Homolog-scanning mutagenesis reveals poliovirus receptor residues important for virus binding and replication.

Poliovirus initiates infection of primate cells by binding to the poliovirus receptor, Pvr. Mouse cells do not bind poliovirus but express a Pvr homolog, Mph, that does not function as a poliovirus receptor. Previous work has shown that the first immunoglobulin-like domain of the Pvr protein contains the virus binding site. To further identify sequences of Pvr important for its interaction with poliovirus, stable cell lines expressing mutated Pvr molecules were examined for their abilities to bind virus and support virus replication. Substitution of the amino-terminal domain of Mph with that of Pvr yields a molecule that can function as a poliovirus receptor. Cells expressing this chimeric receptor have normal binding affinity for poliovirus, yet the kinetics of virus replication are delayed. Results of virus alteration assays indicate that this chimeric receptor is defective in converting native virus to 135S altered particles. This defect is not observed with cells expressing receptor recombinants that include Pvr domains 1 and 2. Because altered particles are believed to be an intermediate in poliovirus entry, these findings suggest that Pvr domains 2 and 3 participate in early stages of infection. Additional mutants were made by substituting variant Mph residues for the corresponding residues in Pvr. The results were interpreted by using a model of Pvr predicted from the known structures of other immunoglobulin-like V-type domains. Analysis of stable cell lines expressing the mutant proteins revealed that virus binding is influenced by mutations in the predicted C'-C" loop, the C" beta-strand, the C"-D loop, and the D-E loop. Mutations in homologous regions of the immunoglobulin-like CD4 molecule alter its interaction with gp120 of human immunodeficiency virus type 1. Cells expressing Pvr mutations on the predicted C" edge do not develop cytopathic effect during poliovirus infection, suggesting that poliovirus-induced cytopathic effect may be induced by the virus-receptor interaction.     

Kinetics of poliovirus uncoating in HeLa cells in a nonacidic environment.

Lysis of HeLa cells infected with poliovirus revealed intact virus; 135S particles, devoid of VP4 but containing the viral RNA; and 80S empty capsids. During infection the kinetics of poliovirus uncoating showed a continuous decrease of intact virus, while the number of 135S particles and empty shells increased. After 1.5 h of infection conformational transition to altered particles resulted in complete disappearance of intact virions. To investigate the mechanism of poliovirus uncoating, which has been suggested to depend on low pH in endosomal compartments of cells, we used lysosomotropic amines to raise the pH in these vesicles. In the presence of ammonium chloride, however, the kinetics of uncoating were similar to those for untreated cells, whereas in cells treated with methylamine, monensin, or chloroquine, uncoating was merely delayed by about 30 min. This effect could be attributed to a delay of virus entry into cells after treatment with methylamine and monensin, whereas chloroquine stabilized the viral capsid itself. Thus, elevation of endosomal pH did not affect virus uncoating. We therefore propose a mechanism of poliovirus uncoating which is independent of low pH.     

Viral aggregation: mixed suspensions of poliovirus and reovirus.

The aggregation of mixtures of two dissimilar viruses, poliovirus I (Mahoney) and reovirus III (Dearing), was followed by electron microscopy under conditions known to induce either aggregation or dispersion of each virus separately. Neither virus aggregated at pH 7 in an appropriate buffer, and no mixed aggregates were formed. Under conditions of lowered ionic strength (by dilution into distilled water) poliovirus became aggregated, whereas reovirus did not, and again no mixed aggregates were formed. At pH 6, however, poliovirus again aggregated and, although reovirus did not, it attached to poliovirus aggregates. Thus, some inducement toward aggregation was necessary to cause formation of mixed aggregates. This inducement probably took the form of a reduction of the ionic double layer surrounding the particles, which is known to occur at low pH. At pH 5 and below both viruses aggregated severely, and large mixed aggregates were formed. These mixed aggregates could be broken up by neutralization of the suspension, although small aggregates of poliovirus remained. Reovirus showed a marked tendency to attach to large clumps of poliovirus, but the reverse tendency was not observed. The results indicate that mixed aggregates may be of significance in the isolation of viruses from water or wastewater.     

Viral aggregation: mixed suspensions of poliovirus and reovirus.

The aggregation of mixtures of two dissimilar viruses, poliovirus I (Mahoney) and reovirus III (Dearing), was followed by electron microscopy under conditions known to induce either aggregation or dispersion of each virus separately. Neither virus aggregated at pH 7 in an appropriate buffer, and no mixed aggregates were formed. Under conditions of lowered ionic strength (by dilution into distilled water) poliovirus became aggregated, whereas reovirus did not, and again no mixed aggregates were formed. At pH 6, however, poliovirus again aggregated and, although reovirus did not, it attached to poliovirus aggregates. Thus, some inducement toward aggregation was necessary to cause formation of mixed aggregates. This inducement probably took the form of a reduction of the ionic double layer surrounding the particles, which is known to occur at low pH. At pH 5 and below both viruses aggregated severely, and large mixed aggregates were formed. These mixed aggregates could be broken up by neutralization of the suspension, although small aggregates of poliovirus remained. Reovirus showed a marked tendency to attach to large clumps of poliovirus, but the reverse tendency was not observed. The results indicate that mixed aggregates may be of significance in the isolation of viruses from water or wastewater.     

Neutralization of poliovirus by cell receptors expressed in insect cells.

To examine the interaction of the poliovirus receptor (PVR) with virus and the role of the PVR in virus entry, the PVR was expressed in insect cells. Poliovirus bound to insect cells infected with a recombinant baculovirus (AcPVR) carrying cDNA encoding the PVR. Antibodies raised against PVR expressed in bacteria immunoprecipitated a 67-kilodalton polypeptide from cytoplasmic extracts of AcPVR-infected cells. Treatment of AcPVR-infected cells with tunicamycin revealed that the PVR is a glycoprotein containing N-glycosidic linkages and that carbohydrate accounts for nearly 50% of its molecular weight as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When PVR was solubilized from AcPVR-infected insect cells and incubated with poliovirus, viral infectivity was neutralized. Sedimentation analysis revealed that irreversibly altered 135S particles were formed after incubation of poliovirus at 37 degrees C with solubilized extracts of AcPVR-infected insect cells. These results demonstrate that poliovirus eclipse may result from interaction with the cell receptor at neutral pH in the absence of membranes and suggest that soluble receptors may be effective antiviral agents against picornaviruses.     

The poliovirus 135S particle is infectious.

The molecular mechanism of cell entry by unenveloped viruses is poorly understood. The picornaviruses poliovirus, human rhinovirus, and coxsackievirus convert to an altered form (the 135S or A particle) upon interaction with receptors on susceptible cells at 37 degrees C. The 135S particle is thought to be a necessary intermediate because it accumulates inside susceptible cells soon after infection and drugs which inhibit conversion of the virus to this form also prevent infection. However, since a variable fraction of the altered 135S particles is reported to elute unproductively from the surface of susceptible cells, their precise role remains unclear. We have found that poliovirus 135S particles can infect Chinese hamster ovary (CHO) and murine L cells, neither of which are susceptible to infection by native poliovirus. The infectivity of the particles in tissue culture appears to be between 10(3) to 10(5) times less than that of poliovirus on HeLa cells. The 135S particle infectivity was not sensitive to RNase but was greatly reduced by proteolytic treatment. Proteolysis specifically removed the newly exposed N terminus of VP1, a feature which has previously been shown to mediate interactions of the particle with lipid membranes. These results demonstrate that although the infectivity of the 135S particle appears to be receptor independent, it nonetheless requires some property associated with the protein coat. In particular, the N terminus of VP1 plays an important role in the infection process. Our findings are consistent with the hypothesis that the 135S particle is an intermediate in the normal cell entry pathway of poliovirus infection.     

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

The effect of a number of drugs and culture conditions on the entry into cells of a strain of poliovirus 1 (Brunende) was tested. The cells were exposed in the dark to light-sensitive, neutral red-containing virus, in the presence of the drug to be tested. Then the cells were exposed to light, transferred to normal medium, and incubated overnight. Cytopathogenic effect was measured as inhibition of [3H]leucine incorporation. Compounds that dissipate proton gradients across membranes, like monensin, protonophores, and amines, and compounds that inhibit the acidification process, such as N,N'- dicyclohexylcarbodiimide (DCCD) and tributyltin, inhibited the entry of virus, but not virus binding. This was also the case with metabolic inhibitors that deplete cells for ATP. The same compounds also inhibited the cell-induced alteration of the virus particles. When cells with surface-bound virus were exposed to low pH, the virus entered efficiently, even in the presence of monensin and DCCD. The results indicate that acidification somehow facilitates the entry of the virus RNA into the cytosol and that under normal conditions the entry occurs from intracellular acidic vesicles.     

Pathway of vesicular stomatitis virus entry leading to infection

The entry of vesicular stomatitis virus into Madin-Darby canine kidney (MDCK) cells was examined both biochemically and morphologically. At low multiplicity and 0 °C, viruses bound to the cell surface but were not internalized. Binding was very dependent on pH. More than ten times more virus bound at pH 6.5 than at higher pH values. At the optimal pH, binding failed to reach equilibrium after more than two hours. The proportion of virus bound was irreproducible and low, relative to the binding of other enveloped viruses. Over 90% of the bound viruses were removed by proteases. When cells with pre-bound virus were warmed to 37 °C, a proportion of the bound virus became protease-resistant with a half-time of about 30 minutes. After a brief lag period, degraded viral material was released into the medium. The protease-resistant virus was capable of infecting the cells and probably did so by an intracellular route, since ammonium chloride blocked the infection and slightly reduced the degradation of viral protein.When the entry process was observed by electron microscopy, viruses were seen bound to the cell surface at 0 °C and, after warming at 37 °C, within coated pits, coated vesicles and larger, smooth-surfaced vesicles. No fusion of the virus with the plasma membrane was observed at pH 7.4.When pre-bound virus was incubated at a pH below 6 for 30 seconds at 37 °C, about 40 to 50% of the pre-bound virus became protease-resistant. On the basis of this result and previously published experiments (White et al., 1981), it was concluded that vesicular stomatitis virus fuses to the MDCK cell plasma membrane at low pH.These experiments suggest that vesicular stomatitis virus enters MDCK cells by endocytosis in coated pits and coated vesicles, and is transported to the lysosome where the low pH triggers a fusion reaction ultimately leading to the transfer of the genome into the cytoplasm. The entry pathway of vesicular stomatitis virus thus resembles that described earlier for both Semliki Forest virus and fowl plague virus.     

The clathrin endocytic pathway in viral infection.

How important is the clathrin-dependent endocytic pathway for entry of viruses into host cells? While it is widely accepted that Semliki Forest virus (SFV), an enveloped virus, requires this pathway there are conflicting data concerning the closely related Sindbis virus, as well as varying results with picornaviruses such as human rhinovirus 14 (HRV 14) and poliovirus. We have examined the entry mode of SFV, Sindbis virus, HRV 14 and poliovirus using a method that identifies single infected cells. This assay takes advantage of the observation that the clathrin-dependent endocytic pathway is specifically and potently arrested by overexpression of dynamin mutants that prevent clathrin-coated pit budding. Using HeLa cells and conditions of low multiplicity of infection to favor use of the most avid pathway of cell entry, it was found that SFV, Sindbis virus and HRV 14 require an active clathrin-dependent endocytic pathway for successful infection. In marked contrast, infection of HeLa cells by poliovirus did not appear to require the clathrin pathway.     

Influence of salts on electrostatic interactions between poliovirus and membrane filters.

Neither solutions of salts nor solutions of detergents or of an alcohol at pH 4 are capable of eluting poliovirus adsorbed to membrane filters. However, solutions containing both a salt, such as magnesium chloride or sodium chloride, and a detergent or alcohol at pH 4 were capable of eluting adsorbed virus. The ability of ions to promote elution of virus at low pH in the presence of detergent or alcohol was dependent on the size of the ions and the ionic strength of the medium. These results suggest that both electrostatic and hydrophobic interactions are important in maintaining virus adsorption to membrane filters. Hydrophobic interactions can be disrupted by detergents or alcohols. It appears that electrostatic interactions can be disrupted by raising the pH of a solution or by adding certain salts. Disruption of either electrostatic or hydrophobic interactions alone does not permit efficient elution of the adsorbed virus at low pHs. However, when both interactions are disrupted, most of the poliovirus adsorbed to membrane filters is eluted, even at pH 4.     

Influence of salts on electrostatic interactions between poliovirus and membrane filters

Neither solutions of salts nor solutions of detergents or of an alcohol at pH 4 are capable of eluting poliovirus adsorbed to membrane filters. However, solutions containing both a salt, such as magnesium chloride or sodium chloride, and a detergent or alcohol at pH 4 were capable of eluting adsorbed virus. The ability of ions to promote elution of virus at low pH in the presence of detergent or alcohol was dependent on the size of the ions and the ionic strength of the medium. These results suggest that both electrostatic and hydrophobic interactions are important in maintaining virus adsorption to membrane filters. Hydrophobic interactions can be disrupted by detergents or alcohols. It appears that electrostatic interactions can be disrupted by raising the pH of a solution or by adding certain salts. Disruption of either electrostatic or hydrophobic interactions alone does not permit efficient elution of the adsorbed virus at low pHs. However, when both interactions are disrupted, most of the poliovirus adsorbed to membrane filters is eluted, even at pH 4.     

Variant influenza virus hemagglutinin that induces fusion at elevated pH.

The hemagglutinin (HA) glycoprotein of influenza virus performs two critical roles during infection: it binds virus to cell surface sialic acids, and under mildly acidic conditions it induces fusion of the virion with intracellular membranes, liberating the genome into the cytoplasm. The pH dependence of fusion varies for different influenza virus strains. Here we report the isolation and characterization of a naturally occurring variant of the X31 strain that fuses at a pH 0.2 units higher than the parent strain does and that is less sensitive to the effects of ammonium chloride, a compound known to elevate endosomal pH. The bromelain-solubilized ectodomain of the variant HA displayed a corresponding shift in the pH at which it changed conformation and bound to liposomes. Cloning and sequencing of the variant HA gene revealed amino acid substitutions at three positions in the polypeptide. Two substitutions were in antigenic determinants in the globular region of HA1, and the third occurred in HA2 near the base of the molecule. By using chimeric HA molecules expressed in CV-1 cells from simian virus 40-based vectors, we demonstrated that the change in HA2 was solely responsible for the altered fusion phenotype. This substitution, asparagine for aspartic acid at position 132, disrupted a highly conserved interchain salt bridge between adjacent HA2 subunits. The apparent role of this residue in stabilizing the HA trimer is consistent with the idea that the trimer dissociates at low pH. Furthermore, the results demonstrate that influenza virus populations contain fusion variants, raising the possibility that such variants may play a role in the evolution of the virus.     

Cold-adapted poliovirus mutants bypass a postentry replication block.

In the current model of poliovirus entry, the initial interaction of the native virion with its cellular receptor is followed by a transition to an altered form, which then acts as an intermediate in viral entry. While the native virion sediments at 160S in a sucrose gradient, the altered particle sediments at 135S, has lost the coat protein VP4, and has become more hydrophobic. Altered particles can be found both associated with cells and in the culture medium. It has been hypothesized that the cell-associated 135S particle releases the viral genome into the cell cytoplasm and that nonproductive transitions to the 135S form are responsible for the high particle-to-PFU ratio observed for polioviruses. At 25 degrees C, a temperature at which the transition to 135S particles does not occur, the P1/Mahoney strain of poliovirus was unable to replicate, and cold-adapted (ca) mutants were selected from the population. These mutants have not gained the ability to convert to 135S particles at 25 degrees C, and the block to wild-type (wt) infection at low temperatures is not at the level of cellular entry. The particle-to-PFU ratio of poliovirus does not change at 25 degrees C in the absence of alteration. Three independent amino acid changes in the 2C coding region were identified in ca mutants, at positions 218 (Val to Ile), 241 (Arg to Ala), and 309 (Met to Val). Introduction of any of these mutations individually into wt poliovirus by site-directed mutagenesis confers the ca phenotype. All three serotypes of the Sabin vaccine strains and the P3/Leon strain of poliovirus also exhibit the ca phenotype. These results do not support a model of poliovirus entry into cells that includes an obligatory transition to the 135S particle.     

Formation and activity of covalent conjugates of poliovirus and ligands binding to cell surface structures

Disulfide-linked conjugates of poliovirus with streptavidin or concanavalin A were formed and the binding of the conjugates to mouse L cells that lack natural poliovirus receptors was studied. The conjugate with streptavidin was specifically bound to biotinylated L cells, but not to unmodified L cells. The conjugate with conA was bound to L cells in the absence of, but not in the presence of alpha-methyl mannoside. Incubation of L cells with bound conjugates did not produce virus, although the conjugates were highly infectious in HeLa cells, containing natural poliovirus receptors. This suggests that the artificially bound virus was unable to penetrate the L cells and start replication. The possibility that binding of the virus to the natural receptor is required for efficient infection is discussed.     

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.     

Requirement for vacuolar proton-ATPase activity during entry of influenza virus into cells.

The role that endosomal acidification plays during influenza virus entry into MDCK cells has been analyzed by using the macrolide antibiotics bafilomycin A1 and concanamycin A as selective inhibitors of vacuolar proton-ATPase (v-[H+]ATPase), the enzyme responsible for the acidification of endosomes. Bafilomycin A1 and concanamycin A, present at the low concentrations of 5 x 10(-7) and 5 x 10(-9) M, respectively, prevented the entry of influenza virus into cells when added during the first minutes of infection. Attachment of virion particles to the cell surface was not the target for the action of bafilomycin A1. N,N'-Dicyclohexylcarbodiimide, a nonspecific inhibitor of proton-ATPases, also blocked virus entry, whereas elaiophylin, an inhibitor of the plasma-proton ATPase, had no effect. The inhibitory actions of bafilomycin A1 and concanamycin A were tested in culture medium at different pHs. Both antibiotics powerfully prevented influenza virus infection when the virus was added under low-pH conditions. This inhibition was reduced if the virus was bound to cells at 4 degrees C prior to the addition of warm low-pH medium. Moreover, incubation of cells at acidic pH potently blocked influenza virus infection, even in the absence of antibiotics. These results indicate that a pH gradient, rather than low pH, is necessary for efficient entry of influenza virus into cells.     

Soluble receptor-resistant poliovirus mutants identify surface and internal capsid residues that control interaction with the cell receptor.

Poliovirus initiates infection by binding to its cell receptor and undergoing a receptor-mediated conformational alteration. To identify capsid residues that control these interactions, we have isolated and characterized poliovirus mutants that are resistant to neutralization by a soluble form of the poliovirus receptor. Twenty one soluble receptor-resistant (srr) mutants were identified which still use the poliovirus receptor to infect cells. All but one srr mutant contain a single amino acid change at one of 13 different positions, either on the surface or in the interior of the virion. The results of binding and alteration assays demonstrate that both surface and internal capsid residues regulate attachment to the receptor and conformational change of the virus. Mutations that reduce alteration also affect receptor binding, suggesting a common structural basis for early events in poliovirus infection.     

Hydrolytic enzymes in KB cells infected with poliovirus and herpes simplex virus

Flanagan, John F. (Duke University School of Medicine, Durham. N.C.). Hydrolytic enzymes in KB cells infected with poliovirus and herpes simplex virus. J. Bacteriol. 91:789-797. 1966.-The effect of poliovirus and herpes simplex virus infection on the activity of five hydrolytic enzymes was studied in tissue culture cells of KB type. During the course of poliovirus infection, the activity of beta-glucuronidase, acid protease, acid ribonuclease, acid deoxyribonuclease, and acid phosphatase in the cytoplasm rose to levels two- to fourfold greater than the activity present in the cytoplasm of uninfected cells. The rise in cytoplasmic activity was accompanied by a concomitant decrease in enzymatic activity bound to cell particles. Shift of enzymatic activity from the particulate to soluble state was first detected at 6 hr after poliovirus infection, coinciding with the appearance of new infectious particles and virus cytopathic effect. No net synthesis of these enzymes after poliovirus infection was found. Hydrocortisone added to the culture medium failed to affect either the titer of virus produced in the cells or the release of hydrolytic enzymes from the particulate state. Herpes simplex infection produced minimal alterations in the state of these enzymes in KB cells. It is hypothesized that the breakdown of lysosomes and release of hydrolytic enzymes accompanying poliovirus infection is produced by alterations in cell membrane permeability during the course of virus replication and by the consequent change in the ionic content of the cell sap.     

Infectious entry pathway of influenza virus in a canine kidney cell line

The entry of fowl plague virus, and avian influenza A virus, into Madin- Darby canine kidney (MDCK) cells was examined both biochemically and morphologically. At low multiplicity and 0 degrees C, viruses bound to the cell surface but were not internalized. Binding was not greatly dependent on the pH of the medium and reached an equilibrium level in 60-90 min. Over 90% of the bound viruses were removed by neuraminidase but not by proteases. When cells with prebound virus were warmed to 37 degrees C, part of the virus became resistant to removal b neuraminidase, with a half-time of 10-15 min. After a brief lag period, degraded viral material was released into the medium. The neuraminidase- resistant virus was capable of infecting the cells and probably did so by an intracellular route, since ammonium chloride, a lysosomotropic agent, blocked both the infection and the degradation of viral protein. When the entry process was observed by electron microscopy, viruses were seen bound primarily to microvilli on the cell surface at 0 degrees C and, after warming at 37 degrees C, were endocytosed in coated pits, coated vesicles, and large smooth-surfaced vacuoles. Viruses were also present in smooth-surfaced invaginations and small smooth-surfaced vesicles at both temperatures. At physiological pH, no fusion of the virus with the plasma membrane was observed. When prebound virus was incubated at a pH of 5.5 or below for 1 min at 37 degrees C, fusion was, however, detected by ferritin immunolabeling. t low multiplicity, 90% of the prebound virus became neuraminidase- resistant and was presumably fused after only 30 s at low pH. These experiments suggest that fowl plague virus enters MDCK cells by endocytosis in coated pits and coated vesicles and is transported to the lysosome where the low pH initiates a fusion reaction ultimately resulting in the transfer of the genome into the cytoplasm. The entry pathway of fowl plague virus thus resembles tht earlier described for Semliki Forest virus.