Unité de Neurovirologie et Régénération du Système Nerveux, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris cedex 15, France1
Author for correspondence: Bruno Blondel (e-mail bblondel{at}pasteur.fr) and T. Couderc (tcouderc@pasteur.fr). Fax +33 1 45 68 87 62.
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Abstract |
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Introduction |
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In many poliomyelitic patients, a period of decades of clinical stability is followed by the development of a disease called post-polio syndrome, characterized notably by slowly progressive muscle weakness (Dalakas et al., 1995 ). The presence of PV RNA sequences or PV-related RNA (Leon-Monzon & Dalakas, 1995
; Muir et al., 1995
; Leparc-Goffart et al., 1996
) and of anti-PV IgM antibodies (Sharief et al., 1991
) suggests that PV persistence may be involved in this syndrome. Moreover, in human cell cultures of neuronal origin, it has been shown that PV can establish persistent infections (Colbère-Garapin et al., 1989
; Pavio et al., 1996
).
Poliomyelitis can be experimentally transmitted to monkeys by inoculation of the CNS with any of the three serotypes of PV, and to mice with mouse-adapted PV strains. We previously isolated and characterized a mutant pathogenic for mice, PV-1/Mah-T1022I, (Couderc et al., 1993 , 1996
). With this model, we have shown that PV persists in the CNS of paralysed mice for over a year after the acute disease (Destombes et al., 1997
). Moreover, PV plus- and minus-strand RNAs have been detected by RTnested PCR in the spinal cord of paralysed mice suggesting continuous PV RNA replication in the CNS. However, although PV particles could be detected in mouse motor neurons by electron microscopy for at least 12 months after the onset of paralysis, infectious PV particles could not be recovered from homogenates of CNS from paralysed mice beyond 20 days post-paralysis (p.p.), suggesting that PV replication was restricted (Destombes et al., 1997
). Limited replication has been shown with other neurotropic viruses including measles virus, mouse hepatitis virus and Sindbis virus, for which viral genomes were detected although infectious virus could rarely, if ever, be isolated from the CNS during persistent infection (Kyuwa & Stohlman, 1990
; Levine & Griffin, 1992
; Schneider-Schaulies & ter Meulen, 1992
). Similarly, non-productive persistent infection has been described with coxsackievirus B, another enterovirus, during virus-induced chronic myocarditis (Klingel et al., 1992
; Tam & Messner, 1999
; Reetoo et al., 2000
).
One of the main mechanisms by which coxsackieviruses persist in vivo is a restriction of viral RNA replication, possibly as a consequence of inhibition of plus-strand RNA synthesis (Cunningham et al., 1990 ; Klingel et al., 1992
; Andréoletti et al., 1997
; Tam & Messner, 1999
; Reetoo et al., 2000
). In contrast, viral RNA replication is not restricted during persistent infection of the CNS by Theilers murine encephalomyelitis virus, another picornavirus, although little infectious virus is detected in the mouse spinal cord (Trottier et al., 2001
). The molecular mechanisms of PV persistence in the mouse CNS have not been elucidated.
To investigate whether restriction of viral RNA synthesis contributes to the decrease in the viral load in the mouse CNS, we analysed the relative levels of genomic and complementary PV RNAs during both the acute phase of poliomyelitis and the subsequent persistent infection. Female OF1 (Iffa-Credo) mice, 28 days old, were inoculated intracerebrally with 3x107 p.f.u. of the mouse-adapted strain PV-1/Mah-T1022I. Total RNA was extracted from mouse spinal cords during acute (day 0 and day 10 p.p.) and persistent (112 months p.p.) phases of PV infection using the RNA PLUS kit (Bioprobe) according to the manufacturers instructions. Total RNA was resuspended in RNase-free H2O (250 ng/µl). A strand-specific semi-quantitative RTnested PCR method was used to assay viral RNA in samples of total RNA. Briefly, serial dilutions (1/3) of total RNA suspension were subjected to eight independent RTnested PCRs to detect plus- and minus-strand RNA, as described (Destombes et al., 1997 ) and PCR products were visualized after electrophoresis on agarose gel. The dilutions of total RNA from mouse spinal cords for which 50% of the RTnested PCR (PCRD50) were positive were calculated according to the Reed and Muench end-point method (Reed & Muench, 1938
) (Table 1a
). Negative controls involved testing total RNA (1 µg) prepared from mock-infected mice, and no PCR product was detected with the primers used, as previously illustrated (Destombes et al., 1997
). To validate the method, the PCRD50 of known amounts of PV RNA of each polarity transcribed in vitro, as well as those of mixes of plus- and minus-strand PV RNA with known ratios (10 and 1) were determined (Table 1b
). The ratios of the PCRD50 for plus-strand RNA to that for minus-strand RNA in the mixes were close to the expected values (8·5 and 0·9, respectively) (Fig. 1
).
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To investigate whether PV variants with altered replication properties arise during PV persistence in the mouse CNS, we analysed the genome of PV present in the spinal cord of a mouse 6 months p.p. The entire RNA genome was amplified as a series of six overlapping fragments, including the 5' and 3 proximal extremities, and sequenced. Surprisingly, we found no deletion or point mutation with reference to the consensus sequence of the viral genome (data not shown). Although we cannot rule out the role of nucleic acid modification such as methylation, this result seems to indicate that the inhibition of viral RNA synthesis in the mouse CNS is not due to viral factors. Similar results have been reported in mice with coxsackievirus-induced chronic myopathy where virus persistence in muscle was not associated with evolution of the viral genome (Tam & Messner, 1999 ). Thus, host factors are presumably the main factors responsible for the restriction of the synthesis of positive-strand RNA during persistent PV infection in the mouse CNS.
As PV is able to persist in human neuroblastoma cell cultures with a reduced virus yield (Colbère-Garapin et al., 1989 ), we investigated whether PV RNA replication was similarly inhibited during PV persistence in vitro. IMR-32 cell cultures persistently infected with the mouse-adapted mutant PV-1/Mah-T1022I were established as previously described (Colbère-Garapin et al., 1989
). A persistent infection was also established with the parental PV-1/Mahoney to test whether, although unlikely, the evolution of viral RNA synthesis was dependent on the substitution that confers the mouse-adapted phenotype to PV-1/Mah-T1022I (Thr 22 of VP1 to Ile). During the 2 weeks following infection, a large proportion of the cells died. Thereafter a few colonies appeared. After about 1 month, cells reached confluency and the virus yield dropped to about 1 TCID50 per cell per week (data not shown), compared to the virus yield in freshly infected IMR-32 (about 1000 TCID50 per cell 12 hours post-infection). The ratio of PV plus- to minus-strand RNA in persistently infected cell cultures was followed from 12 h to 6 months post-infection (p.i.). The PV plus- and minus-strand RNAs were detected independently by a strand-specific slot blot method. This hybridization technique was chosen because with large amounts of viral RNA it is more accurate than the semi-quantitative method, which would require too many dilutions for such RNA samples. Briefly, serial dilutions (1/5) of total RNA preparations (40 ng/µl) were denatured with formaldehyde and each dilution was blotted on to two separate nylon membranes (Hybond-N+, Amersham Pharmacia Biotech) using a Minifold II slot blot apparatus (Schleicher & Schuell) and fixed by UV. Both membranes were prehybridized at 68 °C for 2 h in 6xSSC, 5xDenhardts solution, 0·1% SDS. Probes specific for either plus- or minus-strand PV RNA were obtained from a 1·5 kb fragment of PV-1/Mahoney cDNA (positions 22323677) by using the Riboprobe Combination System in vitro transcription kit (Promega). The specific activity of the 32P-labelled probes was at least 2x108 c.p.m./µg. One membrane was hybridized in the same buffer containing the plus-strand specific riboprobe at 68 °C overnight and the second in the same conditions with the minus-strand specific riboprobe. The membranes were washed twice in 2xSSC, 0·1% SDS at 68 °C for 30 min, twice in 0·2x SSC, 0·1% SDS at 68 °C for 30 min and exposed to a PhosphorImager Storm 820 (Molecular Dynamics). Slot blot signal densities were quantified as average intensity of all the pixels in the spot, in arbitrary units, using the Image Quant software (Molecular Dynamics). Slot blot experiments included a range of known amounts of PV RNA of each polarity transcribed in vitro. Serial dilutions of total RNA prepared from mock-infected cells as described above were used as negative controls. The background was less than 10% of the signal at each respective dilution and was substracted from experimental values before calculating the ratios of plus- to minus-strand RNA in PV persistently infected IMR-32 cells. To validate the method, mixes of plus- and minus-strand PV RNA transcribed in vitro with known ratios of 10 and 1 were tested: the results obtained were as expected (11 and 1, respectively) (Fig. 2
).
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Since host factors are presumably the main factors responsible for restricting the synthesis of positive-strand RNA during persistent PV infection in the mouse CNS, we investigated whether cellular factors were involved in the inhibition of PV RNA synthesis during virus persistence in IMR-32 cells. First, we cured IMR-32 cell cultures persistently infected with PV-1/Mahoney for 6 months (IMR-32/PV-cured) by three passages in the presence of anti-PV-1 rabbit serum. Complete curing of a culture was confirmed by testing both for infectivity from cells disrupted by a freezethaw step and for viral RNA by RTPCR amplification with viral primers.
Persistently infected cells can be cured by treatment with anti-PV rabbit serum and this may suggest that continuous reinfections are required to maintain PV persistence in IMR-32 cells. Although we have found chromatolysis of motor neurons as well as inflammatory cell infiltration in the mouse CNS at all time points investigated after the onset of paralysis, even at 12 months p.p. (Destombes et al., 1997 ), it is difficult to know whether the persistent PV infection involves continuous reinfection of nerve cells.
RNA synthesis of PV-1/Mahoney during a single growth cycle was compared in IMR-32 and in IMR-32/PV-cured cells. Cells were infected at an m.o.i. of 10 TCID50 and the ratio of plus- to minus-strand RNA was evaluated by slot blot assays from 2 to 24 h p.i. The maximal values of the plus- to minus- strand RNA ratios during the single growth cycle experiments in both IMR-32 and IMR-32/PV-cured cell cultures (6 h p.i.) are presented in Fig. 3. The ratio of PV-1/Mahoney plus- to minus-strand RNA was 40% lower in IMR-32/PV-cured cells than in IMR-32 cells. This seems to indicate that cellular factors are involved in restriction of viral RNA synthesis in persistently infected IMR-32 cells. This result does not exclude the possibility that viral factors also contribute to restricting replication of PV in IMR-32 cells.
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Host cell components regulate the amount of plus- and minus-strand PV RNA synthesized according to the host cell type (Lopez-Guerrero et al., 1991 ). However, the mechanisms of the differential control of viral RNA synthesis leading to highly divergent amounts of plus- and minus-strand RNA are poorly understood but probably involve strand-specific initiation processes (for review, see Xiang et al., 1997
). Efficient selection in viral RNA replication is mediated by ribonucleoprotein complexes at the 5' and 3 ends, involving not only the RNA polymerase 3Dpol and a number of other viral proteins but also cellular factors including the poly(rC) binding protein and the poly(A) binding protein 1 (Xiang et al., 1997
; Herold & Andino, 2001
). It is therefore plausible that modification of one or several of these components during persistent infection could lead to the selective restriction of the synthesis of one strand (Andino et al., 1990
; Giachetti & Semler, 1991
).
In IMR-32 cell cultures persistently infected with PV, modified host factors involved in PV RNA replication may have been selected, notably during the cellular crisis period corresponding to the first month p.i. In contrast, genotypic changes in post-mitotic neuronal cells are highly unlikely, but alterations in PV RNA replication in the CNS could be induced by soluble factors, which may result from the immune response during the acute phase of infection and which have antiviral activity. These factors may include interferons (IFNs), which act by inducing a variety of proteins with different antiviral activities in the CNS of persistently infected mice. Indeed, an IFN-inducible protein, MxA, impairs synthesis of coxsackievirus RNA by an as yet unknown mechanism (Chieux et al., 2001 ). Moreover, interferons act synergistically with antibodies to inhibit PV replication (Langford et al., 1983
). During the persistent infection, PV RNA may persist in a double-stranded form, as shown for coxsackievirus (Tam & Messner, 1999
), and these double-stranded molecules could induce continual IFN production maintaining the restriction of PV RNA replication (Jacobs & Langland, 1996
). It would be interesting to use genetically modified mice that are deficient for the IFN receptors to assess the role of IFNs in the gene expression programme of PV-infected neurons in the CNS. This issue could be also investigated in cultured cells. Moreover, taking into account the fact that IMR-32 cells in culture are going across different steps of the complete cell life-cycle, in contrast to post-mitotic neurons in vivo, it would be of interest to examine the contribution of cell life-cycle to the control of PV RNA synthesis in IMR-32 cells.
In conclusion, the restriction of PV plus-strand RNA production may limit virus replication and thereby contribute to the persistence of PV in the CNS of paralysed mice. Although the mechanism involved in this restriction may be different in vitro, the IMR-32 cell model highlights the relevance of a global approach, using oligonucleotide arrays, to identify the cellular factors involved in the inhibition of PV RNA synthesis. Identification and characterization of these factors will improve our understanding of PV RNA replication and PV persistence.
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Acknowledgments |
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Footnotes |
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References |
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Andréoletti, L., Hober, D., Becquart, P., Belaich, S., Copin, M.-C., Lambert, V. & Wattré, P. (1997). Experimental CVB3-induced chronic myocarditis in two murine strains: evidence of interrelationships between virus replication and myocardial damage in persistent cardiac infection. Journal of Medical Virology 52, 206-214.[Medline]
Chieux, V., Chehadeh, W., Harvey, J., Haller, O., Wattré, P. & Hober, D. (2001). Inhibition of coxsackievirus B4 replication in stably transfected cells. Virology 283, 84-92.[Medline]
Colbère-Garapin, F., Christodoulou, C., Crainic, R. & Pelletier, I. (1989). Persistent poliovirus infection of human neuroblastoma cells. Proceedings of the National Academy of Sciences, USA 86, 7590-7594.[Abstract]
Colbère-Garapin, F., Duncan, G., Pavio, N., Pelletier, I. & Petit, I. (1998). An approach to understanding the mechanisms of poliovirus persistence in infected cells of neural or non-neural origin. Clinical and Diagnostic Virology 9, 107-113.[Medline]
Couderc, T., Hogle, J., Le Blay, H., Horaud, F. & Blondel, B. (1993). Molecular characterization of mouse-virulent poliovirus type 1 Mahoney mutants: involvement of residues of polypeptides VP1 and VP2 located on the inner surface of the capsid protein shell. Journal of Virology 67, 3808-3817.[Abstract]
Couderc, T., Delpeyroux, J., Le Blay, H. & Blondel, B. (1996). Mouse adaptation determinants of poliovirus type 1 enhance viral uncoating. Journal of Virology 70, 305-312.[Abstract]
Cunningham, L., Bowles, N. E., Lane, R. J. M. & Dubowitz, V. (1990). Persistence of enteroviral RNA in chronic fatigue syndrome is associated with the abnormal production of equal amounts of positive and negative strands of enteroviral RNA. Journal of General Virology 71, 1399-1402.[Abstract]
Dalakas, M. C., Bartfeld, H. & Kurland, L. T. (editors) (1995). The Post-polio Syndrome. New York: New York Academy of Sciences.
Destombes, J., Couderc, T., Thiesson, D., Girard, S., Wilt, S. G. & Blondel, B. (1997). Persistent poliovirus infection in mouse motorneurons. Journal of Virology 71, 1621-1628.[Abstract]
Giachetti, C. & Semler, B. L. (1991). Role of a viral membrane polypeptide in strand-specific initiation of poliovirus RNA synthesis. Journal of Virology 65, 2647-2654.[Medline]
Herold, J. & Andino, R. (2001). Poliovirus RNA replication requires genome circularization through a proteinprotein bridge. Molecular Cell 7, 581-591.[Medline]
Jacobs, B. L. & Langland, J. O. (1996). When two strands are better than one: the mediators and modulators of the cellular responses to double-stranded RNA. Virology 219, 339-349.[Medline]
Klingel, K., Hohenadl, C., Canu, A., Albrecht, M., Seemann, M., Mall, G. & Kandolf, R. (1992). Ongoing enterovirus-induced myocarditis is associated with persistent heart muscle infection quantitative analysis of virus replication, tissue damage, and inflammation. Proceedings of the National Academy of Sciences, USA 89, 314-318.[Abstract]
Kyuwa, S. & Stohlman, S. A. (1990). Pathogenesis of a neurotropic murine coronavirus, strain JHM in the central nervous system of mice. Seminars in Virology 1, 273-280.
Langford, M. L., Villareal, A. L. & Stanton, G. S. (1983). Antibody and interferon act synergistically to inhibit enterovirus, adenovirus, and herpes simplex virus infection. Infection and Immunity 41, 214-218.[Medline]
Leon-Monzon, M. E. & Dalakas, M. C. (1995). Detection of poliovirus antibodies and poliovirus genome in patients with post-polio syndrome (PPS). In The Post-polio Syndrome , pp. 208-218. Edited by M. C. Dalakas, H. Bartfeld & L. T. Kurland. New York:New York Academy of Sciences.
Leparc-Goffart, I., Julien, J., Fuchs, F., Janatova, I., Aymard, M. & Kopecka, H. (1996). Evidence of presence of poliovirus genomic sequences in cerebrospinal fluid from patients with postpolio syndrome. Journal of Clinical Microbiology 34, 2023-2026.[Abstract]
Levine, B. & Griffin, D. E. (1992). Persistence of viral RNA in mouse brains after recovery from acute alphavirus encephalitis. Journal of Virology 66, 6429-6435.[Abstract]
Lopez-Guerrero, J. A., Martinez-Abarca, F., Fresno, M., Carrasco, L. & Alonso, M. A. (1991). Cell type determines the relative proportion of (-) and (+) strand RNA during poliovirus replication. Virus Research 20, 23-29.[Medline]
Muir, P., Nicholson, F., Sharief, M. K., Thompson, E. J., Cairns, N. J., Lantos, P., Spencer, G. T., Kaminski, H. J. & Banatvala, J. E. (1995). Evidence for persistent enterovirus infection of the central nervous system in patients with previous paralytic poliomyelitis. In The Post-polio Syndrome , pp. 219-232. Edited by M. C. Dalakas, H. Bartfeld & L. T. Kurland. New York:New York Academy of Sciences.
Novak, J. E. & Kirkegaard, K. (1991). Improved method for detecting poliovirus negative strands used to demonstrate specificity of positive-strand encapsidation and the ratio of positive to negative strands in infected cells. Journal of Virology 65, 3384-3387.[Medline]
Pavio, N., Buc-Caron, M.-H. & Colbère-Garapin, F. (1996). Persistent poliovirus infection of human fetal brain cells. Journal of Virology 70, 6395-6401.[Abstract]
Pavio, N., Couderc, T., Girard, S., Sgro, J. Y., Blondel, B. & Colbère-Garapin, F. (2000). Expression of mutated receptors in human neuroblastoma cells persistently infected with poliovirus. Virology 274, 331-342.[Medline]
Reed, L. J. & Muench, M. (1938). A simple method for estimating fifty percent endpoints. American Journal of Hygiene 27, 493-497.
Reetoo, N. K., Osman, S. A., Illavia, S. J., Cameron-Wilson, C. L., Banatvala, J. E. & Muir, P. (2000). Quantitative analysis of viral RNA kinetics in coxsackievirus B3-induced murine myocarditis: biphasic pattern of clearance following acute infection, with persistence of residual viral RNA throughout and beyond the inflammatory phase of disease. Journal of General Virology 81, 2755-2762.
Schneider-Schaulies, S. & ter Meulen, V. (1992). Molecular aspects of measles virus induced central nervous system disease. In Molecular Neurovirology , pp. 419-449. Edited by R. P. Roos. Totowa, NJ:Humana Press.
Sharief, M. K., Hentges, M. R. & Ciardi, M. (1991). Intrathecal immune response in patients with the post-polio syndrome. New England Journal of Medicine 325, 749-755.[Abstract]
Tam, P. E. & Messner, R. P. (1999). Molecular mechanisms of coxsackievirus persistence in chronic inflammatory myopathy: viral RNA persists through formation of a double-stranded complex without associated genomic mutations or evolution. Journal of Virology 73, 10113-10121.
Trottier, M., Kallio, P., Wang, W. & Lipton, H. L. (2001). High number of viral RNA copies in the central nervous system of mice during persistent infection with Theilers virus. Journal of Virology 75, 7420-7428.
Xiang, W., Paul, A. V. & Wimmer, E. (1997). RNA signals in entero- and rhinovirus genome replication. Seminars in Virology 8, 256-273.
Received 25 October 2001;
accepted 8 January 2002.