Human poliovirus receptor gene expression and poliovirus tissue tropism in transgenic mice.

Expression of the human poliovirus receptor (PVR) in transgenic mice results in susceptibility to poliovirus infection. In the primate host, poliovirus infection is characterized by restricted tissue tropism. To determine the pattern of poliovirus tissue tropism in PVR transgenic mice, PVR gene expression and susceptibility to poliovirus infection were examined by in situ hybridization. PVR RNA is expressed in transgenic mice at high levels in neurons of the central and peripheral nervous system, developing T lymphocytes in the thymus, epithelial cells of Bowman's capsule and tubules in the kidney, alveolar cells in the lung, and endocrine cells in the adrenal cortex, and it is expressed at low levels in intestine, spleen, and skeletal muscle. After infection, poliovirus replication was detected only in neurons of the brain and spinal cord and in skeletal muscle. These results demonstrated that poliovirus tissue tropism is not governed solely by expression of the PVR gene nor by accessibility of cells to virus. Although transgenic mouse kidney tissue expressed poliovirus binding sites and was not a site of poliovirus replication, when cultivated in vitro, kidney cells developed susceptibility to infection. Identification of the changes in cultured kidney cells that permit poliovirus infection may provide information on the mechanism of poliovirus tissue tropism.     

Growth of poliovirus in HeLa cells persistently infected with HVJ (Sendai virus)

The growth of poliovirus in a HeLa cell culture persistently infected with the hemagglutinating virus of Japan (HVJ, the Sendai strain of parainfluenza 1 virus) (HeLaHVJ) was studied. Plaques produced by poliovirus on HeLaHVJ cell monolayers were hazier, smaller and fewer than those on HeLa cells. HeLaHVJ cells were indistinguishable from normal HeLa cells with respect to adsorption rate and penetration efficiency of poliovirus. Extracellular yields of poliovirus in HeLaHVJ cells were lower, and the cytopathic changes were less than those in normal HeLa cells, while cell-associated virus growth in HeLaHVJ cells was nearly equal to that in HeLa cells. HeLaHVJ cells responded more effectively to the action of magnesium chloride, which facilitates virus release from infected cells, resulting in an cytopathic effects. No reduction in poliovirus yield could be detected in HeLa cells acutely infected with HVJ. The relationship between the inhibition of the release of poliovirus from HeLaHVJ cells and the persistent infection of the cells with HVJ is discussed.     

Translation of capped viral mRNAs in poliovirus-infected HeLa cells.

HeLa cells doubly infected with Semliki Forest virus (SFV) and poliovirus synthesize either more poliovirus proteins or more SFV late proteins depending on the time of super-infection with poliovirus. Under some conditions, the infected cells translate uncapped poliovirus mRNA and capped 26S mRNA from SFV simultaneously, even though host protein synthesis has been shut down. Vesicular stomatitis virus (VSV) protein synthesis is depressed drastically when VSV-infected cells are super-infected with poliovirus. In cells doubly infected with VSV and encephalomyocarditis (EMC) virus or with VSV and SFV, dominance of one of the viruses depends on the time of addition of the challenge virus. The influence of external conditions on the relative translation of capped or uncapped viral mRNA in doubly infected cells has also been analysed.     

CD44 is not required for poliovirus replication.

The identification of a monoclonal antibody, AF3, which recognizes a single isoform of the cell surface protein CD44 and preferentially blocks binding of serotype 2 poliovirus to HeLa cells, suggested that CD44 might be an accessory molecule to Pvr, the cell receptor for poliovirus, and that it could play a role in the function of the poliovirus receptor site. We show here that only AF3 blocks binding of serotype 2 poliovirus to HeLa cells and, in contrast to a previously published report, that the anti-CD44 monoclonal antibodies A3D8 and IM7 are unable to block binding of poliovirus. To determine whether CD44 is involved in poliovirus infection, we analyzed the replication of all three serotypes of poliovirus in human neuroblastoma cells which lack or express CD44 and in mouse neuroblastoma cells which lack Pgp-1, the mouse homolog of human CD44, and which express Pvr. All three poliovirus serotypes replicate with normal kinetics and to normal levels in the absence or presence of CD44 or in the absence of Pgp-1. Furthermore, the binding affinity constants of all three poliovirus serotypes for Pvr are unaffected by the presence or absence of CD44 in the human neuroblastoma cell line. We conclude that CD44 and Pgp-1 are not required for poliovirus replication and are unlikely to be involved in poliovirus pathogenesis.     

Monensin and nigericin prevent the inhibition of host translation by poliovirus, without affecting p220 cleavage.

Addition of monensin or nigericin after poliovirus entry into HeLa cells prevents the inhibition of host protein synthesis by poliovirus. The infected cells continue to synthesize cellular proteins at control levels for at least 8 h after infection in the presence of the ionophore. Cleavage of p220 (gamma subunit of eukaryotic initiation factor 4 [eIF-4 gamma]), a component of the translation initiation factor eIF-4F, occurs to the same extent in poliovirus-infected cells whether or not they are treated with monensin. Two hours after infection there is no detectable intact p220, but the cells continue to translate cellular mRNAs for several hours at levels similar to those in uninfected cells. Nigericin or monensin prevented the arrest of host translation at all the multiplicities of poliovirus infection tested. At high multiplicities of infection, an unprecedented situation was found: cells synthesized poliovirus and cellular proteins simultaneously. Superinfection of vesicular stomatitis virus-infected HeLa cells with poliovirus led to a profound inhibition of vesicular stomatitis virus protein synthesis, while nigericin partially prevented this blockade. Drastic inhibition of translation also took place in influenza virus-infected Vero cells treated with nigericin and infected with poliovirus. These findings suggest that the translation of newly synthesized mRNAs is dependent on the integrity of p220, while ongoing cellular protein synthesis does not require an intact p220. The target of ionophore action during the poliovirus life cycle was also investigated. Addition of nigericin at any time postinfection profoundly blocked the synthesis of virus RNA, whereas viral protein synthesis was not affected if nigericin was added at 4 h postinfection. These results agree well with previous findings indicating that inhibitors of phospholipid synthesis or vesicular traffic interfere with poliovirus genome replication. Therefore, the action of nigericin on the vesicular system may affect poliovirus RNA synthesis. In conclusion, monensin and nigericin are potent inhibitors of poliovirus genome replication that prevent the shutoff of host translation by poliovirus while still permitting cleavage of p220.     

A poliovirus 2A(pro) mutant unable to cleave 3CD shows inefficient viral protein synthesis and transactivation defects.

Four poliovirus mutants with modifications of tyrosine 88 in 2A(pro) were generated and introduced into the cloned poliovirus genome. Mutants Y88P and Y88L were nonviable, mutant Y88F showed a wild-type (WT) phenotype, and mutant Y88S showed a delayed cytopathic effect and formed small plaques in HeLa cells. Growth of Y88S in HeLa cells was restricted, giving rise to about 20% of the PFU production of the WT poliovirus. The 2A (Y88S) mutant synthesized significantly lower levels of viral proteins in HeLa cells than did the WT poliovirus, while the kinetics of p220 cleavage were identical for both viruses. Strikingly, the 2A (Y88S) mutant was unable to cleave 3CD, as shown by analysis of poliovirus proteins labeled with [35S]methionine or immunoblotted with a specific anti-3C serum. The ability of the Y88S mutant to form infectious virus and cleave 3CD can be complemented by the WT poliovirus. Synthesis of viral RNA was diminished in the Y88S mutant but less than the inhibition of translation of viral RNA. Experiments in which guanidine was used to inhibit poliovirus RNA synthesis suggest that the primary defect of the Y88S mutant virus is at the level of poliovirus RNA translation, while viral genome replication is much less affected. Transfection of HeLa cells infected with the WT poliovirus with a luciferase mRNA containing the poliovirus 5' untranslated sequence gives rise to a severalfold increase in luciferase activity. This enhanced translation of leader-luc mRNA was not observed when the transfected cells were infected with the 2A (Y88S) mutant. Moreover, cotransfection with mRNA encoding WT poliovirus 2A(pro) enhanced translation of leader-luc mRNA. This enhancement was much lower upon transfection with mRNA encoding 2A(Y88S), 2A(Y88L), or 2A(Y88P). These findings support the view that 2A(pro) itself, rather than the 3C' and/or 3D' products, is necessary for efficient translation of poliovirus RNA in HeLa cells.     

Entry of poliovirus into cells is blocked by valinomycin and concanamycin A

Poliovirus contains a virus particle devoid of a lipid envelope that does not require an intact pH to enter into susceptible cells. Thus, the blockade of pH gradient generated in endosomes is not sufficient to impede the translocation of poliovirus particles to the cytoplasm, suggesting that translocation takes place at the plasma membrane. Measuring both viral protein synthesis and eIF4G-1 cleavage mediated by poliovirus protease 2A has been used to monitor productive entry of poliovirus into cells. Translation of the input poliovirus RNA produces enough 2A(pro) to cleave eIF4G-1, providing a sensitive assay to estimate poliovirus RNA delivery to the cytoplasm followed by its translation. Combination of concanamycin A, a vacuolar proton-ATPase inhibitor, and valinomycin, an ionophore that promotes K(+) efflux from cells, powerfully prevented poliovirus infection. Moreover, modifying the ionic conditions of the culture medium (increasing the concentration of K(+) and decreasing the concentration of Na(+)), together with concanamycin A, also significantly interfered with poliovirus entry. These findings suggest that poliovirus RNA requires an intact concentration of K(+) ions inside the cells to be uncoated and to gain access to the cytoplasm. In addition, the actual contribution of concanamycin A (as well as other inhibitors of endocytosis) to the total inhibition of productive poliovirus entry points to the idea that at least some percentage of polioviral subparticles translocates from the endosomes.     

Coinfection with recombinant vaccinia viruses expressing poliovirus P1 and P3 proteins results in polyprotein processing and formation of empty capsid structures.

The assembly process of poliovirus occurs via an ordered proteolytic processing of the capsid precursor protein, P1, by the virus-encoded proteinase 3CD. To further delineate this process, we have isolated a recombinant vaccinia virus which expresses, upon infection, the poliovirus P1 capsid precursor polyprotein with an authentic carboxy terminus. Coinfection of HeLa cells with the P1-expressing vaccinia virus and with a second recombinant vaccinia virus which expresses the poliovirus proteinase 3CD resulted in the correct processing of P1 to yield the three individual capsid proteins VP0, VP3, and VP1. When extracts from coinfected cells were fractionated on sucrose density gradients, the VP0, VP3, and VP1 capsid proteins were immunoprecipitated with type 1 poliovirus antisera from fractions corresponding to a sedimentation consistent for poliovirus 75S procapsids. Examination of these fractions by electron microscopy revealed structures which lacked electron-dense cores and which corresponded in size and shape to those expected for poliovirus empty capsids. We conclude that the expression of the two poliovirus proteins P1 and 3CD in coinfected cells is sufficient for the correct processing of the capsid precursor to VP0, VP3, and VP1 as well as for the assembly of poliovirus empty capsid-like structures.     

Mechanism of Resistance to Enteroviruses of Some Primate Cells in Tissue Culture

Two cell lines, M10-45-2 and L-41, were studied, each of which possessed specific resistance either to poliovirus or to coxsackievirus. Infection of M10-45-2 cells with poliovirus ribonucleic acid (RNA) and L-41 cells with infectious coxsackievirus RNA was accompanied by production of complete viruses in each of the resistant cell lines. During incubation of the cells with the virus to which they were resistant, the amount of infectious virus did not decrease. Treatment with glycine-HCl buffer solution (pH 2.5) of resistant M10-45-2 cells after incubation with poliovirus at 0 C did not result in recovery of infectious virus, although such release did take place after treatment of sensitive M10 cells. Infection of resistant cells with virus containing poliovirus RNA and coxsackievirus proteins resulted in production of poliovirus in M10-45-2 cells but not in L-41 cells. The resistant cells are apparently unable to adsorb the virus to which they are resistant.     

Proteolysis of the p220 component of the cap-binding protein complex is not sufficient for complete inhibition of host cell protein synthesis after poliovirus infection.

Infection of cells with poliovirus results in the complete shutoff of host protein synthesis. It is presumed that proteolysis of the p220 component of the cap-binding protein complex that is required for the translation of host mRNAs is responsible for the shutoff phenomenon. In this paper, we show that when cells are infected with poliovirus in the presence of guanidine or 3-methylquercetin, both inhibitors of poliovirus replication, complete cleavage of p220 occurs by 3.5 h postinfection. However, under these conditions only 55 to 77% of host protein synthesis is suppressed. Results obtained with extracts prepared from poliovirus-infected cells were similar to those obtained in vivo. These results suggest that complete inhibition of host protein synthesis after poliovirus infection requires at least one event in addition to proteolysis of p220. Thus, proteolysis of p220 is probably necessary but not sufficient for total suppression of host protein synthesis after poliovirus infection.     

Subversion of cellular autophagosomal machinery by RNA viruses

Infection of human cells with poliovirus induces the proliferation of double-membraned cytoplasmic vesicles whose surfaces are used as the sites of viral RNA replication and whose origin is unknown. Here, we show that several hallmarks of cellular autophagosomes can be identified in poliovirus-induced vesicles, including colocalization of LAMP1 and LC3, the human homolog of Saccharomyces cerevisiae Atg8p, and staining with the fluorophore monodansylcadaverine followed by fixation. Colocalization of LC3 and LAMP1 was observed early in the poliovirus replicative cycle, in cells infected with rhinoviruses 2 and 14, and in cells that express poliovirus proteins 2BC and 3A, known to be sufficient to induce double-membraned vesicles. Stimulation of autophagy increased poliovirus yield, and inhibition of the autophagosomal pathway by 3-methyladenine or by RNA interference against mRNAs that encode two different proteins known to be required for autophagy decreased poliovirus yield. We propose that, for poliovirus and rhinovirus, components of the cellular machinery of autophagosome formation are subverted to promote viral replication. Although autophagy can serve in the innate immune response to microorganisms, our findings are inconsistent with a role for the induced autophagosome-like structures in clearance of poliovirus. Instead, we argue that these double-membraned structures provide membranous supports for viral RNA replication complexes, possibly enabling the nonlytic release of cytoplasmic contents, including progeny virions, from infected cells.     

Relationship between synthesis and cleavage of poliovirus-specific proteins.

Poliovirus proteinase was studied in vitro in lysates from poliovirus-infected HeLa cells. Preincubation of these lysates caused (i) a reduction in poliovirus proteinase activity and (ii) a partial dependence on exogenous mRNA for optimal translation. Proteins translated from endogenous poliovirus RNA in preincubated extracts from virus-infected HeLa cells are poorly cleaved. This cleavage deficiency is alleviated by adding fresh poliovirus RNA to the translation system, thus, allowing re-initiation to occur. This suggests that the poliovirus proteinase is highly unstable.     

Dendritic cells and macrophages are productively infected by poliovirus

Expression of the poliovirus receptor (PVR) on cells is a major host determinant of infection by poliovirus. Previously, the only immune cell type known to express PVR was the blood-derived monocyte, which is susceptible to infection at very low frequency. We demonstrate that professional antigen-presenting cells-macrophages and dendritic cells, generated upon differentiation of monocytes-retain expression of PVR and are highly susceptible to infection by type 1 Mahoney strain of poliovirus. Maximal cell-associated titers of virus are obtained within 6 to 8 h postinfection, and cell death and lysis occurs within 24 h postinfection. Similar kinetics are observed in cells infected with the Sabin 1 vaccine strain. Although protein synthesis and receptor-mediated endocytosis are inhibited upon poliovirus infection of these critical antigen-presenting cells, we demonstrate for the first time that functional presentation of antigen occurs in these infected cells via the HLA class II pathway.     

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.     

Complementation of a poliovirus defective genome by a recombinant vaccinia virus which provides poliovirus P1 capsid precursor in trans.

Defective interfering (DI) RNA genomes of poliovirus which contain in-frame deletions in the P1 capsid protein-encoding region have been described. DI genomes are capable of replication and can be encapsidated by capsid proteins provided in trans from wild-type poliovirus. In this report, we demonstrate that a previously described poliovirus DI genome (K. Hagino-Yamagishi and A. Nomoto, J. Virol. 63:5386-5392, 1989) can be complemented by a recombinant vaccinia virus, VVP1 (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 65:2088-2092, 1991), which expresses the poliovirus capsid precursor polyprotein, P1. Stocks of defective polioviruses were generated by transfecting in vitro-transcribed defective genome RNA derived from plasmid pSM1(T7)1 into HeLa cells infected with VVP1 and were maintained by serial passage in the presence of VVP1. Encapsidation of the defective poliovirus genome was demonstrated by characterizing poliovirus-specific protein expression in cells infected with preparations of defective poliovirus and by Northern (RNA) blot analysis of poliovirus-specific RNA incorporated into defective poliovirus particles. Cells infected with preparations of defective poliovirus expressed poliovirus protein 3CD but did not express capsid proteins derived from a full-length P1 precursor. Poliovirus-specific RNA encapsidated in viral particles generated in cells coinfected with VVP1 and defective poliovirus migrated slightly faster on formaldehyde-agarose gels than wild-type poliovirus RNA, demonstrating maintenance of the genomic deletion. By metabolic radiolabeling with [35S]methionine-cysteine, the defective poliovirus particles were shown to contain appropriate mature-virion proteins. This is the first report of the generation of a pure population of defective polioviruses free of contaminating wild-type poliovirus. We demonstrate the use of this recombinant vaccinia virus-defective poliovirus genome complementation system for studying the effects of a defined mutation in the P1 capsid precursor on virus assembly. Following removal of residual VVP1 from defective poliovirus preparations, processing and assembly of poliovirus capsid proteins derived from a nonmyristylated P1 precursor expressed by a recombinant vaccinia virus, VVP1 myr- (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 66:4556-4563, 1992), in cells coinfected with defective poliovirus were analyzed. Capsid proteins generated from nonmyristylated P1 did not assemble detectable levels of mature virions but did assemble, at low levels, into empty capsids.(ABSTRACT TRUNCATED AT 400 WORDS)     

A chimeric poliovirus/CD4 receptor confers susceptibility to poliovirus on mouse cells.

The human poliovirus receptor consists of three extracellular immunoglobulinlike domains, a transmembrane domain, and an intracytoplasmic domain. The amino-terminal variable-type domain (V domain) of the human poliovirus receptor is necessary and sufficient for its function as a viral receptor (H.-C. Selinka, A. Zibert, and E. Wimmer, Proc. Natl. Acad. Sci. USA 88:3598-3602, 1991). In this paper, data are presented showing that transfer of the putative poliovirus receptor-binding domain to a truncated receptor for the human immunodeficiency virus results in a functional receptor for poliovirus. After expression in mouse cells, this chimeric protein confers susceptibility to poliovirus. Thus, unlike human immunodeficiency virus, poliovirus can enter mouse cells by way of a truncated CD4 receptor if the specific binding domain for poliovirus is provided.     

Isolation and characterization of HeLa cell lines blocked at different steps in the poliovirus life cycle.

Cotransfection of poliovirus RNA and R1, a poliovirus subgenomic RNA containing a deletion of nearly all of the capsid region, resulted in surviving cells, in contrast to the complete cell death observed after transfection with viral RNA. Cells that survived the cotransfection grew into colonies, produced infectious poliovirus, and underwent cycles of cell lysis (crisis periods) where less than 1% of the cells survived, followed by periods of growth. Poliovirus evolved during the persistent infection as judged by changes in plaque size. After passage for 6 months, a stable line called SOFIA emerged that no longer produced infectious virus and did not contain viral proteins or viral RNA. Cells frozen in liquid N2 while still in crisis and recultured 4 months later (named SOFIA N2) were also stabilized. After infection with poliovirus, SOFIA N2 cells showed a delay in the development of cytopathic effect, viral production, and cellular death when compared with HeLa cells. In contrast, SOFIA cells did not develop cytopathic effect and produced 10,000 times less virus than SOFIA N2 or HeLa cells. Viral production was delayed in SOFIA and SOFIA N2 cells transfected with poliovirus RNA when compared with HeLa cells, suggesting the presence of an intracellular block to poliovirus replication. Analysis of the cellular receptor for poliovirus by virus binding, an enzyme-linked immunosorbent assay, and in situ rosette assays with an antireceptor monoclonal antibody showed that receptors were expressed in SOFIA N2 cells but not in SOFIA cells. Echovirus 6, an enterovirus which uses a different cellular receptor, formed small plaques on SOFIA cells. Vesicular stomatitis virus formed plaques of similar size on SOFIA and HeLa cells, suggesting that the intracellular block was specific for enteroviruses. Cotransfection of the subgenomic replicon R1 with poliovirion RNA therefore resulted in the selection of HeLa cell variants containing blocks to poliovirus replication at the level of receptor and within the cell.     

Inhibitors of poliovirus uncoating efficiently block the early membrane permeabilization induced by virus particles.

The entry of animal viruses into cells is associated with permeabilization of the infected cells to protein toxins such as alpha-sarcin (C. Fernández-Puentes and L. Carrasco, Cell 20:769-775, 1980). This phenomenon has been referred to as "the early permeabilization by animal viruses" (L. Carrasco, Virology 113:623-629, 1981). A number of inhibitors of poliovirus growth such as WIN 51711 6-(3,4-dichlorophenoxy)-3-(ethylthio)-2-pyridincarbonitrile (DEPC) and Ro 09-0410 specifically block the uncoating step of poliovirus but have no effect on attachment or entry of poliovirus particles into cells. These agents are potent inhibitors of the early permeabilization induced by poliovirus to the toxin alpha-sarcin. Thus, the uncoating of poliovirus is required for the permeabilization of cell membranes to proteins. The increased entry of labeled heparin promoted by virus entry is not blocked by these agents, indicating that poliovirus binds to its receptor and is internalized along with heparin in endosomes in the presence of WIN 51711, DEPC, or Ro 09-0410. We conclude that the delivery to the cytoplasm of some molecules that coenter with virion particles does not take place if the uncoating process is hindered.