MDA-5 is cleaved in poliovirus-infected cells

Infections with RNA viruses are sensed by the innate immune system through membrane-bound Toll-like receptors or the cytoplasmic RNA helicases RIG-I and MDA-5. It is believed that MDA-5 is crucial for sensing infections by picornaviruses, but there have been no studies on the role of this protein during infection with poliovirus, the prototypic picornavirus. Beginning at 4 h postinfection, MDA-5 protein is degraded in poliovirus-infected cells. Levels of MDA-5 declined beginning at 6 h after infection with rhinovirus type 1a or encephalomyocarditis virus, but the protein was stable in cells infected with rhinovirus type 16 or echovirus type 1. Cleavage of MDA-5 is not carried out by either poliovirus proteinase 2Apro or 3Cpro. Instead, degradation of MDA-5 in poliovirus-infected cells occurs in a proteasome- and caspase-dependent manner. Degradation of MDA-5 during poliovirus infection correlates with cleavage of poly(ADP) ribose polymerase (PARP), a hallmark of apoptosis. Induction of apoptosis by puromycin leads to cleavage of both PARP and MDA-5. The MDA-5 cleavage product observed in cells treated with puromycin is approximately 90 kDa, similar in size to the putative cleavage product observed in poliovirus-infected cells. Poliovirus-induced cleavage of MDA-5 may be a mechanism to antagonize production of type I interferon in response to viral infection.     

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.     

Rescue of Temperature-sensitive Poliovirus

A temperature-sensitive strain of type 1 poliovirus, LSc, was functionally rescued when infected cells were incubated at 40 C in the presence of Mahoney, a temperature-resistant strain of type 1 poliovirus. The rescue value was 9% of the mutant yield obtained under permissive conditions. Rescued virus underwent replication, because the progeny of (32)P-labeled LSc were not radiosensitive. Serum inactivation studies with Mahoney specific antiserum indicated that a small amount of phenotypic mixing occurred among the rescued particles. The temperature-sensitive event occurred between 2 and 4 hr postinfection in the developmental cycle of LSc. Neither viral polymerase activity nor virus-induced ribonucleic acid could be demonstrated in infected cells between 2 and 4 hr after infection at 40 C with the temperature-sensitive mutant.     

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.     

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.     

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.     

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.     

Interaction of Poliovirus with Its Receptor Affords a High Level of Infectivity to the Virion in Poliovirus Infections Mediated by the Fc Receptor

Poliovirus infects susceptible cells through the poliovirus receptor (PVR), which functions to bind virus and to change its conformation. These two activities are thought to be necessary for efficient poliovirus infection. How binding and conformation conversion activities contribute to the establishment of poliovirus infection was investigated. Mouse L cells expressing mouse high-affinity Fcγ receptor molecules were established and used to study poliovirus infection mediated by mouse antipoliovirus monoclonal antibodies (MAbs) (immunoglobulin G2a [IgG2a] subtypes) or PVR-IgG2a, a chimeric molecule consisting of the extracellular moiety of PVR and the hinge and Fc portion of mouse IgG2a. The antibodies and PVR-IgG2a showed the same degree of affinity for poliovirus, but the infectivities mediated by these molecules were different. Among the molecules tested, PVR-IgG2a mediated the infection most efficiently, showing 50- to 100-fold-higher efficiency than that attained with the different MAbs. A conformational change of poliovirus was induced only by PVR-IgG2a. These results strongly suggested that some specific interaction(s) between poliovirus and the PVR is required for high-level infectivity of poliovirus in this system.     

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.     

Persistence of herpes simplex virus genes in cells of neuronal origin.

The growth characteristics of the KOS strain of herpes simplex virus type 1 (HSV-1) in cell lines of nervous tissues origin were examined in an attempt to develop a tissue culture system mimicking the in vivo state of HSV-1 latency. We have previously reported that the B103 rat brain neuroma cell line is nonpermissive for growth of the KOS strain. In this report, we show that this nonpermissiveness is a temperature- and multiplicity-dependent phenomenon, with minimum virus yields at an elevated temperature and a low multiplicity of infection. Under these conditions, B103 cells survived infection with active wild-type or mutant HSV-1, whereas similarly treated Vero cells were killed. Six independent cultures of B103 cells surviving HSV-1 infection have been established. The surviving cells ceased production of any HSV-1 virus by 14 days postinfection and resumed growth and division at rates comparable to those of uninfected B103 cells. Survivor cells continued to express HSV-1-specific antigens, however, as detected by indirect immunofluorescence and by surface iodination followed by immunoprecipitation and polyacrylamide gel electrophoresis. The survivor cells did not express all of the surface proteins seen on productively infected B103 cells, and they were not susceptible to complement-mediated immune cytolysis with anti-HSV-1 antiserum. These results demonstrate that at least a portion of the HSV-1 genome is being harbored in these survivor cells.     

Vectorial release of poliovirus from polarized human intestinal epithelial cells.

Polarized epithelial cells represent the primary barrier to virus infection of the host, which must also be traversed prior to virus dissemination from the infected organism. Although there is considerable information available concerning the release of enveloped viruses from such cells, relatively little is known about the processes involved in the dissemination of nonenveloped viruses. We have used two polarized epithelial cell lines, Vero C1008 (African green monkey kidney epithelial cells) and Caco-2 (human intestinal epithelial cells), infected with poliovirus and investigated the process of virus release. Release of poliovirus was observed to occur almost exclusively from the apical cell surface in Caco-2 cells, whereas infected Vero C1008 cells exhibited nondirectional release. Structures consistent with the vectorial transport of virus contained within vesicles or viral aggregates were observed by electron microscopy. Treatment with monensin or ammonium chloride partially inhibited virus release from Caco-2 cells. No significant cell lysis was observed at the times postinfection when extracellular virus was initially detected, and transepithelial resistance and vital dye uptake measurements showed only a moderate decrease. Brefeldin A was found to significantly and specifically inhibit poliovirus biosynthetic processes by an as yet uncharacterized mechanism. The vectorial release of poliovirus from the apical (or luminal) surface of human intestinal epithelial cells has significant implications for viral pathogenesis in the human gut.     

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.     

Poliovirus infection results in structural alteration of a microtubule-associated protein.

Poliovirus infection results in profound changes in cellular metabolism and architecture. To identify alterations in cellular proteins following poliovirus infection which might account for these changes, monoclonal antibodies were prepared by screening for differences in antigen pattern in infected and uninfected cell lysates. Further characterization of the antigen of one such antibody (25 C C1) is described in this report. The 25 C C1 antigen is a cytoskeleton-associated protein which decreases in size 4 to 5 h postinfection. It copurifies with some of the protein synthesis initiation factors but not with eucaryotic initiation factor (eIF)-4F, the p220 subunit of which is cleaved following infection (D. Etchison, S. C. Milburn, I. Edery, N. Sonenberg, and J. W. B. Hershey, J. Biol. Chem. 257:14806-14810, 1982). Unlike alteration of p220, alteration of the 25 C C1 antigen is not due to a protease which can be detected by cell lysate mixing experiments. Alteration of the antigen occurs during purification, suggesting progressive proteolysis, but the alteration is more extensive in preparations from infected cells than in those from uninfected cells. A recombinant phage expressing the antigenic determinant was isolated from a human fibroblast cDNA library, and the sequence of the cDNA insert was found to be entirely contained within the established sequence of microtubule-associated protein (MAP) 4 (R. R. West, K. M. Tenbarge, and J. B. Olmsted, J. Biol. Chem. 266:21886-21896, 1991). The antigen distribution, as detected by indirect immunofluorescence, was similar to, but more diffuse than, the distribution of tubulin. The antibody recognized the largest abundant HeLa cell MAP, which copurified with tubulin after three cycles of polymerization-depolymerization, thus confirming the identity of the antigen as MAP 4. These results indicate that poliovirus infection of HeLa cells affects the structural integrity of a cytoskeletal protein, MAP 4.     

Elimination of some enteroviruses in the excrements of experimentally infected rats (Rattus norvegicus) and gulls (Larus ridibundus)

Young rats of both sexes, weight 150-170 g, the first laboratory progeny of captured wild parent pairs, were used throughout this experiment. Rats in two experimental groups comprising a total of 34 animals were infected orally with type 2 poliovirus vaccine strain given in each group at doses of 500, 5000 or 50,000 TCD50. In the first experiment, the presence of poliovirus in rat excrements was detectable irregularly till day 13, in the second experiment till day 2 after infection. Small quantities of virus were also detectable from the colon and cecum wall, exceptionally from the mesenteric lymph node. The third experiment included 8 rats orally infected with 5,000 TCD50 of echovirus 30; at the lower dose of virus all excrement samples were culture-negative, at the higher dose the positive virus recovery was recorded in 3 animals one day after infection. Analogous experiments in the fifth group of rats orally infected with 5,000 TCD50 or 50,000 TCD50 of enterovirus 71 yielded much the same results; organs of further 6 animals infected intranasally with 5,000 TCD50 of this virus were culture-negative and no virus-related changes could be histologically demonstrated in these animals. The second part of this study included the experiments conducted on 17 young Larus gulls bred in the laboratory from eggs collected in a colony of free living birds. Groups of these gulls were orally infected with 500 or 5,000 TCD50 of one of the following viruses: type 1 poliovirus vaccine strain, type 3 poliovirus vaccine strain, echovirus 30, enterovirus 71 and coxsackievirus B4. All samples of gull excrements collected till day 7 or 20 postinfection were culture-negative. These results suggest that wild rats may play some role in the spread of human enteroviruses in the environment, but no such role could be demonstrated in the Larus gull.     

Cholesterol removal by methyl-beta-cyclodextrin inhibits poliovirus entry

Upon binding to the poliovirus receptor (PVR), the poliovirus 160S particles undergo a conformational transition to generate 135S particles, which are believed to be intermediates in the virus entry process. The 135S particles interact with host cell membranes through exposure of the N termini of VP1 and the myristylated VP4 protein, and successful cytoplasmic delivery of the genomic RNA requires the interaction of these domains with cellular membranes whose identity is unknown. Because detergent-insoluble microdomains (DIMs) in the plasma membrane have been shown to be important in the entry of other picornaviruses, it was of interest to determine if poliovirus similarly required DIMs during virus entry. We show here that methyl-beta-cyclodextrin (MbetaCD), which disrupts DIMs by depleting cells of cholesterol, inhibits virus infection and that this inhibition was partially reversed by partially restoring cholesterol levels in cells, suggesting that MbetaCD inhibition of virus infection was mediated by removal of cellular cholesterol. However, fractionation of cellular membranes into DIMs and detergent-soluble membrane fractions showed that both PVR and poliovirus capsid proteins localize not to DIMs but to detergent-soluble membrane fractions during entry into the cells, and their localization was unaffected by treatment with MbetaCD. We further demonstrate that treatment with MbetaCD inhibits RNA delivery after formation of the 135S particles. These data indicate that the cholesterol status of the cell is important during the process of genome delivery and that these entry pathways are distinct from those requiring DIM integrity.     

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.