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Journal of Virology, June 2006, p. 5388-5396, Vol. 80, No. 11
0022-538X/06/$08.00+0     doi:10.1128/JVI.00204-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Roles of Human Parainfluenza Virus Type 3 Bases 13 to 78 in Replication and Transcription: Identification of an Additional Replication Promoter Element and Evidence for Internal Transcription Initiation

Michael A. Hoffman,^* LeeAnne M. Thorson, John E. Vickman, Joseph S. Anderson, Nathan A. May, and Michelle N. Schweitzer

Department of Microbiology, University of Wisconsin—La Crosse, La Crosse, Wisconsin 54601

Received 27 January 2006/ Accepted 14 March 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
The genomic promoter of human parainfluenza virus type 3 (HPIV3) contains multiple cis-elements controlling transcription and replication. Previous work showed that regions 1 to 12 and 79 to 96 were critical in promoting replication of an HPIV3 minireplicon, while the intergenic sequence and N gene start signal (IS/Ngs, bases 49 to 61) were important for transcription. Because these data were collected primarily using point mutations, not every base from position 1 to 96 was analyzed, and some important control elements may have been missed. To clarify the role of bases 13 to 78 in transcription and replication, a series of mutations were made which collectively scanned this entire region. Mutation of bases 13 to 28 resulted in markedly decreased HPIV3 minireplicon replication, indicating these bases constitute an additional cis-element involved in the synthesis of the HPIV3 antigenomic RNA. The position dependence of the IS/Ngs was also examined. Analysis of mutants in which the IS/Ngs was shifted 5' or 3' showed that this segment could be moved without significantly disrupting transcription initiation. Additional mutants which contained two successive IS/Ngs segments were created to test whether the polymerase accessed the gene start signal by proceeding along the template 3' to 5' or by binding internally at the gene start signal. Based on analysis of the double gene start mutants, we propose a model of internal transcription initiation in which the polymerase enters the template at approximately the location of the natural N gene start but then scans the template bidirectionally to find a gene start signal and initiate transcription.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human parainfluenza virus type 3 (HPIV3) is an enveloped, nonsegmented, negative-sense RNA virus within the Respirovirus genus of the subfamily Paramyxovirinae, family Paramyxoviridae, order Mononegavirales. HPIV3 is an important cause of lower respiratory tract infections in young children and is being increasingly recognized as a cause of serious lower respiratory tract infections in the immunocompromised and elderly (4, 14, 15, 35).

During replication of the mononegaviruses, the nucleocapsid (N) protein-encapsidated genomic and antigenomic RNAs serve as templates for the synthesis of cRNA. Specific factors affecting RNA replication vary among the different families and subfamilies of the Mononegavirales. For the Paramyxovirinae, which includes the Respirovirus (HPIV3 and Sendai virus [SeV]), Morbillivirus (measles virus), and Rubulavirus (simian virus 5 [SV5]) genera, these factors include a requirement for specific sequence elements at the 3' ends of the genome and antigenome, proper spacing between sequence elements, and adherence to the rule of six (20, 39).

The genomic and antigenomic promoters (the 3' ends of the genome and antigenome; GP and AGP, respectively) of all members of the Paramyxovirinae studied thus far contain two critical regions: conserved region I (CRI) and conserved region II (CRII) (16, 22, 24, 26, 33, 34, 38). Conserved region I is at the extreme 3' end of the genomic and antigenomic promoters and, for HPIV3, appears to consist of the terminal 12 nucleotides (16). Most of the bases in CRI are highly conserved and, when mutated, result in very poor replication. Conserved region II is found 79 to 96 nucleotides (for HPIV3 and SeV) from the 3' end of the promoter and appears to consist of three consecutive hexamers in which the first position must be a C (3'-CNNNNN-CNNNNN-CNNNNN-5') for replication to occur efficiently. The exact position and sequence composition of CRII varies among the different genera of the Paramyxovirinae (20). One additional sequence element recognized as being involved in replication is bases 51 to 66 in the AGP of SV5 (17). It is not known if this nonconserved 51-66 element is common to all members of the Paramyxovirinae or is Rubulavirus specific.

The spacing between CRI and CRII is also important, as insertions or deletions that alter the natural spacing result in significant reductions in replication (24, 27, 33). In contrast, the 51-66 element of the SV5 AGP is position independent, as the element can be moved (2 to 6 bases) without disrupting replication (17).

The hexameric nature of CRII and the requirement for proper spacing between CRI and CRII may derive from the rule of six. The rule of six appears to hold for all the Paramyxovirinae (7, 25, 29) but stems from two findings made with SeV. Through examination of the SeV N-RNA by electron microscopy, the N protein was estimated to cover 6 nucleotides (8). Furthermore, only SeV minireplicons whose length was a multiple of six could be replicated (3). This length requirement for replication has been interpreted as a need for the N protein to encapsidate exactly 6 nucleotides to form a functional template for replication (19, 20). Recent results support a model in which the viral replication promoter sequences are recognized in the context of bound N protein (27, 37). Thus, if the spacing between CRI and CRII or if the total length of the genome is not a multiple of six, the N protein position on the critical control regions would be out of phase for template recognition.

An additional reason for the spacing requirement between CRI and CRII may relate to the three-dimensional structure of the N RNA template. The N-RNA of the Mononegavirales forms a coiled structure, and with SeV there are an estimated 13 N proteins per turn of the coil (8). If so, this places CRI (hexamers 1 and 2) adjacent to CRII (hexamers 14 to 16) in the coiled structure. Thus, RNA polymerase binding (replication initiation) may involve recognition of discontinuous sequence elements bound by N proteins in a coiled N-RNA complex (19, 20).

The cis-elements involved in transcription initiation are not as well understood as those for replication initiation. This is in part due to increased difficulty in studying transcription with minireplicon systems. Mutations that affect the replication of minireplicons also affect transcription by reducing the amount of template available for transcription. Thus, it has been difficult to clearly determine which cis-elements are important for transcription initiation of the Paramyxovirinae. Thus far, only the intergenic sequence/N gene start (IS/Ngs) segment has been clearly shown as critical for transcription initiation (2, 16). However, recent experiments with vesicular stomatitis virus (VSV) have shed light on possible mechanisms of transcription initiation for the Mononegavirales.

Two models of transcription initiation have been postulated, the 3' entry model and the internal entry model. In the 3' entry model, the viral polymerase initiates RNA synthesis at the 3' end of the template (as is the case for replication) and proceeds to make a short, nontranslated, leader RNA. Following termination of the leader RNA, the polymerase reinitiates transcription at the N gene start signal and then proceeds with transcription of the viral genome (1). While there is experimental evidence for the 3' entry model (9, 18), recent genetic and biochemical experiments have shown that a transcriptase can initiate transcription directly at the N gene start signal, without synthesis of the leader RNA (5, 28, 41). These new findings support a model of internal entry of the RNA polymerase during transcription initiation.

Our previous work has helped define HPIV3 sequence elements important in promoting replication and transcription. Analysis of point mutants of an HPIV3 minireplicon showed that bases 1 to 12 (CRI) and 79, 85, and 91 (CRII) were critical in promoting HPIV3 replication, while the IS/Ngs (bases 49 to 61) was important for transcription but not replication (16). Because much of these data were collected using point mutants, not every base from positions 13 to 78 was analyzed, and some important control elements may have been missed. Additionally, these results contrast slightly with previous findings with SeV, in which bases 1 to 31 were deemed critical for promoting replication (34). SeV, being closely related to HPIV3, should have a GP that resembles that of HPIV3. Therefore, to clarify the role of HPIV3 GP bases 13 to 78 in transcription and replication, further mutagenesis and analysis of this region was done with an HPIV3 minireplicon. This analysis revealed a third domain important in replication, showed that the IS/Ngs was a position-independent element for transcription initiation, and provided evidence for internal initiation of transcription.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Plasmid construction. Construction of the negative-sense minireplicon, pHPIV3MG(-), was described previously (16). The T7 RNA polymerase-produced transcript from pHPIV3MG(-) contains 91 nucleotides from the 5' terminus of the HPIV3 genome, a negative-sense copy of the luciferase gene, 97 nucleotides from the 3' terminus of the genome, and the hepatitis delta virus antigenomic ribozyme. Mutations in pHPIV3MG(-) were made using the QuikChange Multi site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions or by megaprimer PCR-directed mutagenesis (32). Megaprimer PCR products containing mutations were digested with SacI and BstXI and inserted into the same sites of pHPIV3MG(-). All mutants were confirmed by sequencing.

Transfection. HeLa cell monolayers grown on six-well plates to 90% confluence were infected with recombinant vaccinia virus vTF7-3 (13), which expresses T7 RNA polymerase, at a multiplicity of infection of 5. After 1 h at 37°C, the minireplicon and support plasmids were transfected using Lipofectin (Invitrogen) according to the manufacturer's instructions. The plasmid amounts used per well were 1.25 µg pHPIV3-MG(-), 1.13 µg pHPIV3-N, 1.38 µg pHPIV3-P, and 0.35 µg pHPIV3-L. After 24 h the transfection medium was removed and replaced with 1.5 ml of OptiMem containing actinomycin D (2 µg/ml) and 2% fetal bovine serum as described elsewhere (12). After 2 h the actinomycin D-containing medium was replaced with OptiMem containing 2% fetal bovine serum and incubated for an additional 22 h.

RNA isolation. Total RNA was purified directly from transfected HeLa cells with TRIzol (Invitrogen) according to the manufacturer's specifications. For isolation of replication-specific genomic and antigenomic RNAs, the transfected cells were collected and lysed and the nonencapsidated RNA was digested with S7 nuclease (Roche) as described previously (7). Following S7 treatment, protected RNA was then extracted with TRIzol.

Primer extension analysis. Detection of the positive-sense luciferase mRNA and antigenomic RNA was done using a negative-sense, [-³²P]ATP end-labeled oligomer that primes at nucleotide 2 of the luciferase gene. This primer is extended 47 nucleotides when annealed to luciferase mRNA and 101 nucleotides when annealed to the antigenomic RNA. Detection of the genomic RNA was done using an end-labeled oligomer that primes at nucleotide 1649 of the luciferase gene, which is 101 nucleotides from the 5' end of the genomic RNA. These primers were used with 25% of the total RNA extract or 33% of the S7-treated extract in standard reverse transcription reactions with Moloney murine leukemia virus reverse transcriptase (New England Biolabs) at 44°C for 1 h. The extension products were separated on a 6% acrylamide-7 M urea gel and analyzed by autoradiography and/or quantitation on a Molecular Dynamics PhosphorImager.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Analysis of genomic promoter bases 13 to 78 in replication. To clarify the role of bases 13 to 78 in replication, a series of four mutations that collectively scanned this entire region were made in HPIV3 minigenome plasmids. These mutations were made by scrambling the wild-type (WT) sequence to preserve the original base content (Fig. 1). These mutants were then analyzed by transfection (along with support plasmids encoding the N, P, and L proteins) into HeLa cells infected with a recombinant vaccinia virus encoding T7 RNA polymerase. After 48 h, RNA was extracted for analysis by primer extension. Analysis of transcription products was done using total RNA extracts from the transfected cells, while cell lysates treated with S7 nuclease prior to RNA isolation were used to reduce the background of unencapsidated RNA for analysis of replication products. Scrambling the bases of the 13-28 region resulted in a replication level 9% that of WT (Fig. 1). Mutations of bases 29 to 44, 45 to 61, and 62 to 78 had relatively minor effects on replication, resulting in antigenomic RNA synthesis at 45 to 67% of WT. The 45-61 and 62-78 mutants were deficient in transcription, but this was expected, as these mutations overlapped the IS/Ngs. The doublets seen with the detection of the transcription product (Fig. 1B, but seen more clearly in Fig. 4 and 5, below) have been seen previously and are most likely due to premature termination of the reverse transcriptase on methylated transcripts (16).

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FIG. 1. Scanning mutagenesis of the 13-78 region of the HPIV3 genomic promoter. (A) Sequences of the 13-78 scanning mutations and replication and transcription relative to WT. Bases changed for each of the mutants are underlined, and the N gene start signal is boxed. (B) Representative primer extension experiments. For analysis of RNA synthesis, total RNA (for transcription products) or S7-treated RNA (for replication products) was isolated from cells transfected with the indicated minigenome and N-, P-, and L-encoding support plasmids. The RNA was analyzed by primer extension using a negative-sense primer to detect the transcribed luciferase mRNA or replicated antigenomic RNA. Averages were based on three or more experiments with each mutant. The lane marked -L indicates the L plasmid was omitted in a transfection with the WT minigenome.

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FIG. 4. Effects of changing the position of the intergenic sequence/N gene start. A series of minigenome mutants was made in which the intergenic sequence/N gene start was moved toward (-Ngs mutants) or away (+Ngs mutants) from the 3' end of the genome. For analysis of RNA synthesis, total RNA (for transcription products) or S7-treated RNA (for replication products) was isolated from cells transfected with the indicated minigenome and N-, P-, and L-encoding support plasmids. The total RNA and S7-treated RNA extracts were analyzed by primer extension using a negative-sense primer to detect the transcribed luciferase mRNA or replicated antigenomic RNA, respectively. S7-treated RNA was also analyzed by primer extension using a positive-sense primer to detect replicated genomic RNA. (A) Summary of replication and transcription of mutants. The antigenome synthesis is shown as a percentage relative to WT. The percent WT transcription efficiency is the amount of transcript relative to the amount of template (genomic RNA) and was compared to WT. Average RNA amounts were based on at least three determinations of each RNA species. (B) Representative gels showing primer extension experiments detecting the antigenomic RNA (AG), genomic RNA (G), and luciferase mRNA (mRNA). The image showing detection of the antigenome is overexposed to show the low levels of antigenomic RNA synthesis with the –13Ngs mutant. The background seen in the +6Ngs transcription lane was seen occasionally with different mutants, but the nature of this background is unknown. Data from such lanes were not used for quantitation purposes. The lane marked -L indicates the L plasmid was omitted in the transfection with the WT minigenome.

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FIG. 5. Transcription start site usage with the double gene start constructs. (A) Sequence of the double gene start mutants and predicted and actual gene start 1/gene start 2 (GS1/GS2) utilization ratios. The gene start sites are underlined. The calculation of the expected GS1/GS2 ratio with polymerase scanning from the 3' end was done as follows: TEGS1/[(1 – TEGS1)(TEGS2)], where TE refers to the percent WT transcription efficiency of the corresponding + and – Ngs mutants shown in Fig. 4. The denominator in this equation reflects the assumption that transcription initiating at GS1 would not initiate at GS2. The expected GS1/GS2 ratio with internal transcription initiation was TEGS1/TEGS2. The resulting ratios were then normalized so that GS1 was equal to 1. The actual GS1/GS2 ratio was determined by phosphorimager analysis of three or more experiments. (B) Representative gels of primer extension experiments detecting transcription products. The lane marked -L indicates the L plasmid was omitted in the transfection with the WT minigenome.

The 13-28 region was not previously recognized as involved in promoting synthesis of the HPIV3 antigenome. To further delineate the important regions within the 13-28 segment, the original 13-28 mutation sequence was separated into four mutants which scanned this region. None of the four individual mutations resulted in the severe decrease in replication seen with the original 13-28 mutation (Fig. 2A). This was surprising, given that numerous single-base mutations in CRI or CRII completely abrogate replication. Thus, either the 13-28 segment was relatively insensitive to mutations (unlike CRI and CRII) or the original 13-28 mutation was deleterious due to the creation of a sequence that is inhibitory to replication.

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FIG. 2. Defining important bases within the 13-28 region of the genomic promoter. S7-treated RNA extracts from cells transfected with the indicated minigenomes were analyzed by primer extension using a negative-sense primer to detect antigenomic RNA. Bases changed for each of the mutants are underlined. Averages were based on three or more experiments with each mutant. Lanes marked -L indicate the L plasmid was omitted in transfections with the WT minigenome. (A) Sequence and primer extension analysis of minigenome mutants scanning the 13-28 region. (B) Sequence and primer extension analysis of additional 13-28 minigenome mutants.

To distinguish between these possibilities, additional 13-28 mutations were created. If the original 13-28 mutation created an inhibitory sequence, the additional 13-28 mutants would not be expected to do so. The three additional 13-28 mutations included an additional scrambling of the WT sequence (13-28/scrambled), a conversion of the 13-28 WT sequence to the cognate (13-28/cognate), and a random sequence containing increased GC content (13-28/50%GC). All three of these additional mutants resulted in replication comparable to that of the original 13-28 mutation (Fig. 2B). This indicates that the 13-28 segment constitutes a distinct domain that is relatively insensitive to mutation but is important in replication.

An additional mutation confirmed the important nature of the 13-28 segment. In this 13-16/25-28 mutant, the two most severe of the four 13-28 scanning mutations (13-16 and 25-28) were combined (Fig. 2A). This mutation resulted in replication similar to that of the 13-28 mutations, indicating that the poor replication of the 13-28 mutants was due to a cumulative effect of changes throughout the 13-28 sequence.

The position dependence of the 13-28 element was also examined. This was done because the only other sequence element, besides CRI or CRII, recognized as important in Paramyxovirinae replication is the 51-66 element in the AGP of SV5 (17). This SV5 element is position independent. HPIV3 minigenome mutants were created in which the 13-28 element was shifted 3, 6, 9, and 12 bases toward CRII. These mutations were designed by deleting bases immediately downstream of the 13-28 element and inserting them between bases 12 and 13. Analysis of these mutants showed that movement of the 13-28 element was as deleterious for replication as were mutations of the primary 13-28 sequence (Fig. 3A). These results are consistent with the 13-28 element being position dependent; however, other explanations are plausible. Foremost is that it is possible that the 13-28 element extends into bases 1 to 12 (CRI). If so, the mutations in which the 13-28 sequence was shifted contained insertions in the domain being investigated, which may thus account for the poor replication.

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FIG. 3. Positional and functional analysis of the 13-28 element. (A) Position dependence of the 13-28 element. The 13-28 element (underlined) was shifted 3 to 12 bases away from the 3' end of the genomic RNA in a series of minigenome mutants. S7-treated RNA extracts from cells transfected with the mutants were analyzed by primer extension analysis using a negative-sense primer to detect antigenomic RNA. Averages were based on two experiments with each mutant. (B) Primer extension analysis of the 13-28 mutant using total RNA from transfected cells. Total RNA extracts from cells transfected with the WT minigenome or the 13-28 mutant were analyzed by primer extension analysis using a negative-sense primer to detect RNA with a 5' terminus identical to that of the antigenomic RNA. The average was based on three experiments. Lanes marked -L indicate that the L plasmid was omitted in the transfection with the WT minigenome.

This analysis establishes that the 13-28 element has a role in synthesis of the antigenomic RNA, but the exact function of the element is not known. However, the 13-28 element may be similar to an element recently recognized in the respiratory syncytial virus (RSV) GP (23). Replication of RSV (Pneumovirinae subfamily of the Paramyxoviridae) is distinct from the Paramyxovirinae in that RSV does not follow the rule of six and does not have a CRII element (6, 31). The CRI of the RSV GP appears to consist of the first 11 nucleotides (12). Adjacent to RSV CRI is a recently recognized element (bases 16 to 34) that is important for encapsidation of the replication product (23). When the 16-34 element of RSV was mutated, replication initiated but the nascent RNA was not encapsidated, and RNA synthesis terminated after synthesis of a few hundred nucleotides.

To test the HPIV3 13-28 GP mutant for these aborted replication products, primer extension analysis was done with total RNA extracts from transfected cells. With an RSV minigenome containing a 16-34 mutation, similar analysis showed almost-WT levels of replication initiation events (23). For the 13-28 mutant of HPIV3, this analysis showed higher levels (31% of WT) of total 13-28 antigenomic replication product (Fig. 3B) than was seen when encapsidated RNA amounts were analyzed (9% of WT) (Fig. 1). This suggests that some synthesis of unencapsidated replication products may occur. However, this difference, though suggestive, is not large enough to conclusively show that mutation of the HPIV3 13-28 segment results in significant levels of unencapsidated replication product (or RNA having the same 5' terminus as the replication product), as was seen with RSV.

Position dependence of the intergenic sequence/N gene start on transcription. Following the analysis of the 13-78 region of the HPIV3 GP in replication, we focused on the role of this region in control of transcription initiation. We first wanted to know whether the IS/Ngs was a position-dependent or -independent signal for transcription initiation. Because our initial analysis showed that bases 29 to 78 could be altered without significantly decreasing replication, we knew constructs in which the IS/Ngs element was repositioned in this region should replicate. These constructs could therefore be analyzed for transcription efficiency and IS/Ngs position dependence. In a series of minigenome constructs, the IS/Ngs was moved 1, 3, 6, 9, 12, 13, and 15 bases toward the 3' end of the genome and 1, 3, 6, 9, and 12 bases toward the 5' end. To preserve the spacing between CRI and CRII and adherence to the rule of six, these mutations were made by deleting bases from one side of the IS/Ngs and inserting bases on the other side. In these mutants, the sequence 3'-UUGAAUCCUAAUUUCU-5' was kept intact, where the intergenic sequence is in bold and the N gene start is underlined.

The mutant minigenomes were analyzed for antigenomic RNA replication, genomic RNA replication, and transcription (Fig. 4). Most of the mutants had modest defects in antigenomic RNA synthesis, though some (–13Ngs, +6Ngs, +9Ngs, and +12Ngs) were more severe. The severe nature of the –13Ngs mutation may be due to the creation of a sequence inhibitory to RNA synthesis (30). The decreased replication with the +6Ngs, +9Ngs, and +12Ngs mutations may be due to interference created when the N gene start is too close to CRII. In the +12Ngs minigenome, the last base of the N gene start is at position 76, only three bases from the start of CRII.

Transcription efficiency was determined as the amount of transcript produced relative to the amount of template (genomic RNA) and compared to WT (set at 100%). Most of the IS/Ngs mutants were transcribed efficiently, indicating that the IS/Ngs is not a position-dependent element for transcription initiation. The mutations with a significant decrease in transcription efficiency (–13Ngs, +6Ngs, +9Ngs, and +12Ngs) were also those that had a significant decrease in antigenome synthesis. The same explanations may apply: the –13 mutation could be poor in transcription due to the creation of an inhibitory sequence, while the +6Ngs, +9Ngs, and +12Ngs mutations may have the N gene start too close to CRII. Regardless of the exact reason for RNA synthesis defects, there is an obvious correlation between antigenome synthesis and transcription efficiency. This indicates that the signal(s) being affected by these mutations is involved in both replication initiation and transcription initiation.

Additionally, no correlation between transcription efficiency and hexamer phasing was seen. This is significant, as it was previously noted that the first base of the six gene start signals of HPIV3 and SeV corresponds to either the first or second base of the hexamer (19). In these results, the –6Ngs, –12Ngs, +6Ngs, and +12Ngs mutants preserved the hexamer phasing of the N gene start signal but did not have increased transcription efficiency relative to other mutants.

The result that the leader-N gene junction could be moved without severely impacting replication or transcription allowed further analysis of the mechanism of transcription initiation. Toward this goal, minigenome constructs with two consecutive IS/Ngs elements were created. If the transcriptase accessed the N gene start by scanning from the 3' end of the template, the first gene start should be used preferentially. If the transcriptase accessed the N gene start via direct binding, the usage of each gene start signal should correlate with their respective transcription efficiencies. Furthermore, based on the previous determination of the transcription efficiency of each of the + and – Ngs mutants, it was possible to make predictions for both models based on the use of the first gene start relative to the second (Fig. 5; see legend for calculations).

In these double gene start constructs, the sequence kept intact in the + and – Ngs constructs (3'-UUGAAUCCUAAUUUCU-5') was retained for each gene start, except for the –12/+1 double gene start. In the –12/+1 construct, the gene start of the upstream IS/Ngs is immediately adjacent to the intergenic sequence of the downstream IS/Ngs. Thus, the flanking sequence present in the original –12Ngs and +1Ngs mutants was not conserved. To serve as more appropriate controls for the –12/+1 double gene start construct, additional –12Ngs and +1Ngs constructs, containing flanking sequences present in the –12/+1 double gene start, were created and analyzed for replication and transcription (data not shown). Transcription efficiencies from these new constructs were used in the predictions of the gene start usage for the –12/+1 double gene start construct (Fig. 5).

The results did not fit perfectly with either model but seem to favor a model of internal binding for transcription initiation. With the –15/0 and –15/+3 double gene start mutants, the first gene start signal was very poorly recognized, and greater than 90% of the transcription initiation took place at the second gene start signal (Fig. 5). If the transcriptase were scanning from the 3' end of the template, it would be hard to conceive why it would not initiate efficiently at the first gene start, as was the case in the –15Ngs construct (Fig. 4). The –12/+1, –12/+3, and –12/+6 double gene start mutants showed a trend. With the –12/+1 mutant, the first gene start is poorly used. However, as the second IS/Ngs is moved further downstream in the –12/+3 and –12/+6 constructs, the first gene start is increasingly used. A modified version of the internal entry model, to be discussed, may explain these results.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The HPIV3 13-28 element. The initial analysis of HPIV3 GP bases 13 to 78 was done to thoroughly characterize the role of this region in promoting antigenome synthesis and to resolve perceived differences in the replication promoters of HPIV3 and SeV. Through this analysis, an additional promoter element important for antigenomic RNA synthesis was recognized. This 13-28 element is distinct from CRI in that single-base (16) to 4-base changes had only minor effects on replication. Only when 8 or more bases of this segment were altered did the effect on replication become obvious. Recognition of the 13-28 element also resolves the discrepancy between previous findings with HPIV3 and SeV. Most likely, the 13-28 region of both viruses is insensitive to point mutations, as in the previous HPIV3 analysis (16), but is sensitive to more extensive changes, as in the previous SeV analysis (34).

The HPIV3 13-28 element could function in several ways to promote replication. The HPIV3 13-28 element may serve as a binding site in the GP for the RNA polymerase (or an ancillary replication factor) during promoter recognition. However, we believe that it is more likely that the HPIV3 13-28 element is similar to the 16-34 element of RSV and functions as a signal for encapsidation of the nascent RNA during replication. This is because the 13-28 element of HPIV3 is in the same position (immediately following CRI) as the RSV element and because both elements are insensitive to single base substitutions (23). However, mutation of the respective HPIV3 13-28 and RSV 16-34 elements did not give identical results in minigenome replication assays. In both cases, substitution mutations did result in low levels of encapsidated antigenomic RNA, but mutation of the HPIV3 element did not result in the synthesis of high levels of unencapsidated RNA as was the case with mutation of the RSV element. It is possible that the HPIV3 13-28 element is an encapsidation nucleation site during synthesis of the antigenome but when mutated results in poor encapsidation of the nascent transcript and rapid termination of unencapsidated RNAs, so that the nascent RNA rarely extends more than 100 nucleotides (where it would have been detected in our primer extension assays). Further analysis is needed to better define the function of the HPIV3 13-28 element in replication.

We expect that an element analogous to the 13-28 element of the HPIV3 GP will be present in the HPIV3 AGP. This is based on evidence that substitution of bases 1 to 31, but not 1 to 26, of the SeV AGP into the SeV GP conferred a high replication phenotype to the GP (2). Other members of the Paramyxovirinae may also contain an element analogous to the HPIV3 13-28 element; however, experimental evidence is currently lacking. This is, in part, because determining the boundaries between CRI and a possible adjacent element requires the use of point mutations, with CRI being highly sensitive to point mutations and the adjacent element being insensitive. Such mutagenesis has not been done with the genomic or antigenomic promoters of other members of the Paramyxovirinae.

It is also possible that the HPIV3 13-28 element could be analogous to the 51-66 element in the SV5 AGP (17). The two elements are similar in that they are not highly conserved and do not appear to be as critical in replication as CRI and CRII. Their locations in their respective promoters are different, however. But since it is clear that the 51-66 element is position independent, it is possible that the HPIV3 13-28 element is a relocated 51-66 element (or vice versa).

Transcription initiation. By first establishing that changes of bases 29 to 78 would not severely affect replication, we were then able to test additional mutations in this region for transcription effects. Specifically, mutants in which the IS/Ngs was repositioned in this region were tested to determine the position dependence of this element for transcription initiation. We showed that the IS/Ngs could be moved without causing significant decreases in transcription efficiency. Therefore, the HPIV3 IS/Ngs does not have to be positioned precisely relative to CRI or CRII for transcription to initiate effectively. Similar results have been seen with VSV and RSV. Six-base deletions made in a VSV minireplicon resulted in the IS/Ngs being positioned six bases closer to the 3' terminus of the genomic RNA (21). While some of these constructs did have moderate decreases in transcription efficiency, these decreases were due to the removal of sequences that enhance transcription and not the repositioning of the IS/Ngs. In RSV, additional sequence could be inserted 3' of the N gene start and transcription still occurred efficiently (11).

This analysis also further underscores the relative unimportance of sequences flanking the HPIV3 IS/Ngs in transcription initiation. This is most obvious in the –15Ngs construct, in which bases 35 to 49 were deleted yet the repositioned gene start signal was still well-recognized for transcription initiation. This is consistent with previous findings with RSV, in which sequences immediately upstream of the RSV gene start signal could be altered without a significant decrease in transcription efficiency (11). This is in contrast to VSV, in which sequences important for transcription are located just upstream of the N gene start signal (21, 40).

The reduced transcription and replication observed when the IS/Ngs was moved toward CRII may be due to steric interference between transcriptase and replicase complexes binding a common template. If there are separate transcriptase and replicase complexes for the Paramyxovirinae, as there appear to be for VSV (28), there may be competition between the two for binding a common template. Such competition could be enhanced when initiation signals for the two processes are moved closer together, as was the case with the +6, +9, and +12Ngs mutants. Additionally, it is possible that minimization of steric interference between the IS/Ngs and CRII may, in part, account for the natural position of the IS/Ngs.

With the knowledge that the IS/Ngs could function when repositioned in the 29-78 region, additional constructs with tandem IS/Ngs elements were created. These constructs were designed to test the 3'-end entry versus internal initiation models for polymerase accessing the IS/Ngs. The observation that the –15Ngs was used efficiently for transcription initiation when present as the only gene start signal, but not used efficiently when a second IS/Ngs signal was downstream, is strong evidence for internal initiation of transcription. If the polymerase were traveling from the 3' end of the template as in the 3' entry model, the –15Ngs should have been used efficiently regardless of whether a second IS/Ngs was positioned downstream. The increasing utilization of the upstream IS/Ngs that occurs as the second IS/Ngs is moved further away (in the –12/+1, –12/+3, and –12/+6 constructs) from the 3' terminus is intriguing. A modified version of the internal entry model may explain these results. In this model the transcriptase may first associate with the N-bound CRI and/or CRII sequences but then enters the template at approximately the location of the WT N gene start and scans upstream or downstream to find a gene start signal and initiate transcription. This internal initiation with scanning model would explain the preference for the downstream gene start in the –15/0 and –15/+3 constructs and the increasing use of the upstream gene start as the second gene start moves further downstream in the –12/+1, –12/+3, and –12/+6 constructs. This model of internal transcription initiation with scanning is also consistent with recent genetic and biochemical experiments with VSV, which have shown a distinct transcriptase can initiate transcription directly at the N gene start signal (28, 41).

The bidirectional scanning of the transcriptase suggested by this research and incorporated into the proposed model for transcription initiation is not unprecedented in the Paramyxoviridae. During transcription of most paramyxovirus gene junctions, the transcriptase usually scans a short distance after terminating transcription at a gene end signal to reach a downstream gene start signal (20). However, the RSV transcriptase must, after termination of M2 mRNA synthesis, scan upstream to encounter the gene start signal for the L gene (10). We do not know over what distance scanning may occur. For the Paramyxoviridae, the longest intergenic sequence between gene end and gene start signals over which the transcriptase would scan is 56 nucleotides (20). Additionally, recent evidence with SeV suggests that polymerase scanning during transcription initiation may proceed beyond CRII (36). In this experiment, tandem promoters were created in which replication could occur from the first (3'-terminal) promoter, while transcription could occur from the adjacent, second (internal) promoter. When the CRI and CRII elements of the second promoter were deleted, transcription could still initiate from the gene start signal of the second promoter. This initiation could be the result of RNA polymerase binding to the 3'-terminal promoter and scanning beyond the CRII to the gene start signal of the second promoter. Additional experiments creating or relocating single or multiple IS/Ngs elements in the HPIV3 minireplicon may help to better define template requirements, and understanding, of the mechanism of transcription initiation.

 
This work was supported by a UWL Undergraduate Research Award and a Dean's Undergraduate Research Fellowship to J.S.A. and NIH grant AI49961 to M.A.H.

 
* Corresponding author. Mailing address: Department of Microbiology, University of Wisconsin—La Crosse, 1725 State St., La Crosse, WI 54601. Phone: (608) 785-6984. Fax: (608) 785-6460. E-mail: hoffman.mic2{at}uwlax.edu.

Present address: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Virology, June 2006, p. 5388-5396, Vol. 80, No. 11
0022-538X/06/$08.00+0     doi:10.1128/JVI.00204-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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