The alpha/beta interferon response controls tissue tropism and pathogenicity of poliovirus

Poliovirus selectively replicates in neurons in the spinal cord and brainstem, although poliovirus receptor (PVR) expression is observed in both the target and nontarget tissues in humans and transgenic mice expressing human PVR (PVR-transgenic mice). We assessed the role of alpha/beta interferon (IFN) in determining tissue tropism by comparing the pathogenesis of the virulent Mahoney strain in PVR-transgenic mice and PVR-transgenic mice deficient in the alpha/beta IFN receptor gene (PVR-transgenic/Ifnar knockout mice). PVR-transgenic/Ifnar knockout mice showed increased susceptibility to poliovirus. After intravenous inoculation, severe lesions positive for the poliovirus antigen were detected in the liver, spleen, and pancreas in addition to the central nervous system. These results suggest that the alpha/beta IFN system plays an important role in determining tissue tropism by protecting nontarget tissues that are potentially susceptible to infection. We subsequently examined the expression of IFN and IFN-stimulated genes (ISGs) in the PVR-transgenic mice. In the nontarget tissues, ISGs were expressed even in the noninfected state, and the expression level increased soon after poliovirus infection. On the contrary, in the target tissues, ISG expression was low in the noninfected state and sufficient response after poliovirus infection was not observed. The results suggest that the unequal IFN response is one of the important determinants for the differential susceptibility of tissues to poliovirus. We consider that poliovirus replication was observed in the nontarget tissues of PVR-transgenic/Ifnar knockout mice because the IFN response was null in all tissues.     

Expression of the poliovirus receptor in intestinal epithelial cells is not sufficient to permit poliovirus replication in the mouse gut.

Although the initial site of poliovirus replication in humans is the intestine, previously isolated transgenic mice which carry the human poliovirus receptor (PVR) gene (TgPVR mice), which develop poliomyelitis after intracerebral inoculation, are not susceptible to infection by the oral route. The low levels of PVR expressed in the TgPVR mouse intestine might explain the absence of poliovirus replication at that site. To ascertain whether PVR is the sole determinant of poliovirus susceptibility of the mouse intestine, we have generated transgenic mice by using the promoter for rat intestine fatty acid binding protein to direct PVR expression in mouse gut. Pvr was detected by immunohistochemistry in the enterocytes and M cells of transgenic mouse (TgFABP-PVR) small intestine. Upon oral inoculation with poliovirus, no increase in virus titer was detected in the feces of TgFABP-PVR mice, and no virus replication was observed in the small intestine, although poliovirus replicated in the brain after intracerebral inoculation. The failure of poliovirus to replicate in the TgFABP-PVR mouse small intestine was not due to lack of virus binding sites, because poliovirus could attach to fragments of small intestine from these mice. These results indicate that the inability of poliovirus to replicate in the mouse alimentary tract is not solely due to the absence of virus receptor, and other factors are involved in determining the ability of poliovirus to replicate in the mouse gut.     

Heterogeneous expression of poliovirus receptor-related proteins in human cells and tissues.

Portions of the cellular receptor for poliovirus were expressed in Escherichia coli as fusion proteins with the product of the trpE gene. One of two antireceptor antisera obtained by immunizing rabbits with the fusion proteins blocked poliovirus infection. Western immunoblot analyses demonstrated that poliovirus receptor-related proteins were expressed in HeLa cells and a variety of human tissues, including those that are not sites of poliovirus replication. Tissue-specific variation in electrophoretic mobility, immunoreactivity, and subunit arrangement of poliovirus receptor-related proteins was observed. These results demonstrate that poliovirus tissue tropism cannot be explained by a limited distribution of receptor polypeptide, but may be the result of alternative splicing, posttranslational modifications, or both. In addition, the widespread but heterogeneous expression of the receptor suggests that the protein may have an important endogenous function.     

Host range phenotype induced by mutations in the internal ribosomal entry site of poliovirus RNA.

Most poliovirus strains infect only primates. The host range (HR) of poliovirus is thought to be primarily determined by a cell surface molecule that functions as poliovirus receptor (PVR), since it has been shown that transgenic mice are made poliovirus sensitive by introducing the human PVR gene into the genome. The relative levels of neurovirulence of polioviruses tested in these transgenic mice were shown to correlate well with the levels tested in monkeys (H. Horie et al., J. Virol. 68:681-688, 1994). Mutants of the virulent Mahoney strain of poliovirus have been generated by disruption of nucleotides 128 to 134, at stem-loop II within the 5' noncoding region, and four of these mutants multiplicated well in human HeLa cells but poorly in mouse TgSVA cells that had been established from the kidney of the poliovirus-sensitive transgenic mouse. Neurovirulence tests using the two animal models revealed that these mutants were strongly attenuated only in tests with the mouse model and were therefore HR mutants. The virus infection cycle in TgSVA cells was restricted by an internal ribosomal entry site (IRES)-dependent initiation process of translation. Viral protein synthesis and the associated block of cellular protein synthesis were not observed in TgSVA cells infected with three of four HR mutants and was evident at only a low level in the remaining mutant. The mutant RNAs were functional in a cell-free protein synthesis system from HeLa cells but not in those from TgSVA and mouse neuroblastoma NS20Y cells. These results suggest that host factor(s) affecting IRES-dependent translation of poliovirus differ between human and mouse cells and that the mutant IRES constructs detect species differences in such host factor(s). The IRES could potentially be a host range determinant for poliovirus infection.     

N glycosylation of the virus binding domain is not essential for function of the human poliovirus receptor.

The human poliovirus receptor (hPVR) is a glycoprotein with three immunoglobulin-like extracellular domains, of which the N-terminal domain (V-type domain) is necessary and sufficient for virus binding and uptake. The effect of N glycosylation of the V domain of hPVR on binding and entry of poliovirus was studied. Stable mouse L-cell lines were generated that express PVR-specific cDNA. One of the cell lines expressed a mutant of hPVR, in which both asparagine residues of the two N-glycosylation sites of the V domain were changed to aspartate (N105D) and serine (N120S), respectively. In the second mutant cell line, the portion of the cDNA encoding the V domain of hPVR was substituted by the homologous sequence of the recently isolated PVR cDNA from monkey cells. This V domain naturally lacks both N glycosylation sites and encodes D105 and S120 at the respective positions of the open reading frame. Absence of N glycosylation at these sites was demonstrated by in vitro translation of the two mutant coding sequences in the presence of microsomal membranes. Both PVR mutant cell lines were capable of poliovirus binding and replication. However, binding of anti-PVR monoclonal antibody D171 and protection from viral replication by this antibody were observed only with the glycosylation mutant carrying the human V domain. In contrast, infection of the cell line expressing the monkey-human hybrid receptor was not blocked even though monkey cells are fully protected by monoclonal antibody D171. The data suggest that N glycosylation of the V domain of hPVR is not essential for viral replication in human tissues and that differential glycosylation of hPVR at these sites is likely not a determinant of viral tissue tropism. Furthermore, the virus binding site and the epitope recognized by monoclonal antibody D171 do not appear to overlap.     

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.     

Role of the alpha/beta interferon response in the acquisition of susceptibility to poliovirus by kidney cells in culture

Replication of poliovirus (PV) is restricted to a few sites, including the brain and spinal cord. However, this neurotropism is not conserved in cultured cells. Monkey kidney cells become susceptible to PV infection after cultivation in vitro, and cell lines of monolayer cultures from almost any tissue of primates are susceptible to PV infection. These observations suggest that cellular changes during cultivation are required for acquisition of susceptibility. The molecular basis for the cellular changes during this process is not known. We investigated the relationship between PV susceptibility and interferon (IFN) response in primary cultured kidney and liver cells derived from transgenic mice expressing human PV receptor and in several primate cell lines. Both kidneys and liver in vivo showed rapid IFN response within 6 h postinfection. However, monkey and mouse kidney cells in culture and primate cell lines, which were susceptible to PV, did not show such rapid response or showed no response at all. On the other hand, primary cultured liver cells, which were partially resistant to infection, showed rapid IFN induction. The loss of IFN inducibility in kidney cells was associated with a decrease in expression of IFN-stimulated genes involved in IFN response. Mouse kidney cells pretreated with a small dose of IFN, in turn, restored IFN inducibility and resistance to PV. These results strongly suggest that the cells in culture acquire PV susceptibility during the process of cultivation by losing rapid IFN response that has been normally maintained in extraneural tissues in vivo.     

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.     

Mutational analysis of the cellular receptor for poliovirus.

To identify sequences of the cellular poliovirus receptor (PVR) involved in viral infection, mutant PVR cDNAs were constructed and assayed for biological activity in mouse L cells. To confirm that mutant PVRs reached the cell surface, an immunological tag, consisting of part of CH3 from human immunoglobulin G1, was engineered into the PVR. Deletion of PVR amino acids 256 to 320 or 385 to the carboxy terminus yielded receptors that were able to support poliovirus infection. PVRs lacking amino acids 40 to 136 or 137 to 256 were expressed at the cell surface but were not active as receptors for poliovirus. The results show that immunoglobulin-type domain 3 and the extreme carboxy terminus of the PVR are not required for viral receptor function, but sequences within the two amino-terminal domains contribute to the initiation of poliovirus infection.     

Identification of a lens-specific regulatory region (LSR) of the murine alpha B-crystallin gene.

Previous studies have shown that the -661/+44 sequence of the murine alpha B-crystallin gene contains a muscle-preferred enhancer (-426/-257) and can drive the bacterial chloramphenicol acetyltransferase (CAT) gene in the lens, skeletal muscle and heart of transgenic mice. Here we show that transgenic mice carrying a truncated -164/+44 fragment of the alpha B-crystallin gene fused to the CAT gene expressed exclusively in the lens; by contrast mice carrying a -426/+44 fragment of the alpha B gene fused to CAT expressed highly in the lens, skeletal muscle and heart, and slightly in the lung, brain, kidney, spleen and liver. DNase I protection experiments indicated that the -147/-118 sequence is protected by nuclear proteins from alpha TN4-1 lens cell line, but not by nuclear proteins from myotubes of the C2C12 cell line. Site directed mutagenesis of this sequence decreased promoter activity in transiently-transfected lens cells, consistent with this sequence being a lens-specific regulatory region (LSR). We conclude that the -426/-257 enhancer is required for expression in skeletal muscle, heart and possibly other tissues, and that the -164/+44 sequence of the alpha B-crystallin gene is sufficient for expression in the lens of transgenic mice.     

A second gene for the African green monkey poliovirus receptor that has no putative N-glycosylation site in the functional N-terminal immunoglobulin-like domain.

Using cDNA of the human poliovirus receptor (PVR) as a probe, two types of cDNA clones of the monkey homologs were isolated from a cDNA library prepared from an African green monkey kidney cell line. Either type of cDNA clone rendered mouse L cells permissive for poliovirus infection. Homologies of the amino acid sequences deduced from these cDNA sequences with that of human PVR were 90.2 and 86.4%, respectively. These two monkey PVRs were found to be encoded in two different loci of the genome. Evolutionary analysis suggested that duplication of the PVR gene in the monkey genome had occurred after the species differentiation between humans and monkeys. The NH2-terminal immunoglobulin-like domain, domain 1, of the second monkey PVR, which lacks a putative N-glycosylation site, mediated poliovirus infection. In addition, a human PVR mutant without N-glycosylation sites in domain 1 also promoted viral infection. These results suggest that domain 1 of the monkey receptor also harbors the binding site for poliovirus and that sugar moieties possibly attached to this domain of human PVR are dispensable for the virus-receptor interaction.     

Quercetinase pirin makes poliovirus replication resistant to flavonoid quercetin

Flavonoid quercetin and its derivative, methylquercetin, inhibit the replication of poliovirus in several cell lines. Here, we show that replication of poliovirus is inhibited by quercetin and that the extent of this inhibition depends on the intracellular content of pirin, a quercetinase. HeLa cells contain higher content of pirin protein than normal kidney human epithelial (NKE) or 293 cells do. Poliovirus replication in HeLa cells is significantly more resistant to quercetin than its replication in NKE and 293 cells. Overexpression of pirin reduced antiviral inhibitory effect of quercetin, while siRNA-induced suppression of pirin level made poliovirus replication more sensitive to the flavonoid. The results suggest that quercetinase activity of pirin determines the resistance of poliovirus infection to quercetin.     

Changes in Skeletal Muscle and Body Weight on Sleeping Beauty Transposon-Mediated Transgenic Mice Overexpressing Pig mIGF-1

Insulin-like growth factor (IGF-I) is an important growth factor in mammals, but the functions of the local muscle-specific isoform of insulin-like growth factor 1 (mIGF-1) to skeletal muscle development have rarely been reported. To determine the effect of pig mIGF-1 on body development and muscle deposition in vivo and to investigate the molecular mechanisms, the transgenic mouse model was generated which can also provide experimental data for making transgenic pigs with pig endogenous IGF1 gene. We constructed a skeletal muscle-specific expression vector using 5′- and 3′-regulatory regions of porcine skeletal α-actin gene. The expression cassette was flanked with Sleeping Beauty transposon (SB)-inverted terminal repeats. The recombinant vector could strongly drive enhanced green fluorescence protein (EGFP) reporter gene expression specifically in mouse myoblast cells and porcine fetal fibroblast cells, but not in porcine kidney cells. The EGFP level driven by α-actin regulators was significantly stronger than that driven by cytomegalovirus promoters. These results indicated that the cloned α-actin regulators could effectively drive specific expression of foreign genes in myoblasts, and the skeletal muscle-specific expression vector mediated with SB transposon was successfully constructed. To validate the effect of pig mIGF-1 on skeletal muscle growth, transgenic mice were generated by pronuclear microinjection of SB-mediated mIGF-1 skeletal expression vector and SB transposase-expressing plasmid. The transgene-positive rates of founder mice and the next-generation F1 mice were 30% (54/180) and 90.1% (64/71), respectively. The mIGF-1 gene could be expressed in skeletal muscle specifically. The levels of mRNA and protein in transgenic mice were 15 and 3.5 times higher, respectively, than in wild-type mice. The body weights of F1 transgenic mice were significantly heavier than wild-type mice from the age of 8 weeks onwards. The paraffin-embedded sections of gastrocnemius from 16-week-old transgenic male mice showed that the numbers of myofibers per unit were increased in comparison with those in the wild-type mice. mIGF-1 overexpression in mice skeletal muscle may promote myofibers hypertrophy and muscle production, and increased the average body weight of adult mice. Transgenic mice models can be generated by the mediation of SB transposon with high transgene efficiency.     

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.     

Tissue-specific replicating capacity of a chimeric poliovirus that carries the internal ribosome entry site of hepatitis C virus in a new mouse model transgenic for the human poliovirus receptor

Nucleotides (nt) 108 to 742 of an infectious cDNA clone of poliovirus (PV) Mahoney strain, including the corresponding region of the internal ribosome entry site (IRES), was replaced by nt 28 to 710 of hepatitis C virus (HCV) cDNA corresponding to the whole HCV IRES. A chimeric PV (2A-369) was generated by transfecting mammalian cells with an RNA transcribed in vitro from the cDNA. To examine replicating capacity of virus 2A-369 in the brain and liver of a mouse model for poliomyelitis, a new mouse model (MPVRTg25-61) that is transgenic for human PV receptor (hPVR; CD155) was generated in order to obtain a higher expression level of hPVR in the liver than those of hPVRTg mouse lines generated by us so far. The transgene used was constructed by combining a putative regulatory region of the mouse PVR homolog and the whole structural region of the hPVR gene. Virus 2A-369 replicated well in the liver of MPVRTg25-61 but not in the brain, whereas control Mahoney virus replicated well both in the liver and in the brain. The data suggest that the HCV IRES works more efficiently in the liver than in the brain and that PV IRES works well both in the liver and in the brain. The results support the notion that tissue-specific activity of IRES may be reflected in tissue tropism of a virus whose specific translation initiation is driven by IRES, that is, an IRES-dependent virus tropism.     

Metabolic effects of developmental, tissue-, and cell-specific expression of a chimeric phosphoenolpyruvate carboxykinase (GTP)/bovine growth hormone gene in transgenic mice.

Transgenic mice were used to investigate sequences within the promoter of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the rat (EC 4.1.1.32) (PEPCK) which are involved in tissue-specific and developmental regulation of gene expression. Segments of the PEPCK promoter between -2000 and -109 were linked to the structural gene for bovine growth hormone (bGH) and introduced into the germ line of mice by microinjection. Bovine growth hormone mRNA was found in tissues that express the endogenous PEPCK gene, mainly in the liver but to a lesser extent in the kidney, adipose tissue, small intestine, and mammary gland. In the liver the chimeric PEPCK/bGH(460) gene was expressed in periportal cells, which is consistent with the zonation of endogenous PEPCK. The PEPCK/bGH gene was not transcribed in the livers of fetal mice until immediately before birth; at birth the concentration of bGH mRNA increased 200-fold. Our results indicate that the region of the PEPCK promoter from -460 to +73 base pairs contains regulatory sequences required for tissue-specific and developmental regulation of PEPCK gene expression. Mice transgenic for PEPCK/bGH(460) were not hyperglycemic or hyperinsulinemic in response to elevated bGH, as were transgenic mice with the MT/bGH gene. The number of insulin receptors in skeletal muscle was no different in mice transgenic for MT/bGH when compared with mice transgenic for PEPCK/bGH(460) and control animals. However, mRNA abundance for the insulin-sensitive glucose transporter in skeletal muscle was decreased in mice transgenic for the MT/bGH gene. The differences in glucose homeostasis noted with the two types of transgenic mice may be the result of the relative site of expression, the different developmental pattern, or hormonal regulation of expression of the bGH gene.     

The poliovirus receptor protein is produced both as membrane-bound and secreted forms.

Both genomic and complementary DNA clones encoding poliovirus receptors were isolated from genomic and complementary DNA libraries prepared from HeLa S3 cells, respectively. Nucleotide sequence analysis of these cloned DNAs revealed that the poliovirus receptor gene is approximately 20 kb long and contains seven introns in the coding region, and that at least four mRNA isoforms referring to the coding sequence are generated by alternative splicing and appear to encode four different molecules, that is, PVR alpha, PVR beta, PVR gamma and PVR delta. The predicted amino acid sequences indicate that PVR alpha and PVR delta, corresponding to the previously described cDNA clones H20A and H20B, respectively, are integral membrane proteins while the other two molecules described here for the first time lack a putative transmembrane domain. Mouse cell transformants carrying PVR alpha were permissive for poliovirus infection, but those carrying PVR beta were hardly permissive. In contrast to PVR alpha, PVR beta was not detected on the surface of the mouse cell transformants but was detected in the culture fluid by an immunological method using a monoclonal antibody against poliovirus receptor. Three types of splicing products for PVR alpha, PVR beta and PVR gamma were detected by polymerase chain reactions using appropriate primers in poly(A)+ RNAs of the brain, leukocyte, liver, lung and placenta of humans; the choice of primers used did not permit detection of PVR delta. In situ hybridization using a cDNA fragment as a probe demonstrated that the PVR gene is located at the band q13.1----13.2 of human chromosome 19.