Fluorescent poliovirus for flow cytometric cell surface binding studies.

Specific cell-surface binding is the essential first step for cellular invasion by viruses. To understand this process, various methods to evaluate binding properties of viruses to cells have been developed. However, many rely on radioactive labeling or indirect immunofluorescence. The development of a novel fluorescence binding assay for poliovirus is described. Poliovirus (type 1 Mahoney or Sabin) was labeled directly with fluorescein using a commercially available fluoresceination kit. Fluorescently labeled poliovirus was bound to its specific receptor on Hela or U937 cells and detected by flow cytometric analysis. Specific binding and infectivity was retained, although reduced, depending on the extent of fluoresceination. Therefore, depending on the users' requirements, the extent of fluoresceination must be titrated carefully to achieve maximal fluorescence and minimal functional destruction. It is likely that this method may be useful with other viruses.

An improved method for detection and quantification of differential interactions between poliovirus internal ribosome entry site RNA and pyrimidine tract binding protein from primary cells.

Attenuated and pathogenic viral variants are often extremely similar viruses with drastically different replication potentials. Despite precise knowledge of viral residues responsible for poliovirus attenuation/neurovirulence, molecular mechanisms mediating these effects remain poorly understood. Data from numerous sources suggest that a functional difference in translation initiation is one responsible factor. However, direct evidence, as well as a comprehensive model are lacking. Several difficulties, including lack of an assay system to quantify differential internal ribosome entry site/pyrimidine tract binding protein interaction in relevant systems, have precluded progress. A novel assay system that overcomes some difficulties is presented below. The assay uses streptavidin paramagnetic particles, biotinylated RNA and glutathione-S-transferase/pyrimidine tract binding protein fusion to detect nanogram levels of uncloned cellular pyrimidine tract binding protein species that interact with internal ribosome entry site RNA. Using this assay, it was shown that pyrimidine tract binding protein from primary human monocytes binds to internal ribosome entry site RNA from virulent poliovirus better than to that from attenuated virus, while pyrimidine tract binding protein from HeLa cells does not distinguish between the two internal ribosome entry sites. Since primary human monocytes reflect neurovirulence-related differential poliovirus replication, these results suggest that pyrimidine tract binding protein may contribute to differential poliovirus replication in vivo. This assay also has the potential to be applicable broadly to other nucleic acid/protein interactions.

Hematopoietic cells from CD155-transgenic mice express CD155 and support poliovirus replication ex vivo.

Despite identification of the poliovirus (PV) receptor (CD155), mechanisms by which this molecule mediates paralytic disease remain obscure. Unanswered questions include CD155 localization in human tissues, the nature of cells supporting the first round of replication, identity of nonneural replication sites, and route of entry into the CNS. In earlier work, we showed that CD155 is expressed on primary human monocytes and that these cells support low, but statistically significant, levels of PV replication ex vivo without prior culturing. We hypothesize that monocytes support PV replication in vivo and that they contribute to pathogenesis. In the current study, we tested whether CD155-transgenic mouse hematopoietic cells express cell surface CD155 and whether these cells support PV replication. We found that the majority of monocyte/macrophages from peritoneal washes express CD155. In addition, 26-32% of CD155-transgenic bone marrow and spleen cells express CD155 on monocyte/macrophages, T cells and hematopoietic precursor cells. Various tissues supported PV replication without pre-culturing, however, pre-culturing or pre-treatment of mice with thioglycollate increased virus yield. These results are consistent with those from human cells and suggest that the CD155 transgenic mouse model is useful to help understand the role of hematopoietic cells in PV pathogenesis.

Physical association between CD155 and CD44 in human monocytes.

Regulation of CD44-mediated binding to hyaluronan is critical in normal and diseased immune cell function. In earlier work by others (Shepley and Racaniello, J. Virol., 68, 1301 1309), anti-CD44 mAb blocked poliovirus binding to CD155 (the poliovirus receptor) in HeLa cells, suggesting that CD155 and CD44 may be physically associated. Here, we present evidence that CD155 and CD44 are physically associated in human monocytes. In co-modulation experiments in U937 monocytic cells, CD155 and CD44 reciprocally co-modulated. In primary human monocytes, CD 155 syn-capped with CD44. In immunofluorescence flow cytometric experiments, anti CD44 mAb inhibited up to 94% of binding by anti-CD155 mAb which blocks poliovirus binding to CD155. This inhibition was specific for CD155. Culturing monocytes increased the extent of inhibition. In addition, mAb against PRR2, a novel molecule that is related to CD 155, was inhibited by anti-CD44 in a dose-dependent manner, but not by anti-CD14. These data support the interpretation that CD155 (and related proteins) are physically associated with CD44 on monocyte cell surfaces. Although the current study does not address functional significance, we speculate that this interaction may have a role in regulating monocyte CD44 ligand binding which may be critical in pathological processes such as tumor metastasis and arthritis.

CD44 is not required for poliovirus replication in cultured cells and does not limit replication in monocytes.

Although poliovirus receptor is required to mediate poliovirus infection, its role in mediating the tissue specificity of poliovirus replication in natural infections remains unclear due to the presence of this receptor in nonsusceptible cells. It has been hypothesized that CD44 has a role in determining the susceptibility of cell to poliovirus. To test this hypothesis, we determined whether HepG2, a cultured cell line that lacks cell surface CD44, can support poliovirus replication. We found that PV(1) Mahoney, PV(2)W2, and PV(3)Leon strains of poliovirus can replicate in HepG2 cells. Jurkat cells, which also lack CD44, support PV replication. These results suggest that CD44 is not required for poliovirus replication in cultured cells. To examine this question further expression of CD44 in primary human monocytes was examined. Greater than 90% of monocytes express the poliovirus receptor but only 6% of these cells are susceptible to poliovirus infection, making this a good system to study blocks to poliovirus replication. 97% of primary human monocytes reacted with a monoclonal antibody against CD44 that has been shown to block poliovirus binding and replication. This finding suggests that CD44 is present on more cells that poliovirus infects, making CD44 unlikely to be the factor limiting poliovirus replication in human tissues.

Correlation between poliovirus type 1 Mahoney replication in blood cells and neurovirulence.

Poliovirus (PV) is not often described as a monocyte- or macrophage-tropic virus; however, previous work indicated that neurovirulent PV type 1 Mahoney [PV(1)Mahoney] can productively infect primary human monocytes. To determine whether this replication has a functional role in pathogenesis, primary human mononuclear blood cells were infected with pairs of attenuated and neurovirulent strains of PV. Two neurovirulent strains of PV, PV(1)Mahoney and PV(2)MEF-1, replicated faster and to higher titers than attenuated counterparts PV(1)Sabin and PV(2)W-2, respectively, in primary human monocytes, suggesting that this replication may contribute to pathogenesis. PV(3)Leon grew weakly, while PV(3)Sabin, PV(2)Sabin, and PV(2) P712 did not replicate in these cells, perhaps because of their slow replication cycle. In U937 cells, a monocytelike cell line, PV(1)Mahoney replicated but PV(1)Sabin did not, while both grew well in HeLa cells. When molecular recombinants of PV(1)Mahoney and PV(1)Sabin were assessed, a correlation between neurovirulence and the ability to replicate in primary human mononuclear blood cells was found. Surprisingly, infectious centers assays with primary human mononuclear blood cells and U937 cells indicated that despite the lower overall viral yield, more cells are initially infected with the attenuated viruses. These results indicate that there are virulence-specific differences in the ability of PV(1)Mahoney to replicate in monocytes and suggest that there may be factors in monocytes that virulent strains of PV require.

Role of poliovirus receptors in the spread of the infection.

Although the poliovirus receptor (PVR) has been cloned, lack of knowledge of its precise tissue distribution makes assessment of its role in mediating poliomyelitis difficult. Our recent work demonstrated that PVR is expressed on human monocytes and that primary human blood cells can support PV replication. In the current work, we demonstrate that CD14-positive cells (monocytes) support PV replication but that only a minority (< 10%) of the cells become infected. In other preliminary studies, immunocytochemical analyses of human brain tissue demonstrated the presence of PVR in the olfactory bulb, a tissue thought to not support PV replication. Thus, it appears that some apparently "ectopic" sites of PVR expression may in fact be sites for PV replication, whereas other sites may indeed be restricted. The ability of monocytes to replicate PV may pertain to some unexplained phenomena in PV pathogenesis, such as the specific cell type carrying out the initial round of replication in the gut, sites of extraneural replication and transport of the virus into the CNS. Preliminary studies with monocytes from post-polio syndrome patients showed no difference in the levels of PVR relative to control monocytes. In other preliminary work, PVR was shown to be phosphorylated and its expression on monocytes increased by treatment with gamma-interferon. The normal function of PVR is likely to be involved in monocyte function during immune activation.

Low levels of poliovirus replication in primary human monocytes: possible interactions with lymphocytes.

To investigate the molecular mediators of poliovirus tissue tropism, the correlation between poliovirus replication and poliovirus receptor expression was examined in a primary human tissue system. Earlier work [M. Freistadt, H. Fleit, and E. Wimmer, Virology 195: 798-803 (1993)] showed that the cellular receptor for poliovirus is present in 87% of primary human monocytes and that peripheral blood mononuclear cells support poliovirus replication. In the current work, monocytes, obtained by adherence or by a novel negative selection procedure using specific monoclonal antibodies to lymphocyte surface antigens, supported poliovirus replication. However, total virus yield was low and infectious centers assays revealed that a minority (6%) of monocytes become productively infected. Viral yield from monocytes was lower than from the heterogeneous mononuclear cells; however, when uninfected lymphocytes were added back to infected monocytes, the higher viral yield was restored. The purity of the cells did not significantly affect the number of cells infected. These results suggest that more poliovirus is produced per cell from activated rather than unactivated monocytes. Furthermore, poliovirus replication in monocytes may reflect genuine in vivo replication and comprise a system in which to determine molecular mediators of poliovirus tissue tropism.

Poliovirus receptor on human blood cells: a possible extraneural site of poliovirus replication.

In order to determine whether any primary human blood cells have the ability to replicate poliovirus (PV), peripheral blood cell components were isolated and analyzed for their cell surface expression of the poliovirus receptor (PVR). Following two-color immunfluoresence staining with lineage-specific markers, the cells were analyzed by flow-cytometric methods. PVR cell surface expression was detected on most mononuclear cells expressing CD14, a marker for mononuclear phagocytes. There was no PVR cell surface expression on platelets and extremely low levels on polymorphonuclear leukocytes. Mononuclear leukocytes from Ficoll density centrifugation were found to support PV replication. The finding of PVR on mononuclear phagocytes and the ability of primary human blood cells to support PV replication in the absence of cultivation has implications for both the normal and pathogenic role of PVR.

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.

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.

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.

Discontinuously synthesized mRNA from Trypanosoma brucei contains the highly methylated 5' cap structure, m7GpppA*A*C(2'-O)mU*A.

Trypanosoma brucei mRNA is discontinuously synthesized via the 5' addition of a "mini-exon" sequence. The mini-exon-specific cap structure was purified from a complete RNase T2 and phosphatase digest of in vivo 32P-labeled poly(A)+RNA. The purified cap structure was sequenced by a series of partial and complete enzymatic digests by nuclease P1 and venom phosphodiesterase. This approach demonstrated that the T. brucei mini-exon cap structure consists of N7-methylguanosine linked in a conventional 5'-5' triphosphate bond to five nucleotides, in the sequence A*A*C(2'-O)mU*A (asterisks denote modifications that were not fully characterized in this work). 2'-O-methylations and other modifications appear to be present in this novel cap structure, which could have a functional role in the metabolism of the mini-exon.

Direct analysis of the mini-exon donor RNA of Trypanosoma brucei: detection of a novel cap structure also present in messenger RNA.

The mini-exon, a short segment found at the 5' end of trypanosome mRNAs, is contributed by a small RNA, the mini-exon donor (medRNA). In vivo 32P-labeled medRNA, a set of smaller RNAs related to it, and mRNA, were purified from Trypanosoma brucei by hybrid selection and gel electrophoresis. Using RNA fingerprinting and sequencing techniques, mini-exon oligonucleotides were identified and characterized. We detected a novel 5' terminal capped oligonucleotide present in both medRNA and mRNA. This structure contained m7G and at least four modified nucleotides, not identified previously. If the T. brucei mini-exon has exactly four transcribed nucleotides upstream from its originally designated 5' end, it would begin with the sequence: m7GpppA*A*C*U*AA*CG (asterisks denote modification) and medRNA would be 140 nucleotides long, excluding the m7G residue. The mini-exon contains, and retains during its transfer to mRNA, a novel 5' terminal structure whose presence could confer unique functional attributes.