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
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ABSTRACT |
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Results of the measurement of protein synthesis activity directed by the IRES mutants in SK-N-MC cells are available in JGV Online.
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INTRODUCTION |
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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 stemloop V (nt 448556) 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 stemloop 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 1267 % 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 stemloops 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 stemloop 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 stemloop 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).
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METHODS |
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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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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 DEAEdextran 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 BehrensKärber 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), 45 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 (11·5 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 (11·5 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/).
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RESULTS |
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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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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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DISCUSSION |
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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 3267 % 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 (1755 % 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.
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ACKNOWLEDGEMENTS |
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Received 30 October 2003;
accepted 10 March 2004.