Fluorosugars inhibit biological properties of different enveloped viruses.

Both 2-deoxy-2-fluoro-D-glucose and 2-deoxy-2-fluoro-D-mannose were found to be potent inhibitors of the synthesis of infectious Semliki forest and fowl plague virus in chicken embryo cells and also of pseudorabies virus grown in rabbit kidney cells. It was found that the pseudorabies virus-mediated cell fusion and the synthesis of functional hemagglutinin of fowl plague virus were blocked. In all cases the 2-deoxy-2-fluoro-D-mannose-caused inhibition was stronger than the 2-deoxy-2-fluoro-D-glucose- or 2-deoxy-D-glucose-mediated blocks. Studies on the virus-specified proteins from Semiliki forest virus-infected cells grown in the presence of the inhibitors show that the target of the fluorosugar action, parallel to the well-studied effects of 2-deoxy-D-glucose, is the glycoprotein biosynthesis.     

Inhibition of cellular protein synthesis by simultaneous pretreatment of host cells with fowl plague virus and actinomycin D: a method for studying early protein synthesis of several RNA viruses.

A method is described for analysis of viral protein synthesis early after infection when minute amounts of viral proteins are effectively concealed by large amounts of produced host-specific proteins. The method is superior to a radioimmune assay, since all virus-induced proteins can be measured independent of their immunological reactivity. Host-specific protein synthesis can be suppressed by infection with fowl plague virus. Addition of actinomycin C 1.25 h postinfection does not prevent this suppression, but it does block effectively the formation of fowl plague virus-specific proteins. Such cells synthesize only small amounts of cellular proteins, as revealed by polyacrylamide electrophoresis. They can be superinfected with several different enveloped viruses, however, without significant diminution of virus yeilds. In pretreated cells the eclipse is shortened for Semliki Forest virus, Sindbis virus, and vesicular stomatitis virus, but prolonged for Newcastle disease virus. The onset of protein synthesis, specific for the superinfecting virus, could be clearly demonstrated within 1 h after superinfection. At this time, in cells superinfected with Semliki Forest virus, great amounts of NSP 75 (nonstructural protein; molecular weight, 75 X 10(3)) and reduced amounts of the core protein C could be deomonstrated. The precursor glycoprotein NSP 68 is followed by a new polypeptide, NSP 65: three proteins with molecular weights exceeding 100 X 10(3) were observed which are missing later in the infectious cycle. Similar results were obtained after superinfection with Sindbis virus. The formation of a new polypeptide with a molecular weight of about 80 X 10(3) was detected. After superinfection with vesicular stomatis virus or Newcastle disease virus the formation of new proteins, characteristic for the early stage of infeciton, was not observed.     

Different membrane anchors allow the Semliki Forest virus spike subunit E2 to reach the cell surface.

The Semliki Forest virus spike subunit E2, a membrane-spanning protein, was transported to the plasma membrane in BHK cells after its carboxy terminus, including the intramembranous and cytoplasmic portions, was replaced by respective fragments of either the vesicular stomatitis virus glycoprotein or the fowl plague virus hemagglutinin. The hybrid proteins were constructed by cDNA fusion. Upon a transient expression they could be localized at the cell surface by immunofluorescence with specific antibodies directed against any of the protein fragments.     

Polarized sorting of viral glycoproteins to the axon and dendrites of hippocampal neurons in culture

Cultured hippocampal neurons were infected with a temperature-sensitive mutant of vesicular stomatitis virus (VSV) and a wild-type strain of the avian influenza fowl plague virus (FPV). The intracellular distribution of viral glycoproteins was monitored by immunofluorescence microscopy. In mature, fully polarized neurons the VSV glycoprotein (a basolateral protein in epithelial MDCK cells) moved from the Golgi complex to the dendritic domain, whereas the hemagglutinin protein of FPV (an apically sorted protein in MDCK cells) was targeted preferentially, but not exclusively, to the axon. The VSV glycoprotein appeared in clusters on the dendritic surface, while the hemagglutinin was distributed uniformly along the axonal membrane. Based on the finding that the same viral glycoproteins are sorted in a polarized fashion in both neuronal and epithelial cells, we propose that the molecular mechanisms of surface protein sorting share common features in the two cell types.     

Influence of the infection with lipid-containing viruses on the metabolism and pools of phospholipid precursors in animal cells

The influence of infection with three different lipid-containing RNA viruses, Newcastle disease virus, fowl plague virus, and Semliki Forest virus on the phosphatidylcholine precursors of chick embryo cells and of baby hamster kidney (BHK) cells has been measured. In chick embryo cells infection with Newcastle disease virus does not influence the energy charge, or the distribution and absolute pool sizes of the precursors or the choline phosphotransferase activity. In chick embryo cells infected with fowl plague virus the CDP-choline pool increases because of an inhibition of the choline phosphotransferase activity. The phosphorylcholine and CTP pools are smaller in infected cells when compared with mock-infected ones, although the energy charge is not influenced by infection. In chick embryo cells as well as in BHK cells the energy charge is diminished by infection with Semliki Forest virus. Therefore the CTP and phosphorylcholine pools are decreased. The CDP-choline pool in chick embryo cells becomes extremely small after infection with Semliki Forest virus because of a significant stimulation of the choline phosphotransferase. In BHK cells infected with Semliki Forest virus the opposite effect is observed. There are also severe effects on the uptake of the labeled precursors by infection. One and the same virus (Semliki Forest virus) has two completely different effects on the phosphatidylcholine precursors when infecting two different cell types. If one and the same cell type (chick embryo cells) is infected with three different lipid-containing RNA viruses also completely different effects on the phosphatidylcholine precursors were observed. Thus, each virus develops its own strategy to influence the lipid metabolism of the host cell, depending also on the choice of the host. This explains the many disturbing contradictory results described in the literature about the influence of lipid-containing viruses on the lipid metabolism of the host.     

Inhibition of Cellular Protein Synthesis by Simultaneous Pretreatment of Host Cells with Fowl Plague Virus and Actinomycin D: a Method for Studying Early Protein Synthesis of Several RNA Viruses

A method is described for analysis of viral protein synthesis early after infection when minute amounts of viral proteins are effectively concealed by large amounts of produced host-specific proteins. The method is superior to a radioimmune assay, since all virus-induced proteins can be measured independent of their immunological reactivity. Host-specific protein synthesis can be suppressed by infection with fowl plague virus. Addition of actinomycin D 1.25 h postinfection does not prevent this suppression, but it does block effectively the formation of fowl plague virus-specific proteins. Such cells synthesize only small amounts of cellular proteins, as revealed by polyacrylamide electrophoresis. They can be superinfected with several different enveloped viruses, however, without significant diminution of virus yields. In pretreated cells the eclipse is shortened for Semliki Forest virus, Sindbis virus, and vesicular stomatitis virus, but prolonged for Newcastle disease virus. The onset of protein synthesis, specific for the superinfecting virus, could be clearly demonstrated within 1 h after superinfection. At this time, in cells superinfected with Semliki Forest virus, great amounts of NSP 78 (nonstructural protein; molecular weight, 78 × 10³) and reduced amounts of the core protein C could be demonstrated. The precursor glycoprotein NSP 68 is followed by a new polypeptide, NSP 65; three proteins with molecular weights exceeding 100 × 10³ were observed which are missing later in the infectious cycle. Similar results were obtained after superinfection with Sindbis virus. The formation of a new polypeptide with a molecular weight of about 80 × 10³ was detected. After superinfection with vesicular stomatitis virus or Newcastle disease virus the formation of new proteins, characteristic for the early stage of infection, was not observed.     

Intracellular sorting and basolateral appearance of the G protein of vesicular stomatitis virus in Madin-Darby canine kidney cells

The polarity of the surface distribution of viral glycoproteins during virus infection has been studied in the Madin-Darby canine kidney epithelial cell line on nitrocellulose filters. Using a surface radioimmunoassay on Madin-Darby canine kidney strain I cells that had been infected with vesicular stomatitis virus or with avian influenza fowl plague virus, we found that the surface G protein was 97% basolateral, whereas the fowl plague virus hemagglutinin was 88% apical. Newly synthesized, pulse-labeled vesicular stomatitis virus appeared first on the basolateral plasma membrane as measured by an immunoprecipitation assay in which the anti-G protein antibody was applied to the monolayer either from the apical or the basolateral side. Labeled G protein could be accumulated inside the cell at a late stage of transport by decreasing the temperature to 20 degrees C during the chase. Reversal to 37 degrees C led to its rapid and synchronous transport to the basolateral surface at an initial rate 61-fold greater than that of transport to the apical side. These results demonstrate that the newly synthesized G protein is transported directly to the basolateral membrane and does not pass over the apical membrane en route. Since a previous study of the surface appearance of influenza virus hemagglutinins showed that the newly synthesized hemagglutinins were inserted directly from an intracellular site into the apical membrane (Matlin, K., and K. Simons, 1984, J. Cell Biol., 99:2131-2139), we conclude that the divergence of the transport pathway for the apical and basolateral viral glycoproteins has to occur intracellularly, i.e., before reaching the cell surface.     

Glycosylation in vitro of Semliki-Forest-virus and influenza-virus glycoproteins and its suppression by nucleotide-2-deoxy-hexose

Cell-free enzyme preparations from cultured fibroblasts infected with Semliki forest virus or fowl plague virus (an influenza A virus) incorporate [14C]-mannose into dolichol-phosphate-mannose, lipid-linked oligosaccharides and into endogenous virus-specific glycoproteins. When GDP-2-deoxy-D-[14C]glucose serves as substrate 2-deoxy-D-[14C]glucose is transferred to dolichol phosphate yielding dolichol-monophosphate-2-deoxy-D-[14C]glucose. UDP-2-deoxy-D-[14C]glucose gives rise also to a lipid which, however, is not a polyprenol derivative. The transfer of [14C]mannose to lipid-extractable fractions and glycoproteins in vitro is blocked by GDP-2-deoxy-D-glucose. It can be restored by exogenous dolichol monophosphate only with regard to the formation of dolichol-monophosphate-[14C]mannose-labelled oligosaccharides into glycoproteins. UDP-2-deoxy-D-glucose has no inhibitory effect on transfer reactions of [14C]mannose from GDP-[14C]mannose into various lipid fractions or into glycoprotein. It is concluded therefore, that the inhibition of glycosylation brought about by 2-deoxyglucose in vivo is caused by an interference of its GDP derivative with the formation of a correct lipid-oligosaccharide.     

Infectious entry pathway of influenza virus in a canine kidney cell line

The entry of fowl plague virus, and avian influenza A virus, into Madin- Darby canine kidney (MDCK) cells was examined both biochemically and morphologically. At low multiplicity and 0 degrees C, viruses bound to the cell surface but were not internalized. Binding was not greatly dependent on the pH of the medium and reached an equilibrium level in 60-90 min. Over 90% of the bound viruses were removed by neuraminidase but not by proteases. When cells with prebound virus were warmed to 37 degrees C, part of the virus became resistant to removal b neuraminidase, with a half-time of 10-15 min. After a brief lag period, degraded viral material was released into the medium. The neuraminidase- resistant virus was capable of infecting the cells and probably did so by an intracellular route, since ammonium chloride, a lysosomotropic agent, blocked both the infection and the degradation of viral protein. When the entry process was observed by electron microscopy, viruses were seen bound primarily to microvilli on the cell surface at 0 degrees C and, after warming at 37 degrees C, were endocytosed in coated pits, coated vesicles, and large smooth-surfaced vacuoles. Viruses were also present in smooth-surfaced invaginations and small smooth-surfaced vesicles at both temperatures. At physiological pH, no fusion of the virus with the plasma membrane was observed. When prebound virus was incubated at a pH of 5.5 or below for 1 min at 37 degrees C, fusion was, however, detected by ferritin immunolabeling. t low multiplicity, 90% of the prebound virus became neuraminidase- resistant and was presumably fused after only 30 s at low pH. These experiments suggest that fowl plague virus enters MDCK cells by endocytosis in coated pits and coated vesicles and is transported to the lysosome where the low pH initiates a fusion reaction ultimately resulting in the transfer of the genome into the cytoplasm. The entry pathway of fowl plague virus thus resembles tht earlier described for Semliki Forest virus.     

Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease.

Many viruses have membrane glycoproteins that are activated at cleavage sites containing multiple arginine and lysine residues by cellular proteases so far not identified. The proteases responsible for cleavage of the hemagglutinin of fowl plague virus, a prototype of these glycoproteins, has now been isolated from Madin-Darby bovine kidney cells. The enzyme has a mol. wt of 85,000, a pH optimum ranging from 6.5 to 7.5, is calcium dependent and recognizes the consensus sequence R-X-K/R-R at the cleavage site of the hemagglutinin. Using a specific antiserum it has been identified as furin, a subtilisin-like eukaryotic protease. The fowl plague virus hemagglutinin was also cleaved after coexpression with human furin from cDNA by vaccinia virus vectors. Peptidyl chloroalkylketones containing the R-X-K/R-R motif specifically bind to the catalytic site of furin and are therefore potent inhibitors of hemagglutinin cleavage and fusion activity.     

Incorporation of Fowl Plague Virus Hemagglutinin into Murine Leukemia Virus Particles and Analysis of the Infectivity of the Pseudotyped Retroviruses

We describe retrovirus particles carrying the fowl plague virus (FPV) hemagglutinin (HA). When expressed in cells providing Moloney murine leukemia virus (MoMLV) Gag and Pol proteins and a lacZ retroviral vector, FPV HA was found to be efficiently expressed, correctly processed, and stably incorporated into retroviral particles. HA-bearing retroviruses were infectious with a wide host range and were only 10-fold less infectious than retroviruses carrying wild-type MLV retroviral envelopes. We also coexpressed HA proteins in retroviral particles with chimeric MoMLV-derived envelope glycoproteins that efficiently retarget virus attachment but are only weakly fusogenic. Our results suggest that HA can in some cases enhance the fusion ability of these retroviral particles, depending on the cell surface molecule that is used as a receptor.     

Ion channel activity of influenza A virus M2 protein: characterization of the amantadine block.

The influenza A virus M2 integral membrane protein has ion channel activity which can be blocked by the antiviral drug amantadine. The M2 protein transmembrane domain is highly conserved in amino acid sequence for all the human, swine, equine, and avian strains of influenza A virus, and thus, known amino acid differences could lead to altered properties of the M2 ion channel. We have expressed in oocytes of Xenopus laevis the M2 protein of human influenza virus A/Udorn/72 and the avian virus A/chicken/Germany/34 (fowl plague virus, Rostock) and derivatives of the Rostock ion channel altered in the presumed pore region. The pH of activation of the M2 ion channels and amantadine block of the M2 ion channels were investigated. The channels were found to be activated by pH in a similar manner but differed in their apparent Kis for amantadine block.     

Formation of Influenza Virus Proteins

Eight virus-specific proteins have been found in chicken embryo fibroblasts infected with fowl plague virus. Among them are two glycoproteins which are the constituents of the hemagglutinin on the virus particle. They are derived from a large precursor glycoprotein by cleavage of a covalent linkage. The reaction can be blocked by the protease inhibitor diisopropylfluorophosphate and the amino acid analogue fluorophenylalanine. This indicates that a peptide bond is cleaved. If infected cells are kept at 25 C, a temperature at which virus maturation is inhibited, the precursor glycoprotein is cleaved at a significantly slower rate than at 37 C. It appears, however, that a reduced synthesis of the carbohydrate-free envelope protein is responsible for the block of virus maturation at 25 C rather than the lower cleavage rate of the precursor.     

Early synthesis of Semliki Forest virus-specific proteins in infected chicken cells.

Cells preinfected with fowl plague virus followed by treatment with actinomycin D are a suitable system for studying early protein synthesis in cells infected with Semliki forest virus. One and one-half hours after superinfection, three new nonstructural proteins (NVP) were detected: NVP 145, NVP, 112, and NVP 65. They appeared in parallel with a low incorporation of mannose at the beginning of the infectious cycle. Behavior on chasing suggested a precursor relationship of NVP 112 to the envelope glycoproteins. Two kinds of NVP 65 are described, both of which are varieties of NVP 68 with an incomplete mannose content. One type, detected early after infection, was converted into NVP 68 by supplementary glycosylation. The second, late type was stable. It contains fucose and resembles the NVP 65 observed after impairment of glycosylation. The mechanism of NVP 68 glycosylation is discussed. The presence of the complete carbohydrate moiety is crucial for the cleavage of NVP 68 into the envelope proteins E2 and E3 and, thus, for virus maturation. Only the complete form of NVP 68 was precipitated by envelope-specific antisera. A large production of NVP 78 is a further feature of the early events in infected cells. It is not related to the structural proteins.     

The 6-kilodalton membrane protein of Semliki Forest virus is involved in the budding process.

Alphavirus genomes encode a small hydrophobic protein of 6 kDa (the 6K protein) that is expressed as part of a large polyprotein containing the sequences of the two virus transmembranal glycoproteins which form the spikes of the infectious particle. Although made in amounts equivalent to those of the glycoproteins, very little of the 6K protein is found in secreted infectious virions. The role of this protein in virus replication and structure has been studied by use of a variety of mutationally altered forms of 6K, which yield phenotypically distinct viruses. A complete deletion of the gene encoding the 6K protein (delta 6K) of Semliki Forest Virus (SFV) has been constructed from an SFV infectious cDNA and the transcribed RNA-produced progeny virus that closely resembled the normal virus (P. Liljeström, S. Lusa, D. Huylebroeck, and H. Garoff, J. Virol. 65:4107-4113, 1991). Further studies of this mutant have now been performed, and they show that growth of delta 6K has a strong dependency on its host cell, varying from 2 to 50% of the rate of formation of the wild-type SFV. Mammalian cells are much more defective than insect and avian cells in replication of the delta 6K mutant. This mutant is not defective in formation and transport of the glycoproteins or in production of nucleocapsids, which accumulate at the plasma cell membrane in infected BHK cells. The major defect, thus, is in the final assembly and budding of new virus. In BHK cells infected with the delta 6K strain, a relatively large fraction of the total infectious virus formed can be recovered by osmotic lysis of exhaustively washed cells. Infectious SFV totally lacking 6K is identical to wild-type SFV in the early stages of virus replication, i.e., binding and uptake. The particles themselves are more thermolabile than those of wild-type SFV, suggesting that the 6K protein may be a part of the structure of wild-type virus or that the slower budding leads to an altered configuration of the trimeric spikes. These data support other studies that implicate the 6K protein as an important but nonessential component in the assembly and budding of the alphavirus particle, perhaps by affecting the packing of the glycoproteins and their interactions with membrane lipid.     

Inhibitory effect of alpha-methyl-1-adamantane methylamine hydrochloride (rimantadine) on RNA-dependent RNA polymerase induction in culture of cells, infected with influenza virus]

An anti-influenza preparation, rimantadine (alpha-methyl-1-adamantane methylamine hydrochloride) at concentrations of 10--25 mkg/ml depresses the RNA-dependent RNA polymerase induction in a culture of cells infected with influenza virus (fowl plague virus). The inhibitory effect is also observed 2 hours following cell infection. In vitro studies have demonstrated that rimantadine has no effect on the activity of virus-induced RNA-dependent RNA polymerase, as well as on that of RNA-dependent RNA polymerase associated with virus particles.     

Chemical cross-linking of proteins of Semliki Forest virus: virus particles and plasma membranes from BHK-21 cells treated with colchicine or dibucaine.

Chemical cross-linking of the proteins of Semliki Forest virus has been performed in virus particles and in baby hamster kidney-21 (BHK-21) cells infected with Semliki Forest virus. Most of the studies were done with the reversible cross-linkers dimethyl 3,3'-thiobis(propionimidate) and dithiobis(succinimidyl propionate). The identity of the cross-linked species was determined by two-dimensional electrophoresis. The results with virus particles showed extensive cross-linking of the nucleocapsid proteins and the formation of dimers of the two large envelope glycoproteins (E1 and E2). Similar patterns for the cross-linked virus proteins were observed in plasma membranes isolated from BHK-21 cells infected with Semliki Forest virus. No cross-linking of the third envelope glycoprotein (E3) was observed. Also, there was no evidence for significant cross-linking between host and virus proteins. The addition of colchicine, a drug that disrupts microtubules, to infected BHK-21 cells had no effect on the cross-linking of virus proteins in the plasma membrane. In contrast, dibucaine, a local anesthetic, greatly inhibited the formation of envelope dimers (E1-E2) in plasma membranes, but not in virus particles. The implication of these results for the involvement of the cytoskeletal system in the morphogenesis of Semliki Forest virus is discussed.     

Coronavirus proteins: structure and function of the oligosaccharides of the avian infectious bronchitis virus glycoproteins.

The recent finding that the E1 glycoproteins of murine coronaviruses contain only O-linked oligosaccharides suggested that this unusual modification might be a distinguishing feature of coronaviruses and might play an essential role in the life cycle of this family of viruses. To examine these possibilities, we analyzed the oligosaccharide moieties of the membrane proteins of the avian coronavirus infectious bronchitis virus. In addition, we determined the effect of inhibiting the glycosylation of these proteins on viral maturation and infectivity. Infectious bronchitis virus virions contain nine proteins. Four of these proteins, GP36, GP31, GP28, and P23, are closely related structurally and appear to be homologous to the E1 proteins of murine coronaviruses. We found that the oligosaccharides of GP31 and GP28 could be removed with endoglycosidase H and that neither of these glycoproteins was detectable in tunicamycin-treated cells. These two results indicated that GP31 and GP28 contain N-linked oligosaccharides. Therefore, O-linked oligosaccharides are not a universal feature of the small coronavirus membrane glycoproteins. Tunicamycin inhibited glycosylation of all of the viral glycoproteins but did not inhibit production of virions by infectious bronchitis virus-infected cells. The virions released by these cells contained only the three non-glycosylated viral proteins P51, P23, and P14. These particles were not infectious. Therefore, it appears that glycosylated infectious bronchitis virus polypeptides are not required for particle formation. However, the viral glycoproteins are apparently indispensible for viral infectivity.     

Effect of impaired glycosylation on the biosynthesis of Semliki forest virus glycoproteins.

The glycoproteins of Semliki Forest virus, grown in chicken embryo cells, were labeled with radioactive sugars. The data indicate a high mannose content of the nonstructural precursor glycoprotein NSP 63. This protein can also be readily labeled with 2-deoxy-D-glucose. The envelope glycoproteins E1 and E2 are relatively rich in galactose, glucosamine, and fucose. Glycosylation can be impaired by 2-deoxy-D-glucose or D-glucosamine or by omission of sugars in the culture medium. Under these conditions characteristic changes in the electrophoretic profile of the viral polypeptides are observed: in the regions of glycoproteins NSP 97, NSP 63, and E1 and E2 new protein peaks can be detected. These polypeptides seem to be aberrant forms of the glycoproteins. When compared with the normal molecules they have lower molecular weights and contain less carbohydrates, especially mannose. Pulse-chase experiments indicate that the altered glycoproteins are degraded very slowly if at all. If, however, impairment is caused by omission of sugars in the culture medium, the radioactivity is chased after addition of glucose from the region between NSP 63 and E1 + E2 into the E1 + E2 peak. This suggests a completion of the carbohydrate chains under these conditions.