Some factors affecting the production, by cultured baby-hamster kidney cells, of BHK glycoprotein I which cross-reacts immunologically with Tamm-Horsfall glycoprotein.

Cultured baby-hamster kidney cells (BHK-21/C13), which are adapted to grow in suspension (strain 2P), roduce a glycoprotein, termed BHK glycoprotein I, which cross-reacts immunologically with hamster urinary (Tamm-Horsfall glycoprotein. BHK glycoprotein I was isolated in an electrophoretically (sodium dodecyl sulphate/polyacrylamide gel) homogeneous form by application of affinity chromatography to the medium in which cells had been cultured. Insolubilized anti-(Tamm-Horsfall glycoprotein immunoglobulin G) was used as the adsorbent. The amount of BHK glycoprotein I associated with the cultured cells was found by both radioimmunoassay and immunofluorescence to be related to the amount of Ca2+ in the medium and to the particular stage of the cell cycle. 5'-Nucleotidase was also shed by the cells into the culture medium in amounts related to the stage of the cell cycle. The turnover of hamster Tamm-Horsfall glycoprotein in vivo appeared to be considerably more rapid than can be accounted for by cell turnover. Hamster Tamm-Horsfall glycoprotein was shown to be ineffective in inhibiting agglutination of chicken erythrocytes caused by influenza virus.     

The murine coronavirus mouse hepatitis virus strain A59 from persistently infected murine cells exhibits an extended host range.

In murine 17 Cl 1 cells persistently infected with murine coronavirus mouse hepatitis virus strain A59 (MHV-A59), expression of the virus receptor glycoprotein MHVR was markedly reduced (S. G. Sawicki, J. H. Lu, and K. V. Holmes, J. Virol. 69:5535-5543, 1995). Virus isolated from passage 600 of the persistently infected cells made smaller plaques on 17 Cl 1 cells than did MHV-A59. Unlike the parental MHV-A59, this variant virus also infected the BHK-21 (BHK) line of hamster cells. Virus plaque purified on BHK cells (MHV/BHK) grew more slowly in murine cells than did MHV-A59, and the rate of viral RNA synthesis was lower and the development of the viral nucleocapsid (N) protein was slower than those of MHV-A59. MHV/BHK was 100-fold more resistant to neutralization with the purified soluble recombinant MHV receptor glycoprotein (sMHVR) than was MHV-A59. Pretreatment of 17 Cl 1 cells with anti-MHVR monoclonal antibody CC1 protected the cells from infection with MHV-A59 but only partially protected them from infection with MHV/BHK. Thus, although MHV/BHK could still utilize MHVR as a receptor, its interactions with the receptor were significantly different from those of MHV-A59. To determine whether a hemagglutinin esterase (HE) glycoprotein that could bind the virions to 9-O-acetylated neuraminic acid moieties on the cell surface was expressed by MHV/BHK, an in situ esterase assay was used. No expression of HE activity was detected in 17 Cl 1 cells infected with MHV/BHK, suggesting that this virus, like MHV-A59, bound to cell membranes via its S glycoprotein. MHV/BHK was able to infect cell lines from many mammalian species, including murine (17 Cl 1), hamster (BHK), feline (Fcwf), bovine (MDBK), rat (RIE), monkey (Vero), and human (L132 and HeLa) cell lines. MHV/BHK could not infect dog kidney (MDCK I) or swine testis (ST) cell lines. Thus, in persistently infected murine cell lines that express very low levels of virus receptor MHVR and which also have and may express alternative virus receptors of lesser efficiency, there is a strong selective advantage for virus with altered interactions with receptor (D. S. Chen, M. Asanaka, F. S. Chen, J. E. Shively, and M. M. C. Lai, J. Virol. 71:1688-1691, 1997; D. S. Chen, M. Asanaka, K. Yokomori, F.-I. Wang, S. B. Hwang, H.-P. Li, and M. M. C. Lai, Proc. Natl. Acad. Sci. USA 92:12095-12099, 1995; P. Nedellec, G. S. Dveksler, E. Daniels, C. Turbide, B. Chow, A. A. Basile, K. V. Holmes, and N. Beauchemin, J. Virol. 68:4525-4537, 1994). Possibly, in coronavirus-infected animals, replication of the virus in tissues that express low levels of receptor might also select viruses with altered receptor recognition and extended host range.     

Membrane vesicles containing the Sendai virus binding glycoprotein, but not the viral fusion protein, fuse with phosphatidylserine liposomes at low pH

Membrane vesicles containing the Sendai virus hemagglutinin/neuraminidase (HN) glycoprotein were able to induce carboxyfluorescein (CF) release from loaded phosphatidylserine (PS) but not loaded phosphatidylcholine (PC) liposomes. Similarly, fluorescence dequenching was observed only when HN vesicles, bearing self-quenched N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (N-NBD-PE), were incubated with PS but not PC liposomes. Thus, fusion between Sendai virus HN glycoprotein vesicles and the negatively charged PS liposomes is suggested. Induction of CF release and fluorescence dequenching were not observed when Pronase-treated HN vesicles were incubated with the PS liposomes. On the other hand, the fusogenic activity of the HN vesicles was not inhibited by treatment with dithiothreitol (DTT) or phenylmethanesulfonyl fluoride (PMSF), both of which are known to inhibit the Sendai virus fusogenic activity. Fusion was highly dependent on the pH of the medium, being maximal after an incubation of 60-90 s at pH 4.0. Electron microscopy studies showed that incubation at pH 4.0 of the HN vesicles with PS liposomes, both of which are of an average diameter of 150 nm, resulted in the formation of large unilamellar vesicles, the average diameter of which reached 450 nm. The relevance of these observations to the mechanism of liposome-membrane and virus-membrane fusion is discussed.     

Sulfated components of enveloped viruses.

The glycoproteins of several enveloped viruses, grown in a variety of cell types, are labeled with 35SO4(-2), whereas the nonglycosylated proteins are not. This was shown for the HN and F glycoproteins of SV5 and Sendai virus, the E1 and E2 glycoproteins of Sindbis virus, and for the major glycoprotein, gp69, as well as for a minor glycoprotein, gp52, of Rauscher leukemia virus. The minor glycoprotein of Rauscher leukemia virus is more highly sulfated, with a ratio of 35SO4- [3H]glucosamine about threefold greater than that of gp69. The G protein of vesicular stomatitis virus was labeled when virions were grown in the MDBK line of bovine kidney cells, although no significant incorporation of 35SO4(-2) into this protein was observed in virions grown in BHK21-F line of baby hamster kidney cells. In addition to the viral glycoproteins, sulfate was also incorporated into a heterogenous component with an electrophoretic mobility lower than that of any labeled with 35SO4(-2) and [3H]leucine, this component had a much greater 35S-3H ratio than any of the viral polypeptides and thus could not represent aggregated viral proteins. This material is believed to be a cell-derived mucopolysaccharide and can be removed from virions by treatment with hyaluronidase without affecting the amount of sulfate present on the glycoproteins.     

The N-terminal region of the murine coronavirus spike glycoprotein is associated with the extended host range of viruses from persistently infected murine cells

Although murine coronaviruses naturally infect only mice, several virus variants derived from persistently infected murine cell cultures have an extended host range. The mouse hepatitis virus (MHV) variant MHV/BHK can infect hamster, rat, cat, dog, monkey, and human cell lines but not the swine testis (ST) porcine cell line (J. H. Schickli, B. D. Zelus, D. E. Wentworth, S. G. Sawicki, and K. V. Holmes, J. Virol. 71:9499-9507, 1997). The spike (S) gene of MHV/BHK had 63 point mutations and a 21-bp insert that encoded 56 amino acid substitutions and a 7-amino-acid insert compared to the parental MHV strain A59. Recombinant viruses between MHV-A59 and MHV/BHK were selected in hamster cells. All of the recombinants retained 21 amino acid substitutions and a 7-amino-acid insert found in the N-terminal region of S of MHV/BHK, suggesting that these residues were responsible for the extended host range of MHV/BHK. Flow cytometry showed that MHV-A59 bound only to cells that expressed the murine glycoprotein receptor CEACAM1a. In contrast, MHV/BHK and a recombinant virus, k6c, with the 21 amino acid substitutions and 7-amino-acid insert in S bound to hamster (BHK) and ST cells as well as murine cells. Thus, 21 amino acid substitutions and a 7-amino-acid insert in the N-terminal region of the S glycoprotein of MHV/BHK confer the ability to bind and in some cases infect cells of nonmurine species.     

Glutathione inhibits replication and expression of viral proteins in cultured cells infected with Sendai virus.

Addition of reduced glutathione inhibited the production of Sendai virus in African green monkey kidney (AGMK) cells. This result could be accounted for by a direct action of GSH on viral replication. The inhibitory action was associated to an increase of the GSH intracellular level, while the host cell metabolism was unaffected. The antiviral effect was related to decrease and inactivation of the hemagglutinin-neuraminidase (HN) virus glycoprotein.     

Intracellular maturation and transport of the SV5 type II glycoprotein hemagglutinin-neuraminidase: specific and transient association with GRP78-BiP in the endoplasmic reticulum and extensive internalization from the cell surface

The hemagglutinin-neuraminidase (HN) glycoprotein of the paramyxovirus SV5 is a type II integral membrane protein that is expressed at the infected cell surface. The intracellular assembly and transport of HN in CV1 cells was examined using conformation-specific HN mAbs and sucrose density sedimentation analysis. HN was found to oligomerize with a t1/2 of 25-30 min and these data suggest the oligomer is a tetramer consisting primarily of two noncovalently associated disulfide- linked dimers. As HN oligomers could be found that were sensitive to endoglycosidase H digestion and oligomers formed in the presence of the ER to the Golgi complex transport inhibitor, carbonylcyanide m- chlorophenylhydrazone (CCCP), these data are consistent with HN oligomerization occurring in the ER. Unfolded or immature HN molecules that could not be recognized by conformation-specific antibodies were found to specifically associate with the resident ER protein GRP78-BiP. Immunoprecipitation of BiP-HN complexes with an immunoglobulin heavy- chain binding protein (BiP) antibody indicated that newly synthesized HN associated and dissociated from GRP78-BiP (t1/2 20-25 min) in an inverse correlation with the gain in reactivity with a HN conformation- specific antibody, suggesting that the transient association of GRP78- BiP with immature HN is part of the normal HN maturation pathway. After pulse-labeling of HN in infected cells, it was found that HN is rapidly turned over in cells (t1/2 2-2.5 h). This led to the finding that the vast majority of HN expressed at the cell surface, rather than being incorporated into budding virions, is internalized and degraded after localization to endocytic vesicles and lysosomes.     

Structure of Sendai viral proteins in plasma membranes of virus-infected cells.

Purified plasma membranes attached to polycationic polyacrylamide beads by their external surface were isolated from BHK cells infected with Sendai virus. Each of the viral proteins could be identified in the membranes of infected cells. Proteolysis with trypsin, which digests only the cytoplasmic surface of these membranes (because the external surface is protected by its attachment to beads), revealed that the internal proteins, L, P, NP, and M, were present on the cytoplasmic surface of the membrane and that small segments of the viral envelope glycoproteins, HN and F0, were partially exposed on the cytoplasmic surface. Since the major portions of HN and F0 are known to be present on the external membrane surface, these glycoproteins are transmembrane proteins before Sendai virus budding in infected cells.     

Different biosynthetic transport routes to the plasma membrane in BHK and CHO cells

The question of how membrane proteins are delivered from the TGN to the cell surface in fibroblasts has received little attention. In this paper we have studied how their post-Golgi delivery routes compare with those in epithelia] cells. We have analyzed the transport of the vesicular stomatitis virus G protein, the Semliki Forest virus spike glycoprotein, both basolateral in MDCK cells, and the influenza virus hemagglutinin, apical in MDCK cells. In addition, we also have studied the transport of a hemagglutinin mutant (Cys543Tyr) which is basolateral in MDCK cells. Aluminum fluoride, a general activator of heterotrimeric G proteins, inhibited the transport of the basolateral cognate proteins, as well as of the hemagglutinin mutant, from the TGN to the cell surface in BHK and CHO cells, while having no effect on the surface delivery of the wild-type hemagglutinin. Only wild-type hemagglutinin became insoluble in the detergent CHAPS during transport through the BHK and CHO Golgi complexes, whereas the basolateral marker proteins remained CHAPS-soluble. We also have developed an in vitro assay using streptolysin O-permeabilized BHK cells, similar to the one we have previously used for analyzing polarized transport in MDCK cells (Pimplikar, S.W., E. Ikonen, and K. Simons. 1994. J. Cell Biol. 125:1025-1035). In this assay anti-NSF and rab-GDI inhibited transport of Semliki Forest virus spike glycoproteins from the TGN to the cell surface while having little effect on transport of the hemagglutinin. Altogether these data suggest that fibroblasts have apical and basolateral cognate routes from the TGN to the plasma membrane.     

Neutral alpha-glucosidase from human kidney. Molecular and immunological properties. Relationship with intestinal glucoamylase.

Some molecular properties of the purified neutral alpha-glucosidase from human kidney were studied. The enzyme is a glycoprotein with high molecular weight (315000-352000 according to the method used). Its sedimentation coefficient is 12.9S. It exhibits at least three peaks of activity in isoelectric focusing experiments. This heterogeneity appears to be related to sialic acid residues from the carbohydrate moiety. An anti-human renal alpha-glucosidase antiserum was raised from rabbit. The antiserum effect on human intestinal maltases was studied in immunodiffusion experiments. An identity pattern was observed between renal neutral alpha-glucosidase and intestinal glucoamylase. No precipitation occurred with intestinal sucrase. Renal neutral alpha-glucosidase and intestinal glucoamylase were both completely precipitated by the antiserum, their maltase activity being only slightly inhibited in the antigen-antibody complex. From their molecular and immunological properties a large homology appears between human renal alpha-glucosidase and intestinal glycoamylase.     

The interaction of Sendai virus glycoprotein-bearing recombinant vesicles with cell surfaces

Sendai virus glycoproteins HN and F were purified by immunoaffinity chromatography from virions disrupted by beta-D-octylglucoside. The purified glycoproteins were reconstituted in recombinant vesicles with phosphatidylcholine or phosphatidylethanolamine and phosphatidylserine. P815 or EL-4 cells treated with glycoprotein HN/F-phosphatidylcholine recombinant vesicles acquired the glycoproteins and retained them in the plasma membrane for 4 h as demonstrated by surface immunofluorescence specific for each protein. Cells treated with glycoprotein HN-phosphatidylcholine recombinant vesicles initially bore glycoprotein HN on the surface but the protein eluted within 2 h. Surfaces of cells treated with glycoprotein F-phosphatidylcholine recombinant vesicles did not acquire the glycoprotein. Cells treated with glycoprotein HN-phosphatidylethanolamine: phosphatidylserine recombinant vesicles or glycoprotein F-phosphatidylethanolamine: phosphatidylserine recombinant vesicles in the presence of 5 mM Ca2+ acquired each protein for at least 2 h. Experiments showed that the acquired glycoproteins capped with antibody and that when glycoproteins HN and F were together on the surface they co-capped. Acquired viral glycoproteins did not co-cap with intrinsic H-2 glycoproteins.     

Vesicular stomatitis virus envelope glycoprotein alterations induced by host cell transformation

Vesicular stomatitis virus (VSV) contains a single structural glycoprotein in which the sugar sequences are largely host specified. We have used VSV as a probe to study the changes in cell glycoprotein metabolism induced by virus transformation. Analysis of purified VSV grown in baby hamster kidney (BHK) or polyoma transformed BHK cells showed that the virus glycoproteins have identical apparent molecular weights. The glycopeptides derived from the glycoproteins by extensive pronase digestion have an identical molecular weight distribution.On the basis of labeling experiments with fucose, mannose, and glucosamine, the oligosaccharide moieties of the VSV glycoprotein were different in virus from the two cell lines. The VSV glycopeptides from transformed cells showed an increased resistance to cleavage by an endoglycosidase, indicating structural changes in the core region of the oligosaccharides. They also showed an increased ratio of sialic acid to N-acetylglucosamine.VSV grows in a wide variety of cell types, and the carbohydrate structures of its single glycoprotein are amenable to analysis with specific glycosidases. The virus thus provides an excellent tool with which to study alterations induced by cell transformation in the glycosylation of membrane proteins.     

Conformational changes of Newcastle disease virus envelope glycoproteins triggered by gangliosides.

We have investigated the conformational changes of Newcastle disease virus (NDV) glycoproteins in response to receptor binding, using 1,1-bis(4-anilino)naphthalene-5,5-disulfonic acid (bis-ANS) as a hydrophobicity-sensitive probe. Temperature- and pH-dependent conformational changes were detected in the presence of free bovine gangliosides. The fluorescence of bis-ANS was maximal at pH 5. The binding of bis-ANS to NDV was not affected by chemicals that denature the fusion glycoprotein, such as reducing agents, nor by the presence of neuraminidase inhibitors such as N-acetyl neuramicic acid. Gangliosides partially inhibited fusion and hemadsorption, but not neuraminidase hemagglutinin-neuraminidase glycoprotein (HN) activity. A conformational intermediate of HN, triggered by the presence of gangliosides acting as receptor mimics, was detected. Our results indicate that, upon binding to free gangliosides, HN undergoes a certain conformational change that does not affect the fusion glycoprotein.     

Glycopeptides from brain inhibit rates of polypeptide chain elongation

In previous reports, we have identified cell-surface glycopeptides from mouse cerebrum (BCSG) that inhibited protein synthesis and mitosis in several cell types. When baby hamster kidney (BHK)-21 cells were infected with vesicular stomatitis virus (a negative strand RNA virus), BCSG extensively inhibited viral protein synthesis. This inhibition was effective against both protein and glycoprotein synthesis and was independent of amino acid uptake by infected cells, synthesis of viral RNA, and degradation of viral proteins. Analysis of polyribosome profiles in uninfected BHK-21 cells indicated that the degree of cellular protein synthesis inhibition could not be attributed to activation of RNase or solely to a disruption of chain initiation. When added directly to a cell-free protein-synthesizing system derived from BHK-21 cells, BCSG was ineffective, but if the inhibitory material was first allowed to react with cells, cell-free protein synthesis was substantially reduced. When BCSG were reacted with cells for 5 min at 0 degrees C, the cells tested, BHK-21 (a BCSG-sensitive line) and murine fibrosarcoma 2237 (a BCSG-insensitive line), both effectively adsorbed the inhibitor from the medium.     

The paramyxovirus simian virus 5 hemagglutinin-neuraminidase glycoprotein, but not the fusion glycoprotein, is internalized via coated pits and enters the endocytic pathway.

The hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of the paramyxovirus simian virus 5 (SV5) are expressed on the surface of virus-infected cells. Although the F protein was found to be expressed stably, the HN protein was internalized from the plasma membrane. HN protein lacks known internalization signals in its cytoplasmic domain that are common to many integral membrane proteins that are internalized via clathrin-coated pits. Thus, the cellular pathway of HN protein internalization was examined. Biochemical analysis indicated that HN was lost from the cell surface with a t1/2 of approximately 45-50 min and turned over with a t1/2 of approximately 2 h. Immunofluorescent analysis showed internalized SV5 HN in vesicle-like structures in a juxtanuclear pattern coincident with the localization of ovalbumin. In contrast the SV5 F glycoprotein and the HN glycoprotein of the highly related parainfluenza virus 3 (hPIV-3) were found only on the cell surface. Immunogold staining of HN on the surface of SV5-infected CV-1 cells and examination using electron microscopy, showed heavy surface labeling that gradually decreased with time. Concomitantly, gold particles were detected in the endosomal system and with increasing time, gold-labeled structures having the morphology of lysosomes were observed. On the plasma membrane approximately 5% of the gold-labeled HN was found in coated pits. The inhibition of the pinching-off of coated pits from the plasma membrane by cytosol acidification significantly reduced HN internalization. Internalized HN was co-localized with gold-conjugated transferrin, a marker for the early endosomal compartments, and with gold-conjugated bovine serum albumin, a marker for late endosomal compartments. Taken together, these data strongly suggest that the HN glycoprotein is internalized via clathrin-coated pits and delivered to the endocytic pathway.     

Isolation and characterization of a novel large protease accumulated in mammalian cells in the presence of inhibitors

We have isolated and characterized a novel, large, multicatalytic protease from mammalian cells. This protease was designated PABI (protease accumulated by inhibitors). When baby hamster kidney (BHK) cells were grown in medium containing leupeptin, a potent serine-cysteine protease inhibitor, the trypsin-like protease activity (PABI) in the cells increased its level more than 100-fold over the control. This increase was also observed in other cultured cells such as COS, HepG2, and skin fibroblast cells. The activity was also elevated by treatment with other protease inhibitors including chymostatin or trans-epoxysuccinyl-L-leucylamide-(4-guanidino)butane. Immunoblot analysis, by employing antisera prepared against the purified PABI, also showed a concomitant increase of this protein in BHK, COS, and HepG2 cells on leupeptin treatment. PABI was purified to a homogeneous state from leupeptin-treated BHK cells. PABI is a glycoprotein of molecular weight 700,000. PABI was found to be a multimer of a major subunit of apparent Mr of 84,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopic analysis. PABI dissociates into subunits only under reducing conditions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PABI has both trypsin-like and chymotrypsin-like protease activities toward synthetic substrates. Both activities were inhibited by phenylmethanesulfonyl fluoride, aprotinin, bovine pancreas trypsin inhibitor, and chymostatin. Leupeptin inhibited only the trypsin-like activity of PABI. p-Chloromercuribenzoate had no effect on either activity. Furthermore, PABI degraded collagen type I and fibronectin. These results indicate that PABI is a novel protease which differs from any known proteases including cytosolic high molecular weight proteases. The physiological function of PABI is yet to be determined.     

GalNAc-alpha -O-benzyl inhibits sialylation of de Novo synthesized apical but not basolateral sialoglycoproteins and blocks lysosomal enzyme processing in a post-trans-Golgi network compartment

Glycosylation plays an important role in glycoprotein traffic. Our previous work has shown that long term treatment of mucus-secreting HT-29 cells with GalNAc-alpha-O-benzyl reversibly inhibits sialylation and causes the accumulation of apical glycoproteins in cytoplasmic vesicles. We have analyzed at the biochemical level the effects of GalNAc-alpha-O-benzyl on glycoprotein processing. Both apical and basolateral membrane glycoproteins were sialylated, but GalNAc-alpha-O-benzyl selectively inhibited the sialylation of apical glycoproteins. In addition, lysosomal alpha-glucosidase, which is partially targeted to the apical membrane, was abnormally processed leading to the accumulation of an immature molecular species. Several findings support the conclusion that accumulation of this protein occurs in a post-trans-Golgi network (TGN) compartment: 1) it is partially sialylated; 2) it does not occur when glycoprotein exit from the TGN is blocked at 20 degrees C; 3) upon Triton X-114 partition, it distributes to the aqueous phase, a characteristic that is acquired in a post-TGN compartment; and 4) its appearance is inhibited when cells are cultured in the presence of NH(4)Cl. The processing of cathepsin D was also found to be affected by GalNAc-alpha-O-benzyl treatment. In conclusion, GalNAc-alpha-O-benzyl selectively inhibits sialylation of apical glycoproteins and perturbs lysosomal enzyme processing; these effects occur in a post-TGN acidic compartment and are reminiscent of the alterations found in sialic acid storage diseases.     

An Amino Acid Substitution in the Coding Region of the E2 Glycoprotein Adapts Ross River Virus To Utilize Heparan Sulfate as an Attachment Moiety

Passage of Ross River virus strain NB5092 in avian cells has been previously shown to select for virus variants that have enhanced replication in these cells. Sequencing of these variants identified two independent sites that might be responsible for the phenotype. We now demonstrate, using a molecular cDNA clone of the wild-type T48 strain, that an amino acid substitution at residue 218 in the E2 glycoprotein can account for the phenotype. Substitutions that replaced the wild-type asparagine with basic residues had enhanced replication in avian cells while acidic or neutral residues had little or no observable effect. Ross River virus mutants that had increased replication in avian cells also grew better in BHK cells than the wild-type virus, whereas the remaining mutants were unaffected in growth. Replication in both BHK and avian cells of Ross River virus mutants N218K and N218R was inhibited by the presence of heparin or by the pretreatment of the cells with heparinase. Binding of the mutants, but not of the wild type, to a heparin-Sepharose column produced binding comparable to that of Sindbis virus, which has previously been shown to bind heparin. Replication of these mutants was also adversely affected when they were grown in a CHO cell line that was deficient in heparan sulfate production. These results demonstrate that amino acid 218 of the E2 glycoprotein can be modified to create an heparan sulfate binding site and this modification expands the host range of Ross River virus in cultured cells to cells of avian origin.     

Inhibitory activity of cyanidin-3-rutinoside on alpha-glucosidase

Cyanidin-3-rutinoside, a natural anthocyanin, inhibited alpha-glucosidase from baker's yeast in dose-responsive manner. The IC50 value was 19.7 microM +/- 0.24 microM, compared with the IC50 value of voglibose (IC50 = 23.4 +/- 0.30 microM). Cyanidin-3-rutinoside was found to be a non-competitive inhibitor for yeast alpha-glucosidase with a Ki value in the range of 1.31-1.56 x 10(-5)M. These results indicated that cyanidin-3-rutinoside could be classed as a new alpha-glucosidase inhibitor.