Role of Heparan Sulfate in Cellular Infection of Integrin-Binding Coxsackievirus A9 and Human Parechovirus 1 Isolates - PubMed (original) (raw)
Role of Heparan Sulfate in Cellular Infection of Integrin-Binding Coxsackievirus A9 and Human Parechovirus 1 Isolates
Pirjo Merilahti et al. PLoS One. 2016.
Abstract
Heparan sulfate/heparin class of proteoglycans (HSPG) have been shown to function in cellular attachment and infection of numerous viruses including picornaviruses. Coxsackievirus A9 (CV-A9) and human parechovirus 1 (HPeV-1) are integrin-binding members in the family Picornaviridae. CV-A9 Griggs and HPeV-1 Harris (prototype) strains have been reported not to bind to heparin, but it was recently shown that some CV-A9 isolates interact with heparin in vitro via VP1 protein with a specific T132R/K mutation. We found that the infectivity of both CV-A9 Griggs and HPeV-1 Harris was reduced by sodium chlorate and heparinase suggestive of HSPG interactions. We analyzed the T132 site in fifty-four (54) CV-A9 clinical isolates and found that only one of them possessed T132/R mutation while the other nine (9) had T132K. We then treated CV-A9 Griggs and HPeV-1 Harris and eight CV-A9 and six HPeV-1 clinical isolates with heparin and protamine. Although infectivity of Griggs strain was slightly reduced (by 25%), heparin treatment did not affect the infectivity of the CV-A9 isolates that do not possess the T132R/K mutation, which is in line with the previous findings. Some of the HPeV-1 isolates were also affected by heparin treatment, which suggested that there may be a specific heparin binding site in HPeV-1. In contrast, protamine (a specific inhibitor of heparin) completely inhibited the infection of both prototypes and clinical CV-A9 and HPeV-1 isolates. We conclude that T132R/K mutation has a role in heparin binding of CV-A9, but we also show data, which suggest that there are other HSPG binding sites in CV-A9. In all, we suggest that HSPGs play a general role in both CV-A9 and HPeV-1 infections.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Fig 1. Effect of siRNA silencing of exostosin I on CV-A9 (Griggs) infection.
(A) Control cells were transfected with AllStars scramble siRNAs. Infections were followed by immunofluorescence microscopy using CV-A9 specific antibodies. (B) Relative infectivity of CV-A9 in exostosin 1 silenced cells were calculated from the immunofluorescence images as the ratio of infected cells to the total cell number. The AllStars siRNA control cells were given the value of 100%.
Fig 2. Effect of sodium chlorate and heparinase I on CV-A9 (Griggs) and HPeV-1 (Harris) infections.
(A) Cells were grown in medium supplemented with 50 mM NaClO3 for 72 h prior to infection, or incubated with heparinase I for 2 h prior to infection. Infections were allowed to proceed for 6 h followed by antibody staining and immunofluorescence imaging. (B) Relative infectivities of CV-A9 and HPeV-1 were calculated from the immunofluorescence images as the ratio of infected cells to the total cell number. The non-treated control cells were given the value of 100%. Standard deviations are shown in the figure. *** indicates p<0.05.
Fig 3. Sequence analysis of VP1-T132 site within CV-A9 isolates.
The VP1 sequence of CV-A9 Griggs (Griggs_HS) and fifty-four (54) CV-A9 clinical isolates was analyzed to identify VP1-T132 site. Sequences were aligned by ClustalW, translated and trimmed using SeaView and visualized by GeneDoc programs.
Fig 4. Effect of heparin on CV-A9 and HPeV-1 infections.
(A and C) Viruses were incubated with heparin (0.5 and 3 mg/ml) for 2 hours prior to inoculation onto A549 cells. Infections were allowed to proceed for 6 h and followed by antibody staining and immunofluorescence microscopy. Representative samples of prototype viruses and two isolates of both viruses have been chosen to the figures. (B and D) Relative infectivity of CV-A9 and HPeV-1 was calculated from the immunofluorescence images as the ratio of infected cells to the total cell number. The non-treated control cells were given the value of 100%. Standard deviations are shown in the figures and * indicates that p<0.001 and ** indicates that p<0.05.
Fig 5. Effect of protamine on CV-A9 and HPeV-1 infections.
(A and C) A549 cells were incubated with protamine (0.5 and 2 mg/ml) for 2 hours prior to infection. Infections were allowed to proceed for 6 h and followed by antibody staining and immunofluorescence microscopy. (B and D) Relative infectivities of the isolates were calculated from the immunofluorescence images as the ratio of infected cells to the total cell number. The non-treated control cells were given the value of 100%. Standard deviations are shown in the figures. * indicates that p<0.001 in (B), in (D) p-value is <0.001 in all cases.
References
- Turnbull J, Powell A, Guimond S. Heparan sulfate: decoding a dynamic multifunctional cell regulator. Trends Cell Biol. 2001;11: 75–82. - PubMed
- Krusat T, Streckert HJ. Heparin-dependent attachment of respiratory syncytial virus (RSV) to host cells. Arch Virol. 1997;142: 1247–1254. - PubMed
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This work was supported by Academy of Finland (128539, 263255 to P.S.), European Union (AIROPico, FP7-PEOPLE-2013-IAPP Grant no. 612308), Turku Graduate School of Biomedical Sciences, Turku Doctoral Programme of Molecular Medicine, Finnish Culture Foundation, Jane and Aatos Erkko Foundation and Turku University of Applied Sciences (601129 to P.S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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