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.

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.

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)

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.

Specific cell-surface alteration by enteroviruses as reflected by viral-attachment interference.

Crowell, Richard L. (Hahnemann Medical College, Philadelphia, Pa.). Specific cell-surface alteration by enteroviruses as reflected by viral-attachment interference. J. Bacteriol. 91:198-204. 1966.-Exposure of HeLa cells to high levels of coxsackievirus B3 produced cells which were refractory to attachment of coxsackievirus B1, whereas poliovirus T2 attached normally. Under similar conditions, poliovirus T2 was found to interfere with the attachment of poliovirus T1 to HeLa cells without affecting the attachment rate of coxsackievirus B3. The data confirm earlier findings that the receptor sites on HeLa cells, which bind members of group B coxsackieviruses, are distinct from those for polioviruses. Quantitatively, coxsackieviruses B1 and B3 were found to be mutually exclusive in the attachment interference assay to suggest that they compete for the same receptors on the HeLa cell surface. The finding that input multiplicities of B3 virus which exceeded 500 saturated the homologous viral receptors of HeLa cells was unexpected, but was consistent with the results of interference assays. Excessive amounts of input virus did not, however, inhibit eclipse of homologous cell-associated virus. Attachment interference between enteroviruses occurred even though the interfering virus was eclipsed prior to addition of challenge virus. The finding that enterovirus attachment interference was reversible with acid pH suggested that attachment and eclipse of enterovirus does not result in a permanent alteration of the cell membrane and that these events occur at the cell surface.

Immunological studies of the group B coxsackieviruses by the sandwich enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation.

A microplate double antibody sandwich ELISA was employed in an immunological study of the group B Coxsackieviruses. The assay, described in detail, detected high dilutions of virion antigen (less than 10 ng) in purified preparations and in crude infected cell extracts. Furthermore, by using a constant amount of antigen, group B virus antibodies in hamster antisera could be quantified with a sensitivity equivalent to the virus neutralization test. Titrations of virus antigens and antibodies were found to be type-specific when purified virions were employed in the assay. Urea disruption of virions exposed antigens common to all six group B viruses. The heterotypic reactivity of disrupted group B virions did not extend to the other viruses tested. Immunoprecipitation and SDS-PAGE analysis revealed that, of the four virion structural polypeptides (VPI to 4), VPI contained the major common antigenic determinants shared by members of the group B Coxsackieviruses.

Indirect enzyme-linked immunosorbent assay (ELISA) for the detection of Coxsackievirus group B antibodies.

An indirect, solid phase, microplate enzyme-linked immunosorbent assay (ELISA) was found to be highly sensitive and reliable for detecting antibodies to the group B Coxsackieviruses and other picornaviruses. Conditions for obtaining maximum sensitivity and reproducibility of the indirect ELISA are described. Antibody titres were comparable to those obtained by the virus neutralization test and over 50 times higher than those obtained by the complement-fixation test. Purified virions used in the indirect ELISA reacted with low levels of cross-reacting heterotypic antibodies elicited by each of the six group B Coxsackieviruses, although homotypic reactions resulted in highest titres.

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.

Effect of anticellular serum on the attachment of enteroviruses to HeLa cells.

Anticellular serum (ACS), in the absence of an active complement system, was shown to inhibit the attachment of poliovirus types 1 and 2, echovirus type 6, and coxsackievirus types A13, B1, and B3 to viral receptors of live HeLa cells. This is the first report to provide evidence that ACS has an inhibitory effect on the interaction between host cells and coxsackieviruses of group B. The titer of inhibitory activity of ACS varied inversely with the cell concentration used, the reaction being virtually completed after an incubation period of 30 min at 37 C. The inhibitory activity of ACS persisted for more than 4 hr at 37 C, and was shown to be reversible at pH 2.0, revealing that although the receptors for attaching virus were inactivated by ACS the inactivation was not permanent. These findings are consistent with the concept that antibodies in the ACS combine with and blockade viral receptors located at the cell surface. An antiserum with a specificity for inhibiting attachment of coxsackievirus B1 was obtained by dual absorption of ACS with cells saturated with coxsackievirus type B3 and chymotrypsin-treated cells. These findings offer an approach whereby the antigenic relationship of viral receptors to other constituents of the cell surface can be studied.

Comparative studies of the regeneration of HeLa cell receptors for poliovirus T1 and coxsackievirus B3.

Enterovirus receptors of live HeLa cells have been shown to reappear after inactivation by proteolytic enzymes, provided the cells are incubated at 37 C in a nutritionally adequate medium. Regeneration of receptor activity for poliovirus T1 occurred at a significantly faster rate than for coxsackievirus B3. The regenerative process for both types of receptors studied evidently required an active process of protein synthesis, since it was found that reappearance of receptor activity was inhibited by streptovitacin A, puromycin, and actinomycin D. Substitution of p-fluorophenylalanine for the naturally occurring amino acid, at concentrations which inhibited virus synthesis, was without effect on regeneration of receptor activity. It is anticipated that these findings will aid in the study of the biosynthesis and subsequent chemical characterization of viral receptors of living host cells.

Differential inhibition of attachment and eclipse activities of HeLa cells for enteroviruses.

Receptor activities of HeLa cells were evaluated for ability to both attach and eclipse enteroviruses after exposure of cells to acid or heat. A modified procedure of acid (pH 1.5) elution of cell-associated virus, as compared with other procedures, provided a general method for the optimal recovery of receptor-bound enteroviruses. With this procedure, eclipse of virus operationally was considered to be that amount of virus infectivity which was determined initially to be cell-associated and which was not dissociable from the cells. HeLa cells killed by heating at 56 C for 30 min could not attach or eclipse poliovirus T1, but they attached and eclipsed coxsackieviruses B1 and B3, and they attached echovirus 6 but did not eclipse it. HeLa cells treated at pH 2.5 for 10 min at 2 C could not attach or eclipse poliovirus T1, but they attached coxsackieviruses B1 and B3 and echovirus 6, although these viruses were not eclipsed. These results showed that, within the operational definition of virus eclipse, the eclipse activity of HeLa cells for some viruses can be irreversibly inactivated without impairing the activity of the receptors for attaching these viruses. The data provided additional evidence that HeLa cells possess specific receptors for the different enteroviruses.

Susceptibility of differentiating muscle cells of the fetal mouse in culture to coxsackievirus A13.

The interaction of coxsackievirus A13 with differentiating muscle cells, cultured from tissues of the fetal mouse, was studied. Cultures infected at that stage of myogenic differentiation characterized by the rapid formation of multinucleated myotubes produced maximum virus titers of over 10(7) plaque-forming units. Virus-induced cytopathic effect was characterized by a marked diminution in the number of multinucleated cells. The susceptibility of these cultures decreased appreciably when infection was initiated after the majority of the myotubes had formed. The demonstration of newly synthesized A13 virus antigen by immunofluorescence provided direct evidence that A13 virus replication occurred both in myoblasts and myotubes. The synthesis of A13 virus was markedly depressed in muscle cultures in which the formation of multinucleated cells was inhibited by BUDR or by fusion-inhibiting media. After reversal of this inhibition, the cultures acquired the increased susceptibility to A13 virus characteristic of cells undergoing myogenic differentiation. In contrast to the results obtained with coxsackievirus A13, the primary fetal mouse muscle cultures were resistant to poliovirus T1. It is suggested that changes in the surfaces of developing muscle cells may coincide with the formation and disappearance of specific virus receptors and thereby regulate the cell susceptibility to coxsackievirus A13.

Specific alterations of coxsackievirus B3 eluted from HeLa cells.

After the attachment of radioactive coxsackievirus B3 to HeLa cells at 0 C and subsequent incubation at 37 C, 50 to 80% of attached virus radioactivity was eluted from the cells within 1 hr. Eluted virus had a buoyant density of 1.21 in a potassium tartrate gradient, sedimented more slowly than native virus in sucrose gradients, was resistant to ribonuclease, was unstable in CsCl centrifugation, and did not reattach to uninfected cells. Electrophoretic studies of sodium dodecyl sulfate-disrupted B3 virus in sodium dodecyl sulfate-polyacrylamide gels revealed four radioactive virus polypeptides (VP 1 to 4), of which the three largest migrated slightly faster than their poliovirus T1 counterparts. In contrast, electrophoretic analysis of eluted virus, after banding in a tartrate gradient or pelleting by centrifugation, showed the absence of the fastest migrating polypeptide, VP 4. VP 4 was recovered in the supernatant fluid when the eluted virions were removed by high-speed centrifugation. The results indicate that VP 4 is located at the surface of the native virion, and its dissociation from the capsid may represent the first specific alteration of the virion after virus-receptor interaction at the cell surface.

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.

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.

Proteolytic cleavage of VP1 in 'A' particles of coxsackievirus B3 does not appear to mediate virus uncoating by HeLa cells.

'A' particle of Coxsackievirus B3 were generated from native virus by heating and purified by sucrose gradient centrifugation. These particles were found to be similar to 'A' particles formed by elution from cellular receptors of HeLa cells. Electrophoretic analysis of [35S]methionine-labelled 'A' particles revealed that treatment of the particles with chymotrypsin resulted in the cleavage of VP1 and the formation of a cleavage product which migrated between VP2 and VP3. Analysis of the protease-treated material on sucrose gradients revealed a ribonuclease-sensitive particle which sedimented more slowly than an 'A' particle. This particle apparently degraded to release the viral RNA, thereby providing an in vitro model for protease-mediated uncoating of 'A' particles. The subviral particles of Coxsackievirus B3 were found to be immunoprecipitable with heterotypic Coxsackievirus group B antisera, thereby providing a method for the recovery of products produced in the cell early in infection. Infected cells which had been treated to remove unreacted virus were disrupted, an the lysates were reacted with heterotypic antisera. Analysis of the precipitated material revealed that no cleavage products were formed and no polypeptides were lost. Therefore, it appears that proteolysis is not involved in the uncoating of Coxsackievirus B3 in infected cells.

Acquisition of susceptibility to coxsackievirus A2 by the rat L8 cell line during myogenic differentiation.

Coxsackievirus A2 was propagated to high titres in the established rat L8 myogenic cell line during the stage of differentiation characterized by fusion of myoblasts into multinucleated myotubes (192 h post-plating cultures). In contrast, pre-fusion mononucleated L8 cultured infected 24 h after plating were refractory to Coxsackievirus A2 infection, as was a non-fusing clonal variant of the L8 line (L8CL3-U) regardless of the age of the latter cultures. The development of virus susceptibility in the older L8 cultures correlated with a marked virus-specific c.p.e. as evidenced by vacuolated degenerating myotubes with disrupted cytoplasm, whereas no c.p.e. was seen following infection of the young L8 or variant L8CL3-U cultures. The restriction of Coxsackievirus A2 replication in young L8 and L8CL3-U cultures occurred after virus attachment.