing, this complementarity extends for 15, 18, and 19 nucleotides, respectively. We investigated whether the complementarity of 3' and 5' NTRs reflected a functional requirement for terminal cooperation to promote BUNV RNA synthesis or, alternatively, was a consequence of genomic and antigenomic NTRs having similar functions requiring sequence conservation. We show that cooperation between 3'- and 5'-NTR sequences is required for BUNV RNA synthesis, and our results suggest that this cooperation is due to nucleotide complementarity allowing 3' and 5' NTRs to associate through base-pairing interactions. To examine the importance of complementarity in promoting BUNV RNA synthesis, we utilized a competitive replication assay able to examine the replication ability of all possible combinations of interacting nucleotides within a defined region of BUNV 3' and 5' NTRs. We show here that maximal RNA replication was signaled when sequences exhibiting perfect complementarity within 3' and 5' NTRs were selected.


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INTRODUCTION
 
The Bunyaviridae family of enveloped segmented negative-sense RNA viruses comprise five genera, namely, Orthobunyavirus, Hantavirus, Phlebovirus, Nairovirus, and Tospovirus. Many bunyaviruses are serious human pathogens, and in recognition of this fact, several are described by the Centers for Disease Control and Prevention as category A priority pathogens due to their ability to cause lethal hemorrhagic fevers. Examples include Rift Valley fever phlebovirus, Crimean-Congo hemorrhagic fever nairovirus, and the reassortant Garissa Orthobunyavirus. Furthermore, the Bunyaviridae family includes several so-called "emerging" viral pathogens including the Sin Nombre and Andes hantaviruses and the LaCrosse, Oropouche, and Cache Valley orthobunyaviruses.

The type species of the Bunyaviridae family is Bunyamwera orthobunyavirus (BUNV). For many decades, BUNV has been the subject of intense investigation, and it is now one of the most thoroughly characterized members of this family. Many features of BUNV molecular and cellular biology are common to other Bunyaviridae family members, and consequently BUNV is recognized as a model for studying the serious human pathogens within this family of viruses.

The BUNV genome comprises three segments of negative-sense RNA designated the small (S), medium (M), and large (L) segments. The S segment encodes the nucleocapsid (N) and a nonstructural protein (NSs) from overlapping open reading frames (ORFs) on a single mRNA (15, 24-26). The N protein is responsible for encapsidating the BUNV replication products to form ribonucleoprotein (RNP) templates and the NSs protein acts as an interferon antagonist (6, 50) and has also been reported to downregulate reporter gene activity in a minireplicon system (51). The M segment encodes a polyprotein that is cleaved to generate two envelope glycoproteins (G1 and G2) and also the NSm polypeptide of unknown function (13, 25, 27). The L segment encodes the virus-encoded component of the BUNV RNA-dependent RNA polymerase (14).

Surrounding the coding regions of each segment are terminal nontranslated regions (NTRs) that vary in length between 50 and 85 nucleotides (nt) at the genomic 3' ends and between 100 and 174 nt at the genomic 5' ends. Together, these NTRs contain sequences that control two distinct RNA synthetic activities of each segment: (i) transcription to generate a single mRNA and (ii) replication to generate a positive-sense anti-genome that acts as intermediate for synthesis of further genomic strands. The genome and antigenome replication products are exact complementary copies of each other. In contrast, the mRNAs of BUNV and other bunyaviruses are shorter than their respective genomic templates due to 3'-end truncation of approximately 100 nt (S mRNA) or 40 nt (M and L mRNAs) (4, 11, 17, 29). In addition, the 5' ends of BUNV mRNAs possess a 5' extension relative to the genomic template that is between 12 and 17 nt in length and is capped (29), and this feature is a characteristic of many other bunyaviruses (3, 4, 10, 12, 16, 30, 39). These 5' extensions are derived from host cell cytoplasmic mRNAs and are thought to be generated by a "cap-snatching" process similar to that which results in the characteristic 5' cap structures of Influenza orthomyxovirus mRNAs (5, 32, 43).

The nucleotide sequences of the 3' NTRs of all three BUNV genomic segments are identical for the first 11 nt (Fig. 1A), as are the corresponding 5' NTR sequences. In addition, these 3' and 5' sequences exhibit complementarity for all but one of these positions: a conserved U-G pairing at nucleotide position 9. After these 11 nt, the sequence of the S, M, and L segments are unique but still exhibit terminal complementarity up to nucleotide positions 15, 18, and 19, respectively. Based on their degree of sequence conservation, these terminal nucleotides can be divided into the conserved (nt 1 to 11) and variable complementary regions (nt 12 to 15, 12 to 18, and 12 to 19 for the S, M, and L segments, respectively) (Fig. 1A).



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  		FIG. 1. (A) Schematic of the BUNV S, M, and L genomic RNAs. Only the first 25 nt of both 3'- and 5'-terminal regions of BUNV segments are shown. Potential to form Watson-Crick base pairs () or noncanonical U-G pairings (•) is indicated. The first 11 nt of both 3' and 5' termini are conserved between all segments and comprise the conserved complementary region (boxed sequence). The variable complementary region of each segment is shown (shaded sequence). (B) Schematic of plasmids pBUN-S(ren), pBUN-M(ren), and pBUN-L(ren) designed to generate BUNV S-, M-, and L-segment-specific templates BUN-S(ren), BUN-M(ren), and BUN-L(ren), respectively. The nucleotides that comprise the entire 3' and 5' NTRs of each genomic RNA are specified above each corresponding NTR. The entire 3'- and 5'-NTR sequences from each BUNV segment (S, white; M, black; L, gray) were placed flanking a cDNA encoding a heterologous sequence (Renilla luciferase [ren]). NTRs were flanked by the T7 RNA polymerase promoter and the self-cleaving hepatitis deltavirus ribozyme, such that the primary transcript was of antigenomic polarity and contained two additional G residues at its 5' terminus. The positions of the StuI and XbaI restriction enzyme recognition sites common to all three genome analog-expressing plasmids and used to generate exchanged templates are shown.

The potential for nucleotides within the conserved and variable complementary regions to interact is thought to be responsible for allowing bunyavirus RNPs to adopt a circular conformation observed by electron microscopy and detected by biochemical analysis (38, 42, 45, 46). The 3'- and 5'-terminal regions of each influenza virus segment are capable of interacting through base pairing to form partially double-stranded RNA panhandle structures (1, 9, 28, 37, 48, 49). Analysis of the RNA synthesis activity of influenza virus model RNA and RNP templates has further refined this structure and provided evidence for various structural models that describe RNP conformation. Much evidence supports a cork-screw model in which a short hairpin comprising a 2-nt stem and an exposed 4-nt loop exists proximal to both 3' and 5' termini. Additional interactions between complementary nucleotides within the 3' and 5' termini allows for the characteristic influenza virus terminal interaction and panhandle formation (7, 19, 20, 22, 23, 31, 34-36, 44). Whether functional terminal interaction is a feature of all segmented negative-sense RNA viruses is unknown. The terminal regions of Thogoto orthomyxovirus are thought to adopt a conformation closely related to the influenza virus 5' hairpin-loop model (33). It has also been reported that integrity of the predicted panhandle of Lymphocytic choriomeningitis arenavirus is essential for RNA synthesis (41). However, analysis of the RNA synthesis signaling ability of Uukuniemi phlebovirus indicated that increased potential for interaction between 3' and 5' NTRs did not increase RNA synthesis activity (18). We describe here experiments that investigate whether the terminal regions of BUNV must cooperate in order to perform RNA synthesis.

To investigate the requirements of BUNV RNA synthesis, we recently described a BHK cell-based assay that allows analysis of BUNV RNAs synthesized from either authentic cDNA-derived BUNV-segments, or BUNV-specific genome analogs having S-, M-, and L-segment coding regions replaced with a common sequence (Renilla luciferase [ren]) (Fig. 1B) (2). This assay allows direct detection of both replication and transcription products generated by BUNV templates and thus permits us to independently investigate the requirements of these two distinct RNA synthesis activities. This method offers a major advantage over similar systems that use reporter gene expression to analyze RNA synthesis activity, since the requirements of RNA replication and mRNA transcription can be distinguished unambiguously. We recently used this system to determine that the levels of RNA replication of the three BUNV segments were different, with their relative abilities being M > L > S. We further showed that these different replication abilities were due to segment-specific sequences within the 3' and 5' NTRs (2).

Here we demonstrate that 3'- and 5'-terminal NTRs of BUNV S, M, and L segments must cooperate in order to promote RNA synthesis. This represents the first report describing functional terminal cooperation for a member of the Bunyaviridae family. We provide evidence suggesting that terminal cooperation occurs by base-pairing interactions between complementary nucleotides at opposite ends of the BUNV template. We also show that perfect terminal complementarity between 3' and 5' NTRs allows the highest RNA replication activity independent of nucleotide sequence.


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MATERIALS AND METHODS
 
Plasmid constructions. Previously described plasmids pBUN-S(ren), pBUN-M(ren), and pBUN-L(ren) contained the entire 3' and 5' NTR sequences of the BUNV S, M, and L segments, respectively (2). These sequences surrounded a 936-nt cDNA encoding the Renilla luciferase protein and were flanked by sequences representing the T7 RNA polymerase promoter and the hepatitis deltavirus self-cleaving ribozyme (Fig. 1B). After ribozyme cleavage, the T7 RNA polymerase transcript generated from each of these plasmids was an antigenomic-sense RNA with an authentic 3' end and two additional non-BUNV residues (GG) at the 5' end.

Plasmids designed to transcribe the panel of six exchanged genome analogs were constructed by swapping cDNA fragments generated by StuI and XbaI restriction enzyme digestion of parental plasmids pBUN-S(ren), pBUN-M(ren), and pBUN-L(ren) (Fig. 1B). The StuI recognition sequence is located at the junction between the Renilla luciferase cDNA and the 5' genomic NTR (Fig. 1B), and the XbaI recognition sequence is located downstream of the hepatitis deltavirus ribozyme. Digestion with these enzymes released a 406-nt fragment encoding the entire genomic 5' NTR and ribozyme sequences, which was replaced with a corresponding fragment from a different parental plasmid.

Plasmid alterations that restored complementarity of exchanged templates were performed by QuikChange mutagenesis (Stratagene, La Jolla, Calif.) with mutagenic oligodeoxynucleotides (Operon, Inc., Valencia, Calif.). All sequence changes were confirmed as correct by DNA sequence analysis.

QuikChange mutagenesis was also used to generate pools of plasmids with defined genomic 3' nt 12 to 14 but randomized nt 12 to 14 at the 5' genomic terminus (underlined nucleotides denote positions counted from the 5' terminus). Randomization was achieved by incorporating oligodeoxynucleotides with variable nucleotides at the corresponding positions into the QuikChange reaction. Random cDNA plasmid pools were harvested from cultures directly inoculated with bacteria transformed by using the amplified QuikChange PCR product. The number of cDNAs successfully transformed into competent cells was estimated by spreading aliquots representing one-twentieth of the transformed cell volume onto bacterial growth plates. After adjusting for volume, we estimated that between 3,000 and 5,000 successfully transformed bacterial cells were present in the bacterial cultures used to prepare the random plasmid pools.

Transfections. BUNV RNAs were generated from plasmid-derived BUNV genome analogs as described previously (2). Briefly, 2.0-µg quantities of each support plasmids expressing BUNV S- and L-segment ORFs were transfected, along with 6.0 µg of genome analog-expressing plasmid into BHK-21 cells previously infected with vaccinia virus recombinant vTF7-3. BUNV-specific RNAs were metabolically labeled by incubating monolayers with [3H]uridine (33 µCi/ml; Moravek Biochemicals, Inc., Brea, Calif.) and actinomycin D (10 mg/ml; Sigma Chemical Co., St. Louis, Mo.) 12 h after transfection. After a 6-h labeling period, RNAs were harvested by using the RNeasy procedure (Qiagen, Inc., Valencia, Calif.).

RNA analysis. Metabolically labeled BUNV-specific RNAs were visualized by using agarose-urea gel electrophoresis, followed by fluorography and autoradiography.

Positive-sense BUNV RNAs were also detected by primer extension analysis with end-labeled negative-sense oligonucleotide primer 3'RENSEQ(-) (5'-CCTTTGTTCTGGATCATAAACTTTCG-3'), as described previously (2). Labeled primer extension products were electrophoresed on standard 6% polyacrylamide gels and then visualized by autoradiography. The oligodeoxynucleotide primer 3'RENSEQ(-) was also used to generate sequence ladders from appropriate plasmid templates, where necessary, by using modified T7 DNA polymerase (Sequenase 2.0; U.S. Biochemical Corp., Cleveland, Ohio).

Analysis of RNA replication from templates with randomized 5' genomic sequences. Five pools of plasmids designed to transcribe genome analogs with randomized nt 12 to 14 of the 5' genomic NTR were generated and incorporated into our RNA synthesis assay. After RNA replication, we identified the predominant sequence of previously random nt 12 to 14 by determining the nucleotide sequence of a bulk reverse transcription-PCR (RT-PCR) product representing the 5' end of the genomic RNA. Briefly, harvested RNAs were subjected to two successive rounds of RQ1 DNase digestion (Promega, Madison, Wis.) to remove all traces of transfected cDNA template. The resulting RNAs were incubated with positive-sense oligodeoxynucleotide RD(+) (5'-GGCGCTTGTTTGGCATTTCATTATAGC-3') at 100°C for 1 min and then rapidly chilled on ice. The primer was extended by using modified Moloney murine leukemia virus reverse transcriptase (Superscript II; Invitrogen, Carlsbad, Calif.) under the conditions recommended by the manufacturer. The primer extension product was purified by using the QiaQuick purification procedure (Qiagen) and then used as a template for PCR amplification with the negative-sense oligodeoxynucleotide 5'-11 (5'-AGTAGTGTGCT-3') and the positive-sense oligodeoxynucleotide RENSEQ(+) (5'-ATCAAATCGTTCGTTGAGCGAG-3'). The resulting RT-PCR product was sequenced by using RENSEQ(+) as primer.


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RESULTS
 
BUNV 3' and 5' NTRs cooperate to promote RNA synthesis. By analyzing the RNA synthesis activity of genome analog templates BUN-S(ren), BUN-M(ren), and BUN-L(ren), we previously showed that essential signals for the replication and transcription activities of BUNV S, M, and L segments reside within the 3' and 5' NTRs (2). For each of the BUNV S, M, and L segments, these NTRs exhibit terminal nucleotide complementarity for 15, 18, and 19 nt, respectively (Fig. 1A). This observed complementarity has been suggested to allow the 3' and 5' NTRs of BUNV genomic and antigenomic RNAs to associate, and this terminal interaction is thought to be a functional requirement for RNA synthesis. However, it is possible that the BUNV NTRs are independent promoters that do not interact and that the observed terminal complementarity is a consequence of the NTRs having similar functions that require conservation of sequence signals.

To test these possibilities, we generated a panel of six genome analogs derived from parental BUN-S(ren), BUN-M(ren), and BUN-L(ren) templates in which the 3' and 5' NTRs were exchanged to generate all six possible combinations of S-, M-, and L-segment NTRs (Fig. 2A). These templates were generated by exchanging XbaI-StuI restriction enzyme fragments from parental plasmids pBUN-S(ren), pBUN-M(ren), and pBUN-L(ren), as described in Materials and Methods. In this way, each parental 3' NTR was replaced with a different 3' NTR and, conversely, each parental 5' NTR was replaced with a different 5' NTR. We reasoned that if the genomic and antigenomic NTRs acted independently to promote RNA synthesis, then the six exchanged genome analogs would also be active for RNA synthesis since they possessed genomic and antigenomic NTRs that were functional in BUN-S(ren), BUN-M(ren), and BUN-L(ren).



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  		FIG. 2. Schematic of exchanged genome analog templates and analysis of the RNAs they generate. (A) The entire 3' and 5' NTRs of parental genome analogs BUN-S(ren), BUN-M(ren), and BUN-L(ren) were exchanged to generate six genome analogs having all possible combinations of S-, M-, and L-segment 3' and 5' NTRs. (B) The RNA synthesis characteristics of exchanged genome analogs were compared to the parental genome analogs by direct visualization of metabolically labeled actinomycin D-resistant RNAs after agarose-urea gel electrophoresis and autoradiography. The replication and transcription products generated from BUN-S(ren) are marked. The RNAs generated by BUN-M(ren) and BUN-L(ren) are less well resolved due to their migration characteristics in this gel system. (C) Positive-sense RNAs were also analyzed by primer extension analysis with negative-sense oligonucleotide 3'RENSEQ(-). RNAs were harvested from vTF7-3-infected BHK-21 cells transfected with cDNAs expressing a parental or exchanged genome analog and either BUNV S and L support plasmids (+) or the BUNV S support plasmid alone (-). The lanes of the primer extension gel are positioned below the lanes of the corresponding template on the agarose-urea gel. Bands representing mRNAs (vertical bars), T7 RNA polymerase transcripts (white arrowheads), and antigenomic RNA (black arrowheads) generated by each parental template are marked adjacent to the lanes, as are the T7 RNA transcripts made by the exchanged templates. The cDNA expressing BUN-S(ren) was sequenced by using oligonucleotide 3'RENSEQ(-) to act as size marker, and the antigenomic terminal nucleotide is also marked ().

The six exchanged plasmids were each transfected into cells to generate the corresponding genome analogs, as described in Materials and Methods, and the ability of these templates to perform RNA synthesis was compared to that of the parental BUN-S(ren), BUN-M(ren), and BUN-L(ren) templates. RNA synthesis ability was tested by direct analysis by using both metabolic labeling, followed by agarose-urea gel electrophoresis (Fig. 2B), and also primer extension analysis to detect positive-sense RNAs with an end-labeled negative-sense oligonucleotide 3'RENSEQ(-) (Fig. 2C).

As shown previously (2), the parental BUN-S(ren), BUN-M(ren), and BUN-L(ren) genome analogs generated both replication and transcription products, detected by using both metabolic labeling and primer extension analysis (Fig. 2B and C). Primer extension analysis detects two major T7 RNA polymerase products transcribed from each template. These RNAs differ in size by a single nucleotide and are likely due to addition of a 5' cap by the recombinant vTF7-3, which is present in all transfections. In contrast, the six exchanged templates were unable to synthesize any detectable metabolically labeled actinomycin D-resistant RNAs (Fig. 2B). This result was confirmed by primer extension analysis that showed that no BUNV-specific RNAs were detected, and the only positive-sense RNAs generated by the six exchanged templates were the initial T7 RNA polymerase transcripts (Fig. 2C). This finding suggested that the BUNV NTRs do not act as independent RNA synthesis promoters but instead 3' and 5' sequences cooperate to form the functional promoter for both RNA replication and mRNA transcription.

Restoration of partial terminal complementarity also restores RNA synthesis activity. We next investigated why the exchanged templates described above were unable to perform RNA synthesis. One possibility was that loss of RNA synthesis ability was due to the reduced level of terminal complementarity exhibited by each of the exchanged templates. Since the first 11 nt of the 3' and 5' NTRs that make up the conserved complementary region of each segment are identical, terminal exchange did not alter the degree of terminal complementarity within this region (Fig. 1A and 3A, boxed sequences). However, terminal exchange had reduced the degree of terminal complementarity at positions distal to nucleotide position 11, including nucleotide positions within the variable complementary region (Fig. 1A and 3A, shaded sequences). We wanted to test whether the loss of complementarity within the variable complementary region was alone responsible for the loss of RNA synthesis ability of the exchanged templates.



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  		FIG. 3. Schematic of genome analog templates with restored complementarity within the variable complementary region, and analysis of the RNAs they generate. (A) Exchanged genome analogs BUN-S/M(ren), BUN-M/L(ren), and BUN-L/S(ren) were altered within their 5' NTRs to generate the corresponding templates S/M-comp, M/L-comp, and L/S-comp. These alterations are marked (arrowheads), and the variable complementary regions are shaded. Only the first 25 nt of both 3'- and 5'-terminal regions of the genomic strand are shown, and the conserved complementary region is boxed. The potential to form Watson-Crick base pairs () or noncanonical U-G pairings (•) is indicated. (B) The RNA synthesis characteristics of S/M-comp, M/L-comp, and L/S-comp were analyzed by direct visualization of metabolically labeled actinomycin D-resistant RNAs. RNA synthesis activity of parental genome analogs BUN-S(ren), BUN-M(ren), and BUN-L(ren) are shown alongside for comparison, and BUN-S(ren)-specific RNAs are indicated by arrowheads.

We made nucleotide changes within the 5' genomic NTRs of inactive exchanged templates BUN-S/M(ren), BUN-M/L(ren), and BUN-L/S(ren) to restore their terminal complementarity up to nucleotide position 15 (S/M-comp and L/S-comp) or 16 (M/L-comp) (Fig. 3A). The RNA synthesis ability of these repaired genome analogs was analyzed by metabolic labeling, followed by agarose-urea gel electrophoresis. All three restored templates synthesized RNA products of both replication and transcription (Fig. 3B), indicating that restoration of complementarity between nucleotide positions 12 and 15 (or between nucleotide positions 12 and 16) within the variable complementary region alone restored RNA synthesis activity. This shows that additional regions of complementarity internal to these sequences are not essential for BUNV RNA synthesis activity.

The migration pattern of metabolically labeled replication and transcription products synthesized by the three restored genome analogs appeared similar to the corresponding RNAs generated by the three parental templates. The RNAs generated by each restored template most closely matched those of the parental template that contributed the restored template 5' genomic NTR, in terms of mobility. For example, the RNAs generated by the template L/S-comp most closely resembled those generated by BUN-S(ren). This finding is likely due to the presence of sequences located within these 5' NTRs that signal the characteristic 3'-end truncation of S, M, and L mRNAs (J. N. Barr and G. W. Wertz, unpublished data).

Terminal complementarity between alternative sequences at positions 12 to 15 also promotes RNA synthesis. In the previous section, we restored the RNA synthesis activity of exchanged genome analogs by partially or completely restoring the sequences of wild-type S, M, and L segments within the variable complementary region. We next wanted to investigate whether complementary sequences other than those found within the variable complementary regions of the S, M, and L segments would also promote RNA synthesis. If the BUNV RNA synthesis machinery had a strict sequence requirement in this region, it was possible that different sequences would not promote BUNV RNA synthesis.

We generated genome analog 3'+5'-comp having nt 12 to 15 within the 3' genomic variable complementary region of BUN-S(ren) altered from 3'-GGUG-5' to 3'-CCAC-5' and corresponding nt 12 to 15 of the 5' genomic terminus (underlining denotes nucleotides within 5' genomic NTR) altered from 3'-CACC-5' to 3'-GUGG-5' (Fig. 4A, shaded sequence). These sequences are not present within the corresponding locations of either S, M, or L segments (Fig. 1A). In addition, we constructed genome analogs 3'-comp and 5'-comp which had these same altered nucleotides at positions 12 to 15 of either 3' or 5' termini alone, respectively (Fig. 4A, shaded sequence).



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  		FIG.4. Schematic of genome analog templates with alterations within the variable complementary region and analysis of the RNAs they generate. (A) The parental genome analog BUN-S(ren) was altered to generate the templates 3'+5'-comp, 3'-comp, and 5'-comp by making alterations within nt 12 to 15 (arrowheads). The conserved (boxed sequences) and variable complementary regions (shaded sequences) of these templates are shown. Only the first 25 nt of both 3'- and 5'-terminal regions of the genomic strand are shown, and their potential to form Watson-Crick base pairs () or noncanonical U-G pairings (•) is indicated. (B) The RNA synthesis characteristics of 3'+5'-comp, 3'-comp, and 5'-comp were analyzed by direct visualization of metabolically labeled actinomycin D-resistant RNAs. Replication and transcription products are identified (arrowheads). (C) Positive-sense RNAs were also analyzed by primer extension analysis with negative-sense oligonucleotide 3'RENSEQ(-). RNAs were harvested from vTF7-3-infected BHK-21 cells transfected with a parental or altered genome analog expressing cDNA, and either BUNV S and L support plasmids (+), or BUNV S support plasmid alone (-). The cDNA expressing BUN-S(ren) was sequenced with oligonucleotide 3'RENSEQ(-) to act as size marker, and the antigenomic terminal nucleotide is marked ().

We compared the RNA synthesis activity of these three genome analogs to that of parental template BUN-S(ren) by using both metabolic labeling (Fig. 4B) and primer extension analysis (Fig. 4C). This analysis showed that template 3'+5'-comp was active for synthesis of both replication and transcription products, indicating that a variable complementary sequence comprising non-wild-type sequences promoted RNA synthesis. Interestingly, the relative abundance of genome, anti-genome, and mRNA products synthesized by templates 3'+5'-comp and BUN-S(ren) were not identical. Most noticeably, template 3'+5'-comp generated a diminished level of genomic RNA and elevated levels of antigenomic RNA. This indicated that nucleotides within the variable complementary region provided an RNA synthesis signal that did not depend on complementarity alone but also contained a sequence-specific component.

In contrast, analysis of the RNA synthesis activity of templates 3'-comp and 5'-comp failed to detect any BUNV replication or transcription products (Fig. 4B and C), despite these templates having individual genomic and antigenomic NTRs that promoted RNA synthesis activity when present in either BUN-S(ren) or 3'+5'-comp (Fig. 4A). Primer extension analysis of 3'-comp and 5'-comp RNAs identified a low abundance product that comigrated with the antigenomic band of BUN-S(ren) and 3'+5'-comp. However, this band was not BUNV-RNA-dependent RNA polymerase specific and likely corresponded to a T7 RNA polymerase primary transcript (Fig. 4C). The lack of RNA synthesis activity of 3'-comp and 5'-comp templates supports the evidence presented in the above, indicating that BUNV NTRs do not act independently to promote RNA synthesis activity but instead require the cooperation of complementary nucleotides at the opposite end of the template.

Competitive replication of randomized genome analogs reveals that terminal complementarity signals greatest replication ability. The results of the previous sections showed that BUNV RNA synthesis requires cooperation between the 3' and 5' NTRs. These results also showed that RNA synthesis activity required the presence of complementary nucleotides within the 3' and 5' NTRs, specifically within the variable complementary region. To further investigate the role of terminal complementarity in signaling RNA synthesis, we wanted to determine whether perfect complementarity within the variable complementary region signaled most active RNA replication. It was possible that other closely related but noncomplementary sequences within this region would allow greater RNA replication activity.

To accurately perform this analysis, we needed to test the RNA replication ability of all possible sequences within the variable complementary region, and achieving this aim would be difficult due to the high number of individual genome analogs that would have to be constructed. Instead, our solution was to generate all possible template sequences by random mutagenesis and to determine which of these templates was most able to perform RNA replication by identifying the most abundant genome template after RNA synthesis (Fig. 5A). To further simplify our analysis, we chose to restrict the nucleotides subjected to randomization to those at positions 12 to 14 within the genomic 5' NTR, while we kept the corresponding nucleotide positions 12 to 14 within the 3' NTR unaltered. Randomization of these three positions allowed for 64 possible sequence permutations, and by generating a library of over 3,000 randomized cDNAs, we achieved greater than 95% coverage of these sequences. We performed this procedure with five different defined 3' NTR sequences and thus generated five corresponding pools of altered cDNAs. The sequence of defined 3' nt 12 to 14 were chosen such that at least two potential G-C base pairings would be maintained, as is present at the corresponding nucleotide positions of BUN-S(ren).



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  		FIG. 5. Identification of sequences within the variable complementary region that signal most active genome replication. (A) Schematic representation of the procedure used to generate and analyze the replication ability of genome analogs with randomized nucleotides within the variable complementary region. (B) Five different cDNA pools were generated that transcribed genome analogs with defined nt 12 to 14 of the 3' genomic NTR, and randomized nucleotides at the corresponding positions 12 to 14 at the 5' genomic NTR. The nucleotide sequence of each pool of cDNA templates was determined to confirm that randomization had been successful (left boxes). After transfection of randomized cDNAs into BHK cells, the sequence of the same nucleotide region of the corresponding genome analogs was determined to identify sequences that signaled most active RNA replication (right boxes).

We first tested whether random mutagenesis had been achieved by sequencing the portion of the templates containing 5' nt 12 to 14 within each of the cDNA pools. This analysis showed that each nucleotide within positions 12 to 14 was present with comparable abundance, indicating that randomization had been successful (Fig. 5B, left boxed sequence). The five cDNA pools were each individually transfected into BHK cells to generate corresponding pools of RNP templates.

Following RNA synthesis, we performed DNA sequence analysis on bulk RT-PCR amplified cDNAs representing 5' genomic nt 12 to 14, as described in Materials and Methods. This allowed us to identify the most abundant nucleotide at each of the three previously randomized nucleotide positions and thus identify the nucleotide that best signaled RNA replication. We showed that the most abundant 5' nucleotide at positions 12, 13, and 14 from each of the five pools of randomized genome analogs was always complementary to the corresponding defined nucleotide at the 3' end of the genomic RNA. This finding indicates that complementarity between nucleotides within the variable complementary region signaled the greatest replication ability. Minor peaks corresponding to alternative nucleotides were absent at the majority of positions tested. However, in a minority of positions, the presence at low abundance of an alternative nucleotide was noticed, suggesting that, at certain positions, alternative nucleotides may also allow RNA replication. However, since the abundance of these alternative nucleotides was relatively low compared to the more abundant complementary nucleotide, the level of RNA replication they signaled is also likely to be correspondingly low. Interestingly, for both template pools with a U residue within defined nt 12 to 14 (3-GGU-5' and 3'-CUC-5'), a G residue was selected at low abundance after replication at the corresponding position within the randomized 5' nucleotides. This finding suggests that U-G base pairings may also allow RNA replication, but at a lower level than the canonical Watson-Crick U-A base pairing.

These results show that the dominant template sequence within each of the five random pools depended on the identity of the defined 3' genomic nucleotides. Therefore, most active RNA replication depends on the presence of perfect complementarity between 3' and 5' nucleotides within the variable complementary region.


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DISCUSSION
 
As for all negative-sense RNA viruses studied to date, the S, M, and L segments of bunyaviruses exhibit terminal nucleotide complementarity between their 3' and 5' NTRs. This observed complementarity has been suggested to allow the bunyavirus NTRs to associate through Watson-Crick base pairing, thus circularizing each RNP segment. In support of this suggestion, bunyavirus RNPs have been visualized in circular conformations (38, 42, 46), and biochemical analysis has shown that nucleotides within 3' and 5' NTRs are able to base pair with each other (40, 45). Furthermore, it has been speculated that this terminal association may be a functional requirement for bunyavirus RNA synthesis.

Using BUNV, the model bunyavirus, we examined whether the 3' and 5' NTRs of BUNV RNP templates act as independent RNA synthesis promoters or whether they need to cooperate with each other in order to promote RNA synthesis. We exchanged the NTRs of the three parental genome analogs BUN-S(ren), BUN-M(ren), and BUN-L(ren) to generate a panel of six templates with all possible combinations of 3' and 5' termini. The finding that these exchanged templates were not active for RNA synthesis despite their individual 3' and 5' NTRs being active in the parental constructs show that the BUNV NTRs of genomic and antigenomic RNA segments do not act as independent promoters. Instead, our results indicate that the 3' and 5' NTRs cooperate in order to promote RNA synthesis.

We next investigated whether nucleotide complementarity was involved in the observed cooperation of 3' and 5' NTRs in promoting RNA synthesis. We altered the inactive exchanged templates to restore complementarity up to nt 15 or 16 of the variable complementary region. These changes correspondingly restored RNA synthesis activity, irrespective of whether we altered the S, M, or L NTRs. These results indicated that the presence of complementary nucleotides within these sequences was critical for RNA synthesis activity.

Our results show complete correlation between the ability of a template to signal RNA synthesis and the presence of complementary sequences within the variable complementary region. Conversely, our results also show that templates that lack complementarity within this region are not active for RNA synthesis. Although these findings do not prove that complementarity is the driving force behind terminal interaction, our results strongly suggest that nucleotide complementarity is critical for RNA synthesis activity. Indeed, our findings obtained with the competitive replication assay show that most active RNA replication is signaled only when all three selected nucleotides within the variable complementary region exhibit perfect complementarity. Together, these observations are consistent with a model that posits that nucleotide complementarity drives terminal association by allowing canonical Watson-Crick base pairing interactions between nucleotides within 3' and 5' NTRs. We have considered an alternative model in which complementarity-driven base-pairing interactions play no role in promoting RNA synthesis, and instead functional terminal association results from recognition of 3' and 5' sequences by a trans-acting factor of viral or cellular origin. However, we do not favor this model for two reasons. First and most importantly, to fit with our observations, the potential trans-acting factor association would need to implicitly require sequence-independent complementarity for a reason other than providing the potential for Watson-Crick pairings, and it is difficult to envisage what such a requirement might be. Second, any potential trans-acting factor would need to consistently interact with a range of unrelated sequences within individual NTRs, and we consider this to be an unlikely property.

We observed that a variety of different sequences within the variable complementary regions were able to promote RNA synthesis, suggesting that this signal may depend on formation of a structural element that is largely independent of sequence. These nucleotides may form a double-stranded region of RNA that is recognized by a component of the BUNV RNA synthesis machinery or, alternatively, may facilitate interaction of the conserved complementary nucleotides located at the extreme 3' and 5' termini. However, the observation that the relative abundance of replication and transcription products differs slightly according to the identity of the nucleotides within the variable complementary region suggests that this signal may also have a sequence-specific component. Our analysis of the RNA synthesis ability of BUN-S(ren) and 3'+5'-comp suggest that this sequence-specific component is not related to the relative potential of these nucleotides to base pair through Watson-Crick interactions. The nucleotide changes to the variable complementary regions of these templates did not affect the base-pairing potential within this region (three G-C pairs and one A-U pair); however, these templates signal slightly different relative levels of RNA replication and mRNA transcription.

The variable complementary region of the BUNV NTRs shows characteristics similar to those of the terminal-distal duplex region of the well-characterized influenza virus panhandle, which is thought to form an entirely double-stranded RNA structure (1). Nucleotide changes within this duplex region have been shown to affect the RNA synthesis characteristics of altered segments, and recombinant viruses containing these segments exhibit attenuated growth characteristics (8, 21, 47, 52). Since the BUNV S, M, and L segments differ in sequence within the analogous variable complementary regions, and we have shown that nucleotides within these regions affect the RNA synthesis ability of genome analogs (2), it is possible that these sequences play a subtle yet important role in determining the relative gene expression characteristics of the three BUNV segments. Similar to influenza virus, incorporation of these changes into the BUNV genome may allow the generation of growth-attenuated viruses. We are currently investigating the relationship between these sequences and the RNA synthesis activities of BUNV templates.


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ACKNOWLEDGMENTS
 
We thank members of the G. W. Wertz and L. A. Ball laboratories for helpful discussions during the course of this study. We thank the CFAR DNA Sequencing Core of the University of Alabama at Birmingham for sequencing.

This study was supported by an unrestricted infectious disease research award from the Bristol-Meyers-Squibb Foundation to G.W.W.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, University of Alabama School of Medicine, BBRB Rm. 360/17, 845 19th St. South, Birmingham, AL 35294-2170. Phone: (205) 934-0453. Fax: (205) 934-1636. E-mail: jbarr{at}uab.edu. Back


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Journal of Virology, February 2004, p. 1129-1138, Vol. 78, No. 3
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.3.1129-1138.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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