1 Department of Virology and MediCity Research Laboratory, University of Turku, Kiinamyllynkatu 13, FIN-20520 Turku, Finland
2 Department of Pathology, University of Turku and Turku University Hospital, FIN-20520 Turku, Finland
3 Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, UK
4 Department of Medical Microbiology, University of Oulu, FIN-90014 Oulu, Finland
Correspondence
Heli Harvala
heli.harvala{at}utu.fi
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ABSTRACT |
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MAIN TEXT |
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Coxsackieviruses are divided into subgroups A and B, according to pathogenicity in newborn mice: CAVs cause flaccid paralysis; in contrast, CBVs induce spastic paralysis. Accordingly, CAVs affect striated muscle, while CBVs replicate in several tissues, including the central nervous system, pancreas and liver (Hyypiä et al., 1993). CAV9, despite its CAV-like pathogenicity in newborn mice, is genetically more closely related to CBVs than to other CAVs. However, when compared to CBVs, the CAV9 capsid protein VP1 has an apparent insertion of approximately 15 amino acids at its C terminus (Chang et al., 1989
). This insertion contains an arginine-glycine-aspartic acid (RGD) tripeptide, which has been shown to be fully conserved among clinical CAV9 strains isolated from different geographical regions over the past five decades but has not been found in other CAVs (Chang et al., 1992
; Santti et al., 2000
). The RGD motif mediates attachment of CAV9 to the cell surface integrin
V
3 but the virus is able also to utilize alternative pathway(s) in cell entry, since deletion or mutation of the RGD motif does not destroy infectivity completely (Roivainen et al., 1991
, 1994
; Hughes et al., 1995
). The C-terminal region of the VP1 capsid protein has been shown also to be antigenic by peptide scanning but it was found that the RGD motif itself was poorly immunogenic, whereas antibody-binding sites were located at both sides of the motif (Pulli et al., 1998a
, b
).
To analyse the effect of the RGD motif on tissue tropism and pathogenicity in vivo, BALB/c mice were infected with the parental (CAV9) or mutant (CAV9RGE, CAV9d4 and CAV9d12) viruses described previously (Chang et al., 1989; Hughes et al., 1995
). The substitution mutant (CAV9RGE) contains an RGE (arginine-glycine-glutamic acid) motif instead of an RGD motif, whereas in the genomes of deletion mutants, the region deleted included the RGD motif alone (CAV9d4) or the RGD motif and eight additional amino acids located at both sides of the motif (CAV9d12). Transfection of rhabdomyosarcoma (RD) cells with RNA transcripts, generated from the parental and three mutant plasmids, resulted in complete CPE within 3 days. Examination of the growth curves of mutant viruses showed similar production of infectious virus when compared to parental CAV9 (data not shown).
Groups of newborn BALB/c mice (Animal Center of the University of Turku), aged between 8 and 24 h, were infected intraperitoneally with 2x104 p.f.u. of the mutant or parental viruses in 50 µl PBS. The size of groups varied from 10 to 18. The litters within groups were maintained separately and observed daily. Following inoculation, one to six mice from each group were sacrificed after 1 to 5 days and fixed in 10 % formalin. Transverse sections of the head, upper and lower abdomen, and lower limb were embedded in paraffin and used to analyse the presence of viral RNA by in situ hybridization using a radiolabelled CAV9 cDNA probe, as described previously (Harvala et al., 2002). Both uninfected mouse tissue and a plasmid probe were used to control the specificity of the hybridization reactions.
All of the newborn mice infected with CAV9 died between 3 and 5 days post-infection (p.i.), whereas the CAV9-mutants appeared to be less virulent: some mice survived until day 5 p.i. (Fig. 1). After parental CAV9 infection of newborn mice, viral RNA was detectable by in situ hybridization in skeletal muscle (intercostal, platysma, lingual and thigh muscles), whereas CAV9RGE and CAV9d4 RNA was seen only in lingual (CAV9RGE and CAV9d4) or platysma (CAV9RGE) muscle. CAV9d12 mutant RNA was not detected in any tissue (Fig. 2
AH). The signal obtained 3 days after mutant virus infection was much weaker than that observed in CAV9 infection. Surprisingly, the genome of parental CAV9 was also detectable in the exocrine part of the pancreas, in addition to muscle tissue, 3 days after infection (Fig. 2D
). All other tissues studied were negative for viral RNA (data not shown).
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None of the adult mice, infected with either the parental or the mutant CAV9 strains, died during the follow-up period. The parental CAV9 strain replicated to high titres in the pancreas, whereas the RGD mutant viruses did not (Fig. 3). CAV9 was seen to replicate in the pancreas at 1 day p.i. (the tissue of the mouse that was positive contained 1·3x104 p.f.u. g-1), whereas maximal virus titres in the pancreas were detected at 3 days p.i. (mean of three mice, 6·5x104 p.f.u. g-1; range of titres, 3·2x104 p.f.u. g-1 to 8·8x104 p.f.u. g-1). Virus replication in the other tissues and blood remained undetectable. The plaque assay used has a sensitivity of about 100 p.f.u. g-1 and it is possible that small amounts of virus, below this level of detection, were present in these tissues.
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Two CAV9 mutants lacking the RGD motif (CAV9RGE and CAV9d4) were able to infect platysma and lingual muscles in newborn mice, which may be due to the reported capability of CAV9 lacking the RGD motif to interact with the V
3 integrin (Triantafilou et al., 2000
). An alternative explanation could be the use of another, presumably RGD-independent, receptor pathway by the CAV9 mutant viruses. The limited tropism and lower signal, indicative of a reduced ability to infect the tissue, could be because RGD-mediated integrin clustering and signalling may be needed for efficient virus internalization, as has been reported for adenoviruses (Chiu et al., 1999
). In addition, the interaction of integrins with RGD-containing peptides can also activate caspases and lead to apoptosis (Buckley et al., 1999
; Ruoslahti & Reed, 1999
), although it is not known whether viruses themselves can induce such signalling through interaction with integrin. If apoptosis does play a role in CAV9 infection, it may be the case that RGD-less mutants fail to replicate efficiently because they do not induce apoptosis.
It is interesting that, although CAV9RGE and CAV9d4 were able to infect platysma and lingual muscles in newborn mice, the mutant CAV9d12 could not be detected in these tissues. This mutant grows as efficiently in RD cells as the two less radical mutants, suggesting that differences in pathogenicity are not due to markedly diminished particle stability or altered polyprotein processing (data not shown). It is possible that the extra eight amino acids deleted in this mutant may themselves contain determinants that contribute to pathogenicity.
It is well known that CBVs can replicate to high titres in the mouse pancreas and cause acinar cell destruction but, until recently, the ability of CAV9 to replicate in pancreatic tissue has not been documented. In our current work, however, the parental CAV9 strain was seen to replicate in the pancreas, in both newborn and adult mice, while the CAV9 mutants were not detectable in this tissue. The hybridization signal obtained in the pancreas after inoculation of newborn mice with parental CAV9 was relatively weak and virus titres in the pancreas of the CAV9-infected adult mice remained low compared to, for instance, those observed in CBV3-infected mice (data not shown). In addition, our earlier findings suggest that non-capsid determinants contribute to pancreotropism in newborn mice, since a recombinant containing the 5'NCR from CBV3 and the rest of the genome from CAV9 showed substantially greater replication in the pancreas when compared to parental CAV9 infection (Harvala et al., 2002). Nonetheless, the data support the importance of the RGD motif in mouse pancreotropism of CAV9. It is not clear if this observation has implications for human disease, since it is not known whether CAV9 can infect the human pancreas. However, a strain of EV-9, the other enterovirus with an RGD motif in VP1, was isolated recently from a patient at clinical onset of diabetes and was found to be cytolytic for pancreatic
cells (Paananen et al., 2003
).
In addition to CAV9 and EV-9, RGD motifs play important roles in integrinligand interactions among other picornaviruses, namely human parechovirus type 1 (HPEV-1) and foot-and-mouth disease virus (FMDV) (Fox et al., 1989; Jackson et al., 2000a
, b
; Stanway et al., 1994
). The RGD motifs in HPEV-1 and EV-9 are located in the same position in VP1 (C terminus) as that in CAV9, while FMDV contains an RGD tripeptide in the GH loop of the VP1 protein (Acharya et al., 1989
; Forss et al., 1984
; Hyypiä et al., 1992
; Zimmermann et al., 1996
). According to mutagenesis studies, the RGD motif plays a critical role in HPEV-1 infection, since viruses carrying an RGE sequence are not viable and transfection of mutant RNA resulted only in production of revertant viruses with a restored RGD motif (Boonyakiat et al., 2001
). The substitution of the RGD motif by an RGE tripeptide or deletion of the RGD motif does not abolish the infectivity of EV-9 strain in green monkey kidney cells but the presence of an RGD motif correlates with EV-9 pathogenesis in mice (Zimmermann et al., 1997
). Viable FMDV mutants lacking the RGD motif have been characterized also (Baranowski et al., 2000
) but FMDV strains virulent for cattle appeared to utilize the RGD-mediated integrin interaction (Neff et al., 1998
). CAV9, EV-9 and FMDV mutants lacking the RGD tripeptide can be viable in certain cell lines (Baranowski et al., 2000
; Hughes et al., 1995
; Zimmermann et al., 1997
) but the almost ubiquitous occurrence of this motif in isolates suggests again that it is required for natural infections by these viruses. This is consistent with the observations described here, in that the RGD motif influences the tropism and pathogenicity of CAV9 in mice.
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ACKNOWLEDGEMENTS |
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Received 21 March 2003;
accepted 26 May 2003.