ABSTRACT
We have performed an extensive mutational analysis of the proposed promoter region of the phlebovirus Uukuniemi (UUK), a member of the Bunyaviridae family. This was achieved by using a recently developed RNA polymerase I (Pol I)-driven reverse genetics system (R. Flick and R. F. Pettersson, J. Virol. 75:1643-1655, 2001). Chimeric cDNAs containing the coding region for the reporter chloramphenicol acetyltransferase (CAT) in an antisense orientation were flanked by the 5′- and 3′-terminal nontranslated regions of the UUK virus-sense RNA (vRNA) derived from the medium-sized (M) RNA segment. The chimeric cDNAs (Pol I expression cassettes) were cloned between the murine Pol I promoter and terminator, and the plasmids were transfected into BHK-21 cells. CAT activity was determined after cotransfection with viral expression plasmids encoding the RNA-dependent RNA polymerase (L) and the nucleoprotein (N) or, alternatively, after superinfection with UUK virus helper virus. Using oligonucleotide-directed mutagenesis, single point mutations (substitutions, deletions, and insertions) were introduced into the viral promoter region. Differences in CAT activities were interpreted to reflect the efficiency of mRNA transcription from the mutated promoter and the influence on RNA replication. Analysis of 109 mutants allowed us to define two important regulatory regions within the proximal promoter region (site A, positions 3 to 5 and 2 to 4; site B, positions 8 and 8, where underlined nucleotides refer to positions in the vRNA 3′ end). Complementary double nucleotide exchanges in the proximal promoter region, which maintained the possibility for base pairing between the 5′ and 3′ ends, demonstrated that nucleotides in the two described regions are essential for viral polymerase recognition in a base-specific manner. Thus, mere preservation of panhandle base pairing between the 5′ and 3′ ends is not sufficient for promoter activity. In conclusion, we have been able to demonstrate that both ends of the M RNA segment build up the promoter region and are involved in the specific recognition by the viral polymerase.
The Bunyaviridae family of arthropod- or rodent-borne viruses comprises more than 300 viruses (46) classified into five genera, Bunyavirus, Hantavirus, Nairovirus, Phlebovirus, and Tospovirus. Bunyaviridae members all share many common structural, molecular, and cell biological characteristics. They are enveloped viruses containing a tripartite, single-stranded RNA genome of negative polarity. The large (L) RNA segment encodes the RNA-dependent RNA polymerase (L), the medium-sized (M) segment encodes the two surface glycoproteins (G1 and G2) and in some member viruses also a nonstructural protein (NSM), and the small (S) segment encodes the nucleoprotein (N) and in some cases a nonstructural protein (NSS) (40). Upon entry of the virus into cells, the three virion ribonucleoprotein (RNP) species are released into the cytoplasm where the RNP-associated RNA polymerase catalyzes primary transcription, resulting in the synthesis of the individual mRNAs. Once new viral proteins have been translated, the virion RNA (vRNA) segments also serve as templates for the synthesis of full-length cRNAs, which in turn serve as templates for the synthesis of more vRNAs. This replicative cycle is also catalyzed by the viral RNA polymerase (40).
Uukuniemi (UUK) virus (a Phlebovirus), which is nonpathogenic for humans, has for more than three decades been used as an excellent model for studying the molecular and cell biology of the highly pathogenic members of the Bunyaviridae family. Initiation of transcription of the UUK virus-specific mRNAs is primed on vRNA templates by short sequences derived from the 5′ end of host mRNAs (41). This “cap snatching” occurs in the cytoplasm (36), and the endonucleolytic cleavage of the host mRNA some 10 to 20 bases downstream of the 5′ cap structure is probably carried out by the L protein, as described for other bunyaviruses (1, 16, 19, 20, 22, 45). As revealed by electron microscopy, the viral RNAs (18) and RNPs (35) are circular due to base pairing between complementary sequences at the 5′ and 3′ ends of each segment (6). These terminal sequences are conserved between each RNA segment and also between members within the same genus (5, 6). Thus, it is thought that this panhandle structure harbors the promoter elements necessary for initiation of both transcription and replication as well as genome packaging signals.
Bunyaviridae mRNAs derived from each vRNA segment are truncated at the 3′ end, due to as yet poorly characterized transcription termination signals (40). This means that the mRNAs are unable to circularize and thus to serve as templates for replication or transcription. Since they do not associate with the N protein and therefore are not packaged into virions, the encapsidation signal for N protein association is also likely to map to the 5′-3′ panhandle region, a conclusion that has recently also been confirmed experimentally (33, 42).
The lack of an efficient reverse genetics system for Bunyaviridae members has so far precluded the detailed mutational dissection of cis-acting elements in the promoter (panhandle) region of the three RNA genome segments. However, during the past few years the first reports have appeared describing attempts to develop such systems (4, 29, 37). By using the T7-vaccinia virus-based expression system (15), Bridgen and Elliott described a reverse genetics system for Bunyamwera (BUN) virus (Bunyavirus) that allowed the rescue of infectious BUN virus entirely from cloned cDNAs (2). We recently reported an alternative approach (11) based on the RNA polymerase I (Pol I)-driven expression system initially developed for influenza A virus (32, 48). This system allowed us to drive the replication and transcription of a reporter cDNA cassette (chloramphenicol acetyltransferase [CAT] or green fluorescent protein [GFP]) flanked by the 5′ and 3′ nontranslated sequences from the M RNA segment of UUK virus. The transcription of the reporter cassette, flanked by the murine Pol I promoter and terminator, is first carried out in the nucleus by RNA Pol I. This results in an uncapped, nonpolyadenylated, and nonspliced (Flick and Hobom, unpublished data) RNA transcript with the correct viral 5′- and 3′-terminal sequences (8, 48). After transport to the cytoplasm, the reporter RNA is replicated and transcribed by the L and N proteins supplied either by superinfection with UUK helper virus or by L- and N-expressing plasmids alone (11).
Here we have utilized the Pol I system to dissect the importance of the 5′ and 3′ nucleotide sequences of the M RNA segment of UUK virus for replication and transcription. A total of 109 point mutations, deletions, or insertions were introduced in the putative promoter region and their effects on reporter (CAT) expression were determined. The result of these extensive mutational analyses identified two crucial sites in the panhandle region separated by a short spacer region, the length of which is critical.
MATERIALS AND METHODS
Cells and virus.BHK-21 cells (American Type Culture Collection) were grown on plastic dishes in Eagle's minimal essential medium (EMEM) supplemented with 7.5% fetal calf serum (Invitrogen/Life Technologies), 2 mM l-glutamine, 100 IU of penicillin/ml, and 100 μg of streptomycin/ml. The origin and the preparation of stock virus from the prototype strain S23 of UUK virus have been described (34). The stock virus had a titer of 2 × 108 PFU/ml. Cells were infected with a multiplicity of infection of approximately 5 PFU/cell.
Construction of plasmids.Mutations in the 5′ and 3′ vRNA promoter regions were generated by using pRF108 as a basic vector construct. This plasmid contains the ribosomal DNA (rDNA) promoter region (−251 to −1 relative to the 45S pre-rRNA start point) and the rDNA terminator sequence (+571 to +745 relative to the 3′ end of the 28S rDNA) derived from murine rDNA (48). Between these two elements is a spacer region flanked by BsmBI and BbsI sites. After restriction by BsmBI and BbsI, BbsI- or BsmBI-restricted PCR fragments can be inserted in an orientation-specific manner. Using primers carrying the promoter single point mutations, the template pRF200 (Pol I [murine] UUK M-CAT, recently described as pRF33;11) can be PCR amplified, introducing the mutations into the nontranslated chimeric M-segment ends. Double substitution mutants were constructed by ligation of the large BglII/EcoRI fragment (3,200 bp) from the 3′ single mutation constructs and the small BglII/EcoRI fragment (657 bp) from the 5′ single mutation plasmids.
After transfection into BHK-21 cells, the resulting constructs can be transcribed by the cellular RNA Pol I, generating RNA transcripts without any additional nucleotides or modifications at the 5′ or 3′ ends [e.g., cap structure, poly(A) tail]. Consequently, artificial UUK vRNA segments with exchanged open reading frames (ORFs) (glycoprotein precursor replaced with CAT) flanked by promoter mutations are produced.
Whenever PCR was used the sequence of the inserts was checked by dideoxy sequencing with an ABI PRISM3100 sequencer (Applied Biosystems). In cases where a central part of the inserted PCR fragment was exchanged with a nonamplified fragment derived from previously characterized inserts, only the sequences of the flanking regions were checked. The oligonucleotide primers used in the paper are listed in Table 1.
Oligonucleotide primers used to insert UUK promoter mutations and for vRNA and cRNA analyses
Transfection.Subconfluent (60 to 80%) BHK-21 cells (3 × 106) were cotransfected with plasmids (1 μg) containing promoter mutations and viral expression plasmids pCMV UUK-L and pCMV UUK-N (2.4 and 0.3 μg, respectively) (11) by using 20 μl of liposome Plus buffer (Lipofectamine PLUS; Invitrogen/Life Technologies) mixed in serum-free EMEM. After 15 min, 12 μl of liposome reagent was added and incubation was continued for another 15 min. The BHK-21 cells were incubated at 37°C with the DNA-Lipofectamine mixture for 3 to 5 h. After further incubation for 20 h in EMEM containing 7.5% fetal calf serum, the transfected cells were washed with phosphate-buffered saline and harvested for CAT analysis or used for UUK virus superinfection. To determine the efficiency of transfection, the plasmid pHL2823, which contains an enhanced GFP under the cytomegalovirus (CMV) promoter (Flick and Hobom, unpublished data), was similarly transfected.
Superinfection with UUK helper virus.Plasmids containing promoter mutations were transfected into subconfluent BHK-21 cells (6 × 106) by using the technique described above, with the following modified amounts of reagents: 4 μg of the respective plasmid, 20 μl of the liposome Plus buffer (Lipofectamine PLUS; Invitrogen/Life Technologies), and 30 μl of liposome reagent. Twenty hours posttransfection the cells were washed with adsorption medium (EMEM supplemented with 20 mM HEPES and 0.2% bovine serum albumin) and superinfected with UUK virus at a multiplicity of infection of 5 PFU/cell. After a 60-min adsorption period, the cells were washed once and incubated with fresh adsorption medium for 15 to 30 h. A complete replication cycle occurs during this period.
CAT assay.Cell extracts were prepared as described by Gorman et al. (17). In a first experiment, 50% of each cell lysate (prepared from 3 × 106 cells in the case of cotransfection experiments and 6 × 106 cells in superinfection experiments) and, depending on the results, serially diluted samples of the various cell lysates were mixed with 10 μl of acetyl-coenzyme A (4 mM lithium salt; Sigma) and 10 μl of fluorescently labeled chloramphenicol substrate (borondipyrromethane difluoride fluorophore [BODIPY CAM substrate]; Flash CAT kit; Stratagene) and then incubated at 37°C for 2 h. For extraction of reaction products, 0.4 ml of ethyl acetate (Merck) was added, and after centrifugation for 1 min at 15,000 × g, the upper phase containing the reaction products was isolated and the solvent was evaporated. The resulting pellet was resuspended in 20 μl of ethyl acetate, and the reaction products were separated by thin-layer chromatography (20- by 20-cm plates; Silica gel 60; Merck) using a solvent mixture of chloroform and methanol (87:13). Finally, the reaction products were visualized by UV illumination, documented by photography, and evaluated using Quantity One software (Bio-Rad). Ratios of CAT activities were calculated based on at least three independent sets of serial dilutions of cell lysates down to a level of 30 to 50% product formation. The percentages of CAT activity in the figures reflect the ratios between product and substrate relative to the positive control pRF200 (wild-type promoter construct = 100%).
RT-PCR and semiquantitative PCR analysis.BHK-21 cells were transfected with promoter mutant-containing Pol I cassettes and viral expression plasmids for UUK-L and UUK-N (pCMV UUK-L, pCMV UUK-N) as described before (11). Total RNA was isolated (RNeasy mini kit; Qiagen) at 20 h posttransfection and treated with DNase (Amp grade; Gibco BRL). RNA was quantitated by UV absorbance (A260/A280) using an Eppendorf Biophotometer, and 1 μg of total RNA was used as template for the reverse transcription (RT) (rTth kit; Applied Biosystems). Artificial UUK M-CAT cRNA and vRNA molecules were reverse transcribed (30 min, 60°C) using oligonucleotides RF268 and RF267, respectively. Oligonucleotides RF178 or RF177, respectively, were added under the following PCR conditions: 1 min at 94°C; 40 cycles of 30 s at 94°C and 30 s at 60°C; and 75 min at 60°C. The reaction products (for cRNA amplification, a 605-bp fragment; for vRNA amplification, a 608-bp fragment) were analyzed in a 1.5% agarose (Gibco BRL) horizontal gel containing 0.25 μg of ethidium bromide (Gibco BRL) per ml in Tris-borate-EDTA electrophoresis buffer, documented by digital photography, and analyzed using Quantity One software (Bio-Rad).
RESULTS
General strategy for expression of CAT cDNA flanked by UUK M vRNA nontranslated regions (NTRs).The reporter plasmid pRF200 (CAT-M vRNA) and all the 109 promoter mutant constructs described below contained the ORF of the reporter CAT gene in antisense orientation, flanked by the 5′- and 3′-terminal sequences of the UUK virus M vRNA segment. Oligonucleotide-directed mutagenesis of the terminal sequences was carried out by introducing mutations into PCR primers. The chimeric constructs were expressed by using the murine Pol I system (11, 32, 48). Plasmids were introduced into BHK-21 cells by liposome-mediated transfection. Control experiments using a CMV-enhanced GFP expression plasmid (pHL2823) (Flick and Hobom, unpublished data) indicated a reproducible transfection efficiency of approximately 20 to 25% (11). To drive transcription and replication of the chimeric RNAs, cells were either infected with UUK virus 20 to 24 h after transfection or cotransfected with expression plasmids encoding viral L and N proteins (11). The observed CAT activity directly reflects the CAT enzyme concentration, which is dependent on the viral mRNA transcription rate. The latter is in turn dependent on the vRNA promoter activity. Therefore, the CAT activity reflects the interaction between the viral RNA polymerase and the promoter region and this was used to quantify the effect of the introduced promoter mutations on the viral promoter activity. To make it easier for the reader to assess the effect of the mutations on promoter activity, we have used a color code to describe the CAT activity obtained with each mutant. Each mutant was given a number, which appears in the figures showing the quantification of CAT activity (Fig. 1 to 3) and in the summary figures (Fig. 1E and 4) (see figure legends for further details).
Comparative CAT analysis of single nucleotide substitution derivatives of UUK M-CAT minigenomes in the proximal promoter region and summary of the effect on CAT expression of point mutations introduced into the promoter region of the UUK M-segment-based minigenome. BHK-21 cells were transfected with different RNA Pol I-driven mutated promoter constructs containing UUK M-CAT minigenome plasmids and cotransfected with viral expression plasmids pCMV UUK-L and pCMV UUK-N. At 30 h posttransfection, the cells were harvested and analyzed for CAT activity. Acetylated products were separated by thin-layer chromatography. Construct numbers are indicated above each lane. CAT activity, expressed as the percentage relative to the wild-type promoter construct pRF200, is indicated below each lane. (A to D) CAT activity in BHK-21 cells transfected with pCMV UUK-L and pCMV UUK-N expression plasmids and UUK M-CAT minigenomes containing mutations in the 5′ branch of the proximal promoter element (positions 1 to 5 at the 5′ vRNA end) (A), mutations in the 5′ branch of the proximal promoter element (positions 6 to 10 at the 5′ vRNA end) (B), mutations in the 3′ branch of the proximal promoter element (positions 1 to 5 at the 3′ vRNA end) (C), and mutations in the 3′ branch of the proximal promoter element (positions 6 to 10 at the 3′ vRNA end) (D). Colors: red, no detectable CAT activity (<5%); orange, CAT activity between 5 and 49%; yellow, CAT activity between 50 and 80%; green, CAT activity above 80%. The CAT activity of the basic construct pRF200 (UUK M-CAT with wild-type promoter sequence) was set at 100%. Mock, BHK-21 cells transfected only with pRF200. (E) Summary of the effect on CAT expression of point mutations introduced into the promoter region of the UUK M-segment-based minigenome. The nucleotide sequence of the base-paired panhandle configuration formed by the inverted complementary 5′ and 3′ ends of the UUK M vRNA is shown in the middle (in black and white). The compiled CAT expression efficiencies (average of three or more experiments for each mutant) have been tabulated above and below that sequence for a complete set of 77 single-substitution mutants in the 5′ and 3′ arms, respectively. In addition, the results from 15 double-substitution mutants, 14 insertion mutants (hexagons), and 3 deletion mutants (triangles) in the proximal and distal promoter elements as well in the bulge region are listed. CAT activities are relative to the pRF200 reporter gene expression rate (wild-type promoter) as shown in the color legend. For representative experimental data, see panels A to D and Fig. 2. Note that due to the mechanism of RNA Pol I transcription termination, the substitution U1C cannot be analyzed (48).
Mutational analysis of the proximal promoter element—identification of two important promoter elements, A and B.In the first series of mutants, single point mutations were introduced into the proximal promoter region of the UUK virus M segment. The 5′ and 3′ sequences of this region, which encompass positions 1 to 10, are completely complementary (see Fig. 1E, 4, and 6) and are likely to be crucial in the formation of the panhandle structure and the circularization of the vRNA and cRNA species. Analyses of CAT activity in lysates prepared from cells expressing these mutants revealed nucleotide positions which could be mutated with no or only a moderate effect on the promoter activity (e.g., positions 1, 2, 6, 7, 9, and 10 and 1, 5 to 7, 9, and 10, where underlined nucleotides refer to positions in the vRNA 3′ end) and other positions which were very sensitive to any kind of modification (positions 3 to 5 and 8 and 2 to 4 and 8) (Fig. 1A to D; summarized in Fig. 1E). Interestingly, two distinct elements within the proximal promoter part in the panhandle conformation could be identified as important regulatory sites (site A, positions 3 to 5 and 2 to 4; site B, positions 8 and 8) (Fig. 4). These elements are separated by a short stretch of nucleotides that are apparently not recognized by the viral polymerase in a nucleotide-specific manner (positions 6 to 7 and 5 to 7).
To determine if these nucleotides only serve a spacer function to position the important promoter elements A and B to the correct interaction sites on the viral polymerase, additional nucleotides were inserted between positions 5 and 6 into the 5′ or 3′ promoter arms and the new constructs were tested for CAT expression. As a result, a substantial difference between the insertions into the 5′ versus the 3′ part of the UUK vRNA promoter could be demonstrated (Fig. 1E and 2A). Nucleotide insertions between positions 5 and 6 at the 5′ end completely abolished the promoter activity, since no CAT activity could be detected, independent of whether an insertion of one or two A (pRF251, pRF252) or U (pRF253, pRF254) residues was introduced. In contrast, the insertion at the corresponding position at the 3′ end of the vRNA resulted in strong remaining promoter activity, independent of whether one or two A (pRF259, pRF260) or U (pRF257, pRF258) residues were inserted. Only insertion of three or five nucleotides between positions 5 and 6 dramatically decreased reporter expression in the case of A insertions (pRF266, pRF267) or completely abolished promoter activity in the case of U insertions (pRF268 and pRF269) (Fig. 1E and 2A).
Graphic representation of CAT activities from UUK minigenomes containing nucleotide mutations in the 5′ and/or 3′ arms of the UUK M-CAT minigenome promoter region. (A) RNA Pol I-driven UUK M-CAT minigenome plasmids containing nucleotide insertions in the proximal promoter element were transfected into BHK-21 cells and cotransfected with pCMV UUK-L and pCMV UUK-N expression plasmids. At 30 h posttransfection cells were harvested and analyzed for CAT activity. The analyzed constructs are listed on the x axis. Construct numbers are shown on top of the graph, and the number of inserted nucleotides and type of nucleotide are listed below the graph. The y axis shows the CAT activities from each construct compared to that of the wild-type minigenome pRF200. (B) Graphic representation of CAT activities from UUK minigenomes containing compensating double mutations in the proximal promoter element. (C) Analysis of the effect of mutations introduced into the bulge region of the viral M-segment promoter.
The lack of correct interaction between the viral polymerase and the substituted nucleotides could be the result of changing the nucleotide sequence or changing base-pairing potential in the panhandle. Mutants with complementary double substitutions with preserved base pairing were therefore generated to examine the promoter-abolishing effect of the previous introduced single point mutations. As shown in Fig. 2B and summarized in Fig. 1E, complementary double substitution constructs containing an inactivating single point mutation (positions 3 to 5 and 8) resulted in undetectable CAT activity (pRF180, pRF220, pRF178, and pRF177). Thus, the nature of the nucleotide located at a certain promoter position is more important than base pairing per se. All other constructs with altered base pairs showed CAT activity comparable to that obtained for the single point mutations (positions 1, 7, and 9) or had slightly decreased CAT expression levels (positions 2, 6, and 10).
In summary, the mutational analyses of the highly conserved proximal promoter region of the UUK virus M vRNA segment demonstrate that two important polymerase interaction sites (site A and site B) are located within this region. Sites A and B are separated by a stretch of nucleotides which are not recognized by the viral polymerase in a nucleotide-specific manner. The distance between sites A and B seems to be important only for the 5′ part of the promoter, whereas the 3′ part is less sensitive to nucleotide insertions, suggesting a binding mechanism differing between vRNA and cRNA molecules.
Mutational analysis of the bulge region.Nucleotides A11 and C12 at the 5′ end are not complementary to bases at the 3′ end of the UUK M vRNA, while the next 5 nucleotides (which we call the distal promoter region) are complementary. Thus, these two nucleotides are likely to form a bulge (Fig. 1E and 4). This bulge constitutes the main difference between the vRNA and the cRNA promoter, whereas residues 1 to 10 (proximal promoter region) and the distal promoter region at the 5′ and 3′ ends of the RNA in both promoter situations consist of fully complementary nucleotides (Fig. 1E and 4). Therefore, it was expected that these bulge structures should play an important role for the interaction with the viral RNA polymerase, perhaps to discriminate between different RNA species during the packaging process as shown for influenza A virus (43). Using an oligonucleotide-directed mutagenesis approach, single point mutations, including substitutions, deletions, and insertions, were introduced into this bulge region. Interestingly, substitutions maintaining a purine at position 11 (A-to-G substitution; pRF145) or a pyrimidine at position 12 (C-to-U substitution; pRF201) had only minor effects on promoter activity, whereas constructs with substitutions of a pyrimidine at position 11 (pRF133, A11U; pRF144, A11C) or of a purine at position 12 (pRF198, C12A; pRF199, C12G) gave a slight to moderate decrease in CAT activity compared to the wild-type promoter construct pRF200 (Fig. 1E and 2C).
The role of the bulge region was also examined by generating constructs with insertions and deletions. Surprisingly, deletion of the A11 nucleotide (pRF105) as well as the deletion of both bulge nucleotides A11 and C12 (pRF101) had no effect on the promoter activity (Fig. 1E and 2C). Furthermore, nucleotide insertions into the 3′ part of the vRNA promoter opposite to the two bulge nucleotides (between positions 10 and 11) showed minor effects on promoter activity. This was true both in the case of nucleotide insertion with base-pairing potential of the bulge nucleotides A11 and C12 (pRF104) and in the case of insertion without any base-pairing possibility (pRF195) (Fig. 1E and 2C). With the constructs pRF101 (ΔA11C12) and pRF104 (insertion of UG at positions 10 and 11), a UUK promoter sequence was generated that displayed no differences between the vRNA and cRNA promoter situation. In these mutants, either the bulge nucleotides were deleted or nucleotides complementary to the bulge region were inserted, thus generating the possibility for a completely double-stranded promoter region of 15 or 17 bp, respectively.
In conclusion, the results from the single nucleotide exchange experiments suggest that the bulge region is involved in the interaction between the viral polymerase and the promoter region. However, there is no nucleotide-specific recognition by the viral polymerase. Instead, the type of nucleotide (purine or pyrimidine) appears to be important. In contrast, the deletion and insertion experiments demonstrated that the unpaired bulge nucleotides are not necessary for the transcription and replication processes of the UUK minigenomes (Fig. 1E, 2C, and 4). However, their role in the packaging process has to be further analyzed.
Analysis of the distal promoter element.Similar to the mutational analysis of the proximal promoter region, the effect of single point mutations in the distal promoter element (positions 13 to 17 and 11 to 15) was also analyzed. This was followed by an analysis of complementary double substitutions, all of which preserved base pairing. The single nucleotide exchanges were chosen so that the interactions between the nucleotides from the 5′ promoter arm and the 3′ arm within the distal promoter element were either weakened, e.g., G—C→G-U, or destroyed, e.g., G—C→A/C, where “—” indicates a strong bond, “-” indicates a weak bond, and “/” indicates the absence of a bond. It should be noted that in the latter case the cRNA promoter will still carry a week G-U basepair. Furthermore, since the authentic AUG start codon is located at positions 18 to 20 (5′ end of cRNA), single point substitutions were chosen such that the optimal Kozak sequence (23) was not influenced, i.e., keeping a purine at cRNA position 15 (Kozak position −3) and a pyrimidine at cRNA position 14 (Kozak position −4).
Surprisingly, all of the analyzed single point mutation constructs showed a promoter activity similar to that of the wild-type promoter construct pRF200 (not shown, but summarized in Fig. 1E and 4). This suggests that none of the nucleotides in the distal promoter region are recognized by the viral polymerase in a nucleotide-specific manner, nor does the base pairing between the 5′ and 3′ promoter arms in this region seem to play an important role (the A/C situation in the vRNA promoter [pRF189, pRF190, pRF184, pRF182 and pRF186]). CAT expression (not shown, but summarized in Fig. 1E) of complementary double substitution mutants with preserved base pairs between the 5′ and 3′ promoter arms in the distal promoter region was likewise comparable to that of the wild-type promoter (pRF187, pRF188, pRF203, pRF215, and pRF216), demonstrating again that the distal region is not recognized by the viral polymerase in a nucleotide-specific manner.
Mutational analysis combined with UUK superinfection.To demonstrate that the CAT activities determined for the different promoter mutants are not specific to the use of cotransfected plasmids expressing the UUK nucleoprotein (N) and the viral polymerase (L), we repeated the analysis with a set of selected mutants using UUK superinfection as a source for the viral N and L proteins. The mutant constructs selected were pRF120 and pRF169 (wild-type level of CAT activity), pRF126 (moderate reduction of CAT activity), pRF123 (strongly reduced CAT activity), and pRF134 (no detectable CAT expression). As expected, comparable relative CAT activities were observed (Fig. 3), but at a lower level than in the cotransfecting experiments, in agreement with our recently published data (11).
CAT analysis of UUK minigenomes containing representative single point mutations in the proximal promoter element driven by UUK superinfection. BHK-21 cells were transfected with selected minigenomes containing mutated promoter sequences. At 24 h posttransfection cells were superinfected with UUK helper virus and then were harvested at 30 h postinfection and analyzed for CAT expression. CAT activity measured from each lysate is expressed as the percentage of the activity obtained from cells transfected with the wild-type promoter construct pRF200 and superinfected with helper virus (set at 100%) (lane 3).
RNA analysis of promoter-inactivating mutations.The introduced promoter mutations can have effects at different stages of the viral life cycle. They can, for example, influence the overall binding of the viral polymerase to the promoter region, or they can affect the specific interaction with the active center of the polymerase. Different polymerase reactions can also be influenced by these mutations, e.g., transcription, replication, or cap-snatching or endonucleolytic cleavage. In addition, the packaging process may be affected by the altered promoter sequence. Furthermore, nucleotide substitutions can have different effects on the vRNA and cRNA promoters. Therefore, we examined vRNA and cRNA levels in BHK-21 cells transfected with promoter mutants showing no detectable CAT activity. We used a semiquantitative RT-PCR approach to examine the vRNA/cRNA ratios for each analyzed single promoter-inactivating mutant. For the RT reaction, the primer RF267 was used to start the RT reaction at the 3′ end of the vRNA of the M-CAT segment and primer RF268 was used for the RT of minigenome cRNA. For the following PCR amplification step, RF177 (for vRNA detection) or RF178 (for cRNA detection) was used as the reverse primer to amplify a 608- or 605-bp fragment, respectively. Since the 3′ end of the M mRNA has been found to be about 100 nucleotides shorter than the full-length cRNA (R. Rönnholm and R. F. Pettersson, unpublished data), the primer RF268 was designed such that no viral mRNA could be amplified. This ensured that the amplification product represented only the cRNA derived from the artificial UUK M-CAT segment.
Control experiments were conducted to exclude any plasmid DNA contamination (Fig. 5, lane 1) or any cRNA background (Fig. 5, lane 2) by not adding the reverse transcriptase to the RT-PCR or by using total RNA from BHK-21 cells transfected with only the wild-type promoter construct pRF200 (no cotransfected expression plasmids), respectively. As expected, no amplification product could be detected after an RT-PCR control reaction in the absence of reverse transcriptase, showing that no DNA contamination was present (Fig. 5, lane 1). Furthermore, the second control experiment analyzing RNA from cells transfected only with pRF200 (wild-type promoter), omitting the two expression plasmids, demonstrated that only vRNA molecules could be amplified due to the expressed RNA Pol I transcripts, whereas no cRNA could be detected (Fig. 5, lane 2). The different levels of vRNA with and without cotransfected viral N and L expression plasmids (lane 2 versus lane 3) demonstrate clearly the replication activity (vRNA→cRNA→vRNA) in our minigenome rescue system.
The selected mutant constructs that had promoter-abolishing effects were pRF140 (C4A), pRF118 (C4U), pRF119 (A5U), pRF156 (A5C), and pRF129 (G4C) (Fig. 1 and 4). As expected, the vRNA amplification product was easily detectable for all examined promoter mutants due to the RNA transcribed by RNA polymerase I. However, the amount of cRNA was much lower than that of the vRNA, as expected for a negative-strand RNA virus (30) (Fig. 5, upper panel). To determine the effect of the introduced promoter mutations on the viral replication steps (vRNA→cRNA, cRNA→vRNA), the vRNA/cRNA ratio was determined for the five mutants and compared to that derived from the wild-type promoter construct pRF200. In our system, a vRNA/cRNA ratio of about 90:10 was obtained for pRF200 (wild-type promoter) in cells cotransfected with the pCMV UUK-L and pCMV UUK-N expression plasmids (Fig. 5, lower panel, column 3). Surprisingly, similar levels of cRNA synthesis were detected for all five examined promoter mutants compared to the wild-type situation, demonstrating that the first replication step (vRNA→cRNA) is not severely blocked by the introduced mutations (Fig. 5, columns 4 to 8). Furthermore, the vRNA/cRNA ratios, which reflect the effect on both replication steps, were in a close range between 90:10 and 85:15 for all examined promoter mutants, demonstrating no major influence of the nucleotide exchanges on the minigenome replication.
Proposed model for the UUK promoter elements important for the interaction with the viral RNA polymerase as suggested by the CAT activity pattern of single substitutions and supported by complementary double substitutions and nucleotide insertions or deletions. The nucleotide sequence of the panhandle proposed to be formed between the 5′ and 3′ ends of the UUK M vRNA is shown. The proximal and distal promoter regions are shown in gray boxes, separated by the two bulge nucleotides. Promoter elements framed by red and orange boxes represent the nucleotides most important for promoter activity. They are referred to as recognition sites A and B. Green and yellow letters show freely exchangeable promoter nucleotides that, when mutated, have no or little effect on promoter activity.
Analysis of vRNA and cRNA synthesis by CAT-negative promoter mutants. BHK-21 cells were transfected with selected CAT-negative promoter mutant constructs and cotransfected with viral expression plasmids as shown at the top. Total RNA was isolated at 20 h posttransfection and treated with DNase I to destroy the transfected plasmids. Subsequently, 1 μg of total RNA was used as a template for RT, followed by a PCR with UUK-CAT minigenome-specific oligonucleotides (see Table 1). The reaction products (cRNA amplification, 605-bp fragment; vRNA amplification, 608-bp fragment) were analyzed in a 1.5% agarose horizontal gel. The upper panel shows the cRNA amplification product, the middle panel shows the vRNA amplification product, and the lower panel shows the vRNA/cRNA ratio of all examined promoter mutant minigenomes.
Thus, we conclude that the reason for the lack of detectable CAT activity of some promoter mutants is not the result of blocked replication but is rather likely to be due to the inability to initiate and synthesize functional viral mRNA.
DISCUSSION
The development of a reverse genetics system for UUK virus (11) based on the RNA pol I system (32, 48) has opened up the possibility to genetically manipulate this and other members of the Bunyaviridae family. As a first application of this new system, we have introduced a total of 109 mutations into the putative promoter region of the M segment and studied their effects on the promoter function as determined by analyzing the expression of a minigenome encoding CAT. In these analyses, we have focused on the 5′ and 3′ ends of the vRNA molecule, which we call the 5′ and 3′ promoter arms, respectively. The two ends are able to form a stable panhandle structure due to base pairing between inverted complementary nucleotide sequences (5, 6, 18, 39). The two promoter arms are thought to regulate replication and transcription as well as the association of the vRNA with the N protein to form RNPs. Furthermore, they probably contain the putative RNA segment packaging signal.
The results of our mutational analyses can be summarized as follows (Fig. 4). Two critical sites, or elements, called A and B, were identified in the proximal region of the promoter, which comprises the 10 terminal residues of the 5′ and 3′ ends of the vRNA. At the 5′ promoter arm, nucleotides at positions 3 to 5 (site A) and 8 (site B) could not be changed to any other residue without abolishing or substantially reducing promoter activity. Likewise, mutating residues 2 to 4 (site A) and 8 (site B) in the 3′ promoter arm drastically reduced activity, although some changes at positions 3 and 4 were tolerated. The abolishing effect of double mutations in the 5′ and 3′ arms, which preserved base pairing, also underscored the importance of the nucleotide sequence at the two sites. The spacing between sites A and B (two nucleotides) in the 5′ promoter arm was found to be critical, as introduction of just a single base abolished promoter activity. In contrast, the corresponding spacer region (three residues) in the 3′ promoter arm tolerated the introduction of one or two residues without significant loss of activity. Taken together, these results support the notion that the sequence of the 5′ promoter arm is more critical than that of the 3′ promoter arm (see below). A surprising finding was that the bulge consisting of two unpaired residues at positions 11 and 12 (5′ promoter arm) could be deleted, base paired, or mutated without loss of activity. However, a purine at position 11 and a pyrimidine at position 12 were still favored in single point mutation experiments. Finally, each nucleotide of the distal promoter region which has the potential to fully base pair could be mutated without loss of activity, even if base pairs were destroyed. No experimental proof could be found that both RNA segment ends have to be in a panhandle conformation to serve as a functional promoter. However, important residues for the promoter function, which could function independently during the interaction between the viral polymerase and the vRNA or cRNA promoter, are located in each promoter arm.
Our analysis of the UUK M vRNA promoter function is the first extensive in vivo mutagenesis study of a Bunyaviridae member based on the exchange of all nucleotides within the entire promoter region. One previous study on Rift Valley fever (RVF) virus focused on the effect of deletions within the promoter region (37). Here, recombinant vaccinia viruses providing the RVF virus L and N proteins and in vitro synthesized T7 runoff transcripts (minigenomes) were used and only transcription rather than replication and packaging of the minigenomes could be analyzed. The results indicated that the promoter region between nucleotides 3 and 8, as well as the G residue at position 13 in the 3′ promoter arm of the genome-sense RNA (vRNA), was important for transcription initiation. The former region corresponds to our sites A and B, including the spacer (residues 2 to 8), and the results thus support the conclusion of the importance of this region in phlebovirus RNA segments.
Extensive mutagenesis studies similar to the one reported here have during the last decade been carried out with influenza A virus (9, 10, 14, 21, 25-28, 31, 38, 44). These analyses either employed the RNA Pol I-driven reverse genetics system used here (32, 48) or a T7-driven system (15). Because of the similarities between the orthomyxoviruses and Bunyaviridae members, it is of interest to compare the results obtained with these two virus systems. The first conclusion is that the results clearly reveal many common features. Nucleotides important for promoter activity are in both cases mainly located at the 5′ promoter arm. In the case of UUK virus, there were 11 mutations of nucleotide positions in the 5′ promoter sites A and B that completely abolished activity, while only two mutations at the 3′ promoter arm led to no detectable reporter gene activity. For influenza virus, the corresponding numbers were three mutations in the 5′ arm and only one in the 3′ arm (10). Furthermore, two sites sensitive to single point mutations could be defined for the UUK M vRNA promoter, namely site A (positions 3 to 5 [5′ arm] and 2 to 4 [3′ arm]) and site B (position 8 [both 5′ and 3′ arms]) (Fig. 1E and 4). Similarly, two critical sites were identified in the influenza A virus promoter: site A (positions 2, 3, and 5 and 2 and 3) and site B (positions 7 to 9 and 7 to 9). For both viruses, these important sites for promoter activity are separated by a stretch of two to three nucleotides, the sequence of which is not critical for the recognition by the viral polymerase (Fig. 1E) (7). In the case of UUK virus, nucleotide insertions between sites A and B demonstrated that the distance between these sites plays an important role for the 5′ promoter arm (Fig. 1E and 2B) but much less so for the 3′ promoter arm. The fact that this promoter part is very sensitive to nucleotide changes suggests that the viral RNA polymerase interacts very specifically with the 5′ promoter arm.
Besides positions recognized in a nucleotide-specific manner and positions having just a spacer function, the promoter region contains nucleotides that can only be replaced by a similar type of nucleotide (e.g., positions 1, 2, and 11 must be purines and position 12 must be a pyrimidine). Again, similarities can be found by comparison with influenza A virus (9, 10).
Therefore, viral polymerases differentiate between the following three different types of promoter nucleotides: (i) nucleotides that interact in a sensitive and nucleotide-specific manner, (ii) nucleotides for which being a purine or pyrimidine is important, and (iii) a third type of nucleotides which are not directly recognized by the viral polymerase but serve a spacer function to position the functionally important promoter residues for the proper interactions.
The proximal promoter region (the first 10 nucleotides; Fig. 4) of the L, M, and S vRNAs of UUK virus is highly conserved except at position 9 in the 5′ promoter arm. At this position, the L segment has a U, whereas the M and S segments have an A. This natural variation can be understood based on the result of the different CAT expression levels of the single point mutations at position 9. As expected, the two naturally occurring nucleotides showed a similarly high CAT activity (100 and 97%; Fig. 1B and E), whereas an A9C or an A9G substitution resulted in decreased CAT activity (70 and 6%; Fig. 1B and E).
Single nucleotide exchanges leading to an increased CAT expression (promoter up-mutations) may also help us to understand the importance of single nucleotides located in the promoter region. The influenza A virus studies revealed several complementary double mutants with an increased level of CAT expression (9, 10, 31). However, we found only one UUK promoter mutant (A6U) that displayed a 10% higher activity (pRF120; Fig. 1B and E) than the wild-type construct. Since this mutation is located in the spacer region between sites A and B (5′ arm), no further conclusions can be drawn at this point from this result.
The mutational analysis of the bulge region included single nucleotide substitutions as well as insertions and deletions. Single point mutations demonstrated that the type of each of the two nucleotides in the bulge is important (position 11 is a purine and position 12 is a pyrimidine). The insertion of the UG nucleotides in the 3′ promoter arm opposite to the 5′ bulge nucleotides (pRF104) generated a fully complementary promoter region of 17 bp in the panhandle. This mutant displayed a strong CAT activity (94% compared to the wild-type promoter). This is similar to the results obtained for the equivalent mutations in the influenza A virus promoter (construct pHL1140 in references 7 and 31). However, the deletion of one or both bulge nucleotides resulted in no detectable expression (7) or strongly decreased reporter gene expression in the influenza A virus study (43), whereas the UUK promoter could tolerate such deletions (Fig. 2C, pRF101 and pRF105). Therefore, deletions and insertions revealed that the bulge nucleotides are less important for the promoter activity than the proximal two recognition sites A and B. Since our system only allows for an assessment of the effect on transcription and replication, it cannot be excluded that the bulge nucleotides are important for the packaging process, as has been recently shown for influenza A virus (43). Further studies including the passaging of recombinant UUK viruses to fresh cell cultures with subsequent reporter analysis need to be carried out to address this question.
The mutational analysis of the UUK distal promoter element reveals surprisingly major differences compared to studies carried out on the influenza A virus (9, 10) and the RVF virus (37) promoter regions. In our UUK study neither single point substitutions nor changing of panhandle base pairs within the distal promoter element altered the resulting promoter activity. The distal element of the influenza A promoter, on the other hand, plays an important role during the viral polymerase interaction, either as a stabilizing double-stranded promoter element (10, 13, 14, 21) or as a regulatory element for viral mRNA transcription rates (12).
Studies with RVF demonstrated that the purine at position 13 at the 3′ promoter branch, which is highly conserved within the genus Phlebovirus, could not be changed into any other nucleotide without completely abolishing reporter gene expression (37). The UUK mutational analysis, however, showed no effect of any kind of substitution at position 13 (pRF184, G13A; pRF275, G13U; pRF276, G13C; Fig. 1E), suggesting that this position is not specifically recognized during the interaction between the viral polymerase and the UUK minigenome promoter.
To find out why some single point mutations totally abolished the promoter activity, we analyzed the synthesis of vRNA and cRNA. The point mutations could have affected different steps in the replication and transcription processes of the UUK minigenomes. The binding of the viral polymerase to the vRNA or the cRNA templates could be affected and could thereby affect the efficiency of the template-dependent transcription and/or replication steps. Furthermore, the mutations could influence the cap-snatching or endonucleolytic cleavage and priming processes and thereby affect mRNA synthesis. We therefore estimated the amount of vRNA and cRNA in transfected cells by semiquantitative RT-PCR and calculated the vRNA/cRNA ratio. We did not attempt to determine the amount of mRNA, because the nondetectable levels of CAT activity indirectly indicated total absence of functional viral mRNAs. The vRNA/cRNA ratio of about 9:1 obtained for the wild-type promoter construct was expected, since synthesis of cRNA, a replicative intermediate, in most viral systems is substantially lower than that of vRNA synthesis. This result was similar to that obtained for influenza A virus, where a vRNA/cRNA ratio of 10:1 was demonstrated (30). However, only a minor influence on the vRNA/cRNA ratio could be detected for the mutants tested. This could mean that the mutations primarily affect transcription rather than replication. Further experiments will have to be carried out to demonstrate the exact mechanism by which the CAT-negative promoter mutants inhibit the transcription steps within the viral life cycle.
Based on the studies of the influenza A virus promoter elements, a novel corkscrew model was introduced to explain the role of the secondary structure of the influenza A virus promoter region (8, 9, 10, 25, 26). This model underscores the importance of intrastrand stem-loop structures for promoter function. Interestingly, nucleotides that are important for the promoter activity of influenza virus map within these loop parts. The loops presumably facilitate interaction with the viral polymerase complex (9, 10, 25, 26). Recent studies have confirmed that the corkscrew structure also plays an important role for the promoter activity of influenza C viruses (3) and thogoto viruses (24, 47). This suggests that the corkscrew is a common promoter structure for members of the Orthomyxoviridae (7).
Comparison of the promoter sequences of the five genera comprising the Bunyaviridae family revealed for members of four of the genera the possibility for intrastrand stem-loop structures within the proximal promoter region (Fig. 6), in conformity with the corkscrew model (7). This is also true for Tenuiviruses, which share many features common to Bunyaviridae. Interestingly, the only exceptions are the phleboviruses, for which no stem-loop structure(s) could be found. Our mutagenesis studies do not offer an obvious explanation for this unique feature of phleboviruses. Complementary double mutations at positions 4 and 8 that allowed for intrastrand base pairing and the potential to form a corkscrew configuration completely abolished promoter activity (unpublished data). Whether the lack of the potential to form a corkscrew configuration reflects an evolutionary divergence or a different mechanism for polymerase-promoter interaction must await further experimental analyses.
Secondary structure predictions of the highly conserved terminal regions of the genome segments of members of the five Bunyaviridae genera and the Tenuiviruses in comparison to the corkscrew structure of the influenza A virus vRNA segment promoter. In four genera of the Bunyaviridae family, the terminal nucleotides of the RNA genome segments (vRNA and cRNA) can theoretically form intrastrand stem-loop structures, referred to as the corkscrew configuration. The model is based on the experimentally proven promoter structure of the influenza A (9, 10, 25, 26) and C (3) viruses and the thogoto (24, 47) virus.
The reverse genetics system and the results presented here for UUK virus will hopefully be useful as a model for similar approaches in studies of the highly pathogenic members of the Bunyaviridae family.
ACKNOWLEDGMENTS
We thank A. Wallin for expert sequencing assistance, C. Westin for cloning help, and L. Fernando for editorial suggestions.
FOOTNOTES
- Received 1 May 2002.
- Accepted 22 July 2002.
- Copyright © 2002 American Society for Microbiology
