Enterovirus 71 (EV71) is a major causative agent of hand, foot and mouth disease (HFMD), a common febrile disease occurring mainly in young children. Although clinical manifestations of HFMD are usually mild and self limiting, a severe EV71 outbreak can lead to a diverse array of neurological diseases. Identification of the specific cellular receptors is crucial for elucidating the mechanism of early virus-host interactions and the pathogenesis of enteroviruses. Here we identify human P-selectin glycoprotein ligand-1 (PSGL-1; CD162), a sialomucin membrane protein expressed on leukocytes that has a major role in early stages of inflammation, as a functional receptor for EV71 using an expression cloning method by panning. The N-terminal region of PSGL-1 binds specifically to EV71. Stable PSGL-1 expression allowed EV71 entry and replication, and development of cytopathic effects in nonsusceptible mouse L929 cells. Five out of eight EV71 strains bound soluble PSGL-1 and used intact PSGL-1 as the primary receptor for infection of Jurkat T cells. Three other EV71 strains did not use PSGL-1, suggesting the presence of strain-specific replication of EV71 in leukocytes. EV71 replicated in nonleukocyte cell lines in a PSGL-1-independent manner, indicating the presence of alternative receptor(s) for EV71. The identification of PSGL-1 as a receptor for EV71 sheds new light on a role for PSGL-1-positive leukocytes in cell tropism and pathogenesis during the course of HFMD and other EV71-mediated diseases.

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The hepatitis C virus (HCV) nonstructural protein (NS)5AHepatitis C virus (HCV) infects 170 million people worldwide and frequently leads to cirrhosis or hepatocellular carcinoma (6, 29). HCV is classified in the family Flaviviridae and possesses a single-stranded positive-sense RNA with a length of 9.6 kb. The HCV genome encodes a single large precursor polyprotein composed of about 3,000 amino acids (aa) that is processed by cellular and viral proteases, resulting in at least 10 structural and nonstructural (NS) proteins (29). Details of HCV's replication cycle are unknown because of the low viral load in the sera of HCV-infected individuals and the lack of a reliable and robust cell culture system to support HCV infection and replication. The development of HCV RNA replicons in which a synthetic HCV genomic or subgenomic RNA replicates efficiently in the human hepatocarcinoma cell line Huh-7 has enabled the study of viral RNA replication in cell culture (4,20,24). The HCV RNA replication complex, composed of the viral NS proteins and host cellular proteins, replicates the viral RNA genome at the intracellular membrane. Thus far, the HCV replicon system has greatly contributed to the understanding of HCV replication and pathogenesis associated with the expression of viral NS proteins. Replication of positive-strand RNA viruses generally involves certain intracellular membrane structures, including the endoplasmic reticulum (ER), Golgi apparatus, endosome, and lysosome (39).Recently, several groups have succeeded in demonstrating cellfree replication activities of replication complexes in crude membrane fractions of HCV subgenomic replicon cells (2, 3, 14, 53). These cell-free systems provide semi-intact polymerase assays for biochemical dissection of HCV RNA replication and are a useful source for the isolation of HCV replication complexes. Replication complexes were detected in detergent-resistant membrane structures, most likely lipid raft structures (2, 14). Although HCV NS proteins presumably form a membrane-associated RNA replication complex with host proteins, the precise components and mechanisms for replication are poorly understood.HCV NS5A is a phosphoprotein that appears to possess multiple and diverse functions in viral replication, interferon resistance, and pathogenesis (26,35). Cell culture-adaptive mutations have been shown to cluster in the central portion of NS5A in subgenomic HCV replicons, indicating that NS5A is involved in the viral replication process either directly or by interacting with host cellular proteins (4, 55). This observation, together with the modulation of NS5A hyperphosphorylation by NS3, NS4A, and NS4B and physical interaction with other viral NS proteins, strongly supports the notion that NS5A is an essential component of the HCV replication complex (21,30,36). NS5A has been shown to be associated with a range of cellular proteins involved in cellular signaling pathways, such as interferon-induced kinase PKR (11), growth factor receptor-binding protein 2 (Grb2) (45), p53...

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a component of viral replicase and is well known to modulate the functions of several host proteins. Here, we show that NS5A specifically interacts with FKBP8, a member of the FK506-binding protein family, but not with other homologous immunophilins. Three sets of tetratricopeptide repeats in FKBP8 are responsible for interactions with NS5A. The siRNA-mediated knockdown of FKBP8 in a human hepatoma cell line harboring an HCV RNA replicon suppressed HCV RNA replication, and this reduction was reversed by the expression of an siRNA-resistant FKBP8 mutant. Furthermore, immunoprecipitation analyses revealed that FKBP8 forms a complex with Hsp90 and NS5A. Treatment of HCV replicon cells with geldanamycin, an inhibitor of Hsp90, suppressed RNA replication in a dose-dependent manner. These results suggest that the complex consisting of NS5A, FKBP8, and Hsp90 plays an important role in HCV RNA replication.

Some strains of enterovirus 71 (EV71), but not others, infect leukocytes by binding to a specific receptor molecule: the P-selectin glycoprotein ligand-1 (PSGL-1). We find that a single amino acid residue within the capsid protein VP1 determines whether EV71 binds to PSGL-1. Examination of capsid sequences of representative EV71 strains revealed that the PSGL-1-binding viruses had either a G or a Q at residue 145 within the capsid protein VP1 (VP1-145G or Q), whereas PSGL-1-nonbinding viruses had VP1-145E. Using site-directed mutagenesis we found that PSGL-1-binding strains lost their capacity to bind when VP1-145G/Q was replaced by E; conversely, nonbinding strains gained the capacity to bind PSGL-1 when VP1-145E was replaced with either G or Q. Viruses with G/Q at VP1-145 productively infected a leukocyte cell line, Jurkat T-cells, whereas viruses with E at this position did not. We previously reported that EV71 binds to the N-terminal region of PSGL-1, and that binding depends on sulfated tyrosine residues within this region. We speculated that binding depends on interaction between negatively charged sulfate groups and positively charged basic residues in the virus capsid. VP1-145 on the virus surface is in close proximity to conserved lysine residues at VP1-242 and VP1-244. Comparison of recently published crystal structures of EV71 isolates with either Q or E at VP1-145 revealed that VP1-145 controls the orientation of the lysine side-chain of VP1-244: with VP1-145Q the lysine side chain faces outward, but with VP1-145E, the lysine side chain is turned toward the virus surface. Mutation of VP1-244 abolished virus binding to PSGL-1, and mutation of VP1-242 greatly reduced binding. We propose that conserved lysine residues on the virus surface are responsible for interaction with sulfated tyrosine residues at the PSGL-1 N-terminus, and that VP1-145 acts as a switch, controlling PSGL-1 binding by modulating the exposure of VP1-244K.

We determined the complete genomic sequences of nine type 1 immunodeficient vaccine-derived poliovirus (iVDPV) isolates obtained over a 337-day period from a poliomyelitis patient from Taiwan with common variable immunodeficiency. The iVDPV isolates differed from the Sabin type 1 oral poliovirus vaccine (OPV) strain at 1.84% to 3.15% of total open reading frame positions and had diverged into at least five distinct lineages. Phylogenetic analysis suggested that the chronic infection was initiated by the fifth and last OPV dose, given 567 days before onset of paralysis, and that divergence of major lineages began very early in the chronic infection. Key determinants of attenuation in Sabin 1 had reverted in the iVDPV isolates, and representative isolates of each lineage showed increased neurovirulence for PVR-Tg21 transgenic mice. None of the isolates had retained the temperature-sensitive phenotype of Sabin 1. All isolates were antigenic variants of Sabin 1, having multiple amino acid substitutions within or near neutralizing antigenic sites 1, 2, and 3a. Antigenic divergence of the iVDPV variants from Sabin 1 followed two major independent evolutionary pathways. The emergence of distinct coreplicating lineages suggests that iVDPVs can replicate for many months at separate sites in the gastrointestinal tract. Some isolates had mosaic genome structures indicative of recombination across and within lineages. iVDPV excretion apparently ceased after 30 to 35 months of chronic infection. The appearance of a chronic VDPV excretor in a tropical, developing country has important implications for the strategy to stop OPV immunization after eradication of wild polioviruses.The central strategy of the World Health Organization Global Polio Eradication Initiative is widespread use of oral poliovirus vaccine (OPV) at high rates of coverage. This strategy has reduced the global incidence of polio by over 99% since the start of the Initiative in 1988 and restricted wild poliovirus circulation to countries in western and central Africa and southern Asia (87). However, use of OPV is associated with some rare adverse events, including the appearance of cases of vaccine-associated paralytic poliomyelitis among OPV recipients and contacts (76), and the occurrence of polio outbreaks associated with circulating vaccine-derived poliovirus (cVDPV) (36). While cVDPV outbreaks can be prevented by maintenance of high rates of OPV coverage, the occurrence of vaccine-associated paralytic poliomyelitis is associated with the inherent genetic instability of the live, attenuated OPV strains (56).In immunocompetent individuals, the risk of vaccine-associated paralytic poliomyelitis is very low, estimated in the United States at 1 case per 2.4 million OPV doses distributed (75,76).

Correction to: The EMBO Journal (2006) 25, 5015–5025. doi:10.1038/sj.emboj.7601367.

Enterovirus 71 (EV71) is one of the major causative agents of hand, foot, and mouth disease, a common febrile disease in children; however, EV71 has been also associated with various neurological diseases including fatal cases in large EV71 outbreaks particularly in the Asia Pacific region. Recently we identified human P-selectin glycoprotein ligand-1 (PSGL-1) as a cellular receptor for entry and replication of EV71 in leukocytes. PSGL-1 is a sialomucin expressed on the surface of leukocytes, serves as a high affinity counterreceptor for selectins, and mediates leukocyte rolling on the endothelium. The PSGL-1–P-selectin interaction requires sulfation of at least one of three clustered tyrosines and an adjacent O-glycan expressing sialyl Lewis x in an N-terminal region of PSGL-1. To elucidate the molecular basis of the PSGL-1–EV71 interaction, we generated a series of PSGL-1 mutants and identified the post-translational modifications that are critical for binding of PSGL-1 to EV71. We expressed the PSGL-1 mutants in 293T cells and the transfected cells were assayed for their abilities to bind to EV71 by flow cytometry. We found that O-glycosylation on T57, which is critical for PSGL-1–selectin interaction, is not necessary for PSGL-1 binding to EV71. On the other hand, site-directed mutagenesis at one or more potential tyrosine sulfation sites in the N-terminal region of PSGL-1 significantly impaired PSGL-1 binding to EV71. Furthermore, an inhibitor of sulfation, sodium chlorate, blocked the PSGL-1–EV71 interaction and inhibited PSGL-1-mediated viral replication of EV71 in Jurkat T cells in a dose-dependent manner. Thus, the results presented in this study reveal that tyrosine sulfation, but not O-glycosylation, in the N-terminal region of PSGL-1 may facilitate virus entry and replication of EV71 in leukocytes.