A Small Interfering RNA Targeting Coxsackievirus B
http://www.100md.com
病菌学杂志 2005年第13期
Departments of Microbiology
Anatomy & Cell Biology
Research Institute for Biomacromolecules, University of Ulsan College of Medicine
Department of Medicine, Sungkyunkwan University School of Medicine
Cardiac and Vascular Center, Samsung Medical Center, Seoul, Korea
ABSTRACT
We examined the ability of small interfering RNAs (siRNAs) to disrupt infection by coxsackievirus B3 (CVB3). The incorporation of siRNAs dramatically decreased cell death in permissive HeLa cells in parallel with a reduction in viral replication. Three of four siRNAs had potent anti-CVB3 activity. The present study thus demonstrates that the antiviral effect is due to the downregulation of viral replication. In addition, an effective CVB3-specific siRNA had similar antiviral effects in other related enteroviruses possessing sequence homology in the targeted region. Because the CVB3-specific siRNA is effective against other enteroviruses, siRNAs have potential for a universal antienterovirus strategy.
TEXT
Coxsackievirus B3 (CVB3) is a major causative agent of many human diseases, such as meningioencephalitis and myocarditis (16-18, 20, 21). CVB3 is a member of the Picornaviridae family and consists of a positive single-stranded RNA genome coated with capsid proteins, including VP1-4 (Fig. 1A) (23). Previous studies by our group and others have implied that CVB3 infection causes productive virus replication, which results in cell death in both permissive and target cells (1, 2, 9), a process closely associated with CVB3-related human illness (6, 27, 28). Thus, directly blocking viral replication may be an effective strategy for treating the clinical symptoms of CVB3 infection.
RNA interference (RNAi) has emerged as a selective gene-silencing technique for inducing sequence-specific degradation of homologous RNA (8, 10). Posttranscriptional downregulation of gene expression is achieved by a small interfering RNA (siRNA), a 21- to 26-nucleotide RNA duplex (3, 7). Recent studies have reported that RNAi is effective against diverse viruses, such as human immunodeficiency virus and hepatitis C virus (4, 24, 30). To investigate the antiviral potential of RNAi for CVB3 infection, we analyzed the effects of siRNAs specific to various regions of CVB3w (CVB3 woodruff strain) on permissive HeLa cells (Fig. 1A).
Using oligofectamine reagent (Invitrogen, Carlsbad, CA), we transfected the cells with synthetic siRNAs tagged at their 3' end with tetramethylrhodamine (TAM). We began to detect a fluorescent signal in the cytoplasm within a few hours after addition of siRNA polymerase (POL). Then, the signal reached a plateau 8 to 12 h after transfection (Fig. 1B, TAM). More than 90% of the cells expressed the fluorescence, indicating effective siRNA transport. Infection with CVB3w-green fluorescent protein (GFP) led to a bright green color upon viral replication but in many fewer cells (Fig. 1B, GFP) than with infected cells without siRNA (Fig. 2, virus only). In addition to the CVB3 woodruff genome, CVB3w-GFP encoded GFP directly after the 5' untranslated region (UTR), followed by a viral protease recognition sequence (Fig. 1A). These results indicate that the siRNA significantly decreased the GFP signal (Fig. 1B, merge). Later we incubated the cells with 100 nM siRNA for 12 h prior to virus infection for all following experiments.
We first investigated the effect of siRNA on viral cytotoxicity. CVB3w-GFP infection was cytopathic within 12 h postinfection, as indicated by rounding up of the cells and by the presence of heavily condensed Hoechst 33342-stained nuclei (Fig. 2, virus only). Preincubation of the cells with siRNAs, except VP1-b and unmatched control siRNA, dramatically reduced the cytopathic effects (Fig. 2).
The pattern of GFP expression in Fig. 2 (middle column) suggests that a decrease in viral replication occurred in the presence of the siRNAs. We quantified the influence of different siRNAs on viral replication by analyzing the production of VP1 protein, progeny virus, and viral genome. For real-time reverse transcription-PCR of the viral genome, viral cDNA was synthesized using Superscript III reverse transcriptase (Invitrogen). Then PCR was performed with the TaqMan PCR master mix (Applied Biosystems, Foster City, CA). Figure 3 shows that the siRNAs lowered the chances of productive infection occurring. VP1 was detected only in the absence of siRNA or in the presence of control or VP1-b siRNAs. A significant reduction of progeny virus production and genome amplification was consistently observed following pretreatment with VP1-a, VP1-c, or POL. A combination of siRNAs did not further downregulate virus or genome production. Together, these findings suggest that CVB3-specific siRNA efficiently protects the HeLa cells from viral challenge by inhibiting viral replication.
CVB1-6 belongs to the enterovirus genus, which includes coxsackievirus A and echovirus (Echo) (29). To examine the possibility of using CVB3-specific siRNA for other related enteroviruses, we challenged HeLa cells with various viruses, including the CVB3 Nancy strain (CVB3n), after POL siRNA treatment (Fig. 4). We found that CVB3w-targeting siRNA had anticytopathic effects against CVB1, CVB5, CVB6, coxsackievirus A9, and Echo6, similar to that of CVB3w-GFP. In contrast, protective effects were not observed in CVB2, CVB4, Echo7, or even CVB3n. A comparison of the siRNA target sequence reveals that there were mismatches at several positions that had differential effects on the antiviral potency. A switch from C to T at position 17 seemed to be crucial in this regard. This was confirmed by the finding that a POL siRNA with a C-to-T switch (POL-T) exhibited anticytotoxicity opposite to that of the POL siRNA.
Collectively, our results demonstrate that (i) preincubation of HeLa cells with CVB3-specific siRNA prior to infection blocks viral replication and results in a potent antiviral effect; (ii) the interfering effect can vary among siRNAs even if they are perfectly homologous to the target sequence; (iii) combinations of siRNAs do not enhance antiviral potency; and (iv) CVB3-specific siRNA has antiviral effects in other related enteroviruses, depending on the homology of the target sequence. We found that the VP1-b siRNA had nearly no antiviral activity even though the sequence was 100% homologous with the target in CVB3w, whereas the POL siRNA was anticytopathic in other enteroviruses despite the presence of some mismatches. The siRNAs were designed to satisfy properties critical for siRNA functionality (http://www.ambion.com and http://www.oligoengine.com). This means that the ineffectiveness of VP1-b siRNA is not due to improper design (14, 19) or inaccessibility to the target sequence by a stem-loop structure (http://www.bioinfo.rpi.edu/applications/mfold) (31). Thus, VP1-b might have no interfering ability due to other reasons; for example, the inaccessibility of the target site by the binding of RNA-binding protein (7, 25). This also could be due to the unavailability of siRNA to RNA-inducing silencing complex, based on thermodynamic differences (26).
In agreement with reports for other related picornaviruses (5, 11, 13), our study demonstrates that the ability of the CVB3-specific siRNA to inhibit viral replication directly correlated with its antiviral potency. Additionally, a combination of siRNAs had no additive antiviral effect, similar to the results with other picornaviruses (11, 13). Lack of an additive effect might originate from their genome feature, a multicistronic single-stranded RNA genome. Nevertheless, combined treatment might prolong the antiviral effect because it could reduce neutralization by the emergence of siRNA escape mutants. There has been several reports demonstrating that productive CVB3 infection causes diverse human illnesses following substantial losses in functional primary target cells (6, 12, 22). In conclusion, the present study strongly supports the idea that siRNA has an excellent potential for a novel antiviral therapeutic strategy.
The results suggest that siRNAs designed for CVB3 could also have similar antiviral effects in closely related enteroviruses as long as they encode proper target sequences. Thus, a universal and efficient antiviral siRNA against closely related enteroviruses could be generated by careful design and subsequent screening for antiviral characteristics. Maintenance of functional siRNAs in a proper cellular location is a necessary prerequisite. However, synthetic siRNAs are transiently retained, mainly due to constant degradation by lysosomes and cell division (7, 15). We are currently investigating the possibility of overcoming this limitation by an optimal expression of short hairpin siRNA using various gene transfer vehicles.
ACKNOWLEDGMENTS
This work was supported by grants 03-PJ1-PG10-20200-0004 from the Ministry of Health & Welfare and R01-2005-000-10668-0 from Korea Science and Engineering Foundation to H. Lee and 01-PJ11-PG9-01BT00B-0019 (the International Mobile Telecommunications 2000 R&D Project) from the Ministry of Information & Communication to Y. K. Kim, Republic of Korea.
REFERENCES
Ahn, J., J. Choi, C. H. Joo, I. Seo, D. H. Kim, S. Y. Yoon, Y. K. Kim, and H. Lee. 2004. Susceptibility of coxsackievirus B in mouse primary cortical neural cell cultures. J. Gen. Virol. 85:1555-1564.
Ahn, J., C. H. Joo, I. Seo, D. Kim, H. N. Hong, Y. K. Kim, and H. Lee. 2003. Characteristics of apoptotic cell death induced by coxsackievirus B in permissive Vero cells. Intervirology 46:245-251.
Caplen, N. J., S. Parrish, F. Imani, A. Fire, and R. A. Morgan. 2001. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. USA 98:9742-9747.
Carmichael, G. G. 2002. Medicine: silencing viruses with RNA. Nature 418:379-380.
Chen, W., W. Yan, Q. Du, L. Fei, M. Liu, Z. Ni, Z. Sheng, and Z. Zheng. 2004. RNA interference targeting VP1 inhibits foot-and-mouth disease virus replication in BHK-21 cells and suckling mice. J. Virol. 78:6900-6907.
Cherry, J. D. 1981. Non-polio enteroviruses: coxsackieviruses, echoviruses and enteroviruses, p. 1316-1365. In R. D. Feigin and J. Cherry (ed.), Textbook of pediatric infectious diseases. W. B. Saunders, Philadelphia, Pa.
Dykxhoorn, D. M., C. D. Novina, and P. A. Sharp. 2003. Killing the messenger: short RNAs that silence gene expression. Nat. Rev. Mol. Cell Biol. 4:457-467.
Elbashir, S. M., J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, and T. Tuschl. 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494-498.
Feuer, R., I. Mena, R. R. Pagarigan, S. Harkins, D. E. Hassett, and J. L. Whitton. 2003. Coxsackievirus B3 and the neonatal CNS: the roles of stem cells, developing neurons, and apoptosis in infection, viral dissemination, and disease. Am. J. Pathol. 163:1379-1393.
Fire, A., S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver, and C. C. Mello. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806-811.
Gitlin, L., S. Karelsky, and R. Andino. 2002. Short interfering RNA confers intracellular antiviral immunity in human cells. Nature 418:430-434.
Huber, S. A., R. C. Budd, K. Rossner, and M. K. Newell. 1999. Apoptosis in coxsackievirus B3-induced myocarditis and dilated cardiomyopathy. Ann. N. Y. Acad. Sci. 887:181-190.
Kanda, T., Y. Kusov, O. Yokosuka, and V. Gauss-Muller. 2004. Interference of hepatitis A virus replication by small interfering RNAs. Biochem. Biophys. Res. Commun. 318:341-345.
Khvorova, A., A. Reynolds, and S. D. Jayasena. 2003. Functional siRNAs and miRNAs exhibit strand bias. Cell 115:209-216.
Lingor, P., U. Michel, U. Scholl, M. Bahr, and S. Kugler. 2004. Transfection of "naked" siRNA results in endosomal uptake and metabolic impairment in cultured neurons. Biochem. Biophys. Res. Commun. 315:1126-1133.
Muir, P. 1993. Enteroviruses and heart disease. Br. J. Biomed. Sci. 50:258-271.
Nigrovic, L. E. 2001. What's new with enteroviral infections? Curr. Opin. Pediatr. 13:89-94.
Pallansch, M. A., and R. P. Roos. 2001. Enteroviruses: poliovirus, coxsackieviruses, echoviruses, and newer enteroviruses, p. 723-776. In P. M. Howley and D. M. Knipe (ed.), Fields virology, vol. 1. Lippincott Williams & Wilkins, New York, N.Y.
Reynolds, A., D. Leake, Q. Boese, S. Scaringe, W. S. Marshall, and A. Khvorova. 2004. Rational siRNA design for RNA interference. Nat. Biotechnol. 22:326-330.
Roivainen, M. 1999. Enteroviruses and myocardial infarction. Am. Heart J. 138:S479-S483.
Rotbart, H. A. 1995. Enteroviral infections of the central nervous system. Clin. Infect. Dis. 20:971-981.
Rotbart, H. A., P. J. Brennan, K. H. Fife, J. R. Romero, J. A. Griffin, M. A. McKinlay, and F. G. Hayden. 1998. Enterovirus meningitis in adults. Clin. Infect. Dis. 27:896-898.
Rueckert, R. R. 1996. Picornaviridae: the viruses and their replication, p. 609-654. In B. Fields, D. M. Knipe, and P. M. Howley (ed.), Fields virology. Raven, New York, N.Y.
Saksela, K. 2003. Human viruses under attack by small inhibitory RNA. Trends Microbiol. 11:345-347.
Sharp, P. A. 2001. RNA interference—-2001. Genes Dev. 15:485-490.
Tomari, Y., C. Matranga, B. Haley, N. Martinez, and P. D. Zamore. 2004. A protein sensor for siRNA asymmetry. Science 306:1377-1380.
Tracy, S., N. M. Chapman, J. Romero, and A. I. Ramsingh. 1996. Genetics of coxsackievirus B cardiovirulence and inflammatory heart muscle disease. Trends Microbiol. 4:175-179.
Tracy, S., N. M. Chapman, and Z. Tu. 1992. Coxsackievirus B3 from an infectious cDNA copy of the genome is cardiovirulent in mice. Arch Virol. 122:399-409.
van Regenmortel, M. H. V., C. M. Fanquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lomon, J. Maniloft, M. A. Mayo, C. R. Pringe, and R. B. Wickner (ed.). 2000. Virus taxonomy. Academic Press, San Diego, Calif.
Wang, Q. C., Q. H. Nie, and Z. H. Feng. 2003. RNA interference: antiviral weapon and beyond. World J. Gastroenterol. 9:1657-1661.
Yoshinari, K., M. Miyagishi, and K. Taira. 2004. Effects on RNAi of the tight structure, sequence and position of the targeted region. Nucleic Acids Res. 32:691-699.(Jeonghyun Ahn, Eun Seok J)
Anatomy & Cell Biology
Research Institute for Biomacromolecules, University of Ulsan College of Medicine
Department of Medicine, Sungkyunkwan University School of Medicine
Cardiac and Vascular Center, Samsung Medical Center, Seoul, Korea
ABSTRACT
We examined the ability of small interfering RNAs (siRNAs) to disrupt infection by coxsackievirus B3 (CVB3). The incorporation of siRNAs dramatically decreased cell death in permissive HeLa cells in parallel with a reduction in viral replication. Three of four siRNAs had potent anti-CVB3 activity. The present study thus demonstrates that the antiviral effect is due to the downregulation of viral replication. In addition, an effective CVB3-specific siRNA had similar antiviral effects in other related enteroviruses possessing sequence homology in the targeted region. Because the CVB3-specific siRNA is effective against other enteroviruses, siRNAs have potential for a universal antienterovirus strategy.
TEXT
Coxsackievirus B3 (CVB3) is a major causative agent of many human diseases, such as meningioencephalitis and myocarditis (16-18, 20, 21). CVB3 is a member of the Picornaviridae family and consists of a positive single-stranded RNA genome coated with capsid proteins, including VP1-4 (Fig. 1A) (23). Previous studies by our group and others have implied that CVB3 infection causes productive virus replication, which results in cell death in both permissive and target cells (1, 2, 9), a process closely associated with CVB3-related human illness (6, 27, 28). Thus, directly blocking viral replication may be an effective strategy for treating the clinical symptoms of CVB3 infection.
RNA interference (RNAi) has emerged as a selective gene-silencing technique for inducing sequence-specific degradation of homologous RNA (8, 10). Posttranscriptional downregulation of gene expression is achieved by a small interfering RNA (siRNA), a 21- to 26-nucleotide RNA duplex (3, 7). Recent studies have reported that RNAi is effective against diverse viruses, such as human immunodeficiency virus and hepatitis C virus (4, 24, 30). To investigate the antiviral potential of RNAi for CVB3 infection, we analyzed the effects of siRNAs specific to various regions of CVB3w (CVB3 woodruff strain) on permissive HeLa cells (Fig. 1A).
Using oligofectamine reagent (Invitrogen, Carlsbad, CA), we transfected the cells with synthetic siRNAs tagged at their 3' end with tetramethylrhodamine (TAM). We began to detect a fluorescent signal in the cytoplasm within a few hours after addition of siRNA polymerase (POL). Then, the signal reached a plateau 8 to 12 h after transfection (Fig. 1B, TAM). More than 90% of the cells expressed the fluorescence, indicating effective siRNA transport. Infection with CVB3w-green fluorescent protein (GFP) led to a bright green color upon viral replication but in many fewer cells (Fig. 1B, GFP) than with infected cells without siRNA (Fig. 2, virus only). In addition to the CVB3 woodruff genome, CVB3w-GFP encoded GFP directly after the 5' untranslated region (UTR), followed by a viral protease recognition sequence (Fig. 1A). These results indicate that the siRNA significantly decreased the GFP signal (Fig. 1B, merge). Later we incubated the cells with 100 nM siRNA for 12 h prior to virus infection for all following experiments.
We first investigated the effect of siRNA on viral cytotoxicity. CVB3w-GFP infection was cytopathic within 12 h postinfection, as indicated by rounding up of the cells and by the presence of heavily condensed Hoechst 33342-stained nuclei (Fig. 2, virus only). Preincubation of the cells with siRNAs, except VP1-b and unmatched control siRNA, dramatically reduced the cytopathic effects (Fig. 2).
The pattern of GFP expression in Fig. 2 (middle column) suggests that a decrease in viral replication occurred in the presence of the siRNAs. We quantified the influence of different siRNAs on viral replication by analyzing the production of VP1 protein, progeny virus, and viral genome. For real-time reverse transcription-PCR of the viral genome, viral cDNA was synthesized using Superscript III reverse transcriptase (Invitrogen). Then PCR was performed with the TaqMan PCR master mix (Applied Biosystems, Foster City, CA). Figure 3 shows that the siRNAs lowered the chances of productive infection occurring. VP1 was detected only in the absence of siRNA or in the presence of control or VP1-b siRNAs. A significant reduction of progeny virus production and genome amplification was consistently observed following pretreatment with VP1-a, VP1-c, or POL. A combination of siRNAs did not further downregulate virus or genome production. Together, these findings suggest that CVB3-specific siRNA efficiently protects the HeLa cells from viral challenge by inhibiting viral replication.
CVB1-6 belongs to the enterovirus genus, which includes coxsackievirus A and echovirus (Echo) (29). To examine the possibility of using CVB3-specific siRNA for other related enteroviruses, we challenged HeLa cells with various viruses, including the CVB3 Nancy strain (CVB3n), after POL siRNA treatment (Fig. 4). We found that CVB3w-targeting siRNA had anticytopathic effects against CVB1, CVB5, CVB6, coxsackievirus A9, and Echo6, similar to that of CVB3w-GFP. In contrast, protective effects were not observed in CVB2, CVB4, Echo7, or even CVB3n. A comparison of the siRNA target sequence reveals that there were mismatches at several positions that had differential effects on the antiviral potency. A switch from C to T at position 17 seemed to be crucial in this regard. This was confirmed by the finding that a POL siRNA with a C-to-T switch (POL-T) exhibited anticytotoxicity opposite to that of the POL siRNA.
Collectively, our results demonstrate that (i) preincubation of HeLa cells with CVB3-specific siRNA prior to infection blocks viral replication and results in a potent antiviral effect; (ii) the interfering effect can vary among siRNAs even if they are perfectly homologous to the target sequence; (iii) combinations of siRNAs do not enhance antiviral potency; and (iv) CVB3-specific siRNA has antiviral effects in other related enteroviruses, depending on the homology of the target sequence. We found that the VP1-b siRNA had nearly no antiviral activity even though the sequence was 100% homologous with the target in CVB3w, whereas the POL siRNA was anticytopathic in other enteroviruses despite the presence of some mismatches. The siRNAs were designed to satisfy properties critical for siRNA functionality (http://www.ambion.com and http://www.oligoengine.com). This means that the ineffectiveness of VP1-b siRNA is not due to improper design (14, 19) or inaccessibility to the target sequence by a stem-loop structure (http://www.bioinfo.rpi.edu/applications/mfold) (31). Thus, VP1-b might have no interfering ability due to other reasons; for example, the inaccessibility of the target site by the binding of RNA-binding protein (7, 25). This also could be due to the unavailability of siRNA to RNA-inducing silencing complex, based on thermodynamic differences (26).
In agreement with reports for other related picornaviruses (5, 11, 13), our study demonstrates that the ability of the CVB3-specific siRNA to inhibit viral replication directly correlated with its antiviral potency. Additionally, a combination of siRNAs had no additive antiviral effect, similar to the results with other picornaviruses (11, 13). Lack of an additive effect might originate from their genome feature, a multicistronic single-stranded RNA genome. Nevertheless, combined treatment might prolong the antiviral effect because it could reduce neutralization by the emergence of siRNA escape mutants. There has been several reports demonstrating that productive CVB3 infection causes diverse human illnesses following substantial losses in functional primary target cells (6, 12, 22). In conclusion, the present study strongly supports the idea that siRNA has an excellent potential for a novel antiviral therapeutic strategy.
The results suggest that siRNAs designed for CVB3 could also have similar antiviral effects in closely related enteroviruses as long as they encode proper target sequences. Thus, a universal and efficient antiviral siRNA against closely related enteroviruses could be generated by careful design and subsequent screening for antiviral characteristics. Maintenance of functional siRNAs in a proper cellular location is a necessary prerequisite. However, synthetic siRNAs are transiently retained, mainly due to constant degradation by lysosomes and cell division (7, 15). We are currently investigating the possibility of overcoming this limitation by an optimal expression of short hairpin siRNA using various gene transfer vehicles.
ACKNOWLEDGMENTS
This work was supported by grants 03-PJ1-PG10-20200-0004 from the Ministry of Health & Welfare and R01-2005-000-10668-0 from Korea Science and Engineering Foundation to H. Lee and 01-PJ11-PG9-01BT00B-0019 (the International Mobile Telecommunications 2000 R&D Project) from the Ministry of Information & Communication to Y. K. Kim, Republic of Korea.
REFERENCES
Ahn, J., J. Choi, C. H. Joo, I. Seo, D. H. Kim, S. Y. Yoon, Y. K. Kim, and H. Lee. 2004. Susceptibility of coxsackievirus B in mouse primary cortical neural cell cultures. J. Gen. Virol. 85:1555-1564.
Ahn, J., C. H. Joo, I. Seo, D. Kim, H. N. Hong, Y. K. Kim, and H. Lee. 2003. Characteristics of apoptotic cell death induced by coxsackievirus B in permissive Vero cells. Intervirology 46:245-251.
Caplen, N. J., S. Parrish, F. Imani, A. Fire, and R. A. Morgan. 2001. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. USA 98:9742-9747.
Carmichael, G. G. 2002. Medicine: silencing viruses with RNA. Nature 418:379-380.
Chen, W., W. Yan, Q. Du, L. Fei, M. Liu, Z. Ni, Z. Sheng, and Z. Zheng. 2004. RNA interference targeting VP1 inhibits foot-and-mouth disease virus replication in BHK-21 cells and suckling mice. J. Virol. 78:6900-6907.
Cherry, J. D. 1981. Non-polio enteroviruses: coxsackieviruses, echoviruses and enteroviruses, p. 1316-1365. In R. D. Feigin and J. Cherry (ed.), Textbook of pediatric infectious diseases. W. B. Saunders, Philadelphia, Pa.
Dykxhoorn, D. M., C. D. Novina, and P. A. Sharp. 2003. Killing the messenger: short RNAs that silence gene expression. Nat. Rev. Mol. Cell Biol. 4:457-467.
Elbashir, S. M., J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, and T. Tuschl. 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494-498.
Feuer, R., I. Mena, R. R. Pagarigan, S. Harkins, D. E. Hassett, and J. L. Whitton. 2003. Coxsackievirus B3 and the neonatal CNS: the roles of stem cells, developing neurons, and apoptosis in infection, viral dissemination, and disease. Am. J. Pathol. 163:1379-1393.
Fire, A., S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver, and C. C. Mello. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806-811.
Gitlin, L., S. Karelsky, and R. Andino. 2002. Short interfering RNA confers intracellular antiviral immunity in human cells. Nature 418:430-434.
Huber, S. A., R. C. Budd, K. Rossner, and M. K. Newell. 1999. Apoptosis in coxsackievirus B3-induced myocarditis and dilated cardiomyopathy. Ann. N. Y. Acad. Sci. 887:181-190.
Kanda, T., Y. Kusov, O. Yokosuka, and V. Gauss-Muller. 2004. Interference of hepatitis A virus replication by small interfering RNAs. Biochem. Biophys. Res. Commun. 318:341-345.
Khvorova, A., A. Reynolds, and S. D. Jayasena. 2003. Functional siRNAs and miRNAs exhibit strand bias. Cell 115:209-216.
Lingor, P., U. Michel, U. Scholl, M. Bahr, and S. Kugler. 2004. Transfection of "naked" siRNA results in endosomal uptake and metabolic impairment in cultured neurons. Biochem. Biophys. Res. Commun. 315:1126-1133.
Muir, P. 1993. Enteroviruses and heart disease. Br. J. Biomed. Sci. 50:258-271.
Nigrovic, L. E. 2001. What's new with enteroviral infections? Curr. Opin. Pediatr. 13:89-94.
Pallansch, M. A., and R. P. Roos. 2001. Enteroviruses: poliovirus, coxsackieviruses, echoviruses, and newer enteroviruses, p. 723-776. In P. M. Howley and D. M. Knipe (ed.), Fields virology, vol. 1. Lippincott Williams & Wilkins, New York, N.Y.
Reynolds, A., D. Leake, Q. Boese, S. Scaringe, W. S. Marshall, and A. Khvorova. 2004. Rational siRNA design for RNA interference. Nat. Biotechnol. 22:326-330.
Roivainen, M. 1999. Enteroviruses and myocardial infarction. Am. Heart J. 138:S479-S483.
Rotbart, H. A. 1995. Enteroviral infections of the central nervous system. Clin. Infect. Dis. 20:971-981.
Rotbart, H. A., P. J. Brennan, K. H. Fife, J. R. Romero, J. A. Griffin, M. A. McKinlay, and F. G. Hayden. 1998. Enterovirus meningitis in adults. Clin. Infect. Dis. 27:896-898.
Rueckert, R. R. 1996. Picornaviridae: the viruses and their replication, p. 609-654. In B. Fields, D. M. Knipe, and P. M. Howley (ed.), Fields virology. Raven, New York, N.Y.
Saksela, K. 2003. Human viruses under attack by small inhibitory RNA. Trends Microbiol. 11:345-347.
Sharp, P. A. 2001. RNA interference—-2001. Genes Dev. 15:485-490.
Tomari, Y., C. Matranga, B. Haley, N. Martinez, and P. D. Zamore. 2004. A protein sensor for siRNA asymmetry. Science 306:1377-1380.
Tracy, S., N. M. Chapman, J. Romero, and A. I. Ramsingh. 1996. Genetics of coxsackievirus B cardiovirulence and inflammatory heart muscle disease. Trends Microbiol. 4:175-179.
Tracy, S., N. M. Chapman, and Z. Tu. 1992. Coxsackievirus B3 from an infectious cDNA copy of the genome is cardiovirulent in mice. Arch Virol. 122:399-409.
van Regenmortel, M. H. V., C. M. Fanquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lomon, J. Maniloft, M. A. Mayo, C. R. Pringe, and R. B. Wickner (ed.). 2000. Virus taxonomy. Academic Press, San Diego, Calif.
Wang, Q. C., Q. H. Nie, and Z. H. Feng. 2003. RNA interference: antiviral weapon and beyond. World J. Gastroenterol. 9:1657-1661.
Yoshinari, K., M. Miyagishi, and K. Taira. 2004. Effects on RNAi of the tight structure, sequence and position of the targeted region. Nucleic Acids Res. 32:691-699.(Jeonghyun Ahn, Eun Seok J)