Characterization of in vitro and in vivo phenotypes of poliovirus type 1 mutants with reduced viral protein synthesis activity

Minetaro Arita, Hiroyuki Shimizu and Tatsuo Miyamura

Department of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama-shi, Tokyo 208-0011, Japan

Correspondence
Minetaro Arita
minetaro{at}nih.go.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Sabin vaccine strains of poliovirus (PV) contain major attenuation determinants in the internal ribosomal entry site (IRES), an area that directs viral protein synthesis. To examine the effect of reduced viral protein synthesis on PV neurovirulence, spacer sequences, consisting of short open reading frames of different lengths, were introduced between the IRES and the initiation codon of viral polyprotein, resulting in PV mutants with reduced viral protein synthesis. These PV mutants had a viral protein synthesis activity 8·8–55 % of that of the parental Mahoney strain as measured in HeLa S3 cells. Only viruses with more than 28 % of the wild-type activity had intact spacer sequences following plaque purification. Mutants with 17 % or 21 % of the wild-type activity were unstable and a mutant with 8·8 % was lethal. The neurovirulence of PV mutants was evaluated in transgenic mice carrying the human PV receptor gene. In this test, mutants with more than 28 % of the wild-type activity remained neurovirulent, while a mutant with 17 % of wild-type activity exhibited a partially attenuated phenotype. This mutant stably replicated in the spinal cord; however, the stability was severely affected during the course of virus infection from the cerebrum to the spinal cord. These results suggest that reduced viral protein synthesis activity as measured in cultured cells (17–55 % of the wild-type activity) is not the main determinant of PV attenuation.

Results of the measurement of protein synthesis activity directed by the IRES mutants in SK-N-MC cells are available in JGV Online.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Poliovirus (PV) is a small non-enveloped virus belonging to the family Picornaviridae with a single positive-stranded genomic RNA; it is known as the causative agent of poliomyelitis. The PV genome is about 7500 nucleotides (nt) with a 5' non-translated region (nt 1–742 in the Mahoney strain) including a cloverleaf structure (Andino et al., 1990) and an internal ribosomal entry site (IRES) (Jang et al., 1988; Pelletier & Sonenberg, 1988). Motor neurons are the target of PV in the central nervous system (CNS). The tropism of PV infection to target cells in the CNS is, in part, attributable to the specific expression of the PV receptor in neurons (Koike et al., 1994; Ren & Racaniello, 1992).

The effect of viral protein synthesis on PV neurovirulence has mainly been studied on live attenuated strains of PV (Sabin 1, 2 and 3) that are widely used as oral PV vaccines (Sabin, 1965). Attenuation determinants of Sabin strains have been defined throughout the virus genome in detail (reviewed by Minor, 1992), including mutations of the 5' non-translated region, and the coding region of the capsid proteins and polymerase (Bouchard et al., 1995; Cann et al., 1984; Horie et al., 1994; Macadam et al., 1993; Omata et al., 1986). Among these attenuation determinants, a major determinant is mapped on stem–loop V (nt 448–556) of the IRES element in all Sabin strains (nt 480 in Sabin 1, nt 481 in Sabin 2 and nt 472 in Sabin 3) (Cann et al., 1984; Evans et al., 1985; Kawamura et al., 1989; Macadam et al., 1991). The introduction of these attenuation determinants into the wild-type IRES element resulted in a decrease in in vitro viral protein synthesis activity (Muzychenko et al., 1991; Svitkin et al., 1985, 1990), possibly due to destabilization of the stem–loop V structure and/or that of the entire IRES element (Macadam et al., 1994; Malnou et al., 2002; Rowe et al., 2001). Furthermore, the introduction of attenuation determinants into the IRES also caused a cell type-specific decrease in IRES activity in a neuroblastoma cell line or cell lysate compared with the parental IRES activity (Gutierrez et al., 1997; Haller et al., 1996). To date, and pioneered by the study of Svitkin et al., Sabin IRES activities have been found to be 12–67 % of the parental or virulent revertant IRES activity (Gutierrez et al., 1997; Haller et al., 1996; Muzychenko et al., 1991; Svitkin et al., 1985, 1990). A study on the neurovirulence of Theiler's murine encephalomyelitis virus (GDVII strain) showed that a putative host factor (neural-specific homologue of pyrimidine tract-binding protein) affects both the viral protein synthesis and neurovirulence (Pilipenko et al., 2001).

Genetically manipulated PV IRES mutants and their revertant viruses have been used to analyse the role of PV IRES, mainly focusing on stem–loops II and V, and the oligopyrimidine/cryptic AUG motif (Haller et al., 1996; Iizuka et al., 1989; Shiroki et al., 1997; Slobodskaya et al., 1996). The stem–loop V structure was found to be involved in a neuronal cell-specific IRES activity and in virus release from cells (Haller et al., 1996; Stewart & Semler, 1999), and the importance of the cryptic AUG (nt 586 to 588 in the Mahoney strain) in neurovirulence has been reported (Iizuka et al., 1989; Slobodskaya et al., 1996). The stem–loop II structure has a role in PV host-range phenotype by modulating viral protein synthesis (Shiroki et al., 1997). A cis-element for replication (Borman et al., 1994; Shiroki et al., 1993, 1995) and an encapsidation signal (Johansen & Morrow, 2000) have been suggested to exist in the IRES element. These observations suggest that a small structural disturbance in the 5' non-translated region of the PV genome could have pleiotropic effects on the virus life cycle.

In this study, we examined the significance of viral protein synthesis on type 1 PV neurovirulence. We constructed PV mutants with reduced viral protein synthesis activity in HeLa S3 cells, and examined their neurovirulence in transgenic mice expressing the human PV receptor (hPVR).


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and viruses.
HEp-2c and HeLa S3 cells were cultured as monolayers in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5 % fetal calf serum (FCS). Viruses were prepared in HEp-2c cells incubated at 35 °C by RNA transfection of transcripts synthesized from corresponding infectious clones by the DEAE–dextran method, until all the cells showed a cytopathic effect (CPE) (Lu et al., 1995). Virus stocks were stored at –70 °C.

General methods of molecular cloning.
Escherichia coli strain XL10gold (Stratagene) was used for plasmid transformation and propagation. DNA fragments were ligated using a Quick Ligation kit (NEB). Site-directed mutagenesis (SDM) was performed by PCR following a standard procedure using cloned Pfu DNA polymerase (Stratagene) or KOD plus DNA polymerase (TOYOBO) (Sambrook & Russell, 2001). The Titan one-tube RT-PCR system (Roche) was used for RT-PCR. PCR products were purified using a QIAquick PCR Purification kit (QIAGEN). Samples for sequencing were prepared using a BigDye Terminator v3.0 Cycle Sequencing Ready Reaction kit (ABI) and analysed using the ABI PRIZM 310 Genetic Analyser (ABI). Primers used for DNA construction are listed in Table 1.


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Table 1. Primers used in this study

 
Construction of dicistronic replicons.
Firefly luciferase (Fluc) cDNA was obtained from plasmid (Gtx133-141)10(SI)9b/RPh by PCR amplification using primers Fluc-H(+) and Fluc-SacI-T(–) (Chappell et al., 2000). The DNA fragments encoding the PV IRES site were obtained by PCR amplification using primers PV IRES-SalI-H(+) and PV IRES-Fluc-T(–) with pT7PV1M (a generous gift from Dr E. Wimmer), an infectious clone of type 1 Mahoney strain, as the template. Next, these DNA fragments were fused together and reamplified by PCR with primers PV IRES-SalI-H(+) and Fluc-SacI-T(–). The resulting DNA fragment was digested with SalI and SacI, and then cloned into the HRPF-luc vector (Zhao & Wimmer, 2001). Next, the Renilla luciferase (Rluc) DNA fragment was obtained by PCR amplification using primers EcoRI-SmaI-Rluc-H(+) and Rluc-SalI(–) with plasmid (Gtx133-141)10(SI)9b/RPh as the template. The DNA fragment was digested with EcoRI and SalI, and then cloned into the above construct. The resulting dicistronic vector was named (–)IRES-PV dc. EcoRI and XmaI sites were used for the construction of IRES mutants (Fig. 1A). Next, the spacer sequence was obtained from the pEGFP-C1 vector (Clontech) using EcoRI-EGFP-H(+) and SmaI-EGFP-T(–), digested with EcoRI and XmaI, and then cloned into (–)IRES-PV dc. The 232 nt spacer sequence or 139 nt spacer sequence contained part of the coding region for enhanced green fluorescence protein (EGFP) (nt 99–327 or nt 99–234, respectively) followed by three nucleotides (AAG) just upstream of the initiation codon of Rluc. The PV IRES sequence obtained by PCR amplification using PV IRES-EcoRI-H(+) and PV IRES-SacI-SmaI-T(–) primers was digested with EcoRI and MfeI, and then cloned into the above construct. The resulting dicistronic vector was named PV-232(+6+14aa)-PV dc and had two short open reading frames (sORFs) on the spacer sequence, which were out-of-frame compared to the original EGFP coding. Based on this construct, sORFs were introduced on the spacer sequence by the introduction of an initiation codon (AUG) and a termination codon by SDM. PV-232(–)-PV dc, PV-232(+6aa)-PV dc and PV-232(+6+4aa)-PV dc were obtained by SDM from PV-232(+6+14aa)-PV dc. To construct PV-232(+6aa)-PV dc, successive SDM steps were performed with primer set EGFP3-H(+) and EGFP3-T(–), followed by a second round of SDM using primer set EGFP6-1-H(+) and EGFP6-1-T(–) and the plasmid obtained from the first round of SDM. PV-232(–)-PV dc was obtained from PV-232(+6aa)-PV dc by SDM using EGFP6-2-H(+) and EGFP6-2-T(–) primers. PV-232(+6+4aa)-PV dc was obtained from PV-EGFP(+6aa)-PV dc by SDM using EGFP7-H(+) and EGFP7-T(–) primers. PV-232(+14aa)-PV dc was obtained by successive SDM from PV-232(+6aa)-PV dc using the primer sets AUG(1+)+ and AUG(1+)–, 10AA+ and 10AA–, as well as 14AA+ and 14AA– for each round of SDM. DNA fragments used for the construction of PV-139(–)-PV dc, PV-139(+6aa)-PV dc, PV-139(+14aa)-PV dc and PV-139(+25aa)-PV dc were obtained by PCR amplification using primers PV IRES-EcoRI-H(+) and space107– with PV-232(–)-PV dc, PV-232(+6aa)-PV dc, PV-232(+14aa)-PV dc and PV-139(+25aa) mc as the templates, respectively. These fragments were digested with EcoRI and XmaI, and then cloned into PV-232(–)-PV dc. For the construction of Mahoney-PV dc and Sabin 1-PV dc, IRES sequences were obtained by PCR or RT-PCR amplification using primers PV110(+) and PV-SmaI(–) with pT7PV1M or the Sabin 1 virus genome as the template, respectively. The DNA fragments were digested with EcoRI and XmaI, and then cloned into (–)IRES-PV dc.



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Fig. 1. Schematic view of spacers and luciferase replicons. (A) Schematics of spacer sequences with sORFs and restriction enzyme sites used for construction are shown. Sequence-flanking spacers are shown with initiation codons and restriction sites. Spacers of 232 nt or 139 nt were used with or without sORFs, and the number depicted above the spacer sequence represents the nucleotide number on the spacer sequence and in the coding region. Bars in the spacer represent sORFs and the number below each bar represents the number of amino acid residues they encode. Small closed boxes represent AUG codons on the spacer sequences. (B) Schematics of luciferase replicons.

 
Construction of monocistronic replicons.
Plasmid PVM/Luc, which encodes a monocistronic Fluc PV replicon (Li et al., 2001), was a generous gift from Dr E. Wimmer. Monocistronic replicons for IRES mutants were constructed as outlined below. The Fluc-coding region was obtained by PCR amplification using primers Fluc(+) and Fluc(–) with the PV-232(–)-PV dc as the DNA template. The DNA fragment obtained was digested with XmaI and SacI, and then cloned into PV-232(–)-PV dc. The resultant monocistronic replicon was named PV-232(–) mc. For the construction of PV-232(+6aa) mc, PV-232(+6+4aa) mc and PV-232(+14aa) mc, DNA fragments were obtained using primers PV110(+) and RIPO669(–) with PV-232(+6aa)-PV dc, PV-232(+6+4aa)-PV dc and PV-232(+14aa)-PV dc as the templates, respectively. These PCR products were digested with EcoRI and XmaI, and then cloned into PV-232(–) mc. PV-139(–) mc, PV-139(+6aa) mc and PV-139(+14aa) mc were constructed as described above for monocistronic replicons with a 232 nt spacer, but using space107– primer instead of RIPO669(–) primer for the PCR amplification. PV-139(+25aa) mc was obtained by SDM using a primer set 25+ and 25– with PV-232(+14aa) mc as the template.

Construction of infectious clones of PV mutants with reduced viral protein synthesis activity.
A SacI site was introduced into pT7PV1M by SDM using primers MahSacI+ and MahSacI–. The resultant construct was named pMah-SacI. For the construction of infectious clones of the 232(–), 232(+6aa), 232(+6+4aa) and 232(+14aa) viruses, a DNA fragment encoding the IRES and a spacer sequence was obtained by PCR amplification using primers PV110(+) and SacI(–), digested with AgeI and SacI, and then cloned into pMah-SacI. For the construction of infectious clones of the 139(–), 139(+6aa), 139(+6+4aa), 139(+14aa) viruses, a DNA fragment encoding the IRES and spacer sequence was obtained by PCR amplification using primers PV110(+) and spa107-s, digested with AgeI and SacI, and then cloned into pMah-SacI. The parental Mahoney strain used in this study was prepared from RNA transcripts synthesized from pMah-SacI.

RNA transfection.
RNA transcripts were obtained using a RiboMAX Large-Scale RNA Production System T7 kit (Promega) with DraI-linearized DNA as the template. RNA transcripts were transfected to a monolayer of HEp-2c or HeLa S3 cells by the DEAE–dextran method (Lu et al., 1995). Confluent (100 %) HEp-2c cells in six-well plates (Falcon) incubated at 35 °C in 2 ml of 5 % FCS/DMEM per well were used for virus preparation. Confluent HeLa S3 cells in 96-well plates (Falcon) incubated at 37 °C in 150 µl of 5 % FCS/DMEM per well were used for the luciferase assay.

Luciferase assay.
After RNA transfection to HeLa S3 cells, the cells were harvested at the times indicated by adding 20 µl of passive lysis buffer per well (Promega). Luciferase activity was measured with a Dual Luciferase kit (Promega) using a TR717 Microplate luminometer (ABI) according to the manufacturer's instructions.

Virus titration.
Plaque assay was performed in six-well plates (Falcon) containing HEp-2c cell monolayers. Virus solution (100 µl) was inoculated into each well and incubated for 30 min at 36 °C, then 2 ml of 5 % FCS/DMEM containing 0·5 % agarose ME (Iwai Chemicals Company Ltd, Tokyo, Japan) was added and the plates were further incubated for 24 h at 36 °C. The same medium with agarose ME and 0·005 % neutral red was overlaid on the first layer of agarose gel, and the plates were further incubated for 4 days at 36 °C. For the 50 % cell culture infective dose (CCID50) measurements, virus solution was diluted with 5 % FCS/DMEM, inoculated into HEp-2c cell monolayers on 96-well plates (Falcon) and then incubated at 36 °C for 1 week. CCID50 was calculated according to the Behrens–Kärber method (Karber, 1931).

Neurovirulence test in hPVR-expressing transgenic mice.
hPVR-expressing transgenic mice, ICR TgPVR21 (TgPVR21) (Central Laboratory of Experimental Animals, Kanagawa, Japan) (Abe et al., 1995), 4–5 weeks old, were used for the measurement of 50 % paralytic doses (PD50) of mutant viruses and virus growth in the spinal cord. For the measurement of PD50, 10-fold serial dilutions of virus solution were made from the stock virus solution of each mutant, and then 30 µl of the virus solution was intracerebrally inoculated. Six TgPVR21 mice (three males and three females) were used for the injection with each dose. The inoculated TgPVR21 mice were observed for paralysis, weakness and death up to 14 days. Mice that showed paralysis were counted for the calculation of PD50 values. For the intraspinal inoculation, 5 µl of virus solution was inoculated into the spinal cord of TgPVR21 mice.

Isolation of viral genomic RNA from TgPVR21 mice.
The viral genomic RNA was directly extracted from the spinal cords of inoculated TgPVR21 mice that showed paralysis or death. A part of the spinal cord (1–1·5 cm in length) was recovered from the mice, and then subjected to homogenization in 200 µl of 5 % FCS/DMEM. The homogenate was centrifuged at 10 000 g for 5 min at 4 °C, and then a 50 µl aliquot of the supernatant was used for the isolation of the viral genomic RNA using a High-pure-viral RNA purification kit (Roche). Isolated virus genomic RNA was used as the RNA template in the RT-PCR for sequence analysis.

In vivo replication of 139(+25aa) mutant.
The stock solution of 139(+25aa) mutant was prepared by RNA transfection in HEp-2c cells incubated for 14 h at 37 °C in 1 ml of 5 % FCS/DMEM per well. After freezing and thawing, the collected supernatant was centrifuged at 10 000 g for 5 min at 4 °C, then stored at –70 °C before use in the measurement of PD50 and for the intraspinal inoculation. The virus was recovered from a section of the spinal cord (1–1·5 cm) of TgPVR21 mice at the indicated days post-inoculation. Titration of 139(+25aa) mutant was performed by CCID50 measurement and the titre was determined from the CPE appearance, and also from the results of RT-PCR analysis of the cell suspension in each well after freezing and thawing, using primers PV110(+) and RIPO669(–) to detect the virus genome.

Computer-based secondary structure prediction of 139 nt spacer sequence.
The secondary structure model of a spacer sequence was obtained using the MFOLD 3.1 program and selected constraints on base pairing as indicated. The program is run on a server at The Bioinformatics Center at Rensselaer and Wadsworth (http://www.bioinfo.rpi.edu/applications/mfold/).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Construction of PV IRES mutants with reduced translation activity
First, we constructed PV IRES mutants with reduced protein synthesis activity but without direct modification of the IRES core structure. For this purpose, we introduced a series of spacer sequences between the intact PV IRES element and the initiation codon using the MfeI site (at nt 736) with or without sORFs of different lengths (Fig. 1A). We used a spacer sequence from the EGFP gene as an arbitrary sequence, with or without the introduction of sORFs encoding six, 14 or 25 amino acid residues for constructs containing spacers 232(+6aa) and 139(+6aa), or spacers 232(+14aa) and 139(+14aa), or spacer 139(+25aa), respectively. For spacers 232(+6+14aa) and 232(+6+4aa), two sORFs were introduced.

Protein synthesis directed by the PV IRES with sORFs was quantified using dicistronic PV replicons (Fig. 1B). Dicistronic replicons express Renilla luciferase as the first cistron directed by the PV IRES with sORFs, and firefly luciferase as the second cistron directed by the wild-type PV IRES that acts as an internal control of protein synthesis. For the evaluation of the protein synthesis activity of sORF mutants, luciferase activities were measured 2 h post-transfection of corresponding RNA transcripts in HeLa S3 cells, i.e. before starting the replication process of the replicons. The introduction of spacer sequences without sORFs did not affect the Renilla luciferase activity relative to the firefly luciferase activity, consistent with previous observations (Table 2) (Hellen et al., 1994; Kuge et al., 1989; Poyry et al., 2001). The introduction of sORFs into these spacer sequences resulted in moderate to marked changes in the relative Renilla luciferase activity. For the 139 nt spacer, the introduction of sORFs encoding six, 14 or 25 amino acid residues resulted in protein synthesis activity of 45 %, 28 % or 17 % of the wild-type activity, respectively (Table 2). We also measured the Sabin 1 IRES activity and observed slightly less IRES activity (88 %) than that observed for the parental Mahoney IRES. These results showed that the regulation of viral protein synthesis was achieved by introducing spacer sequences with sORFs between the IRES element and the initiation codon.


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Table 2. Protein synthesis directed by mutants and the wild-type PV IRES

 
Effect of viral protein synthesis activity reduction on the time-course of luciferase activity of PV luciferase replicons
We then analysed the effect of reduced viral protein synthesis activity on an in vitro phenotype of PV monocistronic luciferase replicons. Spacer sequences with or without sORFs were introduced between the PV IRES and the initiation codon of firefly luciferase (Fig. 1). The luciferase activity was measured as an indication of replication (Herold & Andino, 2000). As a result, the phenotypes determined by the luciferase activity were classified into four groups. The 139 nt spacer without a sORF [139(–)] did not affect the kinetics of luciferase activity, although the 232 nt spacer [232(–)] slightly reduced it at the maximum point (group I phenotype) (Fig. 2). The introduction of sORFs encoding six and 14 amino acid residues resulted in a moderate reduction in the maximum luciferase activity (10-fold reduction in the maximum activity compared with the wild-type level), with a slight delay in the peak luciferase activity observed at 12 h post-transfection [group II phenotype, 232(+6aa), 232(+14aa), 139(+6aa) and 139(+14aa) spacers]. The introduction of a 25 amino acid-encoding sORF or the tandem insertion of two sORFs (encoding six and four amino acid residues) markedly affected both the maximum luciferase activity and its kinetics, with an approximately 100-fold reduction compared with the wild-type level and the peak activity at 14 h post-transfection [group III phenotype, 139(+25aa) and 232(+6+4aa) spacers, Fig. 2]. The insertion of two sORFs (encoding six and 14 amino acid residues) further reduced the maximum luciferase activity, but an increase in luciferase activity was still detectable [group IV phenotype, 232(+6+14aa) mutant, Fig. 2C]. These results showed that the kinetics and the level of luciferase activity of luciferase replicons could be classified into four groups in a sORF-length-dependent manner, presumably due to reduction of viral protein synthesis.



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Fig. 2. Time-course of luciferase activity of monocistronic luciferase replicons with mutant IRES. Relative light units of firefly luciferase activity used as a reporter were measured for (A) mutants with the 232 nt spacer, (B) mutants with the 139 nt spacers and (C) mutants with delayed kinetics. Based on the kinetics of the increase and the peak of luciferase activity, mutants were classified into four groups (group I, II, III, IV). The group numbers of each mutant are indicated on the right side of each graph.

 
In vitro phenotypes of PV mutants with reduced viral protein synthesis activity
To analyse the phenotype of spacer-containing mutant viruses, we attempted to recover PV mutants with the spacers using an infectious clone of the type 1 PV (Mahoney) (Fig. 3). After RNA transfection of transcripts from corresponding infectious clones to HEp-2c cells, viruses were obtained for mutants 232(–), 232(+6aa), 232(+14aa), 232(+6+4aa), 139(–), 139(+6aa), 139(+14aa) and 139(+25aa), but not for mutant 232(+6+14aa) (Fig. 3 and Table 3). The plaque phenotypes of mutant viruses showed some heterogeneity as observed previously (Burns et al., 1992). Mutants 232(–) and 139(–) formed medium-sized plaques that were smaller than those formed by the parental Mahoney strain. Mutants 232(+6aa), 232(+14aa), 139(+6aa) and 139(+14aa) formed small plaques. Mutants 232(+6+4aa) and 139(+25aa) formed large plaques. After plaque purification, IRES mutants 232(–), 232(+6aa), 232(+14aa), 139(–), 139(+6aa) and 139(+14aa) retained intact spacer sequences, but mutants 232(+6+4aa) and 139(+25aa) showed a deletion in, or mutation of, the spacer sequence which disrupted the sORF (data not shown). Thus, the latter two mutants showed unstable virus production in contrast to that observed for the other six mutants. PV mutants exhibiting more than 28 % of the wild-type protein synthesis activity were capable of supporting stable virus production; however, mutants with less than 21 % of the wild-type activity showed unstable virus production or a lethal phenotype in vitro.



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Fig. 3. Plaque phenotype of PV mutants. HEp-2c cells inoculated with virus solution were incubated for 1 day at 36 °C after the addition of 5 % FCS/DMEM containing 0·5 % agarose ME. The same medium with agarose ME and 0·005 % neutral red was overlaid and the plates were further incubated for 4 days at 36 °C for staining. (A) Plaque phenotype of PV mutants with a 232 nt spacer. (B) Plaque phenotype of PV mutants with a 139 nt spacer.

 

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Table 3. Summary of in vitro phenotype and neurovirulence of PV mutants

 
In vivo phenotypes of PV mutants with reduced viral protein synthesis activity
Next, we examined the neurovirulence of mutant viruses that showed stable virus production in vitro, by the intracerebral inoculation of transgenic mice (TgPVR21) (Koike et al., 1991). The neurovirulence of 139 nt spacer mutants [139(–), 139(+6aa) and 139(+14aa) mutants] and the parental Mahoney strain were compared. Results showed that all of the spacer mutants examined retained high neurovirulence (PD50=3·2 to 3·6 log10CCID50) but were slightly attenuated compared with that of the parental strain (PD50=2·6 log10CCID50). To confirm that the observed neurovirulence was not due to the emergence of revertant viruses, we determined the stability of the introduced spacer sequence or mutation in mutants 139(–), 139(+6aa) and 139(+14aa) during in vivo replication. After intracerebral inoculation of each virus mutant to TgPVR21 mice, the spinal cords were collected from those which had exhibited paralysis and then the virus particles were recovered from the tissue (see Methods). Introduced spacers and sORFs were retained in the virus genomes of all three mutants for 5–14 days post-inoculation (data not shown). These results showed that the virulence of PV mutants only slightly decreased as the translational activity decreased.

Characterization of mutant 139(+25aa)
To further define the viral protein synthesis activity required for an attenuated phenotype of PV, we examined the in vivo replication phenotype of mutant 139(+25aa) which showed unstable virus production in vitro. We prepared 139(+25aa) virus by collecting particles after a single round of replication to reduce the possibility of emergence of revertant viruses (see Methods). To determine the in vivo stability of the mutant, the replication phenotype in the spinal cord was determined by intraspinal inoculation of TgPVR21 mice. After inoculation, the mice showed signs of paralysis or died within 7 days post-inoculation (Table 4). For all nine inoculated mice, the virus genome recovered from the spinal cords retained an intact spacer sequence, suggesting that mutant 139(+25aa) was stable during replication in the spinal cords of the TgPVR21 mice.


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Table 4. Replication of 139(+25aa) mutant after intraspinal or intracerebral inoculation

 
Next, we determined the neurovirulence of mutant 139(+25aa) following intracerebral inoculation of TgPVR21 mice. The virus showed a partially attenuated phenotype (PD50=5·1 log10CCID50) (Table 3). The 139(+25aa) viruses recovered from the spinal cords were mostly revertants (six out of nine mice) with a disrupted initiation AUG codon of the sORF or with deletion of the spacer sequence (Fig. 4). This instability is in marked contrast with that observed for the intraspinal inoculation. This suggests that low viral protein synthesis activity was a limiting factor in the pathway of PV infection from the cerebrum to the spinal cord in the TgPVR21 mouse. These results suggest that PV mutants with reduced viral protein synthesis activity observed in cultured cells (17–55 % of the wild-type activity) were not attenuated completely in these mice, although their translational activity in the cultured cells is lower than that of Sabin 1 IRES. This indicates that the translational activity observed in cell culture does not correlate with the neurovirulence of PV mutants.



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Fig. 4. RNA structural model of spacer sequence of 139(+25aa) mutant. (A) A model of secondary structure of the spacer sequence of the 139(+25aa) mutant estimated by MFOLD 3.1. (B) Schematics of spacer sequences of revertant viruses isolated from mice inoculated intracerebrally with the 139(+25aa) mutant (Table 3).

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
To determine the effect of viral protein synthesis on PV infection, we constructed a series of PV mutants showing reduced viral protein synthesis activity in HeLa S3 cells. We used the dicistronic PV replicon for the measurement of viral protein synthesis. We applied the reinitiation mechanism of ribosomes (Kozak, 1987), by introducing a sORF in a spacer sequence between the IRES element and the initiation codon to construct PV mutants (Fig. 1). The enterovirus genome has an endogenous spacer sequence well conserved in length (approx. 150 nt) without a putative primary or secondary RNA structure between the core IRES structure (from the 5' end of the genome to the last cryptic AUG) and the initiation codon, although the biological role of this spacer remains to be elucidated (Hellen et al., 1994; Kuge et al., 1989; Kuge & Nomoto, 1987). Recognition of an initiation codon downstream of the PV IRES by ribosomes has been demonstrated to utilize both scanning and shunting mechanisms through this endogenous spacer sequence (Hellen et al., 1994; Kuge et al., 1989; Poyry et al., 2001). The efficiency of reinitiation is influenced by several factors, such as the length of the upstream ORF (Kozak, 1987; Luukkonen et al., 1995), the distance between the upstream ORF and the initiation codon (Kozak, 1987) and the presence of translation initiation factors (Garcia-Barrio et al., 1995; reviewed by Kozak, 1999). We used part of a sequence derived from the EGFP gene as the spacer to introduce a sORF between the IRES element and the intiation codon, i.e. following the endogenous spacer sequence of the PV IRES (Fig. 1, 4). This spacer sequence has a secondary structure consisting of two stem–loop structures with a total free energy of –50·8 kcal mol–1 (–212·5 kJ mol–1) (Fig. 4). In a previous report, a single stem–loop structure with a free energy of –50 or –60 kcal mol–1 markedly inhibited translation mediated by the scanning mechanism, but a stem–loop structure with –30 kcal mol–1 did not (Kozak, 1986). The introduction of spacer sequences without sORFs had a minor effect, if any, on protein synthesis (Fig. 1, Table 3), possibly because of loose secondary structures that could not affect the ribosome scanning or shunting on the PV IRES (Hellen et al., 1994; Kuge et al., 1989; Poyry et al., 2001). However, the introduction of the spacer sequence affected the plaque phenotype of mutant viruses (Fig. 3), possibly because of the uncoating process and/or the encapsidation process via an unknown mechanism. The introduction of sORFs severely affected protein synthesis in a manner similar to that of the leaky scanning model (Fig. 1, Table 2). Consequently, we obtained PV IRES mutants that showed reductions in viral protein synthesis activity to between 8·8 % and 55 % of that of the parental Mahoney strain (Fig. 1, Table 2).

We recovered PV mutants with reduced protein synthesis activity (Fig. 3). There was a good correlation among this activity, monocistronic replicon phenotype and the stability of mutant viruses (Fig. 2, Tables 2 and 3). This decreased activity resulted in phenotypes showing delayed kinetics and levels of replication and in instability or lethality of virus production. We found that protein synthesis activity from 17 to 21 % of the wild-type level was on the boundary between stable virus production and the lethal phenotype in vitro.

We analysed the in vivo phenotype of PV mutants to evaluate their neurovirulence using a transgenic mouse model (TgPVR21) (Abe et al., 1995). The Sabin 1 IRES activity has been determined to have a range of around 32–67 % of the IRES activity of the parental strain (Haller et al., 1996; Muzychenko et al., 1991; Svitkin et al., 1985, 1990). Therefore, it was of interest to evaluate the neurovirulence of the type 1 PV mutants with reduced viral protein synthesis. Among those which showed stable virus production in vitro, mutant 139(+14aa) showed the lowest protein synthesis activity (28 % of the wild-type value) (Fig. 3). Despite this low activity, the mutant was highly neurovirulent in TgPVR21 mice (PD50=3·6 log10CCID50), only slightly attenuated compared with the parental strain (PD50=2·6 log10CCID50). To further define the viral protein synthesis activity required for the attenuation of type 1 PV, we examined mutant 139(+25aa), which showed the lowest viral protein synthesis activity with unstable virus production, and observed partial attenuation of its neurovirulence (PD50=5·1 log10CCID50). The apparent attenuation of 139(+25aa) was supported in part by the probability of emergence of revertants caused by the low viral protein synthesis activity as described below (Fig. 4, Table 4). A high ratio of emergent revertants might result in an overestimation of the neurovirulence of this mutant. These results showed that the effect of reduced viral protein synthesis activity in the range examined on the attenuation of PV was limited, hence other steps in the infection process may play major roles in the attenuation. The mutants examined in this study make a contrasting example to the RIPO mutant, which is a PV mutant with rhinovirus IRES that replicated well in HeLa cells but was not neurovirulent in PV receptor-expressing transgenic mice (Gromeier et al., 1996).

Following intracerebral inoculation of mice with mutant 139(+25aa), revertants with a disrupted sORF emerged rapidly (Fig. 4, Table 4). The revertants had either deletion or substitution mutations that disrupted the sORF (Fig. 4). This suggests that it is not the instability of the spacer sequence but the low protein synthesis activity that causes the emergence of revertants. Therefore, PV infection was limited to the stage of viral protein synthesis in some specific sites in the CNS other than the spinal cord. In contrast to the intracerebral inoculation results, mutant 139(+25aa) exhibited partially stable replication in the spinal cord when inoculated intraspinally into the TgPVR21 mice (Table 4). This observation suggests that motor neurons in the spinal cord might provide a ‘rich’ environment for PV replication that may possibly alleviate constraints on viral protein synthesis during PV infection. This suggests that different parts of the CNS provide different limitations on the replication of PV at the step of viral protein synthesis.

In summary, we found that the PV mutant with 17 % of the protein synthesis activity of the parental Mahoney strain was attenuated to only a minor extent. Therefore, the viral protein synthesis activity observed in cultured cells (17–55 % of the wild-type activity) did not determine strong attenuation of PV. This confirmed that reduced in vitro translational activity did not cause the attenuation of PV. The nature of in vivo PV infection remains to be further elucidated, but the IRES mutants constructed in this study would serve as useful tools for exploring the link in vivo between viral protein synthesis and pathogenesis in PV infection.


   ACKNOWLEDGEMENTS
 
We are grateful to Dr Eckard Wimmer for providing us with the infectious clone pT7PV1M and the PVM/Luc luciferase replicon. We are also grateful to Dr Akio Nomoto for continuous support in our study. A series of dicistronic replicons with a 239 nt spacer were constructed by M. A. in Dr Wimmer's laboratory. We thank Dr Noriyo Nagata for the intraspinal inoculation of the TgPVR21 mice. This study was supported in part by a fellowship from The Naito Foundation and by Grants-in-Aid for the Promotion of Polio Eradication from the Ministry of Health, Labour and Welfare, Japan.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 30 October 2003; accepted 10 March 2004.