Following ribonuclease digestion of methyl-3H-labeled B77 avian sarcoma virus RNA subunits, methylated oligonucleotides were isolated by diethylaminoethylcellulose chromotogrpahy. Partial nucleotide sequences were deduced from the known enzymatic specificities of the ribonucleases. In addition to methylated nucleosides in the 5'-terminal cap structure, m7G(5')GmpCp, N6-methyladenosine(m6A) was found to be present in only two internal sequences of the RNA molecule, Gpm6ApC and Apm6ApC. The average numbers of methylated nucleosides per RNA subunit are about 12-13 in Gpm6ApC, 1-2 in Apm6ApC, and 2 in m7GpppGmpCp. The sequences containing m6A in B77 sarcoma virus RNA are identical to m6A-containing sequences previously reported for the bulk mRNA from HeLa cells (Wei, C.M., Gershowitz, A., and Moss, B. (1976), Biochemistry 15, 397-401). Analysis of the oligonucleotides produced by RNase A digestion indicated that the sequence of bases on the 5' side of these trinucleotides is not specific. The oligonucleotide profile, however, was highly reproducible in different virus preparations. This suggests that the methylations occur at specific positions on the RNA molecule. Some of the methylated oligonucleotides produced by RNase A digestion appear to be present in less than molar amounts. Several hypotheses are proposed to explain this result.

The interaction of viruses with host cell receptors is the initial step in viral infection and is an important determinant of virus host range, tissue tropism, and pathogenesis. The complement regulatory protein decayaccelerating factor (DAF/CD55) is an attachment receptor for enterovirus 70 (EV70), a member of the Picornaviridae, commonly associated with an eye infection in humans known as acute hemorrhagic conjunctivitis. In early work, the EV70 receptor on erythrocytes, responsible for its hemagglutinating activity, was shown to be sensitive to neuraminidase, implying an essential role for sialic acid in virus attachment. Here, we extend these results to show that cell surface sialic acid is required for EV70 binding to nucleated cells susceptible to virus infection and that sialic acid binding is important in productive infection. Through the use of site-directed mutagenesis to eliminate the single N-linked glycosylation site of DAF and of a chimeric receptor protein in which the O-glycosylated domain of DAF was replaced by a region of the HLA-B44 molecule, a role in EV70 binding for the sialic acid residues of DAF was excluded, suggesting the existence of at least one additional, sialylated EV70-binding factor at the cell surface. Treatment of cells with metabolic inhibitors of glycosylation excluded a role for the N-linked oligosaccharides of glycoproteins but suggested that O-linked glycosylation is important for EV70 binding.Enterovirus 70 (EV70) is an unusual human pathogen of the Enterovirus genus of the family Picornaviridae. Unlike the majority of human enteroviruses, EV70 apparently does not replicate in the enteric tract but rather is primarily associated with an infection of the eye commonly referred to as acute hemorrhagic conjunctivitis (AHC) (for a review, see reference 49). Since the emergence of the virus in western Africa in 1969, it has been responsible for two pandemics and numerous smaller outbreaks and has been implicated in roughly 100 million cases of AHC; most recently, occurrences have been reported in India (24, 47), Japan (44), and Israel (38). In rare instances, a neurological illness that resembles acute poliomyelitis follows, usually within several weeks of the onset of the conjunctivitis (13), suggesting that the tissue tropism of EV70 also includes the central nervous system (CNS). It has also been observed that, whereas the majority of human enteroviruses are restricted in vitro to replication in cells of human or primate origin, EV70 replicates with various efficiencies in cells derived from a wide variety of mammalian species (50).The first step in a viral infection is the binding of the virus to a specific cell surface receptor. This step is often an important determinant of virus host range, tissue tropism, and pathogenesis (for a review see reference 34) and may contribute to the molecular basis for the unusual tissue tropism and host range of EV70. Previous work in our laboratory demonstrated that EV70 binds to the complement control protein decay-accelerating factor (D...

Enterovirus 70 (EV70) is a recently emerged human pathogen belonging to the family Picornaviridae. The ability of EV70 to infect a wide variety of nonprimate cell lines in vitro is unique among human enteroviruses. The importance of virus receptors as determinants of viral host range and tropism led us to study the host cell receptor for this unusual picornavirus. We produced a monoclonal antibody (MAb), EVR1, which bound to the surface of HeLa cells and protected them against infection by EV70 but not by poliovirus or by coxsackievirus B3. This antibody also inhibited the binding of [ 35 S]EV70 to HeLa cells. MAb EVR1 did not bind to monkey kidney (LLC-MK 2) cells, nor did it protect these cells against virus infection. In Western immunoassays and in immunoprecipitations, MAb EVR1 identified a HeLa cell glycoprotein of approximately 75 kDa that is attached to the cell membrane by a glycosyl-phosphatidylinositol (GPI) anchor. Decay-accelerating factor (DAF, CD55) is a 70-to 75-kDa GPI-anchored membrane protein that is involved in the regulation of complement and has also been shown to function as a receptor for several enteroviruses. MAb EVR1 bound to Chinese hamster ovary (CHO) cells constitutively expressing human DAF. Anti-DAF MAbs inhibited EV70 binding to HeLa cells and protected them against EV70 infection. Transient expression of human DAF in murine NIH 3T3 cells resulted in binding of labelled EV70, and stably transformed NIH 3T3 cells expressing DAF were able to support virus replication. These data indicate that the HeLa cell receptor for EV70 is DAF.

Sialic acids are a ubiquitous family of negatively charged sugar molecules found on the surfaces of mammalian cells, usually at the termini of glycans attached to glycoproteins, glycosphingolipids, and the proteoglycan keratan sulfate (2, 10, 72). Sialic acid is an essential component of cell surface receptors for a variety of microorganisms and microbial toxins (2,34,47). Members of at least eight different virus families-Ortho-

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