SUMMARYCell lines of primate origin carry receptors on their plasma membrane which are responsible for the specific binding of poliovirus.This paper describes the isolation and characterization of a monoclonal antibody reacting with the plasma membrane of HeLa ceils. The antibody (D171) was selected for its protection of HeLa cells against the cytopathic effect of poliovirus type 1. This protection was found to extend to all three viral serotypes, while the replication of five other viruses in HeLa cells was not affected. The 125i_labelled purified antibody did not react with cell lines derived from pig, dog or rodents but bound specifically to all lines of human or primate origin. Immunoglobulin or Fab fragments of DI71 prevented the binding of 35S-labelled poliovirus to HeLa cells. Conversely, nearly all binding sites of 125I-labelled D171 immunoglobulins or Fab fragments could be blocked after preincubation of HeLa cells with poliovirus. These results indicate that D 171 recognizes the poliovirus receptor site on different susceptible cells and that practically all D171 binding sites are involved in the specific attachment of poliovirus to the plasma membrane. To determine whether the epitope recognized by D171 could be separated from the receptor for poliovirus, human-mouse cell hybrids were prepared and analysed. In all 40 clones tested, the susceptibility to poliovirus correlated with the binding of D171.

Protein synthesis, amino acid uptake, membrane potential, cell volume, Na+ and K+ levels, and ATPase (Na+,K+ activated; EC 3.6.1.3) activity were investigated in control and poliovirus-infected HeLa cells. Inhibition of protein synthesis was first observed 60 min postinfection and reached a maximum at 120 min. The onset of protein synthesis inhibition coincided with a decrease in cell volume and with an elevation of ATPase activity in isolated HeLa cell membranes. Some 3 h after virus adsorption, ATPase activity was inhibited, the Na+-K+ gradient of the cell collapsed, both membrane potential-dependent tetraphenylphosphonium ion uptake and amino acid uptake were reduced, and the cell volume increased. These results provide further experimental support for the hypothesis that modification of the cell membrane plays an important role in the strategy of cytopathogenic viruses in the shutoff of host metabolism and cell death.

SUMMARYSeveral triphosphates (TP) of 5-substituted deoxyuridine (dU), like 5-ethyl (Et), 5-n-propyl (n-Pr), 5-iso-propyl (iso-Pr), 5-n-hexyl (n-Hx), and 5-trifluorothymidine (Fa-dT) were used as substrates for HeLa DNA polymerase a and for two herpes simplex virus (HSV)-coded DNA polymerases isolated from HeLa cells infected with HSV-1, strain C42 (wild-type), or its mutant resistant to phosphonoformate (PFAr). All polymerases were purified up to the DNA-cellulose column step and they showed comparable specific activities. The incorporation into DNA studied with all the alkyl analogues of dUTP is several times higher with the virus enzymes than with DNA polymerase a. The DNA polymerase of the mutant virus incorporates dUTP analogues to a lower extent than the wild-type polymerase. The two virus enzymes also differ in the K m and Vma X values for different substrates, indicating that the mutation to PFA r has affected the structure of the virus DNA polymerase. Surprisingly, all three enzymes use Fa-dTTP as substrate for DNA synthesis to an equal but limited extent.

After exposure of HeLa cells to poliovirus there is a rapid decline (within minutes) in fluorescence polarization of DPH (1,6-diphenyl-1,3,5-hexatriene). Within one hour after infection the (Na+/K+)ATPase activity of an isolated plasma-membrane-rich fraction is enhanced, the cell volume decreases, and the intracellular concentration of a potent low-molecular-weight inhibitor of host protein synthesis increases.

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