Georg Herrler

Isolated HE-protein from hemagglutinating encephalomyelitis virus and bovine coronavirus has receptor-destroying and receptor-binding activity

Abstract Bovine coronavirus (BCV) and hemagglutinating encephalomyelitis virus (HEV) from swine were found to grow to high titers in MDCK I cells, a subline of Madin Darby canine kidney cells. Virus grown in these cells was used to isolate and purify the HE-protein. This protein has been shown recently to have acetylesterase activity and to function as the receptor-destroying enzyme of BCV. Here we show that HEV contains this enzyme, too. The glycoproteins were solubilized by treatment of virions with octylglucoside. Following centrifugation through a sucrose gradient the surface proteins S and HE (hemagglutinin-esterase) were obtained in purified form. After removal of the detergent by dialysis, HE formed rosettes as shown by electron microscopy. The purified HE protein retained acetylesterase activity and was able to function as a receptor-destroying enzyme rendering red blood cells resistant against agglutination by both coronaviruses. HE protein released from the viral membrane failed to agglutinate red blood cells. However, it was found to recognize glycoconjugates containing N-acetyl-9-O-acetylneuraminic acid as indicated by a binding assay with rat serum proteins blotted to nitrocellulose and by its ability to inhibit the hemagglutinating activity of BCV, HEV, and influenza C virus. The purified enzyme provides a useful tool for analyzing the cellular receptors for coronaviruses.

Recombinant measles virus requiring an exogenous protease for activation of infectivity.

Proteolytic cleavage of the fusion protein (F) is an important control mechanism of the biological activity of paramyxoviruses. The sequence R-R-H-K-R(112) at the cleavage site of the F protein of measles virus (MV) was altered by site-directed mutagenesis to R-N-H-N-R(112), which is not recognized by the ubiquitous cellular protease furin. When transiently expressed in cell cultures standard F protein was cleaved, whereas the mutant remained in the uncleaved form. Syncytium formation by the mutant that was analysed after coexpression with haemagglutinin protein depended on the presence of trypsin. Recombinant MV containing the mutation required trypsin activation for fusion and infectivity in cell culture. Intranasal infection of transgenic mice susceptible to MV infection (Ifnar(tm)-CD46Ge) resulted in a moderately productive infection and inflammation of the lung. In contrast to parental virus, intracerebral inoculation did not induce neural disease. The possible effects of the change in cleavage activation on tissue tropism and pathogenicity are discussed.

Neuraminidase treatment of avian infectious bronchitis coronavirus reveals a hemagglutinating activity that is dependent on sialic acid-containing receptors on erythrocytes.

Abstract The interaction of infectious bronchitis virus (IBV) with erythrocytes was analyzed. The binding activity of IBV was not sufficient to agglutinate chicken erythrocytes. However, it acquired hemagglutinating activity after treatment with neuraminidase to remove α2,3-linked N-acetylneuraminic acid from the surface of the virion. Pretreatment of erythrocytes with neuraminidase rendered the cells resistant to agglutination by IBV. Susceptibility to agglutination was restored by resialylation of asialo-erythrocytes to contain α2,3-linked sialic acid. These results indicate that IBV attaches to receptors on erythrocytes, the crucial determinant of which is sialic acid α2,3-linked to galactose. In contrast to other enveloped viruses with such a binding specificity (influenza viruses and paramyxoviruses) IBV lacks a receptor-destroying enzyme.

Structure and Function of the Hef Glycoprotein of Influenza C Virus

Publisher Summary Soon after the first isolation of an influenza C virus from a patient, it became obvious that this virus differs from other myxoviruses in several aspects. Pronounced differences have been observed in the interactions between the virus and cell surfaces, suggesting that influenza C virus attaches to the receptors different from those recognized by other myxoviruses. While influenza A and B viruses agglutinate erythrocytes from many species, including humans, the spectrum of erythrocytes agglutinated by influenza C virus is much more restricted. Erythrocytes from rats, mice, and adult chickens are suitable for hemagglutination and hemadsorption tests; cells from other species, however, react not at all or only poorly with influenza C virus. Differences are also observed so far as hemagglutination inhibitors are concerned. A variety of glycoproteins have been shown to prevent influenza A and B viruses from agglutinating erythrocytes. In the case of influenza C virus, rat serum was for a long time the only known hemagglutination inhibitor. A difference in the receptors for influenza C virus and other myxo-viruses was also suggested by studies on the receptor-destroying enzyme. The ability of influenza C virus to inactivate its own receptors was reported soon after the first isolation of this virus from a patient. However, the influenza C enzyme did not affect the receptors of other myxoviruses and, conversely, the receptor-destroying enzyme of either of the latter viruses was unable to inactivate the receptors for influenza C virus on erythrocytes. While the enzyme of influenza A and B virus was characterized as a neuraminidase in the 1950s, even with refined methodology no such activity was detectable with influenza C virus. It is now known that both the receptor-binding and receptor-destroying activities, as well as the fusion activity of influenza C virus are mediated by the only glycoprotein present on the surface of the virus particle. The structure and functions of this protein, which is designated as HEF, are reviewed in this chapter.

Hemagglutinating encephalomyelitis virus attaches to N-acetyl-9-O-acetylneuraminic acid-containing receptors on erythrocytes: comparison with bovine coronavirus and influenza C virus.

Abstract The receptors for the hemagglutinating encephalomyelitis virus (HEV, a porcine coronavirus) on chicken erythrocytes were analyzed and compared to the receptors for bovine coronavirus (BCV) and influenza C virus. Evidence was obtained that HEV requires the presence of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) on the cell surface for agglutination of erythrocytes as has been previously shown for BCV and influenza C virus: (i) Incubation of red blood cells with sialate 9-O-acetylesterase, the receptor-destroying enzyme of influenza C virus, rendered the erythrocytes resistant against agglutination by each of the three viruses; (ii) Human erythrocytes which are resistant to agglutination by HEV acquire receptors for HEV after resialylation with Neu5,9Ac2. Sialylation of red blood cells with limiting amounts of sialic acid indicated that strain JHB/1/66 of influenza C virus requires less Neu5,9Ac2 for agglutination of erythrocytes than the two coronaviruses, both of which were found to be similar in their reactivity with Neu5,9Ac2-containing receptors.

Characterization of the sialic acid binding activity of transmissible gastroenteritis coronavirus by analysis of haemagglutination-deficient mutants.

Transmissible gastroenteritis coronavirus (TGEV) agglutinates erythrocytes of several species by virtue of sialic acid binding activity of the surface protein S. We have isolated and characterized five haemagglutination-defective (HAD) mutants. In contrast to the parental virus, the mutants were unable to bind to porcine submandibulary mucin, a substrate rich in sialic acid. Each of the mutants was found to contain a single point mutation in the S protein (Cys155Phe, Met195Val, Arg196Ser, Asp208Asn or Leu209Pro), indicating that these amino acids are affecting the sialic acid binding site. In four of the HAD mutants a nearby antigenic site is affected in addition to the sialic acid binding site, as indicated by reactivity with monoclonal antibodies. The parental virus was found to have an increased resistance to the detergent octylglucoside compared to the HAD mutants. This effect depended on cellular sialoglycoconjugates bound to the virion. If the binding of sialylated macromolecules was prevented by neuraminidase treatment, the parental virus was as sensitive to octylglucoside as were the HAD mutants. We discuss the possibility that the sialic acid binding activity helps TGEV to resist detergent-like substances encountered during the gastrointestinal passage and thus facilitates the infection of the intestinal epithelium. An alternative function of the sialic acid binding activity - accessory binding to intestinal tissues - is also discussed.

Sialic Acid as Receptor Determinant of Ortho- and Paramyxoviruses

The designation myxoviruses has been chosen historically for a group of viruses comprising influenza viruses, Newcastle disease virus, and mumps virus, because they were able to interact with mucins (see Chapter 5). A characteristic feature of these viruses was the presence of two activities that appeared to counteract each other. On the one hand, the viruses bind to receptors present on erythrocytes, resulting in a hemagglutination reaction. On the other hand, they contain a receptor-destroying enzyme rendering erythrocytes resistant to the viral agglutinating activity. The receptor determinant recognized by this group of viruses turned out to be sialic acid, and the receptor-destroying enzyme has been characterized as a sialidase and a sialate O-acetylesterase. Despite the similarities, members of the myxovirus group differ in several fundamental aspects, e.g., the presence of a segmented or a nonsegmented genome. Therefore, they have been grouped into two taxonomic families. Influenza A, B, and C viruses belong to the family Orthomyxoviridae. Viruses such as mumps virus, Newcastle disease virus, Sendai virus, and other parainfluenza viruses are members of the family Paramyxoviridae. In addition to the receptor-binding and the receptor-destroying activity, these viruses have a fusion activity. They differ, however, in the distribution of the three activities on the viral surface glycoproteins. With influenza A and B viruses, receptor-binding and fusion activity are functions of the hemagglutinin (HA). A second glycoprotein (NA) is responsible for the sialidase activity. In the case of paramyxoviruses, the receptor-binding and the sialidase activity are combined on one type of glycoprotein designated HN, whereas the fusion activity is localized on a separate glycoprotein (F). Influenza C viruses have only a single glycoprotein that is responsible for all three activities. They also differ from the other viruses in that they recognize N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) rather than N-acetylneuraminic acid (Neu5Ac) as receptor determinant. Furthermore, the receptor-destroying enzyme of influenza C viruses is a sialate 9-O-acetylesterase. In this chapter, the interaction of viral proteins with sialic acid and the biological significance of the receptor-binding and the receptor-destroying activity are discussed.

The hemagglutinating glycoproteins of influenza B and C viruses are acylated with different fatty acids

We present evidence that the hemagglutinin (HA) of influenza B virus and the glycoprotein of influenza C virus (HEF) are acylated. The fatty acid linkage is sensitive to treatment with hydroxylamine and mercaptoethanol, which points to a labile thioester-type linkage. The HA of influenza B virus contains mainly palmitic acid, whereas the HEF glycoprotein of influenza C virus is acylated with stearic acid which has not been observed before as the prevailing fatty acid in viral or cellular acyl proteins.

A single point mutation of the influenza C virus glycoprotein (HEF) changes the viral receptor-binding activity

Abstract From strain JHB/1/66 of influenza C virus a mutant was derived with a change in the cell tropism. The mutant was able to grow in a subline of Madin-Darby canine kidney cells (MDCK II) which is resistant to infection by the parent virus due to a lack of receptors. Inactivation of cellular receptors by either neuraminidase or acetylesterase and generation of receptors by resialylation of cells with N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) indicated that 9-O-acetylated sialic acid is a receptor determinant for both parent and mutant virus. However, the mutant required less Neu5,9Ac2 on the cell surface for virus attachment than the parent virus. The increased binding efficiency enabled the mutant to infect cells with a low content of 9-O-acetylated sialic acid which were resistant to the parent virus. By comparing the nucleotide sequences of the glycoprotein (HEF) genes of the parent and the mutant virus only a single point mutation could be identified on the mutant gene. This mutation at nucleotide position 872 causes an amino acid exchange from threonine to isoleucine at position 284 on the amino acid sequence. Sequence similarity with a stretch of amino acids involved in the receptor-binding pocket of the influenza A hemagglutinin suggests that the mutation site on the influenza C glycoprotein (HEF) is part of the receptor-binding site.

Analysis of Cellular Receptors for Human Coronavirus OC43

Bovine coronavirus (BCV), human coronavirus OC43 (HCV-OC43) and hemagglutinating encephalomyelitis virus (HEV) are serologically related viruses that all have hemagglutinating activity. The receptor determinant for attachment to erythrocytes has been shown to be N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2). We compared the ability of the three coronaviruses to recognize 9-O-acetylated sialic acid and found that they all bind to Neu5,9Ac2 attached to galactose in either A2,3 or A2,6-linkage. There are, however, some differences in the minimum amount of sialic acid that is required on the cell surface for agglutination by these viruses. Evidence is presented that HCV-OC43 uses Neu5,9Ac2 as a receptor determinant not only for agglutination of erythrocytes but also for attachment to and infection of a cultured cell line, MDCK I cells.