Mode of initiation of cell infection with sludge-associated poliovirus.

Association with sludge solids did not allow poliovirus to enter cells other than through the normal receptor-mediated route. This implies that the infectivities of sludge-encapsulated virions are masked until their exposure permits binding to cell surface receptors.     

Temperature-sensitive mouse cell factors for strand-specific initiation of poliovirus RNA synthesis.

Two cell lines, TgSVA and TgSVB, were established from the kidneys of transgenic mice carrying the human gene encoding poliovirus receptor. The cells were highly susceptible to poliovirus infection, and a large amount of infectious particles was produced in the infected cells at 37 degrees C. However, the virus yield was greatly reduced at 40 degrees C. This phenomenon was common to all mouse cells tested. To identify the temperature-sensitive step(s) of the virus infection cycle, different steps of the infection cycle were examined for temperature sensitivity. The results strongly suggested that the growth restriction observed at 40 degrees C was due to reduced efficiency of the initiation process of virus-specific RNA synthesis. Furthermore, this restriction appeared to occur only on the synthesis of positive-strand RNA. Virus-specific RNA synthesis in crude replication complexes was not affected by the nonpermissive temperature of 40 degrees C. In vitro uridylylation of VPg seemed to be temperature sensitive only after prolonged incubation at 40 degrees C. These results indicate that a specific host factor(s) is involved in the efficient initiation process of positive-strand RNA synthesis of poliovirus and that the host factor(s) is temperature sensitive in TgSVA and TgSVB cells.     

Identification of the initiation site of poliovirus polyprotein synthesis.

The complete nucleotide sequence of poliovirus RNA has a long open reading frame capable of encoding the precursor polyprotein NCVP00. The first AUG codon in this reading frame is located 743 nucleotides from the 5' end of the RNA and is preceded by eight AUG codons in all three reading frames. Because all proteins that map at the amino terminus of the polyprotein (P1-1a, VP0, and VP4) are blocked at their amino termini and previous studies of ribosome binding have been inconclusive, direct identification of the initiation site of protein synthesis was difficult. We separated and identified all of the tryptic peptides of capsid protein VP4 and correlated these peptides with the amino acid sequence predicted to follow the AUG codon at nucleotide 743. Our data indicate that VP4 begins with a blocked glycine that is encoded immediately after the AUG codon at nucleotide 743. An S1 nuclease analysis of poliovirus mRNA failed to reveal a splice in the 5' region. We concluded that synthesis of the poliovirus polyprotein is initiated at nucleotide 743, the first AUG codon in the long open reading frame.     

Internal translation initiation on poliovirus RNA: further characterization of La function in poliovirus translation in vitro.

Initiation of poliovirus RNA translation by internal entry of ribosomes is believed to require the participation of trans-acting factors. The mechanism of action of these factors is poorly defined. The limiting amount of one of these factors, La protein, in rabbit reticulocyte lysates (RRL) has been postulated to partially explain the inefficient translation of poliovirus RNA in this system. To further characterize La activity in translation and to identify other potential limiting factors, we assayed the ability of La protein as well as purified initiation factors, eIF-2, guanine nucleotide exchange factor (GEF), eIF-4A, eIF-4B, eIF-4F, and eIF-3, to stimulate the synthesis of P1, the capsid precursor protein, in poliovirus type 1 (Mahoney) RNA-programmed RRL. Of the proteins tested, only La, GEF, and to some extent eIF-2 stimulated the synthesis of P1. The enhanced translation of P1 in response to La occurred concomitantly with the inhibition of synthesis of most aberrant polypeptides, resulting from initiation in the middle of the genome. Deletion of the carboxy-terminal half (214 amino acids) of La did not decrease its binding to the poliovirus 5' untranslated region but abrogated the stimulatory and correcting activity in translation. In contrast to La, GEF and eIF-2 stimulated the overall translation and increased the synthesis of aberrant products as well as P1. Neither La, GEF, nor any other factor stimulated translation of encephalomyocarditis virus RNA in RRL. The implications of these findings for the mechanism of internal translation initiation on picornavirus RNAs are discussed.     

Enhanced intracellular calcium concentration during poliovirus infection.

The infection of human fibroblasts by poliovirus leads to a notable increase in the intracellular calcium concentration, [Ca2+]i, measured by microfluorimetry or by flow cytometry. [Ca2+]i increases from 2 to 3 h postinfection, and by the fifth hour there is a 5- to 10-fold increase in [Ca2+]i. At this time postinfection there is active viral protein synthesis. The modifications in [Ca2+]i are not observed in the presence of cycloheximide, guanidine, or Ro 09-0179, indicating that virus gene expression is required for the increase in [Ca2+]i. Attempts to identify the source of the intracellular Ca2+ by using different inhibitors of calcium fluxes suggest that calcium enters from the culture medium through voltage-sensitive calcium channels.     

Persistent poliovirus infection of human fetal brain cells.

It has been suggested that poliovirus (PV), the causative agent of poliomyelitis, could persist in surviving patients. We have previously shown that PV can persistently infect some human cell lines in vitro, particularly neuroblastoma cell lines. We report here an ex vivo model in which PV can persistently infect primary cultures of human fetal brain cells. Two mutations involving capsid residues 142 of VP2 and 95 of VP1 were repeatedly selected during the persistent infections. These residues are located in capsid regions known to be involved in interactions between PV and its receptor. During the first week after infection, viral antigens were found in cells of both the neuronal and glial lineages. In contrast, 2 weeks after infection, viral antigens were detected almost exclusively in cells of the neuronal lineage. They were detected predominantly in cells expressing a marker of early commitment to the neuronal lineage, MAP-5, particularly in neuroblasts. Viral antigens were also found in immature progenitors expressing a neuroepithelium marker, nestin, and in cells expressing a marker of postmitotic neurons, MAP-2. The presence of viral antigens in postmitotic neurons suggests that PV can persist in neurons of patients who have survived poliomyelitis.     

Shutoff of host translation by encephalomyocarditis virus infection does not involve cleavage of the eucaryotic initiation factor 4F polypeptide that accompanies poliovirus infection.

Studies were conducted to determine whether encephalomyocarditis virus infection causes proteolytic cleavage of any of the polypeptides which comprise eucaryotic initiation factor 4F. Since no such alterations in the components of the initiation factor were detected, these observations confirmed that the mechanisms whereby encephalomyocarditis virus and poliovirus shut off host translation are different.     

Functional interaction of heterogeneous nuclear ribonucleoprotein C with poliovirus RNA synthesis initiation complexes

We had previously demonstrated that a cellular protein specifically interacts with the 3' end of poliovirus negative-strand RNA. We now report the identity of this protein as heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Formation of an RNP complex with poliovirus RNA was severely impaired by substitution of a lysine, highly conserved among vertebrates, with glutamine in the RNA recognition motif (RRM) of recombinant hnRNP C1, suggesting that the binding is mediated by the RRM in the protein. We have also shown that in a glutathione S-transferase (GST) pull-down assay, GST/hnRNP C1 binds to poliovirus polypeptide 3CD, a precursor to the viral RNA-dependent RNA polymerase, 3D(pol), as well as to P2 and P3, precursors to the nonstructural proteins. Truncation of the auxiliary domain in hnRNP C1 (C1DeltaC) diminished these protein-protein interactions. When GST/hnRNP C1DeltaC was added to in vitro replication reactions, a significant reduction in RNA synthesis was observed in contrast to reactions supplemented with wild-type fusion protein. Indirect functional depletion of hnRNP C from in vitro replication reactions, using poliovirus negative-strand cloverleaf RNA, led to a decrease in RNA synthesis. The addition of GST/hnRNP C1 to the reactions rescued RNA synthesis to near mock-depleted levels. Furthermore, we demonstrated that poliovirus positive-strand and negative-strand RNA present in cytoplasmic extracts prepared from infected HeLa cells coimmunoprecipitated with hnRNP C1/C2. Our findings suggest that hnRNP C1 has a role in positive-strand RNA synthesis in poliovirus-infected cells, possibly at the level of initiation.     

Inhibition of HeLa cell protein synthesis following poliovirus infection correlates with the proteolysis of a 220,000-dalton polypeptide associated with eucaryotic initiation factor 3 and a cap binding protein complex

Following poliovirus infection of HeLa cells, the synthesis of cellular proteins is inhibited but translation of poliovirus mRNA proceeds. The defect in the recognition of host cell mRNA may be due to a change in a cap recognition complex which, when added to an infected cell lysate, restores the ability to translate capped mRNAs. We employed immunoblotting techniques to examine initiation factors in crude lysates from uninfected and poliovirus-infected HeLa cells. Using an antiserum against eucaryotic initiation factor 3, we detected an antigen of approximate molecular weight 220,000 in uninfected cell lysates but not in infected cell lysates. Antigenically related polypeptides of 100,000 to 130,000 daltons, presumably degradation products, were detected in the infected cell lysate. The time course for degradation of the 220,000-dalton polypeptide correlates with that for inhibition of cellular protein synthesis in vivo. A portion of the population of 220,000-dalton polypeptides apparently associates with initiation factor eIF3 but is readily dissociated in buffers containing high salt. Affinity-purified antibodies against the polypeptide recognize a protein of the same size in a purified preparation of a cap binding protein complex obtained by cap-affinity chromatography. We postulate that the 220,000-dalton polypeptide is an essential component of the cap recognition complex and that its degradation in poliovirus-infected cells results in the inhibition of host cell translation. These results are in the first demonstration of a specific structural defect in an initiation factor resulting from poliovirus infection.     

Role of a viral membrane polypeptide in strand-specific initiation of poliovirus RNA synthesis.

A molecular genetic analysis has been combined with an in vitro biochemical approach to define the functional interactions required for nucleotidyl protein formation during poliovirus RNA synthesis. A site-directed lesion into the hydrophobic domain of a viral membrane protein produced a mutant virus that is defective in RNA synthesis at 39 degrees C. The phenotypic expression of this lesion affects initiation of RNA synthesis, in vitro uridylylation of the genome-linked protein (VPg), and the in vivo synthesis of plus-strand viral RNAs. Our results support a model that employs a viral membrane protein as carrier for VPg in the initiation of plus-strand RNA synthesis. Our data also suggest that a separate mechanism could be used in the initiation of minus-strand RNA synthesis, thereby providing a means for strand-specific regulation of picornavirus RNA replication.     

Peptide-chain initiation with Lsc poliovirus is intrinsically more resistant to hypertonic environment than is peptide-chain initiation with Mahoney virus and deletion mutants of Mahoney virus.

Peptide-chain initiation with LSc poliovirus was more resistant to hypertonic medium than peptide-chain initiation with Mahoney poliovirus. Protein synthesis of LSc virus retained its relative resistance to high osmolarity created by the addition of excess NaCl to the medium in cells coinfected with Mahoney virus. The data indicate that peptide-chain initiation with LSc virus is intrinsically more resistant to high osmolarity than that of Mahoney virus rather than reflecting different permeability changes in cells after infection. Two Mahoney virus mutants harboring deletions at the 5' end of the viral chromosome exhibited the same sensitivity to excess NaCl as parent virus, suggesting that the original chromosomal region for peptide-chain initiation has not been severely altered by the deletions.     

Impaired binding of standard initiation factors mediates poliovirus translation attenuation

In the oral poliovirus vaccine, three attenuated virus strains generated by Albert Sabin are used. However, insufficient genetic stability of these strains causes major problems in poliovirus eradication. In infected cells, translation of the plus-strand poliovirus RNA genome is directed by the internal ribosome entry site (IRES), a cis-acting RNA element that facilitates the cap-independent binding of ribosomes to an internal site of the viral RNA. In each Sabin vaccine strain, a single point mutation in the IRES secondary-structure domain V is a major determinant of neurovirulence attenuation. Here we report how these decisive mutations in the IRES confer a reduction in poliovirus translation efficiency. These single-nucleotide exchanges impair the interaction of the standard translation initiation factor eIF4G with the IRES domain V. Moreover, binding of eIF4B and the polypyrimidine tract-binding protein and the association of ribosomes with the viral RNA are affected by these mutations. However, the negative effects of the IRES mutations are completely relieved by addition of purified eIF4F. This indicates that eIF4G is the crucial factor that initially binds to the poliovirus IRES and recruits the IRES to the other components of the translational apparatus, while impaired binding of eIF4G plays a key role in attenuation of poliovirus neurovirulence.     

Molecular mechanism of tissue-specific infection of poliovirus

Poliovirus is the causative agent of an acute disease of the central nervous system, poliomyelitis. Poliovirus will be eradicated in the near future by a world-wide vaccination program. Poliovirus is a neurotropic virus that produces severe lesions selectively in the CNS. However, a basic question why poliovirus exhibits neurotropic property has not been elucidated. Poliovirus receptor and host factors involved in the translation initiation of viral protein, which are required for virus replication, play important roles in determining tissue tropism. We found that type I interferon response is also an important determinant of poliovirus tissue tropism. Type I interferon inhibits viral replication in the non-target tissues. The tissue tropism of poliovirus may be determined based on the balance of these mechanisms.     

Protein synthesis eukaryotic initiation factors 4A and 4B are not altered by poliovirus infection of HeLa cells

Infection of HeLa cells by poliovirus results in the inhibition of translation of capped cellular mRNA. A plausible mechanism for this inhibition is that the structure of one or more initiation factors involved in the recognition of capped mRNA is altered. Eukaryotic initiation factor (eIF) 4A and eIF-4B are implicated in mRNA binding to 40 S ribosomal subunits and can be cross-linked to oxidized capped mRNA. We examined these factors in HeLa cell lysates by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. No alterations in the number of molecules/cell, in the molecular size, or in extents of covalent modification were detected when lysates from infected and mock-infected cells were compared. The integrity of eIF-2 and several eIF-3 polypeptides was also examined and likewise no alterations were detected. The failure of the translational machinery to recognize capped mRNA therefore is not due to a change in the structure of these initiation factors.