The murine CAR homolog is a receptor for coxsackie B viruses and adenoviruses.

Complementary DNA clones encoding the murine homolog (mCAR) of the human coxsackievirus and adenovirus receptor (CAR) were isolated. Nonpermissive CHO cells transfected with mCAR cDNA became susceptible to infection by coxsackieviruses B3 and B4 and showed increased susceptibility to adenovirus-mediated gene transfer. These results indicate that the same receptor is responsible for virus interactions with both murine and human cells. Analysis of receptor expression in human and murine tissues should be useful in defining factors governing virus tropism in vivo.

Clinical coxsackievirus B isolates differ from laboratory strains in their interaction with two cell surface receptors.

Coxsackie B viruses interact with two putative cell surface receptor molecules. Experiments with prototype laboratory strains suggest that all 6 coxsackie B serotypes interact with a 46-kDa protein recognized by the monoclonal antibody RmcB, whereas CB1, CB3, and CB5 may also bind to decay accelerating factor. Antireceptor monoclonal antibodies were used to study interactions between low-passage clinical coxsackie B virus isolates and the two receptors. In contrast to observations made with single prototype strains, these data indicate that receptor use by clinical isolates is not strictly related to serotype and that even prototype strains with different passage histories may differ in receptor use. Within a given serotype, variation exists in the capacity of individual virus isolates to bind to specific receptors, and variants with altered receptor specificity may arise during infection in humans and in tissue culture.

Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5.

A complementary DNA clone has been isolated that encodes a coxsackievirus and adenovirus receptor (CAR). When transfected with CAR complementary DNA, nonpermissive hamster cells became susceptible to coxsackie B virus attachment and infection. Furthermore, consistent with previous studies demonstrating that adenovirus infection depends on attachment of a viral fiber to the target cell, CAR-transfected hamster cells bound adenovirus in a fiber-dependent fashion and showed a 100-fold increase in susceptibility to virus-mediated gene transfer. Identification of CAR as a receptor for these two unrelated and structurally distinct viral pathogens is important for understanding viral pathogenesis and has implications for therapeutic gene delivery with adenovirus vectors.

A short history and introductory background on the coxsackieviruses of group B.

The past 50 years have revealed an array of significant developments in our documentation and understanding of viruses and their associated diseases. The CVB, as enteroviruses, were discovered in the search for poliomyelitis-related viruses by the inoculation of newborn mice. Future strategies for the discovery of additional viruses will undoubtedly come through the application of differentiating cell culture systems with increased susceptibility to infection by specific viruses. Developments in regulation of the cell cycle also will contribute to the better definition of events controlling persistent infections caused by the CVB. Methods utilizing molecular biological probes in situ will prove to be major aids in identifying the molecular events in CVB pathogenesis. Virology of the CVB continues to be an exciting area for research and application of preventive measures to lesson human suffering. The chapters in this book which follow will amplify most of the themes briefly presented here.

Expression and distribution of the receptors for coxsackievirus B3 during fetal development of the Balb/c mouse and of their brain cells in culture.

This study was designed mainly to determine the relationships between the expression and distribution of the cellular receptor proteins for coxsackievirus B3 (CVB3) and susceptibility of mouse brain cells during fetal development of Balb/c mice. Immunoblot analysis of fetal extracts demonstrated that the CVB3 receptor proteins were first expressed at day 14 of the fetal stage, and that maximal expression of the cellular receptor occurred at near term or newborn stage. Results also suggested that newborn mouse brain tissue expressed much larger quantities of viral receptor proteins, compared to other tissues. In vitro studies showed that both mouse neurons and astrocytes could be infected by two CVB3 strains, pantropic CVB3 Nancy strain (CVB3N) and myocardiotropic CVB3 Woodruff strain (CVB3W). CVB3N, however, replicated and grew to high titer in primary astrocyte cultures and in primary neuron cultures, whereas, primary astrocyte cultures were relatively resistant to CVB3W. Virus binding assays revealed that CVB3N bound faster and in greater amounts to mouse brain cells than CVBW. These two virus strains, however, were found to share the same receptor specificity by virus competition assays. The number of virus binding sites for CVB3 on newborn mouse brain cells was approximately 1.8 x 10(4) per cell. The data suggested that preferential expression of the cellular receptors on newborn mouse brain cells may be related to their high susceptibilities to CVB3 infection.

Receptor proteins on newborn Balb/c mouse brain cells for coxsackievirus B3 are immunologically distinct from those on HeLa cells.

Newborn Balb/c mice are highly susceptible to infection by the six coxsackievirus serotypes of group B (CVB) and it is known that receptor for these viruses are in highest concentration in the brain as compared to other tissues. Therefore, proteins from the brain tissues of these animals were solubilized (Brain-Ext) and characterized for the identification of mouse brain receptor (MBR) proteins. Virus-blot analyses of Brain-Ext suggested that each of three virus variants of CVB3-(N, W and RD) recognized four receptor proteins designated p46, p44, p36 and p33 according to their molecular size. Similar analyses of cultured neurons from newborn Balb/c mice revealed the presence of the same four receptor proteins, while astrocytes appeared to possess only p46 and/or p44. Isoelectric focusing of Brain-Ext, focused MBR proteins in the pH range 4.0-8.5, with a peak around pH 5.7. P46 was found to be neuraminidase sensitive. A polyclonal rat antiserum (anti-MBR) protected cultured neurons and astrocytes against infection by CVB3, inhibited virus binding to these cells and recognized the same four receptor proteins on western-blots as detected on virus-blots by CVB3. However, a rabbit polyclonal anti-HeLa cell antiserum, which strongly binds to HeLa cells and protects them from CVB3 infection, neither recognized any of the receptor proteins in western-blot analyses of Brain-Ext nor inhibited CVB3 infection on cultured neurons and astrocytes. Conversely, anti-MBR did not recognize any of the receptor proteins by western-blot analysis of HeLa cell extracts nor did it inhibit CVB3 infection of HeLa cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Coxsackievirus B3 adapted to growth in RD cells binds to decay-accelerating factor (CD55).

A coxsackievirus B3 (CB3) isolate adapted to growth in RD cells shows an alteration in cell tropism as a result of its capacity to bind a 70-kDa cell surface molecule expressed on these cells. We now show that this molecule is the complement regulatory protein, decay-accelerating factor (DAF) (CD55). Anti-DAF antibodies prevented CB3 attachment to the cell surface. Radiolabeled CB3 adapted to growth in RD cells bound to CHO cells transfected with human DAF, whereas CB3 (strain Nancy), the parental strain, did not bind to DAF transfectants. These results indicate that growth of CB3 in RD cells selected for a virus strain that uses DAF for cell surface attachment.

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.

Mapping of the RD phenotype of the Nancy strain of coxsackievirus B3.

The RD variants of group B coxsackieviruses differ from their parental strains in having the ability to replicate in a human rhabdomyosarcoma cell line, RD. The nucleotide sequence of the P1 region of the RD variant of coxsackievirus B3 strain Nancy (CB3NRD) was determined by sequencing cloned cDNAs, obtained by PCR amplification. A comparison between the established nucleotide sequence and that of the P1 region from the parental virus revealed 12 point mutations which corresponded to six amino acid replacements. To identify if the P1 region is responsible for the phenotype of CB3NRD, a chimeric virus was constructed, using an infectious cDNA clone of CB3. The P1 region of the infectious cDNA was replaced by cDNA fragments from CB3N (parental strain Nancy) or CB3NRD and the resulting recombinants were assayed for their ability to infect and replicate in RD cells. The results showed that the RD phenotype of CB3NRD maps in the P1 region. Furthermore, a chimera which only contained the 5' part of the P1 region derived from CB3NRD and the remaining P1 sequence from CB3N was able to replicate in RD cells, suggesting that the VP2 polypeptide contains at least one determinant for the RD phenotype.

Induction of heterotypic virus resistance in adult inbred mice immunized with a variant of Coxsackievirus B3.

Infection of adult male C3H/HeJ mice with a host range variant of Coxsackievirus B3 (CB3W-RD) induced resistance in these mice to an otherwise lethal dose of Coxsackievirus B1 (CB1). The protective effect induced by CB3W-RD was detectable as early as 1 day post-vaccination and was still present 10 weeks later. While untreated mice infected with CB1 died within 5 days because of massive hepatic necrosis, the liver was spared in mice immunized with CB3W-RD and then challenged with CB1. In general, CB1 titers in heart, liver, and pancreas of CB3W-RD-vaccinated animals were lower than that found in unvaccinated animals. Virus neutralizing antibody was not a mediator of this heterotypic, virus-induced protective effect. In addition, the outcome of CB1 infection could be modified if superinfection with CB3W-RD took place within 1-4 days following CB1 infection. In this regard, maximum therapeutic efficacy was observed when CB1 infected mice were superinfected 2 days after CB1 infection. CB1-infected mice that survived as a result of treatment with CB3W-RD exhibited liver regeneration but did develop myocardial necrosis.

Characterization of a YAC-1 mouse cell receptor for group B coxsackieviruses.

A receptor on YAC-1 cells, a mouse T-lymphoma cell line, bound all six serotypes of the group B coxsackieviruses (CVB). In addition, the cells produced infectious virus. Each of the CVB competed for the same receptor on YAC-1 cells. CVB3 bound relatively slowly to YAC-1 cells (k = 4 x 10(-11) min-1 cell-1), and there were only 500 attachment sites per cell. A rabbit antiserum prepared against the HeLa cell receptor protein Rp-a specifically inhibited the binding of CVB1 and CVB3. A virus-receptor complex with CVB3 could be isolated from detergent (0.5% sodium deoxycholate, 1% Triton X-100)-solubilized YAC-1 plasma membranes. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the iodinated virus-receptor complex revealed a band with the same mobility as Rp-a. The results suggested that the YAC-1 receptor for CVB resembles that of the HeLa cell receptor.

Pathogenesis of acute myocardial necrosis in inbred mice infected with coxsackievirus B3.

The pathogenesis of myocardial necrosis due to CB3W infection was studied in BALB/c and C3H/HeJ mice. BALB/c mice infected with 5 x 10(4) pfu were found to die of massive hepatic coagulative necrosis before myocardial changes occurred. Reducing the inoculum size to 5 x 10(2) pfu resulted in sublethal hepatic involvement and multifocal myocardial coagulative necrosis by day 7 p.i. In contrast, C3H/HeJ mice survived infection and developed multifocal myocardial coagulative necrosis, but not liver disease following inoculation with as much as 5 x 10(6) pfu of CB3W. As with BALB/c mice infected with 5 x 10(2) pfu, myocardial lesions became apparent in C3H/HeJ mice a few days after peak cardiac virus titer was attained. Minimal inflammatory infiltrate was seen following development of cellular necrosis and was restricted to the areas of virus-induced pathologic change. However, no evidence was found for virus-specific cytotoxic T cell activity or for delayed type hypersensitivity responses. Furthermore, myocardial necrosis in CB3W-infected, T cell-depleted C3H/HeJ mice was as severe as in CB3W-infected, immunocompetent mice. These data have led us to conclude that cardiac lesions were due to virus-induced cytopathology rather than immunopathogenic mechanisms.

A monoclonal antibody specific for the cellular receptor for the group B coxsackieviruses.

A 50-kilodalton receptor protein (Rp-a) for the group B coxsackieviruses (CB) was isolated in a virus-receptor complex from detergent-solubilized HeLa cells (J. E. Mapoles, D. L. Krah, and R. L. Crowell, J. Virol. 55:560-566, 1985). It was used as an immunogen for preparation of a mouse monoclonal antibody (RmcB) which protected HeLa cells and Buffalo green monkey kidney cells from infection by all six serotypes of CB. RmcB did not protect HeLa cells from infection by poliovirus, echovirus 6, or coxsackievirus A18. This monoclonal antibody differed in receptor epitope specificity from a previously isolated antibody (RmcA) (R. L. Crowell, A. K. Field, W. A. Schleif, W. L. Long, R. J. Colonno, J. E. Mapoles, and E. A. Emini, J. Virol. 57:438-445, 1986) which blocked receptors only for type 1 CB (CB1), CB3, CB5, and echovirus 6. RmcA and RmcB recognized two distinct saturable receptors on HeLa cells, designated HR2 and HR1, respectively. Human rhabdomyosarcoma (RD) cells have the HR2 receptor for CB3-RD (a variant of CB3), but lack the HR1 receptor for CB3. Therefore, RD cells were resistant to infection by CB3. Although binding of CB3-RD to the HR2 receptor on RD cells can lead to infection, binding of CB3-RD to the HR2 receptor on HeLa cells did not lead to infection. Apparently, both CB3 and CB3-RD use only the HR1 receptor for infection of HeLa cells. Thus, a given virus may use two distinct receptors to bind to cells when only one virus-receptor interaction leads to infection.

Monoclonal antibody that inhibits infection of HeLa and rhabdomyosarcoma cells by selected enteroviruses through receptor blockade.

BALB/c mice were immunized with HeLa cells, and their spleen cells were fused with myeloma cells to produce hybridomas. Initial screening of culture fluids from 800 fusion products in a cell protection assay against coxsackievirus B3 (CB3) and the CB3-RD virus variant yielded five presumptive monoclonal antibodies with three specificities: protection against CB3 on HeLa, protection against CB3-RD on rhabdomyosarcoma (RD) cells, and protection against both viruses on the respective cells. Only one of the monoclonal antibodies (with dual specificity) survived two subclonings and was studied in detail. The antibody was determined to have an immunoglobulin G2a isotype and protected cells by blockade of cellular receptors, since attachment of [35S]methionine-labeled CB3 was inhibited by greater than 90%. The monoclonal antibody protected HeLa cells against infection by CB1, CB3, CB5, echovirus 6, and coxsackievirus A21 and RD cells against CB1-RD, CB3-RD, and CB5-RD virus variants. The monoclonal antibody did not protect either cell type against 16 other immunotypes of picornaviruses. The monoclonal antibody produced only positive fluorescence on those cells which were protected against infection, and 125I-labeled antibody confirmed the specific binding to HeLa and RD cells. The results suggest that this monoclonal antibody possesses some of the receptor specificity of the group B coxsackieviruses.

Purification of a HeLa cell receptor protein for group B coxsackieviruses.

Coxsackievirus B3 (CB3) firmly attaches to HeLa cells, forming a specific complex between the virus and its receptor on the cell surface. We extracted this virus-receptor complex (VRC) with the detergents sodium deoxycholate and Triton X-100. The VRC was identified by its sedimentation coefficient (140S), which was less than that of virions (155S). Formation of the VRC from cell lysates and 3H-CB3 occurred with the same specificity as did attachment of virions to cells, in that its formation was blocked by unlabeled CB3 but not by poliovirus. The VRC was purified 30,000-fold by differential and sucrose gradient centrifugation. Iodination with Na125I revealed that the purified VRC consisted of the normal CB3 proteins and one additional protein (RP-a) with an approximate molecular weight of 49,500. RP-a was eluted from the VRC and was shown to rebind with CB3 and CB1 virions but not with poliovirus type 1. We propose that Rp-a is a protein in the plasma membrane receptor complex which is responsible for the specific recognition and binding of the group B coxsackieviruses.

Properties of the deoxycholate-solubilized HeLa cell plasma membrane receptor for binding group B coxsackieviruses.

Physical and chemical properties of deoxycholate-solubilized HeLa cell plasma membrane receptors for binding group B coxsackieviruses were determined. Receptors eluted from Sepharose 4B with an apparent molecular weight of 275,000 and sedimented with an S value of between 14.7 and 4.9 and a buoyant density of 1.06 to 1.10 g/cm3. Virus-binding activity was destroyed after treatment with proteases, glycosidases, and periodate but was unaffected by lipases or reducing or alkylating agents. Additionally, lectins, including concanavalin A, adsorbed receptors and inhibited virus attachment. The composite data suggested that glycoprotein is an integral part of the receptors for binding virus.

Altered receptor specificity of coxsackievirus B3 after growth in rhabdomyosarcoma cells.

Serial "blind" passages in human rhabdomyosarcoma (RD) cells of prototype viruses from each of the six immunotypes of the group B coxsackieviruses (CB) resulted in the isolation of intratypic variants of CB1, CB3, CB5, and CB6. Each variant virus strain acquired the capacity to agglutinate human erythrocytes and produce small plaques on HeLa cells, although their serological specificity remained unchanged. An alteration in VP1 mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was noted for CB3-RD. The CB3-RD variant was plaque purified on RD cells and studied for receptor interactions on both HeLa and RD cells. An attachment restriction appeared to exist for prototype CB3 on RD cells, whereas CB3-RD attached well to both cells. In attachment interference assays, HeLa cells saturated with CB3-RD blocked the attachment of CB3. In contrast, saturation of cells with CB1 (which shares a common receptor with parental CB3) failed to block the attachment of CB3-RD. This unidirectional receptor blockade suggested that a second site for the attachment of virions to receptors was acquired by the CB3-RD variant. Thus, more than one virus receptor specificity may be operative in the selection of host range virus mutants. The implications of this phenomenon as they may relate to pathogenesis are discussed.

Eclipse of coxsackievirus infectivity: the restrictive event for a non-fusing myogenic cell line.

Coxsackieviruses A2, A5 and B3 did not replicate in L8CL3-U cells (a non-fusing variant of the rat L8 myogenic cell line) although these cells possessed a common receptor for coxsackieviruses A2 and A5, and a different receptor for coxsackievirus B3. The restriction in replication was identified as a block in viral eclipse, since 6 M-LiCl treatment permitted recovery of the coxsackievirus A2 inoculum from L8CL3-U cells after 2 h at 37 degrees C, and the cells could be transfected by viral RNA. Cellular fusion which was induced in L8CL3-U cultures by herpes simplex virus type 1 (HF strain) facilitated coxsackievirus A2 and A5 replication. Differentiating myogenic L8 cells acquired full susceptibility to infection concurrently with the appearance of acetylcholine receptors, the muscle-specific isoenzyme of creatine phosphokinase, prominent myotube formation and the acquired capacity of the cells to eclipse virus.