Physical and metabolic requirements for early interaction of poliovirus and human rhinovirus with HeLa cells

Abstract

Attachment, "tight binding," and eclipse of radioactive poliovirus 2 (P2) and human rhinovirus 2 (HRV2) were investigated. The activation energy for attachment of both HRV2 and P2 was about 13 kcal/mol. HRV2 differed from P2 in two respects: the Arrhenius plot for attachment of HRV2 showed a break at 15 to 19°C when the cells were first treated several hours at 0°C, and attachment of HRV2 was inhibited by treatment of cells with metabolic poisons able to reduce cellular ATP by more than 90%. Tight binding was determined by isolation of a specific P2-membrane complex or by loss of EDTA dissociability of HRV2. Tight binding of both viruses was slowed by 0.01 M iodoacetamide but not by 0.02 M F-; F-plus 0.002 M CN-slowed tight binding of HRV2 but not of P2. Eclipse, the irreversible alteration of parental virions, was detected by isolation of cellassociated subviral particles or by loss of cell-associated infectious virus. Eclipse of both viruses is slowed by iodoacetamide or FIt It seems likely that the early steps of infection with picornaviruses may be sensitive to alterations in the cell membrane produced by metabolic inhibitors or by treatment at low temperature.

Mechanism of entry into the cytosol of poliovirus type 1: requirement for low pH.

“…When cells are depleted of ATP by treatment with 2deoxyglucose and NaN3, infection did not occur, indicating that virus did not enter. Similar findings were earlier made with poliovirus type 2 (15). Most likely the protective effect ofATP-depletion is due to inhibition of the endocytic uptake of the virus and inhibition of vesicle acidification .…”

“…The nuclei were removed by centrifugation for 3 min in an Eppendorf centrifuge. SDS (0 .2% wt/vol) was added to the supernatant which was then layered on top of sucrose gradients (15-30% wt/vol) in 0.14 M NaCl, 10 mM Na-phosphate, pH 7.4 (15) . The gradients were centrifuged for I h at 234,000g in a Beckman SW 50.1 rotor (Beckman Instruments, Inc., Palo Alto, CA).…”

Cold-adapted poliovirus mutants bypass a postentry replication block

“…Second, since antiviral compounds which inhibit 135S particle formation also inhibit poliovirus infectivity (6,16,38,56), how does an entry model which does not involve alteration explain the action of these compounds? Alteration to 135S particles has been observed only under artificial conditions, with high concentrations of either cultured cells (29) or solubilized receptor protein (8,23,55), and so it is not known whether 135S particle formation has been subjected to selective pressure in vivo. Second, picornaviruses are small, highly constrained physical entities, and if alteration does occur in normal infections, it may be a by-product of the structural flexibility required to permit cell entry.…”

Poliovirus and its cellular receptor: a molecular genetic dissection of a virus/receptor affinity interaction

“…The effects of virus concentration, buffer components and toxins have been reported (Lonberg-Holm and Philipson, 1980;Crowell and Landau, 1983). For example, increasing the temperature of a PV/host cell binding reaction from 4 to 25°C increases the association rate of PV for its cell surface binding sites (Crowell, 1973;Lonberg-Holm and Whiteley, 1976;Bibb et al, 1994b). It was also determined that there are approximately 3000 PV binding sites on the surface of an HeLa cell, a human cell line that is susceptible to all three viral serotypes (Lonberg-Holm and Philipson, 1974).…”

“…It was also determined that there are approximately 3000 PV binding sites on the surface of an HeLa cell, a human cell line that is susceptible to all three viral serotypes (Lonberg-Holm and Philipson, 1974). In addition, the equilibrium dissociation constant (K d ), the association and disassociation rates and the half-life of the interaction of PV with its cell surface binding site have been determined (Bachtold et al, 1957;McLaren et al, 1959;Holland and McLaren, 1961;Lonberg-Holm and Whiteley, 1976;Bibb et al, 1994b). Most recently, Bibb et al (1994b) re-examined these properties using a receptor/excess silicone oil partition assay.…”