Platelets interact with Coxsackieviruses B and have a critical role in the pathogenesis of virus-induced myocarditis.

BACKGROUND: To further understand the role of platelets in the pathogenesis of viral infections we explored platelet interaction with Coxsackieviruses B (CVB) 1 and 3. CVB is a group of viruses that cause the majority of human enterovirus-related viral myocarditis; their receptor (CAR) is expressed on the platelet surface and there is a well-characterized CVB3-induced myocarditis murine model. METHODS: Human platelets were infected with CVB1 and 3 and viruses were detected in pellets and in supernatants. C57BL/6J mice with or without platelet depletion were inoculated with CVB3 and peripheral blood and heart samples collected at different times post-infection. RESULTS: CVB1 and 3 RNA and a capsid protein were detected in infected platelets. Despite the fact that titration assays in Vero cells showed increasing infectivity titers over time, supernatants and pellets from infected platelets showed similar levels, suggesting that platelets were not susceptible to a replicative infectivity cycle. CVB binding was CAR-independent and resulted in P-selectin and phosphatidylserine (PS) exposure. CVB3-infected mice showed a rapid thrombocytopenia that correlated with an increase in platelet PS exposure and platelet-leukocyte aggregates without modification of platelet P-selectin expression or von Willebrand factor levels. Mortality, viremia, heart viral titers and myocarditis were significantly higher in platelet-depleted than normal animals. Type I IFN levels were not changed but IgG levels were lower in infected and platelet-depleted mice. CONCLUSIONS: Our data reveal that platelets play a critical role in host survival and immune response against CVB3 infection.

Attempts to purify a second cellular receptor for a coxsackievirus B3 variant, CB3-RD from HeLa cells.

A coxsackievirus B3 variant, CB3-RD, isolated on rhabdomyosarcoma (RD) cells is known to bind HeLa cells at two different receptor protein sites, HR1 and HR2. Since HR2 occurs in almost 50 fold excess of HR1 in HeLa cells, purification of HR2 was attempted, to obtain its partial N-terminal amino acid sequence and its further characterization. This study describes the purification of HR2 from octylthioglucoside solubilized HeLa cell membranes (HeLa-OTG) by preparative isoelectric focusing (IEF) followed by either preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or affinity chromatography on immobilized receptor monoclonal antibody, RmcA (RmcA-agarose). IEF of HeLA-OTG showed that both HR2 and HR1 could be well separated by this technique and focused with peak maxima around pH 3.7 and 6.7, respectively. Both RmcA and CB3-RD recognized HR2 as doublet bands (60 kD major polypeptide and a minor 55 kD polypeptide) on electroblots under non-reducing conditions. Preparative SDS-PAGE of the pool of IEF fractions containing HR2 (IEF pool) and simultaneous elution of polypeptides from the bottom of the gel during electrophoresis, is shown to be a useful technique in purifying HR2 with only one contaminating polypeptide (65 kD). However, affinity chromatography of the IEF pool on RmcA-agarose yielded HR2 without any detectable contaminating polypeptide. A quantitative chemiluminescence assay was developed to estimate the amount of HR2 on HeLa cells and in solution, when dot blotted on polyvinylidene difluoride (PVDF) membranes and probed with RmcA. Assays revealed that about 1.2% of the total HR2 present on HeLa cells could be obtained by IEF followed by affinity chromatography. Efforts are continuing to obtain sufficient quantities of purified HR2 for partial N-terminal amino acid sequencing.