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Journal of Virology, August 2002, p. 8244-8251, Vol. 76, No. 16
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.16.8244-8251.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

The Long Noncoding Region of the Human Parainfluenza Virus Type 1 F Gene Contributes to the Read-Through Transcription at the M-F Gene Junction

Tatiana Bousse,1 Tatyana Matrosovich,1,{dagger} Allen Portner,1,2 Atsushi Kato,3 Yoshiyuki Nagai,4 and Toru Takimoto1*

Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, 38105,1 Department of Pathology, University of Tennessee, Memphis, Tennessee 38163,2 National Institute of Infectious Diseases, Musashimurayama, Tokyo 208-0011, Japan,3 Toyama Institute of Health, Toyama 939-0363, Japan4

Received 27 February 2002/ Accepted 10 May 2002


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ABSTRACT
 
Sendai virus (SV) and human parainfluenza virus type 1 (hPIV1) have genomes consisting of nonsegmented negative-sense RNA in which the six genes are separated by well-conserved intergenic (IG) sequences and transcriptional start (S) and end signals. In hPIV1-infected cells, transcriptional termination at the M-F gene junction is ineffective; a large number of M-F read-through transcripts are produced (T. Bousse, T. Takimoto, K. G. Murti, and A. Portner, Virology 232:44-52, 1997). In contrast, few M-F read-through transcripts are detected in SV-infected cells. Sequence analysis indicated that the hPIV1 IG and S sequences in the M-F junction differ from those of SV. Furthermore, the hPIV1 F gene contains an unusually long noncoding sequence. To identify the cis-acting elements that prevent transcriptional termination at the M-F junction, we rescued recombinant SV (rSVhMFjCG) in which its M-F gene junction was replaced by that of hPIV1. Cells infected with rSVhMFjCG produced an abundance of M-F read-through transcripts; this result indicated that the hPIV1 M-F junction is responsible for inefficient termination. When one or both of the IG and S sites in rSVhMFjCG were replaced by those of SV, the efficiency of transcriptional termination increased but not to the level observed in wild-type SV-infected cells. Deletion of most of the long noncoding region of the hPIV1 F gene in rSVhMFjCG in addition to the mutations in IG and S signals resulted in efficient termination that was equivalent to the level observed in wild-type virus-infected cells. Therefore, the long noncoding sequence of the hPIV1 F gene contains cis-acting element(s) that affects transcriptional termination. Our evaluation of the effect of inefficient transcriptional termination on viral replication in culture revealed that cells infected with rSVhMFjCG produced less F protein than cells infected with wild-type SV and that assembly of the recombinant SV in culture was less efficient. These phenotypes seem to be responsible for the extended survival of mice infected with rSVhMFjCG.


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INTRODUCTION
 
Parainfluenza viruses are members of the genus Respirovirus and of the family Paramyxoviridae. The genome of parainfluenza viruses consists of nonsegmented, negative-sense RNA that contains six genes: 3'-NP-P-M-F-HN-L-5'. Transcription of the genome starts at the single promoter, which is located at the 3' end of the genome, and proceeds to the 5' end. Synthesis of mRNA for each gene is the result of a stop-start transcriptional mechanism. The cis-acting signals that regulate the production of mRNA are located within each of the five gene junctions. These signals contain transcription start (S) and end (E) sequences. A trinucleotide intergenic (IG) sequence is located between the E and S sequences and is not transcribed (7). For many paramyxoviruses, the sequences at the end of the gene and in the IG and S regions are conserved throughout the genome (17). In the closely related human parainfluenza virus type 1 (hPIV1) and Sendai virus (SV), the consensus E signal is 3'-UUAUUCUUUUU-5' and the consensus S sequence is 3'-UCCCUNUUUC-5'. The trinucleotide IG sequence of hPIV1 and SV is 3'-GAA-5', except for the sequence between the HN and L genes of SV (3'-GGG-5') and the one between the M and F genes of hPIV1 (3'-GCA-5') (5, 15, 19).

In addition to synthesizing monocistronic mRNA, the viral polymerase in some cases ignores the transcriptional termination and reinitiation signals and continues transcription. Thus, this polymerase synthesizes read-through products that contain sequences derived from two adjacent cistrons (10, 29, 30). Several studies have shown that the production of read-through transcripts is associated with nucleotide substitutions in the conserved S (13), E (2, 20), or IG sequences (1, 24).

We previously showed that an abundant number of bicistronic M-F transcripts are present in culture cells infected with hPIV1. However, the level of M-F read-through transcription is not substantial in cells infected with SV (5). In the present study, we used an SV reverse-genetics system to identify elements of the hPIV1 M-F gene junction that are involved in read-through transcription. We rescued recombinant SV (rSV) from cells transfected with full-genome SV cDNA that contains the hPIV1 M-F junction. Cells infected with the rescued rSV produced an abundant number of M-F read-through transcripts, a result indicating that the hPIV1 M-F junction is responsible for the inefficient transcriptional termination. Simultaneous substitutions in the IG and S sequences of the hPIV1 F gene substantially reduced M-F read-through transcription. Moreover, we demonstrated that changes in the sequence of the F gene, which is transcribed after the M gene, could affect termination of transcription of the M gene. We also found that cultured cells infected with an rSV produced less F protein than cells infected with wild-type SV, probably because of inefficient reinitiation of F gene transcription. This altered reinitiation may have reduced the pathogenicity of rSV in mice.


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MATERIALS AND METHODS
 
Cells and viruses. LLC-MK2 cells were maintained in Eagle's minimal essential medium (MEM) supplemented with 5% fetal bovine serum. The hPIV1 strain C35 was grown in LLC-MK2 cells in MEM supplemented with trypsin (2 µg/ml). Wild-type SV and mutant SVs rescued from pSeV(+) or mutant full-genome cDNAs were grown in embryonated hen eggs. The recombinant vaccinia virus vTF-7.3 was grown in TK-143 cells as described previously (3, 9).

cDNAs. The plasmid pSeV(+)hMFjCG, from which the rSV containing the hPIV1 M-F junction was produced, was constructed by using PCR-based gene splicing and overlap extension (12) to modify pSeV(+) (14). The same methods were used to construct pSeV(+)hMFj(-nc)CG. The constructions of pTF1SVNP and pTF1SVP were described previously (26). The cDNA clone of the L gene from the SV Enders strain was synthesized from viral RNA by using the Great Lengths cDNA Synthesis kit (Clontech) (26). The cDNA was inserted into pTF1, and that plasmid was designated pTF1SVL.

Site-directed mutagenesis. The cDNA inserts in pSeV(+)hMFjCG or pSeV(+)hMFj(-nc)CG were removed from the plasmids by digestion with SalI and ClaI, and the fragments containing the hPIV1 M-F junctions were subcloned into the same restriction sites in the plasmid pTF1 (25). Mutations in the M-F IG sequence of hPIV1 (3'-GCA-5' to 3'-GAA-5') and in the S site of the hPIV1 F gene (3'-UCCCUGUUU-5' to 3'-UCCCUAUUU-5') were made by using the QuikChange Site-Directed Mutagenesis kit (Stratagene). The mutated fragments were then subcloned from pTF1 into pSeV(+) at the same restriction sites. Mutations were verified by sequencing the full-genome cDNA.

Recovery of recombinant viruses. The rSVs were rescued by a reverse genetic system from cDNAs of full-length SV genomes (14). Briefly, 293T cells in six-well culture plates were infected with UV-inactivated vTF7-3, which expresses T7 RNA polymerase. Using 8 µl of Lipofectamine (Life Technologies, Grand Island, N.Y.), we transfected the cells with 1 µg of wild-type or mutated full-genome SV cDNA together with supporting plasmids pTF1SVNP (1 µg), pTF1SVP (1 µg), and pTF1SVL (0.1 µg). The cells were maintained in Dulbecco's MEM supplemented with 5% fetal bovine serum for 40 h. The cells were suspended in 100 µl of phosphate-buffered saline and lysed by three cycles of freezing and thawing. The lysates were then used to inoculate 10-day-old embryonated eggs. Seventy-two hours later, the allantoic fluid was harvested. In each case, recombinant virus was successfully recovered and cloned by plaque purification on LLC-MK2 cells. The stock viruses were grown in the embryonated eggs. To confirm mutations, reverse transcription-PCR was carried out by using the Titan One-Tube RT-PCR System (Roche) with viral RNA extracted from purified virus used as a template, and the PCR products were sequenced.

Northern blot analysis. Total cellular RNA was isolated 24 h after infection by acid phenol-guanidinium thiocyanate extraction (8) by using RNAzol B (Biotex Laboratories) according to the manufacturer's instruction. Polyadenylated RNA was isolated from the total RNA by oligo(dt)-cellulose chromatography [Poly(A) tract mRNA Isolation system; Promega]. The extracted mRNAs were separated by agarose gel electrophoresis on a 1.2% agarose gel containing 2.2 M formaldehyde, transferred to a GeneScreen Plus membrane (New England Nuclear), and hybridized with 32P-labeled DNA probes corresponding to the SV or hPIV1 NP, P, M, F, HN, or L genes. 32P-labeled probes were prepared by using a random-priming DNA labeling kit (Roche). Each gene in pTF1 was removed by cleavage at restriction sites unique to the insert, and the fragments were purified by treatment with the GeneClean II kit after electrophoresis on an agarose gel. These purified cDNAs were used as templates from which the 32P-labeled DNA probes were prepared.

The relative amounts of monocistronic and dicistronic M or F transcripts were quantified by using a PhosphorImager (Molecular Dynamics). Transcriptional termination at the M-F gene junction was calculated as the ratio of monocistronic M or F transcripts to the sum of the monocistronic M or F transcripts plus the M-F read-through products.

Radioimmunoprecipitation. LLC-MK2 cells were infected with wild-type SV or rSVhMFjCG (multiplicity of infection [MOI], 10 PFU) and incubated for 24 h in MEM supplemented with 5% fetal bovine serum. The cells were labeled with 100 µCi of Tran35S label (ICN)/ml in methionine- and cysteine-free medium (ICN) for 4 h and lysed in 200 µl of TNE buffer (10 mM Tris, pH 7.4; 150 mM NaCl; 0.5% NP-40). Labeled viral proteins in the cytoplasmic extracts were immunoprecipitated by using 20 µl of Dynabeads (Dynal, Lake Success, N.Y.) that had been preincubated with monoclonal antibodies specific for SV F, SV NP, or SV HN as described previously (4). The immunocomplexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the relative amounts of radiolabeled protein within the complexes were quantified by analysis with the PhosphorImager.

Kinetics of viral replication in LLC-MK2 cells. Duplicate wells of six-well plates were infected with wild-type SV or rSVhMFjCG (MOI, 5 or 0.01 PFU) in a total volume of 1 ml for 1 h at room temperature. Then the cells were washed three times. For cells that were to support one cycle of viral growth, the medium was replaced with MEM supplemented with 5% fetal bovine serum. For cells that were to support multiple cycles of viral replication, the medium was replaced with MEM supplemented with 0.15% bovine serum albumin and 2 µg of trypsin/ml. At the indicated time points, 200-µl aliquots of medium were taken and replaced with equal volumes of fresh medium. The amount of infectious virus in each sample of medium was determined by titration in a plaque assay with LLC-MK2 cells.

Virus infection in mice. Seven-week-old female 129X1/SvJ mice (Jackson Laboratory, Bar Harbor, Maine) were used in the experiment. Groups of 4 mice, which were under light anesthesia induced by isoflurane, were infected intranasally with 104, 105, 106, or 107 PFU of wild-type SV or rSVhMFjCG. One group of mice was inoculated with phosphate-buffered saline and served as a negative control. Weight loss and mortality were monitored daily throughout the 16-day experiment.


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RESULTS
 
Expression of hPIV1 and SV read-through transcripts. SV and hPIV1 are highly similar in genome structure and sequence (26). However, it was previously found that in hPIV1-infected cells, abundant numbers of bicistronic M-F read-through transcripts are produced (5). In contrast, transcription of the F gene in SV-infected cells generally produced monocistronic mRNA. To characterize the transcriptional regulation of each gene of SV and hPIV1, we performed Northern blot analysis of the transcripts of all the viral genes present in SV- or hPIV1-infected cells. Each 32P-labeled DNA probe for the NP, P, M, F, HN, or L gene hybridized to the blots of mRNA from LLC-MK2 cells infected with SV or hPIV1. Most of the SV transcripts isolated from infected cells were monocistronic; very little dicistronic mRNA was detected (Fig. 1). The sizes of the monocistronic mRNAs in the agarose gel corresponded well to the appropriate length of the homologous gene. In contrast, abundant M-F read-through transcripts were found in the hPIV1-infected cells (Fig. 1), as was reported previously (5). Transcriptional termination between other genes was very efficient: NP-P, P-M, F-HN, and HN-L read-through transcripts each accounted for less than 5% of the corresponding monocistronic mRNAs. Analysis of other hPIV1 strains isolated from infected patients during different years (11) revealed the same results, showing that the inefficient transcriptional termination at the M-F gene junction is a general characteristic of hPIV1 (data not shown).



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  		FIG. 1. Northern blot analysis of mRNA extracted from LLC-MK2 cells infected with SV or hPIV1. Polyadenylated RNAs were hybridized with 32P-labeled NP, P, M, F, HN, or L probes (as indicated at the top of each blot). The identity and position of the hybridizing mRNAs are marked. The positions to which 28S and 18S rRNA migrated are shown.

Sequence differences in M-F gene junctions of hPIV1 and SV. Each gene of type 1 parainfluenza viruses is separated from its neighboring gene by a well-conserved IG sequence that is composed of three nucleotides. Transcription is initiated at the S signal and terminated at the E signal. The production of abundant M-F read-through transcripts in hPIV1-infected cells suggests that the sequence of the hPIV1 M-F gene junction differs from the conserved consensus sequence. A comparison of the E, S, and IG sequences at the M-F gene junction of SV and of hPIV1 is shown in Fig. 2A. No differences were observed in the E signals of the SV and hPIV1 genes. However, one nucleotide difference was found in the M-F IG sequence of hPIV1 (3'-GCA-5') and SV (3'-GAA-5'). Also, there was one nucleotide difference in the S signal. In addition to the differences in the conserved IG and S sequences, the hPIV1 F gene contained a long noncoding region that was composed of 264 nucleotides; the corresponding noncoding region of the SV F gene consisted of 43 nucleotides.



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  		FIG. 2. Nucleotide sequences at the junction between M and F genes (A) and at the origins of genes of recombinant SV (B). E, IG, and S sequences at the M-F gene junction of the SV and hPIV1 genomes are shown in the viral RNA sense orientation. Nucleotide differences are indicated by lowercase letters. NC, noncoding sequence. (C) Nucleotide sequence of the noncoding region of the hPIV1 F gene. The nucleotides truncated from this region in SVhMFj(-nc)CG are shown in bold italics.

Rescue and characterization of rSV containing the hPIV1 M-F gene junction sequence. To determine whether the sequence of the M-F gene junction in the hPIV1 genome is responsible for the production of dicistronic read-through transcripts, we rescued rSV whose M-F gene junction (including the 5' noncoding region of the M gene and the 3' noncoding region of the F gene, in the viral RNA sense orientation) was replaced with that of hPIV1 (Fig. 2B). To characterize the M or F transcripts produced by rSVhMFjCG, we extracted total mRNA from LLC-MK2 cells infected with rSVhMFjCG and hybridized the mRNA to the 32P-labeled M or F probes (Fig. 3). As a control, mRNA from cells infected with wild-type SV was also analyzed. Quantitative analysis showed that in the cells infected with wild-type SV, 98% of the M transcripts were monocistronic mRNAs and only 2% were M-F read-through transcripts. However, in cells infected with rSVhMFjCG, only 77% of the M transcripts were monocistronic and 23% were dicistronic M-F read-through transcripts. Ninety-six percent of the F mRNAs in wild-type SV-infected cells were monocistronic, whereas only 63% of F transcripts were monocistronic in rSVhMFjCG-infected cells. This substantial increase in the amount of read-through transcripts clearly shows that the sequence at the M-F gene junction in hPIV1 is responsible for the high level of read-through transcription.



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  		FIG. 3. Analysis of M and F transcripts extracted from cells infected with wild-type SV (SVwt) or various rSVs. Polyadenylated RNA was isolated from SV-infected cells and transferred to Northern blots, which were then hybridized with a 32P-labeled M probe (top left panel) and with a 32P-labeled F probe (bottom left panel). The blots were exposed to X-ray film, and the resulting autoradiographs are shown. The ratios of M mRNA to M-F mRNA (top right panel) and of F mRNA to M-F mRNA (bottom right panel) were quantified by using the Storm Image System. The percentages of mRNA that were monocistronic (white bars) or read-through M-F transcripts (black bars) are shown. The averages of four independent experiments (± standard deviations) are shown.

Effect of mutations in conserved sequences on M-F transcription. Unlike the well-conserved IG sequence between the M and F genes of SV (3'-GAA-5'), the IG sequence between the hPIV1 M and F genes is 3'-GCA-5'. The S signal of the hPIV1 F gene is 3'-UCCCUGUUUC-5', but the SV F gene contains the conserved sequence 3'-UCCCUAUUUC-5'. To determine the effect(s) that mutations in the IG sequence of the hPIV1 M-F gene junction have on the termination of transcription, we introduced mutations into the full-genome rSVhMFjCG cDNA and rescued the mutant viruses. The IG sequence in rSVhMFjAG was replaced by that of SV (GCA to GAA). In rSVhMFjCA, the S signal of hPIV1 was changed to that of SV (UCCCUGUUUC to UCCCUAUUUC). The IG and S signals in rSVhMFjAA were replaced by those of SV (Fig. 2B). The efficiency of transcriptional termination at the M-F gene junction of these rescued viruses was analyzed (Fig. 3, lanes 3 to 5). Quantitative analysis of M gene transcription showed that a single nucleotide substitution in either the IG or the S sequence did not markedly alter transcription (Fig. 3). Changing the IG sequence from GCA to GAA or substituting a nucleotide in the S signal resulted in 14% of the hybridizing M transcripts being read-through transcripts; this proportion of M read-through transcripts was less than the proportion observed in rSVhMFjCG-infected cells (23%). In cells infected with rSVhMFjAA, which possessed simultaneous substitutions in both the IG and S signals, the proportion of M read-through transcripts was only 8%.

When the F probe was used in hybridization, individual mutations at either the IG or the S signal reduced the fraction of read-through transcripts to 30%; in contrast, the proportion of F read-through transcripts in cells infected with rSVhMFjCG was 37% (Fig. 3). Mutations in both the IG and S signals resulted in 21% of the mRNAs being read-through transcripts. Although rSVhMFjAA contained all the IG, S, and E signals of SV, the recombinant virus produced an amount of read-through transcripts that was markedly greater than that produced by wild-type SV (M read-through transcripts, 2%; F read-through transcripts, 5%). These results suggest that another region(s) in the M-F gene junction also affects transcriptional termination.

Effect of the 3' noncoding region of hPIV1 F on M-F transcription. Because replacement of the hPIV1 IG and S signals with those of SV did not reduce the level of read-through transcripts to the level associated with wild-type SV, we next analyzed the nonconserved sequences at the M-F gene junction. The most striking difference in the M-F gene junction of SV and of hPIV1 involves the 3' noncoding region of the F genes. The SV F gene contains only a 43-bp noncoding region, whereas the hPIV1 F gene contains a 264-nucleotide sequence that is 3' of the F coding sequence. This long noncoding region contains repeat motifs (3'-GUUUUU-5' and 3'-CUUU-5') that are not found in the corresponding region of the SV F gene (Fig. 2C).

To evaluate the effect of the noncoding sequence of the hPIV1 F gene on transcriptional termination, we rescued rSVhMFj(-nc)CG, in which the 264-bp 3' noncoding region of the hPIV1 F gene was reduced to 31 bp (Fig. 2C). Cells were infected with rSVhMFj(-nc)CG, and the M and the F transcripts were analyzed as described in the preceding paragraphs (Fig. 4). The percentage of F transcripts that were dicistronic was only 18%; in contrast, 37% of the F transcripts were dicistronic in cells infected with rSVhMFjCG, which contains all of the M-F junction sequence from hPIV1 (Fig. 4). Of the M transcripts that were analyzed, 23% in rSVhMFjCG-infected cells were dicistronic, but only 6% in rSVhMFj(-nc)CG-infected cells were dicistronic.



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  		FIG. 4. Effect of long noncoding sequence of the hPIV1 F gene on the production of M-F read-through transcripts in infected cells. Polyadenylated RNA was isolated from LLC-MK2 cells infected with wild-type SV (SVwt), rSVhMFjCG, rSVhMFj(-nc)CG, or rSVhMFj(-nc)AA (10 PFU per cell). Northern blots of the RNA were analyzed by hybridization with 32P-labeled M or F probes. The ratios of M to M-F mRNA and of F to M-F mRNA for each experiment were quantified, and the average ratios of four independent experiments (± standard deviations) are shown.

Further mutations were created in the IG and S signals of rSVhMFj(-nc)CG so that the resulting rSV, which was designated rSVhMFj(-nc)AA, contained the same IG and S signals as wild-type SV. Approximately 2.7% of the M mRNAs in rSVhMFj(-nc)AA-infected cells were read-through transcripts, and 6.5% of the F transcripts were read-through transcripts. These values are almost equivalent to those associated with wild-type SV (Fig. 4). Our findings indicate that the long noncoding region of the hPIV1 F gene, in addition to the IG and S signals at the M-F junction of hPIV1, affects the transcriptional termination of the M and F genes of type 1 parainfluenza viruses.

F protein expression in cells infected with wild-type SV and with rSVhMFjCG. We next examined the biological significance of M-F read-through transcription in viral growth. First, we determined the expression level of the F protein because the failure of transcriptional termination and reinitiation at the M-F junction results in the reduced production of monocistronic F mRNA. Cells were infected with wild-type SV or rSVhMFjCG, and the F protein was immunoprecipitated (Fig. 5). For comparison, we also determined the levels of NP and HN expression. The amounts of NP and HN synthesized by rSVhMFjCG-infected cells were almost equivalent to those synthesized by cells infected with wild-type SV. In contrast, the amount of F protein in rSVhMFjCG-infected cells was about one third of that in wild-type SV-infected cells. These results indicate that failure in transcriptional termination at the M-F gene junction results in a substantial reduction of F protein synthesis in infected cells.



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  		FIG. 5. Radioimmunoprecipitation of F, NP, and HN proteins. LLC-MK2 cells were infected with wild-type SV (SVwt) or rSVhMFjCG. Twenty-four hours after infection, cells were radiolabeled for 4 h. Proteins in cell lysates were immunoprecipitated with specific anti-F, anti-HN, or anti-NP monoclonal antibodies and analyzed by electrophoresis through a sodium dodecyl sulfate-polyacrylamide gel.

Comparison of growth of wild-type SV and rSVhMFjCG in cultured cells. We further analyzed the effect of reduced F protein production on the growth of SV. LLC-MK2 cells were infected with wild-type SV or rSVhMFjCG (MOI, 0.01 or 5), and the amount of infectious virus produced by the cells was quantified by plaque assay (Fig. 6). In each case, the titer of infectious rSVhMFjCG was lower than that of wild-type SV. An analysis of multiple-step virus growth showed that rSVhMFjCG grew more slowly than wild-type SV. This slow growth of rSVhMFjCG seemed to be due to reduced virus production because analysis of single-step virus growth showed that the amount of rSVhMFjCG at each time point (up to 72 h after infection) was approximately 10-fold lower than that of wild-type SV.



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  		FIG. 6. Kinetics of virus growth. Multiple-step (A) or single-step (B) growth curves of wild-type SV (SVwt) and rSV in LLC-MK2 cells are shown. Cells were infected with 0.01 PFU per cell (A) or 5 PFU per cell (B). Aliquots of the medium were harvested every 24 (A) or 12 (B) h and replaced with equal volumes of fresh medium. The quantity of infectious virus in the samples was determined by plaque assays with LLC-MK2 cells.

Comparison of the pathogenicity of wild-type SV and rSVhMFjCG in mice. We next compared the pathogenicity of wild-type SV with that of rSVhMFjCG in mice, natural hosts of SV. Mice (4 per group) were infected with intranasally administered wild-type SV or rSVhMFjCG (dose of 104, 105, 106, or 107 PFU) and observed for 16 days to determine the amount of body weight that was lost and the number of mice that died. The mean number of days of survival are reported in Fig. 7 and Table 1. Except for one, all mice infected with 105, 106, or 107 PFU of either virus lost 30 to 35% of their body weight and died by day 13 after infection. One mouse infected with 105 PFU of rSVhMFjCG recovered after it had lost 18% of its starting body weight by day 9. All mice infected with 104 PFU of wild-type SV or rSVhMFjCG lost as much as 10% of their initial body weight by day 8, but all recovered thereafter. The 50% lethal doses (LD50s) were not markedly different: 3.2 x 104 PFU for wild-type SV and 4.6 x 104 PFU for rSVhMFjCG. However, mice infected with rSVhMFjCG survived longer than wild-type SV-infected mice (Table 1); this result suggests rSVhMFjCG spread more slowly in mice than did wild-type virus.



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  		FIG. 7. Change in body weight of 129X1/SvJ mice infected with wild-type SV (SVwt) or rSVMFjCG. Groups of 4 mice were inoculated intranasally with various doses of virus (104 to 107 PFU per mouse). The weight gain of each mouse was measured every day for up to 16 days after inoculation. The symbols, which indicate the dose of virus given or type of infection, are as follows: {blacksquare}, 107 PFU of virus; {triangleup}, 106 PFU of virus; {blacktriangleup}, 105 PFU of virus; {circ}, 104 PFU of virus; •, mock infection. The body weight of one mouse that survived infection is shown by a dotted line.


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  		TABLE 1. Mean survival and LD50s for 129X1/SvJ mice infected with wild-type SV and rSVhMFjCG


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DISCUSSION
 
Cells infected with hPIV1 produce an abundance of M-F read-through transcripts (5). Disproportionate M-F read-through transcription has also been observed in cells infected with other paramyxoviruses, including hPIV3 (22), measles virus (6), simian virus 41 (SV41) (28), and SV5 (20). However, cells infected with SV, which is closely related to hPIV1, do not produce large quantities of M-F read-through transcripts (5) (Fig. 1).

In the present study, we used an SV reverse genetic system to identify the cis-acting elements in the hPIV1 M-F gene junction that affect transcriptional termination, and we determined the effect of increased read-through transcription on the growth and pathogenicity of SV. Substitution of the SV M-F gene junction sequence for that of hPIV1 resulted in a marked increase in M-F read-through transcription; such an increase suggests that sequences in the hPIV1 M-F gene junction are responsible for the failure of transcriptional termination. Mutations in the conserved gene junction are most likely responsible for M-F read-through transcription. For most paramyxoviruses, production of M-F read-through transcripts is due to mutations in the E signal of the M gene. For example, the E signal of the hPIV3 M gene contains an 8-nucleotide insertion, and the SV41 M gene lacks the entire E signal. These nonconsensus sequences are thought to be responsible for M-F read-through transcription (18, 22, 28). In SV5-infected cells, production of M-F read-through transcripts is associated with the shortest U tract at the end of the M gene and with a mutation in the E signal of the M gene. Mutational analysis with a bicistronic SV5 RNA analog showed that a single nucleotide substitution immediately 3' of the U tract at the end of the M gene is sufficient to convert the E sequence to an efficient signal for polyadenylation (20). However, it is unlikely that the E signal of the hPIV1 M gene is responsible for the production of M-F read-through transcripts because the E signal (3'-UUAUUCUUUUU-5') is not different from the consensus sequence of type 1 parainfluenza viruses (Fig. 2A).

Comparison of the sequence at the M-F gene junction in hPIV1 with that in SV revealed that the IG sequence and S signal of hPIV1 contain mutations. Replacing the mutated IG sequence (3'-GCA-5') with the conserved sequence (3'-GAA-5') improved the efficiency of termination (Fig. 3); this improvement indicates that the mutation in the IG sequence is partly responsible for the read-through transcription. Previous studies showed that the role of the IG sequence in transcriptional termination differs between viruses. For vesicular stomatitis virus, the dinucleotide IG sequence (GA) is highly conserved and nucleotide changes in the sequence result in the alteration of transcriptional termination (2, 24). In contrast, the IG sequence of respiratory syncytial virus is diverse and has little or no influence on transcription (16). Similarly, the IG sequence of SV5 is not conserved between genes and seems to have little effect on transcriptional termination (20, 21).

In addition to the IG sequence, we found that one nucleotide mutation in the S signal of the hPIV1 F gene affects the transcriptional termination of the M gene (Fig. 3). Substitution of an A for a G at the sixth position of the S signal reduced the proportion of M read-through transcripts to 14%, a proportion similar to that observed in cells infected with rSVhMFjAG, in which the only substitution is in the IG sequence. Simultaneous nucleotide substitution in both the IG and S signals reduced the level of M read-through transcripts to 8%. Thus, both IG and S signals appear to be involved in the transcriptional termination of type 1 parainfluenza viruses.

Cells infected with rSVhMFjAA, which contains consensus IG and S signals still produced more M-F read-through transcripts than did cells infected with wild-type SV (Fig. 3). One striking difference between the F gene of hPIV1 and that of SV is that the hPIV1 F gene contains a long (264 bp) noncoding region that follows the S signal. The 3' noncoding region of the SV F gene has only 43 nucleotides. Interestingly, the long noncoding region of the hPIV1 F gene includes a repeated motif composed of 3'-GUUUUU-5' (Fig. 2C). Another member of the human Respirovirus genus, hPIV3, also contains a long noncoding sequence in its F gene; this noncoding sequence includes six 3'-GUUUU-5' and 3'-GUUUUU-5' motifs (23). It is unclear whether these motifs play a role in regulating transcription. However, reducing the length of the noncoding region from 264 to 31 nucleotides resulted in a reduction in the proportion of M read-through transcripts from 23 to 6% (Fig. 4). This truncation of the long noncoding region together with the mutations in the IG and S signals reduced the proportion of read-through transcripts to a level almost identical to that of wild-type SV (Fig. 4). These results suggest that, in addition to the conserved IG and S signals, the sequence that follows the S signal affects the transcription termination of the M gene of type 1 parainfluenza viruses. The mechanism by which the noncoding region of the hPIV1 F gene affects transcriptional termination is not clear. However, the conservation of the motif in the F noncoding region in both hPIV1 and hPIV3, both of which produce read-through M-F transcripts (5, 22), may suggest a functional role for these motifs in the regulation of transcription.

As a consequence of the abundant production of M-F read-through transcripts, the synthesis of F protein in cells infected with rSVhMFjCG was significantly reduced (Fig. 5); a similar reduction was also observed in hPIV1-infected cells (5). Reduced F expression may be responsible for the reduced production of virus particles by infected cells (Fig. 6). It was recently shown that the SV F protein is involved in the budding and formation of virus particles (27). Expression of F protein alone induces the formation and release of virus-like particles bearing F spikes into culture medium; this finding suggests that the F protein drives the budding of SV. Therefore, limited expression of F protein may cause the limited production of progeny virion by rSVhMFjCG-infected cells.

The mean number of survival days of rSVhMFjCG-infected mice was greater than that of wild-type SV-infected mice. Thus, the spread of the rSV appears to be slower than that of wild-type SV, probably because fewer infectious rSV particles were released from infected cells. This result agrees with that of a previous report: rSV that expressed a larger amount of F protein than wild-type SV replicated faster than wild-type SV in tissue culture and in mice (13). The level of F protein expression seems to control the production of progeny virion by infected cells and, as a consequence, the pathogenicity of the virus.


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ACKNOWLEDGMENTS
 
This work was supported by grants AI-38956 and AI-11949 from the National Institute of Allergy and Infectious Diseases, by Cancer Center Support (CORE) grant CA-21765 from the National Cancer Institute, and by the American Lebanese Syrian Associated Charities (ALSAC).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Infectious Diseases, Mail Stop 330, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794. Phone: (901) 495-3375. Fax: (901) 523-2622. E-mail: toru.takimoto{at}stjude.org. Back

{dagger} Present address: Institut fur Virologie, Philipps-Universitat, 35037 Marburg, Germany. Back


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Journal of Virology, August 2002, p. 8244-8251, Vol. 76, No. 16
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.16.8244-8251.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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