- Email: [email protected]
Mutations at nucleotides 573 and 579 within 5′-untranslated region augment the virulence of coxsackievirus B1
Virus Research 135 (2008) 255–259
Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Mutations at nucleotides 573 and 579 within 5 -untranslated region augment the virulence of coxsackievirus B1 Zhaohua Zhong a,∗ , Xiaobo Li a , Wenran Zhao b , Lei Tong a , Jian Liu c , Shuaiqin Wu a , Lexun Lin a , Zhonghai Zhang a , Ye Tian c , Fengmin Zhang a a b c
Department of Microbiology, Harbin Medical University, Harbin 150081, Heilongjiang, China Department of Cell Biology, Harbin Medical University, Harbin 150081, Heilongjiang, China Department of Cardiology, Harbin Medical University, Harbin 150081, Heilongjiang, China
a r t i c l e
i n f o
Article history: Received 26 December 2007 Received in revised form 29 March 2008 Accepted 1 April 2008 Available online 2 June 2008 Keywords: Coxsackievirus B1 5 -Untranslated region Mutation Virulence Pathogenesis
a b s t r a c t Two mutants of coxsackievirus B1 (CVB1), CVB1c and CVB1e, with mutations in the stem-loop H of 5 UTR were generated by site-directed mutagenesis. The A at nt579 of CVB1c was substituted by G. The U at nt573 and A at nt579 of CVB1e were substituted by A and G. The virulences of these mutants had been assessed by means of cytopathic effect (CPE), plaque formation, one-step growth curve, and 50% lethal dose (LD50) assays. The pathogenesis of these mutants was evaluated by attacking suckling Balb/c mice. Plaque assay and one-step growth curve showed that the replication of CVB1c and CVB1e on HeLa cells was significantly faster than that of their prototype CVB1n. Data of CPE assay, LD50, and pathological examination showed that CVB1c and CVB1e were more virulent than CVB1n. These data showed that mutation at nt579 (A → G) alone and mutations at nt579 (A → G) and nt573 (U → A) together within 5 UTR caused significant augment of the virulence and pathogenesis of coxsackievirus B1, and suggested that nt573 and nt579 might be molecular determinants for the virulence of coxsackievirus B1. © 2008 Elsevier B.V. All rights reserved.
1. Instruction Coxsackieviruses are members of the Enterovirus genus of the Picornaviridae family. Coxsackievirus group B type 1 (CVB1) is one of the six CVB serotypes. Other members of this genus include coxsackievirus A, poliovirus, and echovirus serotypes. CVB1 has been associated with human cases of pleurodynia, aseptic meningitis, meningoencephalitis, and myocarditis (Bowles et al., 1986; Baboonian et al., 1997). Experimental studies proved that CVB1 can induce diabetes mellitus (Tracy and Drescher, 2007), myocarditis, hepatitis, pancreatitis, and encephalitis in various strains of mice (Minnich and Ray, 1980). CVB genomic RNA is also detectable in myocardial tissues of patients with dilated cardiomyopathy (Satoh et al., 1994; Kuethe et al., 2007). All enteroviruses have a positive-sense RNA genome of approximately 7400 nucleotides (nt) with a similar genetic organization. The viral genome is translated as a polyprotein in one long open
∗ Corresponding author at: Department of Microbiology, Harbin Medical University, No. 194, Xuefu Road, Harbin 150081, Heilongjiang, China. Tel.: +86 451 8668 5122; fax: +86 451 8668 5122. E-mail addresses: [email protected], [email protected] (Z. Zhong). 0168-1702/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2008.04.012
reading frame (Racaniello, 2001). The first 650 nts of enterovirus genome forms a lengthy 5 -untranslated region (5 -UTR) with a highly ordered secondary structure, which is believed to be the internal ribosome entry site (IRES) for cellular ribosome (Jang et al., 1988, 1989), and plays an important role in controlling viral replication and translation (Ishii et al., 1999). Researches showed there are some sequence elements in the IRES are important for the virulence and pathogenesis of CVB (Yang et al., 1997; Liu et al., 1999). Replacement of the whole 5 -UTR (Rinehart et al., 1997; Dunn et al., 2000) or even substitution of nucleotides (M’hadheb-Gharbi et al., 2007) can significantly affect the virulence of CVB. An artificial mutation study showed that the core sequence of the IRES was located at nts 432–639 (Liu et al., 1999). CVB3 viral infectivity could be abolished and viral protein translation was also undetectable when the stem-loops G and H in the IRES were deleted. A 14-nts pyrimidine-rich tract between stem-loop G and stem-loop H (nts 563–576) is essential for the viral translation and infectivity (Liu et al., 1999). This finding suggested that stem-loops G and H were the crucial sequences of IRES and important for the ribosomal internal initiation of translation and infectivity of coxsackieviruses. Previously, Archard et al. (1998) reported that there were two mutations located in the stem-loop H and the pyrimidine-rich tract between stem-loops G and H were frequently detected in the CVB
256
Z. Zhong et al. / Virus Research 135 (2008) 255–259
digested with PstI and ClaI. The PstI-ClaI fragments of amplification products were ligated into the PstI-ClaI site of pNI4. Two plasmids, pCVB1c and pCVB1e, were generated and confirmed by restricted digestions and sequencing. 2.3. In vitro transfection and virus purification
Fig. 1. The putative secondary structure of CVB1 5 -UTR and the target mutations. The nucleotide location refers to GenBank accession no. NC 001472. The closed nucleotides located at nts 563–573 is the pyrimidine-rich tract. The closed nucleotides from nt591 to nt593 is a conserved AUG approximately 150 nts upstream of the initiation codon AUG. The A at nt579 was substituted by G in mutant CVB1c. The U at nt573 and the A at nt579 were substituted by A and G, respectively, in mutant CVB1e.
HeLa cells in 6-well plate were transfected with 0.75 g of pCVB1c, pCVB1e, and pNI4 with Tfx-50 reagent (1:4 ratio) (Promega), respectively. After incubation for 5–7 days, the transfected cells which showed typical cytopathic effects were frozen and thawed three times. The recovered viruses were purified with plaque forming assay. The purified viruses were designated as CVB1c, CVB1e, and CVB1n. The total RNAs were extracted from HeLa cells infected with CVB1c, CVB1e, and CVB1n, repectively, by TRIzol reagent (Invitrogen). 5 ng of total RNAs were reverse trancripted with primer P2 and ThermoScript reverse trancriptase (Invitrogen). The 5 -UTR DNAs were amplified with Platinum Taq DNA polymerase high fidelity (Invitrogen) and primers (P1, P2) from the cDNAs generated before and then sequensed (Applied Biosystems 377). The MFOLD, a web-based RNA secondary structrural analysis program, was used to analyze the 5 -UTR structures. 2.4. Virus infectivity assays
genomic RNA obtained from myocardial tissues of patients with dilated cardiomyopathy. The A at nt579 (A579) was substituted by G, and the U at nt573 (U573) was substituted by A. These mutations added an AUG triplet downstream of the pyrimidine-rech tract. To identify the roles of these mutations on the phenotypes of CVB, two CVB1 mutants with these mutations were developed by site-directed mutagenesis, and the replication and virulence of these mutants had been tested. Our data showed that these mutations significantly increased the virulence of CVB1, and nt573 and nt579 might be the molecular determinant for the virulence and pathogenesis of CVB1. 2. Materials and methods 2.1. Cell HeLa cells were used for transfection, virus propagation, and virulence determination. 2.2. Construction of CVB1 mutants with 5 -UTR mutations Plasmid pNI4, a pUC18 which carried a full-length cDNA of CVB1 genome (GenBank accession no. NC 001472), was kindly offered by Dr. Narushi Iizuka, Tokyo Metropolitan Institute of Medical Science, Japan (Iizuka et al., 1991). PCR primers and site-directed mutagenesis primers were designed to substitute one or two bases at the stem-loop H of CVB1 5 -UTR (Fig. 1). The primers and target mutations refer to Table 1. Plasmid pNI4 was lineared by EcoRV digestion and PCR amplified with primers Pc-P2 or Pe-P2. The amplifed products were purified and used as downstream primers for further amplification of CVB1 5 -UTR with primer P1. The final amplifed products and pNI4 were
MTT (thiazolyl blue tetrazolium bromide) assay, plaque forming assay, and one-step growth curve were used to evaluate the virulences of the newly generated CVB1 mutants. 2.4.1. MTT assay Confluent HeLa cells cultured in 96-well plate were infected with 100 multiplicity of infection (MOI) of CVB1c, CVBe, and CVB1n, respectively (6 wells for each strain) and cultured for 72 h (37 ◦ C, 5% CO2 ). 25 l of MTT (5 mg/ml in PBS) was added to each well and incubated for 4 h. The optical density at 490 nm (OD490) was measured after mixing with 150 l of dimethyl sulfoxide (DMSO) for 10 min. 2.4.2. Plaque forming assay The plaque assay was tested on HeLa cells as previously described (La Monica et al., 1986). Briefly, confluent HeLa cells cultured in 6-well plate were incubated with 25 PFU/well of CVB1c, CVBe, and CVB1n, respectively (three plates for each strain) for 1 h (37 ◦ C, 5% CO2 ). After removing the medium, the infected cells were covered with 0.8% agarose and cultured for 34, 46, and 58 h (37 ◦ C, 5% CO2 ) (one plate for each timepoint). Finally the cells were stained with 0.05% neutral red and the numbers and diameters of the plaques were measured. 2.4.3. One-step growth curve The one-step growth curves for these CVB1 strains on HeLa cells were performed with 100 PFU of each virus as previously described (Jang et al., 1989; Kong et al., 1994). Briefly, confluent HeLa cells cultured in 6-well plate were incubated with 100 PFU/well of CVB1c, CVBe, and CVB1n, respectively for 3, 5, and 7 h (37 ◦ C, 5% CO2 , two wells for each timepoint). After washing with 0.1 ml of PBS, the cells
Table 1 The primers used for site-directed mutagenesis of CVB1 5 -UTR Primer
Sequence (mutations showed as bold, underlined letters)
Location or target mutation
P1, sense P2, anti-sense Pc, mutagenesis Pe, mutagenesis
5 -GAGAAAACGTTCGTTACCCGGC-3 5 -CACACTCTTCGGCAGAGGGGG-3 5 -ATTTTATTCCTATGCTGGCTGCTTATG-3 5 -GTTTCATTTTATACCTATGCTGGCTGCTTAT-3
nts 219–241 nts 950–970 A579 → G579 T573 → A573, A579 → G579
Z. Zhong et al. / Virus Research 135 (2008) 255–259
257
in paraffin, and 5-m sections were stained with hematoxylin and eosin for histopathological examination. 2.6. Statistical analysis Data were expressed as mean ± standard deviation. The statistical significance was evaluated with one-way ANOVA by SigmapStat 3.1 (Systat Software Inc.). 3. Results 3.1. Sequences of CVB1 5 -UTRs Sequencing showed that the 5 -UTR sequences of the newly generated CVB1c and CVB1e were identical to our design (Fig. 2). The A579 of CVB1c was replaced by G. Coincidentally, this substitution added an AUG triplet between the pyrimidine-rich tract and the AUG at nts 591–593. The T573 and A579 of CVB1e were replaced by A and G. The sequence of CVB1n was identical to that of pNI4. 3.2. Viral replication Fig. 2. Sequensing results showed that the mutations in the recovered progeny CVB1c and CVB1e were identical to that of the mutagesis in the cloned genomic cDNA of CVB1. The putative secondary structures were showed on the right side. Mutation in CVB1c did not change the secondary structure of stem-loop H, however, mutation at nt573 shortened the stem of stem-loop H.
were frozen and thawed three times. The live viral particles were counted by plaque forming assay. The growth curve for each virus was illustrated based on its Log10 (PFU). 2.5. Animal inoculations Newborn (<24-hour-old) Balb/c mice were i.p. inoculated with 0.1 ml of 10-fold dilutions of viruses (n = 4). Four mice were inoculated with 0.1 ml of RPMI1640 as control. The mice were observed for 2 weeks. The death happened within 2 days after inoculation was excluded since it might be caused by inappropriate manipulations. 50% lethal dosage (LD50) was tested with a 50% endpoint method as previously described (Rinehart et al., 1997). Newborn Balb/c mice were i.p. inoculated with 4-fold LD50 of viruses (n = 6). The skeletal muscle and myocardial tissues were collected at the 14th day postinoculation (p.i.) and fixed in 10% neutral buffered formalin. The fixed tissues were then embedded
Plaque forming assay showed that CVB1c produced abundant plaques at 34 h p.i. Only a few of plaques could be observed in CVB1e-inoculated cells. The average diameter of plaques produced by CVB1c was significantly larger than that by CVB1e (P < 0.05). No plaque could be found in HeLa cells inoculated with CVB1n at 34 h p.i. (Fig. 3). However, all tested viruses produced plaques at 46 h p.i. The amount and average diameter of plaques produced by CVB1c were significantly larger than that by other viruses at 46 h p.i. (P < 0.05). There was no significant difference among the plaque diameters of CVB1e and CVB1n (P > 0.05), though the amount of plaques produced by CVB1e was significantly larger than that by CVB1n (P < 0.05). At 58 h p.i., both CVB1e and CVB1n produced numerous plaques, but no increase of plaque amount could be observed in CVB1c-inoculated cells. The plaque diameters of all tested viruses at 58 h p.i. were significantly larger than that at 34 and 46 h p.i. (P < 0.05). This suggested that CVB1c has reached its maximum of plaque forming at 46 h p.i. One-step growth curve (Fig. 4) showed that there was a significant difference between the Log10 (PFU/ml) of CVB1c and that of CVB1n at 7 h p.i. (P < 0.05), and also between the Log10(PFU/ml) of CVB1c and that of CVB1n at 7 h p.i. (P < 0.05), though there were no significant difference at 3 and 5 h. It suggested that CVB1c and CVB1e replicated faster than their prototype CVB1n.
Fig. 3. The plaque forming in HeLa cells inoculated with CVB1c, CVB1e, and CVB1n. Confluent HeLa cells were incubated with 25 PFU/well of CVB1c, CVBe, and CVB1n, respectively for 1 h at 37 ◦ C. After removing the medium, the cells were covered with 0.8% agarose and cultured for 34, 46, and 58 h (37 ◦ C, 5% CO2 ). The cells were stained with 0.05% neutral red, and the numbers and sizes of the plaques were measured. (A) plaque counts and (B) plaque sizes.
258
Z. Zhong et al. / Virus Research 135 (2008) 255–259
Fig. 4. One-step growth curve of CVB1 viruses in HeLa cells. Confluent HeLa cells cultured in 6-well plate were incubated with 100 PFU/well of CVB1c, CVBe, and CVB1n, respectively for 3, 5, and 7 h (37 ◦ C, 5% CO2 ). After washing with 0.1 ml of PBS, the cells were frozen and thawed. The live viral particles were counted by plaque forming assay. *P<0.05 compared between CVB1c and CVB1n, and between CVB1e and CVB1n.
Fig. 6. The LD50s of CVB1c, CVB1e, and CVB1n for Balb/c mice. Newborn Balb/c mice were i.p. inoculated with 0.1 ml of 10-fold dilutions of viruses (n = 4). Four mice were inoculated with 0.1 ml of RPMI1640 as control. The mice were observed for 2 weeks. 50% lethal dosage (LD50) was tested with a 50% endpoint method.
4. Discussion 3.3. Virulence of CVB1 strains MTT assay showed that the OD490s of CVB1c, CVB1e, CVB1n, and normal control were 0.602 ± 0.128, 0.710 ± 0.074, 0.812 ± 0.092, and 1.359 ± 0.109, respectively. The virulences of CVB1c and CVB1e were significantly higher than that of CVB1n (P < 0.05). There was no significant difference between the virulences of CVB1c and CVB1e (P > 0.05) (Fig. 5). The LD50s of CVB1c, CVB1e, and CVB1n for Balb/c mice were 1.89 × 103 PFU/ml, 1.8 × 103 PFU/ml, and 1.26 × 104 PFU/ml, respectively (Fig. 6), suggested that CVB1c and CVB1e were more virulent than CVB1n. Pathological examination on the skeletal muscle and myocardial tissues collected at the 14th day p.i. showed that there was no pathological sign could be found in Balb/c mice inoculated with RPMI1640. Inflammation and necrosis could be found in the skeletal muscle tissues of Balb/c mice inoculated with various CVB1 strains. Lymphocyte infiltration could also be observed in the myocardial tissues of the mice inoculated with all strains. However, necrosis and vacuolar degeneration could only be observed in the myocardial tissues of the mice inoculated with CVB1c and CVB1e. These data indicated that the cardiovirulence of CVB1n was weaker than that of CVB1c and CVB1e.
Fig. 5. Virulence comparison of CVB1 mutants inoculated on HeLa cells. Confluent HeLa cells cultured in 96-well plate were infected with 100 MOI of CVB1c, CVBe, and CVB1n, respectively. The inoculated cells were cultured for 72 h (37 ◦ C, 5% CO2 ). 25 l of MTT (5 mg/ml in PBS) was added to each well and incubated for 4 h. The optical density at 490 nm (OD490) was measured after mixing with 150 l of DMSO for 10 min. NC represents normal control. *Compared with CVB1n, P < 0.05.
Coxsackievirus B is the most common infectious cause of human acute myocarditis. 45% of symptomatic patients with acute myocarditis or its longterm sequela, dilated cardiomyopathy (DCM), had detectable coxsackievirus RNA in myocardial biopsy specimens, compared with none of the controls (Whitton et al., 2005). CVB1n is a low cardiovirulent CVB strain. A substitution of the whole 5 -UTR and part of the P1 gene of CVB1n by the corresponding sequence of the myotropic CVB1 (Tucson strain) could augment more than 90% of its virulence (Rinehart et al., 1997), and indicated that there are molecular determinants in 5 -UTR for the virulence of CVB1. Study proved that the stem-loop H and the pyrimidine-rich tract between stem-loop G and H within 5 UTR were essential elements for the internal ribosomal binding of CVB, therefore these sequences were crucial for the replication and translation of CVB (Liu et al., 1999). Previously Archard et al. (1998) reported there were frequent mutations at nt573 and nt579 of CVB 5 -UTR amplified from the myocardial tissues with dilated cardiomyopathy. Since these mutations located at stemloop H (Fig. 1) and were essential sequence of 5 -UTR of CVB, we suspected that these mutations might affect the phenotypes, such as viral replication and virulence, of CVB. To determine whether these mutations affect the virulence of CVB or not, two artificial mutants which simulated the natural mutations in a cloned CVB1 genome were developed by site-directed mutagenesis. The A579 of CVB1c had been substituted by G. The U573 and A579 of CVB1e had been subtituted by A and G. Plaque assay showed that the amounts and diameters of plaques produced by CVB1c and CVB1e on HeLa cells were significantly larger than that of CVB1n. One-step growth curve showed that CVB1c and CVB1e replicated faster than CVB1n. Both suggested that these mutations had speeded up the replications of CVB1c and CVB1e. Data of CPE assay showed that CVB1c and CVB1e had increased cytopathic abilities on HeLa cells compaired with their prototype CVB1n. The LD50s of CVB1c and CVB1e were significantly lower than that of CVB1n. Both suggested that CVB1c and CVB1e were more virulent than CVB1n. Histological examination at the 14th day p.i. showed all strains caused inflammation and necrosis in the skeletal muscles of Balb/c mice, and lymphocyte infiltration could also be observed in the myocardial tissues of the mice inoculated with all strains, but necrosis and vacuolar degeneration could only be
Z. Zhong et al. / Virus Research 135 (2008) 255–259
observed in the myocardial tissues of the mice inoculated with CVB1c and CVB1e. Our data suggested that mutation at nt579 (A → G) alone and mutations at nt579 (A → G) and nt573 (U → A) together within 5 -UTR augmented the cardiovirulence of CVB1n. The increased cardiovirulence of CVB1 might contribute to its pathogenesis of myocarditis and dilated cardiomyopathy. More in vivo experimental evidences are needed to confirm this hypothesis. Based on the putative secondary structure, the G579 located at the loop region of stem-loop H. This mutation did not change the shape of stem-loop H. Why this substitution can cause a significant deviation of replication and virulence phenotypes of CVB1 is unknown. Previously studies showed that there is a conserved AUG triplet at nts 591–593 of CVB and the corresponding locations of other picornaviruses. This AUG triplet can not be used as an initiation codon for picornaviruses (Meerovitch et al., 1991; Brown et al., 1994), but it works as part of the ribosome-landing site and can cause the augment of translation of poliovirus 2 (Meerovitch et al., 1991). Introducing an AUG triplet at the corresponding location in the IRES of eIF4G mRNA can markedly decrease its translation efficiency (Gan et al., 1998). A deletion of 46-nt segment containing the pyrimidine-rich tract and this AUG triplet can lead to a complete lost of IRES activity of CVB3 (Liu et al., 1999). These data suggested that the AUG triplet at nts 591–593 may play an important role on the ribosome binding and replication of CVB. Interestingly, the substitution of A579 → G actually added another AUG triplet (nts 577–579) located at approximately 10-nt upstream of the AUG triplet at nts 591–593 in CVB1c and CVB1e. Furthermore, the new AUG triplet did match the open reading frame of CVB1 and provided a possible new initiation codon for the polypeptide translation of CVB1c and CVB1e. It needs more experiments to identify the role of the new AUG triplet. The substitution of U573 → A shortened the length of stem-loop H due to base mismatch, and replaced one uracil of the pyrimidinerich tract with adenine (Fig. 2). Our data showed this mutation had countered the augement effects of G579 on the replication and translation of CVB1, though the virulence of CVB1e was still much higher than that of CVB1n. This result further confirmed the crucial role of the pyrimidine-rich tract at nts 563–573 for the replication and virulence of CVB1. In sum, our data indicated that nt579 and nt573 in the stemloop H of 5 -UTR might be molecular determinants for the virulence and pathogenesis of CVB1. Mutations on these locations can significantly affect the virulence of CVB1. Acknowledgements We thank Dr. Narushi Iizuka, Department of Microbiology, Tokyo Metropolitan Institute of Medical Science, Japan for kindly providing us the cDNA of CVB1n. This study has been supported by grants from Heilongjiang Provincial Fund (No. LC01C05, No. 11511159), and Harbin Municipal Fund (No. 2005AA9CS116-16), China.
259
References Archard, L.C., Khan, M.A., Soteriou, B.A., Zhang, H., Why, H.J., Robinson, N.M., Richardson, P.J., 1998. Characterization of Coxsackie B virus RNA in myocardium from patients with dilated cardiomyopathy by nucleotide sequencing of reverse transcription-nested polymerase chain reaction products. Hum. Pathol. 29, 578–584. Baboonian, C., Davies, M.J., Booth, J.C., McKenna, W.J., 1997. Coxsackie B viruses and human heart disease. Curr. Top. Microbiol. Immunol. 223, 31–52. Bowles, N.E., Richardson, P.J., Olsen, E.G., Archard, L.C., 1986. Detection of CoxsackieB-virus-specific RNA sequences in myocardial biopsy samples from patients with myocarditis and dilated cardiomyopathy. Lancet 1, 1120–1123. Brown, E.A., Zajac, A.J., Lemon, S.M., 1994. In vitro characterization of an internal ribosomal entry site (IRES) present within the 5 nontranslated region of hepatitis A virus RNA: comparison with the IRES of encephalomyocarditis virus. J. Virol. 68, 1066–1074. Dunn, J.J., Chapman, N.M., Tracy, S., Romero, J.R., 2000. Genomic determinants of cardiovirulence in coxsackievirus B3 clinical isolates: localization to the 5 nontranslated region. J. Virol. 74, 4787–4794. Gan, W., LaCelle, M., Rhoads, R.E., 1998. Functional characterization of the internal ribosome entry site of eIF4G mRNA. J. Biol. Chem. 273, 5006–5012. Iizuka, N., Yonekawa, H., Nomoto, A., 1991. Nucleotide sequences important for translation initiation of enterovirus RNA. J. Virol. 65, 4867–4873. Ishii, T., Shiroki, K., Iwai, A., Nomoto, A., 1999. Identification of a new element for RNA replication within the internal ribosome entry site of poliovirus RNA. J. Gen. Virol. 80, 917–920. Jang, S.K., Davies, M.V., Kaufman, R.J., Wimmer, E., 1989. Initiation of protein synthesis by internal entry of ribosomes into the 5 nontranslated region of encephalomyocarditis virus RNA in vivo. J. Virol. 63, 1651–1660. ¨ Jang, S.K., Krausslich, H.G., Nicklin, M.J., Duke, G.M., Palmenberg, A.C., Wimmer, E., 1988. A segment of the 5 nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J. Virol. 62, 2636–2643. Kong, W.P., Ghadge, G.D., Roos, R.P., 1994. Involvement of cardiovirus leader in host cell-restricted virus expression. Proc. Natl. Acad. Sci. U.S.A. 91, 1796–1800. ¨ Kuethe, F., Sigusch, H.H., Hilbig, K., Tresselt, C., Gluck, B., Egerer, R., Figulla, H.R., 2007. Detection of viral genome in the myocardium: lack of prognostic and functional relevance in patients with acute dilated cardiomyopathy. Am. Heart J. 153, 850–858. La Monica, N., Meriam, C., Racaniello, V.R., 1986. Mapping of sequences required for mouse neurovirulence of poliovirus type 2 Lansing. J. Virol. 57, 515–525. Liu, Z., Carthy, C.M., Cheung, P., Bohunek, L., Wilson, J.E., McManus, B.M., Yang, D., 1999. Structural and functional analysis of the 5 untranslated region of coxsackievirus B3 RNA: In vivo translational and infectivity studies of full-length mutants. Virology 265, 206–217. Meerovitch, K., Nicholson, R., Sonenberg, N., 1991. In vitro mutational analysis of cisacting RNA translational elements within the poliovirus type 2 5 untranslated region. J. Virol. 65, 5895–5901. M’hadheb-Gharbi, M.B., Paulous, S., Aouni, M., Kean, K.M., Gharbi, J., 2007. The substitution U475 → C with Sabin3-like mutation within the IRES attenuate Coxsackievirus B3 cardiovirulence. Mol. Biotechnol. 36, 52–60. Minnich, L.L., Ray, C.G., 1980. Variable susceptibility of mice to group B coxsackievirus infections. J. Clin. Microbiol. 11, 73–75. Racaniello, V.R., 2001. Picornaviridae: the virus and their replication. In: Fields, B.N., Knipe, D.M., Howley, P.M., Griffin, D.E. (Eds.), Fields Virology, Fourth ed. Lippincott Williams & Williams, Philadelphia, pp. 685–722. ´ Rinehart, J.E., Gomez, R.M., Roos, R.P., 1997. Molecular determinants for virulence in coxsackievirus B1 infection. J. Virol. 71, 3986–3991. Satoh, M., Tamura, G., Segawa, I., Hiramori, K., Satodate, R., 1994. Enteroviral RNA in dilated cardiomyopathy. Eur. Heart J. 15, 934–939. Tracy, S., Drescher, K.M., 2007. Coxsackievirus infections and NOD mice: relevant models of protection from, and induction of, type 1 diabetes. Ann. NY Acad. Sci. 1103, 143–151. Whitton, J.L., Cornell, C.T., Feuer, R., 2005. Host and virus determinants of picornavirus pathogenesis and tropism. Nat. Rev. Microbiol. 3, 765–776. Yang, D., Wilson, J.E., Anderson, D.R., Bohunek, L., Cordeiro, C., Kandolf, R., McManus, B.M., 1997. In vitro mutational and inhibitory analysis of the cis-acting translational elements within the 5 untranslated region of coxsackievirus B3: potential targets for antiviral action of antisense oligomers. Virology 228, 63–73.
Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Mutations at nucleotides 573 and 579 within 5 -untranslated region augment the virulence of coxsackievirus B1 Zhaohua Zhong a,∗ , Xiaobo Li a , Wenran Zhao b , Lei Tong a , Jian Liu c , Shuaiqin Wu a , Lexun Lin a , Zhonghai Zhang a , Ye Tian c , Fengmin Zhang a a b c
Department of Microbiology, Harbin Medical University, Harbin 150081, Heilongjiang, China Department of Cell Biology, Harbin Medical University, Harbin 150081, Heilongjiang, China Department of Cardiology, Harbin Medical University, Harbin 150081, Heilongjiang, China
a r t i c l e
i n f o
Article history: Received 26 December 2007 Received in revised form 29 March 2008 Accepted 1 April 2008 Available online 2 June 2008 Keywords: Coxsackievirus B1 5 -Untranslated region Mutation Virulence Pathogenesis
a b s t r a c t Two mutants of coxsackievirus B1 (CVB1), CVB1c and CVB1e, with mutations in the stem-loop H of 5 UTR were generated by site-directed mutagenesis. The A at nt579 of CVB1c was substituted by G. The U at nt573 and A at nt579 of CVB1e were substituted by A and G. The virulences of these mutants had been assessed by means of cytopathic effect (CPE), plaque formation, one-step growth curve, and 50% lethal dose (LD50) assays. The pathogenesis of these mutants was evaluated by attacking suckling Balb/c mice. Plaque assay and one-step growth curve showed that the replication of CVB1c and CVB1e on HeLa cells was significantly faster than that of their prototype CVB1n. Data of CPE assay, LD50, and pathological examination showed that CVB1c and CVB1e were more virulent than CVB1n. These data showed that mutation at nt579 (A → G) alone and mutations at nt579 (A → G) and nt573 (U → A) together within 5 UTR caused significant augment of the virulence and pathogenesis of coxsackievirus B1, and suggested that nt573 and nt579 might be molecular determinants for the virulence of coxsackievirus B1. © 2008 Elsevier B.V. All rights reserved.
1. Instruction Coxsackieviruses are members of the Enterovirus genus of the Picornaviridae family. Coxsackievirus group B type 1 (CVB1) is one of the six CVB serotypes. Other members of this genus include coxsackievirus A, poliovirus, and echovirus serotypes. CVB1 has been associated with human cases of pleurodynia, aseptic meningitis, meningoencephalitis, and myocarditis (Bowles et al., 1986; Baboonian et al., 1997). Experimental studies proved that CVB1 can induce diabetes mellitus (Tracy and Drescher, 2007), myocarditis, hepatitis, pancreatitis, and encephalitis in various strains of mice (Minnich and Ray, 1980). CVB genomic RNA is also detectable in myocardial tissues of patients with dilated cardiomyopathy (Satoh et al., 1994; Kuethe et al., 2007). All enteroviruses have a positive-sense RNA genome of approximately 7400 nucleotides (nt) with a similar genetic organization. The viral genome is translated as a polyprotein in one long open
∗ Corresponding author at: Department of Microbiology, Harbin Medical University, No. 194, Xuefu Road, Harbin 150081, Heilongjiang, China. Tel.: +86 451 8668 5122; fax: +86 451 8668 5122. E-mail addresses: [email protected], [email protected] (Z. Zhong). 0168-1702/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2008.04.012
reading frame (Racaniello, 2001). The first 650 nts of enterovirus genome forms a lengthy 5 -untranslated region (5 -UTR) with a highly ordered secondary structure, which is believed to be the internal ribosome entry site (IRES) for cellular ribosome (Jang et al., 1988, 1989), and plays an important role in controlling viral replication and translation (Ishii et al., 1999). Researches showed there are some sequence elements in the IRES are important for the virulence and pathogenesis of CVB (Yang et al., 1997; Liu et al., 1999). Replacement of the whole 5 -UTR (Rinehart et al., 1997; Dunn et al., 2000) or even substitution of nucleotides (M’hadheb-Gharbi et al., 2007) can significantly affect the virulence of CVB. An artificial mutation study showed that the core sequence of the IRES was located at nts 432–639 (Liu et al., 1999). CVB3 viral infectivity could be abolished and viral protein translation was also undetectable when the stem-loops G and H in the IRES were deleted. A 14-nts pyrimidine-rich tract between stem-loop G and stem-loop H (nts 563–576) is essential for the viral translation and infectivity (Liu et al., 1999). This finding suggested that stem-loops G and H were the crucial sequences of IRES and important for the ribosomal internal initiation of translation and infectivity of coxsackieviruses. Previously, Archard et al. (1998) reported that there were two mutations located in the stem-loop H and the pyrimidine-rich tract between stem-loops G and H were frequently detected in the CVB
256
Z. Zhong et al. / Virus Research 135 (2008) 255–259
digested with PstI and ClaI. The PstI-ClaI fragments of amplification products were ligated into the PstI-ClaI site of pNI4. Two plasmids, pCVB1c and pCVB1e, were generated and confirmed by restricted digestions and sequencing. 2.3. In vitro transfection and virus purification
Fig. 1. The putative secondary structure of CVB1 5 -UTR and the target mutations. The nucleotide location refers to GenBank accession no. NC 001472. The closed nucleotides located at nts 563–573 is the pyrimidine-rich tract. The closed nucleotides from nt591 to nt593 is a conserved AUG approximately 150 nts upstream of the initiation codon AUG. The A at nt579 was substituted by G in mutant CVB1c. The U at nt573 and the A at nt579 were substituted by A and G, respectively, in mutant CVB1e.
HeLa cells in 6-well plate were transfected with 0.75 g of pCVB1c, pCVB1e, and pNI4 with Tfx-50 reagent (1:4 ratio) (Promega), respectively. After incubation for 5–7 days, the transfected cells which showed typical cytopathic effects were frozen and thawed three times. The recovered viruses were purified with plaque forming assay. The purified viruses were designated as CVB1c, CVB1e, and CVB1n. The total RNAs were extracted from HeLa cells infected with CVB1c, CVB1e, and CVB1n, repectively, by TRIzol reagent (Invitrogen). 5 ng of total RNAs were reverse trancripted with primer P2 and ThermoScript reverse trancriptase (Invitrogen). The 5 -UTR DNAs were amplified with Platinum Taq DNA polymerase high fidelity (Invitrogen) and primers (P1, P2) from the cDNAs generated before and then sequensed (Applied Biosystems 377). The MFOLD, a web-based RNA secondary structrural analysis program, was used to analyze the 5 -UTR structures. 2.4. Virus infectivity assays
genomic RNA obtained from myocardial tissues of patients with dilated cardiomyopathy. The A at nt579 (A579) was substituted by G, and the U at nt573 (U573) was substituted by A. These mutations added an AUG triplet downstream of the pyrimidine-rech tract. To identify the roles of these mutations on the phenotypes of CVB, two CVB1 mutants with these mutations were developed by site-directed mutagenesis, and the replication and virulence of these mutants had been tested. Our data showed that these mutations significantly increased the virulence of CVB1, and nt573 and nt579 might be the molecular determinant for the virulence and pathogenesis of CVB1. 2. Materials and methods 2.1. Cell HeLa cells were used for transfection, virus propagation, and virulence determination. 2.2. Construction of CVB1 mutants with 5 -UTR mutations Plasmid pNI4, a pUC18 which carried a full-length cDNA of CVB1 genome (GenBank accession no. NC 001472), was kindly offered by Dr. Narushi Iizuka, Tokyo Metropolitan Institute of Medical Science, Japan (Iizuka et al., 1991). PCR primers and site-directed mutagenesis primers were designed to substitute one or two bases at the stem-loop H of CVB1 5 -UTR (Fig. 1). The primers and target mutations refer to Table 1. Plasmid pNI4 was lineared by EcoRV digestion and PCR amplified with primers Pc-P2 or Pe-P2. The amplifed products were purified and used as downstream primers for further amplification of CVB1 5 -UTR with primer P1. The final amplifed products and pNI4 were
MTT (thiazolyl blue tetrazolium bromide) assay, plaque forming assay, and one-step growth curve were used to evaluate the virulences of the newly generated CVB1 mutants. 2.4.1. MTT assay Confluent HeLa cells cultured in 96-well plate were infected with 100 multiplicity of infection (MOI) of CVB1c, CVBe, and CVB1n, respectively (6 wells for each strain) and cultured for 72 h (37 ◦ C, 5% CO2 ). 25 l of MTT (5 mg/ml in PBS) was added to each well and incubated for 4 h. The optical density at 490 nm (OD490) was measured after mixing with 150 l of dimethyl sulfoxide (DMSO) for 10 min. 2.4.2. Plaque forming assay The plaque assay was tested on HeLa cells as previously described (La Monica et al., 1986). Briefly, confluent HeLa cells cultured in 6-well plate were incubated with 25 PFU/well of CVB1c, CVBe, and CVB1n, respectively (three plates for each strain) for 1 h (37 ◦ C, 5% CO2 ). After removing the medium, the infected cells were covered with 0.8% agarose and cultured for 34, 46, and 58 h (37 ◦ C, 5% CO2 ) (one plate for each timepoint). Finally the cells were stained with 0.05% neutral red and the numbers and diameters of the plaques were measured. 2.4.3. One-step growth curve The one-step growth curves for these CVB1 strains on HeLa cells were performed with 100 PFU of each virus as previously described (Jang et al., 1989; Kong et al., 1994). Briefly, confluent HeLa cells cultured in 6-well plate were incubated with 100 PFU/well of CVB1c, CVBe, and CVB1n, respectively for 3, 5, and 7 h (37 ◦ C, 5% CO2 , two wells for each timepoint). After washing with 0.1 ml of PBS, the cells
Table 1 The primers used for site-directed mutagenesis of CVB1 5 -UTR Primer
Sequence (mutations showed as bold, underlined letters)
Location or target mutation
P1, sense P2, anti-sense Pc, mutagenesis Pe, mutagenesis
5 -GAGAAAACGTTCGTTACCCGGC-3 5 -CACACTCTTCGGCAGAGGGGG-3 5 -ATTTTATTCCTATGCTGGCTGCTTATG-3 5 -GTTTCATTTTATACCTATGCTGGCTGCTTAT-3
nts 219–241 nts 950–970 A579 → G579 T573 → A573, A579 → G579
Z. Zhong et al. / Virus Research 135 (2008) 255–259
257
in paraffin, and 5-m sections were stained with hematoxylin and eosin for histopathological examination. 2.6. Statistical analysis Data were expressed as mean ± standard deviation. The statistical significance was evaluated with one-way ANOVA by SigmapStat 3.1 (Systat Software Inc.). 3. Results 3.1. Sequences of CVB1 5 -UTRs Sequencing showed that the 5 -UTR sequences of the newly generated CVB1c and CVB1e were identical to our design (Fig. 2). The A579 of CVB1c was replaced by G. Coincidentally, this substitution added an AUG triplet between the pyrimidine-rich tract and the AUG at nts 591–593. The T573 and A579 of CVB1e were replaced by A and G. The sequence of CVB1n was identical to that of pNI4. 3.2. Viral replication Fig. 2. Sequensing results showed that the mutations in the recovered progeny CVB1c and CVB1e were identical to that of the mutagesis in the cloned genomic cDNA of CVB1. The putative secondary structures were showed on the right side. Mutation in CVB1c did not change the secondary structure of stem-loop H, however, mutation at nt573 shortened the stem of stem-loop H.
were frozen and thawed three times. The live viral particles were counted by plaque forming assay. The growth curve for each virus was illustrated based on its Log10 (PFU). 2.5. Animal inoculations Newborn (<24-hour-old) Balb/c mice were i.p. inoculated with 0.1 ml of 10-fold dilutions of viruses (n = 4). Four mice were inoculated with 0.1 ml of RPMI1640 as control. The mice were observed for 2 weeks. The death happened within 2 days after inoculation was excluded since it might be caused by inappropriate manipulations. 50% lethal dosage (LD50) was tested with a 50% endpoint method as previously described (Rinehart et al., 1997). Newborn Balb/c mice were i.p. inoculated with 4-fold LD50 of viruses (n = 6). The skeletal muscle and myocardial tissues were collected at the 14th day postinoculation (p.i.) and fixed in 10% neutral buffered formalin. The fixed tissues were then embedded
Plaque forming assay showed that CVB1c produced abundant plaques at 34 h p.i. Only a few of plaques could be observed in CVB1e-inoculated cells. The average diameter of plaques produced by CVB1c was significantly larger than that by CVB1e (P < 0.05). No plaque could be found in HeLa cells inoculated with CVB1n at 34 h p.i. (Fig. 3). However, all tested viruses produced plaques at 46 h p.i. The amount and average diameter of plaques produced by CVB1c were significantly larger than that by other viruses at 46 h p.i. (P < 0.05). There was no significant difference among the plaque diameters of CVB1e and CVB1n (P > 0.05), though the amount of plaques produced by CVB1e was significantly larger than that by CVB1n (P < 0.05). At 58 h p.i., both CVB1e and CVB1n produced numerous plaques, but no increase of plaque amount could be observed in CVB1c-inoculated cells. The plaque diameters of all tested viruses at 58 h p.i. were significantly larger than that at 34 and 46 h p.i. (P < 0.05). This suggested that CVB1c has reached its maximum of plaque forming at 46 h p.i. One-step growth curve (Fig. 4) showed that there was a significant difference between the Log10 (PFU/ml) of CVB1c and that of CVB1n at 7 h p.i. (P < 0.05), and also between the Log10(PFU/ml) of CVB1c and that of CVB1n at 7 h p.i. (P < 0.05), though there were no significant difference at 3 and 5 h. It suggested that CVB1c and CVB1e replicated faster than their prototype CVB1n.
Fig. 3. The plaque forming in HeLa cells inoculated with CVB1c, CVB1e, and CVB1n. Confluent HeLa cells were incubated with 25 PFU/well of CVB1c, CVBe, and CVB1n, respectively for 1 h at 37 ◦ C. After removing the medium, the cells were covered with 0.8% agarose and cultured for 34, 46, and 58 h (37 ◦ C, 5% CO2 ). The cells were stained with 0.05% neutral red, and the numbers and sizes of the plaques were measured. (A) plaque counts and (B) plaque sizes.
258
Z. Zhong et al. / Virus Research 135 (2008) 255–259
Fig. 4. One-step growth curve of CVB1 viruses in HeLa cells. Confluent HeLa cells cultured in 6-well plate were incubated with 100 PFU/well of CVB1c, CVBe, and CVB1n, respectively for 3, 5, and 7 h (37 ◦ C, 5% CO2 ). After washing with 0.1 ml of PBS, the cells were frozen and thawed. The live viral particles were counted by plaque forming assay. *P<0.05 compared between CVB1c and CVB1n, and between CVB1e and CVB1n.
Fig. 6. The LD50s of CVB1c, CVB1e, and CVB1n for Balb/c mice. Newborn Balb/c mice were i.p. inoculated with 0.1 ml of 10-fold dilutions of viruses (n = 4). Four mice were inoculated with 0.1 ml of RPMI1640 as control. The mice were observed for 2 weeks. 50% lethal dosage (LD50) was tested with a 50% endpoint method.
4. Discussion 3.3. Virulence of CVB1 strains MTT assay showed that the OD490s of CVB1c, CVB1e, CVB1n, and normal control were 0.602 ± 0.128, 0.710 ± 0.074, 0.812 ± 0.092, and 1.359 ± 0.109, respectively. The virulences of CVB1c and CVB1e were significantly higher than that of CVB1n (P < 0.05). There was no significant difference between the virulences of CVB1c and CVB1e (P > 0.05) (Fig. 5). The LD50s of CVB1c, CVB1e, and CVB1n for Balb/c mice were 1.89 × 103 PFU/ml, 1.8 × 103 PFU/ml, and 1.26 × 104 PFU/ml, respectively (Fig. 6), suggested that CVB1c and CVB1e were more virulent than CVB1n. Pathological examination on the skeletal muscle and myocardial tissues collected at the 14th day p.i. showed that there was no pathological sign could be found in Balb/c mice inoculated with RPMI1640. Inflammation and necrosis could be found in the skeletal muscle tissues of Balb/c mice inoculated with various CVB1 strains. Lymphocyte infiltration could also be observed in the myocardial tissues of the mice inoculated with all strains. However, necrosis and vacuolar degeneration could only be observed in the myocardial tissues of the mice inoculated with CVB1c and CVB1e. These data indicated that the cardiovirulence of CVB1n was weaker than that of CVB1c and CVB1e.
Fig. 5. Virulence comparison of CVB1 mutants inoculated on HeLa cells. Confluent HeLa cells cultured in 96-well plate were infected with 100 MOI of CVB1c, CVBe, and CVB1n, respectively. The inoculated cells were cultured for 72 h (37 ◦ C, 5% CO2 ). 25 l of MTT (5 mg/ml in PBS) was added to each well and incubated for 4 h. The optical density at 490 nm (OD490) was measured after mixing with 150 l of DMSO for 10 min. NC represents normal control. *Compared with CVB1n, P < 0.05.
Coxsackievirus B is the most common infectious cause of human acute myocarditis. 45% of symptomatic patients with acute myocarditis or its longterm sequela, dilated cardiomyopathy (DCM), had detectable coxsackievirus RNA in myocardial biopsy specimens, compared with none of the controls (Whitton et al., 2005). CVB1n is a low cardiovirulent CVB strain. A substitution of the whole 5 -UTR and part of the P1 gene of CVB1n by the corresponding sequence of the myotropic CVB1 (Tucson strain) could augment more than 90% of its virulence (Rinehart et al., 1997), and indicated that there are molecular determinants in 5 -UTR for the virulence of CVB1. Study proved that the stem-loop H and the pyrimidine-rich tract between stem-loop G and H within 5 UTR were essential elements for the internal ribosomal binding of CVB, therefore these sequences were crucial for the replication and translation of CVB (Liu et al., 1999). Previously Archard et al. (1998) reported there were frequent mutations at nt573 and nt579 of CVB 5 -UTR amplified from the myocardial tissues with dilated cardiomyopathy. Since these mutations located at stemloop H (Fig. 1) and were essential sequence of 5 -UTR of CVB, we suspected that these mutations might affect the phenotypes, such as viral replication and virulence, of CVB. To determine whether these mutations affect the virulence of CVB or not, two artificial mutants which simulated the natural mutations in a cloned CVB1 genome were developed by site-directed mutagenesis. The A579 of CVB1c had been substituted by G. The U573 and A579 of CVB1e had been subtituted by A and G. Plaque assay showed that the amounts and diameters of plaques produced by CVB1c and CVB1e on HeLa cells were significantly larger than that of CVB1n. One-step growth curve showed that CVB1c and CVB1e replicated faster than CVB1n. Both suggested that these mutations had speeded up the replications of CVB1c and CVB1e. Data of CPE assay showed that CVB1c and CVB1e had increased cytopathic abilities on HeLa cells compaired with their prototype CVB1n. The LD50s of CVB1c and CVB1e were significantly lower than that of CVB1n. Both suggested that CVB1c and CVB1e were more virulent than CVB1n. Histological examination at the 14th day p.i. showed all strains caused inflammation and necrosis in the skeletal muscles of Balb/c mice, and lymphocyte infiltration could also be observed in the myocardial tissues of the mice inoculated with all strains, but necrosis and vacuolar degeneration could only be
Z. Zhong et al. / Virus Research 135 (2008) 255–259
observed in the myocardial tissues of the mice inoculated with CVB1c and CVB1e. Our data suggested that mutation at nt579 (A → G) alone and mutations at nt579 (A → G) and nt573 (U → A) together within 5 -UTR augmented the cardiovirulence of CVB1n. The increased cardiovirulence of CVB1 might contribute to its pathogenesis of myocarditis and dilated cardiomyopathy. More in vivo experimental evidences are needed to confirm this hypothesis. Based on the putative secondary structure, the G579 located at the loop region of stem-loop H. This mutation did not change the shape of stem-loop H. Why this substitution can cause a significant deviation of replication and virulence phenotypes of CVB1 is unknown. Previously studies showed that there is a conserved AUG triplet at nts 591–593 of CVB and the corresponding locations of other picornaviruses. This AUG triplet can not be used as an initiation codon for picornaviruses (Meerovitch et al., 1991; Brown et al., 1994), but it works as part of the ribosome-landing site and can cause the augment of translation of poliovirus 2 (Meerovitch et al., 1991). Introducing an AUG triplet at the corresponding location in the IRES of eIF4G mRNA can markedly decrease its translation efficiency (Gan et al., 1998). A deletion of 46-nt segment containing the pyrimidine-rich tract and this AUG triplet can lead to a complete lost of IRES activity of CVB3 (Liu et al., 1999). These data suggested that the AUG triplet at nts 591–593 may play an important role on the ribosome binding and replication of CVB. Interestingly, the substitution of A579 → G actually added another AUG triplet (nts 577–579) located at approximately 10-nt upstream of the AUG triplet at nts 591–593 in CVB1c and CVB1e. Furthermore, the new AUG triplet did match the open reading frame of CVB1 and provided a possible new initiation codon for the polypeptide translation of CVB1c and CVB1e. It needs more experiments to identify the role of the new AUG triplet. The substitution of U573 → A shortened the length of stem-loop H due to base mismatch, and replaced one uracil of the pyrimidinerich tract with adenine (Fig. 2). Our data showed this mutation had countered the augement effects of G579 on the replication and translation of CVB1, though the virulence of CVB1e was still much higher than that of CVB1n. This result further confirmed the crucial role of the pyrimidine-rich tract at nts 563–573 for the replication and virulence of CVB1. In sum, our data indicated that nt579 and nt573 in the stemloop H of 5 -UTR might be molecular determinants for the virulence and pathogenesis of CVB1. Mutations on these locations can significantly affect the virulence of CVB1. Acknowledgements We thank Dr. Narushi Iizuka, Department of Microbiology, Tokyo Metropolitan Institute of Medical Science, Japan for kindly providing us the cDNA of CVB1n. This study has been supported by grants from Heilongjiang Provincial Fund (No. LC01C05, No. 11511159), and Harbin Municipal Fund (No. 2005AA9CS116-16), China.
259
References Archard, L.C., Khan, M.A., Soteriou, B.A., Zhang, H., Why, H.J., Robinson, N.M., Richardson, P.J., 1998. Characterization of Coxsackie B virus RNA in myocardium from patients with dilated cardiomyopathy by nucleotide sequencing of reverse transcription-nested polymerase chain reaction products. Hum. Pathol. 29, 578–584. Baboonian, C., Davies, M.J., Booth, J.C., McKenna, W.J., 1997. Coxsackie B viruses and human heart disease. Curr. Top. Microbiol. Immunol. 223, 31–52. Bowles, N.E., Richardson, P.J., Olsen, E.G., Archard, L.C., 1986. Detection of CoxsackieB-virus-specific RNA sequences in myocardial biopsy samples from patients with myocarditis and dilated cardiomyopathy. Lancet 1, 1120–1123. Brown, E.A., Zajac, A.J., Lemon, S.M., 1994. In vitro characterization of an internal ribosomal entry site (IRES) present within the 5 nontranslated region of hepatitis A virus RNA: comparison with the IRES of encephalomyocarditis virus. J. Virol. 68, 1066–1074. Dunn, J.J., Chapman, N.M., Tracy, S., Romero, J.R., 2000. Genomic determinants of cardiovirulence in coxsackievirus B3 clinical isolates: localization to the 5 nontranslated region. J. Virol. 74, 4787–4794. Gan, W., LaCelle, M., Rhoads, R.E., 1998. Functional characterization of the internal ribosome entry site of eIF4G mRNA. J. Biol. Chem. 273, 5006–5012. Iizuka, N., Yonekawa, H., Nomoto, A., 1991. Nucleotide sequences important for translation initiation of enterovirus RNA. J. Virol. 65, 4867–4873. Ishii, T., Shiroki, K., Iwai, A., Nomoto, A., 1999. Identification of a new element for RNA replication within the internal ribosome entry site of poliovirus RNA. J. Gen. Virol. 80, 917–920. Jang, S.K., Davies, M.V., Kaufman, R.J., Wimmer, E., 1989. Initiation of protein synthesis by internal entry of ribosomes into the 5 nontranslated region of encephalomyocarditis virus RNA in vivo. J. Virol. 63, 1651–1660. ¨ Jang, S.K., Krausslich, H.G., Nicklin, M.J., Duke, G.M., Palmenberg, A.C., Wimmer, E., 1988. A segment of the 5 nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J. Virol. 62, 2636–2643. Kong, W.P., Ghadge, G.D., Roos, R.P., 1994. Involvement of cardiovirus leader in host cell-restricted virus expression. Proc. Natl. Acad. Sci. U.S.A. 91, 1796–1800. ¨ Kuethe, F., Sigusch, H.H., Hilbig, K., Tresselt, C., Gluck, B., Egerer, R., Figulla, H.R., 2007. Detection of viral genome in the myocardium: lack of prognostic and functional relevance in patients with acute dilated cardiomyopathy. Am. Heart J. 153, 850–858. La Monica, N., Meriam, C., Racaniello, V.R., 1986. Mapping of sequences required for mouse neurovirulence of poliovirus type 2 Lansing. J. Virol. 57, 515–525. Liu, Z., Carthy, C.M., Cheung, P., Bohunek, L., Wilson, J.E., McManus, B.M., Yang, D., 1999. Structural and functional analysis of the 5 untranslated region of coxsackievirus B3 RNA: In vivo translational and infectivity studies of full-length mutants. Virology 265, 206–217. Meerovitch, K., Nicholson, R., Sonenberg, N., 1991. In vitro mutational analysis of cisacting RNA translational elements within the poliovirus type 2 5 untranslated region. J. Virol. 65, 5895–5901. M’hadheb-Gharbi, M.B., Paulous, S., Aouni, M., Kean, K.M., Gharbi, J., 2007. The substitution U475 → C with Sabin3-like mutation within the IRES attenuate Coxsackievirus B3 cardiovirulence. Mol. Biotechnol. 36, 52–60. Minnich, L.L., Ray, C.G., 1980. Variable susceptibility of mice to group B coxsackievirus infections. J. Clin. Microbiol. 11, 73–75. Racaniello, V.R., 2001. Picornaviridae: the virus and their replication. In: Fields, B.N., Knipe, D.M., Howley, P.M., Griffin, D.E. (Eds.), Fields Virology, Fourth ed. Lippincott Williams & Williams, Philadelphia, pp. 685–722. ´ Rinehart, J.E., Gomez, R.M., Roos, R.P., 1997. Molecular determinants for virulence in coxsackievirus B1 infection. J. Virol. 71, 3986–3991. Satoh, M., Tamura, G., Segawa, I., Hiramori, K., Satodate, R., 1994. Enteroviral RNA in dilated cardiomyopathy. Eur. Heart J. 15, 934–939. Tracy, S., Drescher, K.M., 2007. Coxsackievirus infections and NOD mice: relevant models of protection from, and induction of, type 1 diabetes. Ann. NY Acad. Sci. 1103, 143–151. Whitton, J.L., Cornell, C.T., Feuer, R., 2005. Host and virus determinants of picornavirus pathogenesis and tropism. Nat. Rev. Microbiol. 3, 765–776. Yang, D., Wilson, J.E., Anderson, D.R., Bohunek, L., Cordeiro, C., Kandolf, R., McManus, B.M., 1997. In vitro mutational and inhibitory analysis of the cis-acting translational elements within the 5 untranslated region of coxsackievirus B3: potential targets for antiviral action of antisense oligomers. Virology 228, 63–73.