Antimycotic-antibiotic amphotericin B promotes influenza virus replication in cell culture

In general, antibiotics are not rated as substances that inhibit or support influenza virus replication. We describe here the enhancing effect of the polyene antibiotic amphotericin B (AmB) on influenza virus growth in Vero cells. We show that isolation rates of influenza A and B viruses from clinical samples can be dramatically enhanced by adding AmB to the culture medium. We demonstrate that AmB promotes the viral uptake and endocytic processing of the virus particles. This effect is specific for Vero and human nasal epithelial cells and was not observed in Madin-Darby canine kidney cells. The effect of AmB was subtype specific and more prominent for human seasonal influenza strains but absent for H5N1 human viruses. The AmB-enhancing effect seemed to be solely due to the viral hemagglutinin function. Our results indicate that the use of AmB may facilitate influenza virus isolation and production in Vero cells.     

Surface expression of influenza virus neuraminidase, an amino-terminally anchored viral membrane glycoprotein, in polarized epithelial cells.

We have investigated the site of surface expression of the neuraminidase (NA) glycoprotein of influenza A virus, which, in contrast to the hemagglutinin, is bound to membranes by hydrophobic residues near the NH2-terminus. Madin-Darby canine kidney or primary African green monkey kidney cells infected with influenza A/WSN/33 virus and subsequently labeled with monoclonal antibody to the NA and then with a colloidal gold- or ferritin-conjugated second antibody exhibited specific labeling of apical surfaces. Using simian virus 40 late expression vectors, we also studied the surface expression of the complete NA gene (SNC) and a truncated NA gene (SN10) in either primary or a polarized continuous line (MA104) of African green monkey kidney cells. The polypeptides encoded by the cloned NA cDNAs were expressed on the surface of both cell types. Analysis of [3H]mannose-labeled polypeptides from recombinant virus-infected MA104 cells showed that the products of cloned NA cDNA comigrated with glycosylated NA from influenza virus-infected cells. Both the complete and the truncated glycoproteins were found to be preferentially expressed on apical plasma membranes, as detected by immunogold labeling. These results indicate that the NA polypeptide contains structural features capable of directing the transport of the protein to apical cell surfaces and the first 10 amino-terminal residues of the NA polypeptide are not involved in this process.     

Influenza virus hemagglutinin expression is polarized in cells infected with recombinant SV40 viruses carrying cloned hemagglutinin DNA

Primary cell cultures of African Green monkey kidney (AGMK) contain polarized epithelial cells in which influenza virus matures predominantly at the apical surfaces above tight junctions. Influenza virus glycoproteins were found to be localized at the same membrane domain from which the virus budded. When polarized primary AGMK cells were infected with recombinant SV40 viruses containing DNA coding for either an influenza virus H1 or H2 subtype hemagglutinin (HA), the HA proteins were preferentially expressed at the apical surface in a manner identical to that observed in influenza virus-infected cells. Thus, cellular mechanisms for sorting membrane glycoproteins recognize some structural feature of the HA glycoprotein itself, and other viral proteins are not necessary for this process.     

Properties of influenza C virus grown in cell culture.

Influenza C virus was propagated successfully in primary chicken embryo lung (CEL) and fibroblast cells and in Madin-Darby canine kidney (MDCK) cells. In other cell lines, either no virus or only noninfectious hemagglutinin (HA) was produced. In productively infected cells (CEL), HA and infectious virus appeared by 24 h and reached a maximum by 36 to 48 h, cell-associated virus remaining at a constant low level. Infected Vero cells produced noninfective HA by 24 h which also remained predominantly cell associated until 60 to 72 h, when the cells disintegrated. Viral antigen was demonstrable on membranes of both CEL- and Vero-infected cells at 24 h; Vero cells yielded membrane vesicles containing HA, but none of the spherical or filamentous viral particles synthesized in CEL cells. Influenza C virus produced in cell culture or in eggs differed in several important respects from A and B viruses and from Newcastle diseases virus. All influenza C preparations, regardless of infectivity or source, lacked detectable neuraminidase activity, yet retained the ability specifically to inactivate receptors only for influenza C. Influenza C HA was not inhibited by soluble glycoproteins highly active against HA of A virus. A rat serum glycoprotein uniquely inhibited influenza C by binding to the surface components of virious.     

Modulation of glycosylation and transport of viral membrane glycoproteins by a sodium ionophore

Analysis of viral glycoprotein expression on surfaces of monensin- treated cells using a fluorescence-activated cell sorter (FACS) demonstrated that the sodium ionophore completely inhibited the appearance of the vesicular stomatitis virus (VSV) G protein on (Madin- Darby canine kidney) MDCK cell surfaces. In contrast, the expression of the influenza virus hemagglutinin (HA) glycoprotein on the surfaces of MDCK cells was observed to occur at high levels, and the time course of its appearance was not altered by the ionophore. Viral protein synthesis was not inhibited by monensin in either VSV- or influenza virus-infected cells. However, the electrophoretic mobilities of viral glycoproteins were altered, and analysis of pronase-derived glycopeptides by gel filtration indicated that the addition of sialic acid residues to the VSV G protein was impaired in monensin-treated cells. Reduced incorporation of fucose and galactose into influenza virus HA was observed in the presence of the ionophore, but the incompletely processed HA protein was cleaved, transported to the cell surface, and incorporated into budding virus particles. In contrast to the differential effects of monensin on VSV and influenza virus replication previously observed in monolayer cultures of MDCK cells, yields of both viruses were found to be significantly reduced by high concentrations of monensin in suspension cultures, indicating that cellular architecture may play a role in determining the sensitivity of virus replication to the drug. Nigericin, an ionophore that facilitates transport of potassium ions across membranes, blocked the replication of both influenza virus and VSV in MDCK cell monolayers, indicating that the ion specificity of ionophores influences their effect on the replication of enveloped viruses.     

Inhibition of influenza virus A/WSN replication by a trypsin inhibitor, 6-amidino-2-naphthyl p-guanidinobenzoate

A trypsin inhibitor, 6-amidino-2-naphthyl p-guanidinobenzoate (FUTHAN) reduced both the number and size of plaques of influenza virus A/WSN/33 (H1N1) that can grow without trypsin treatment in MDCK cells. The resulting virus particles with uncleaved hemagglutinin (HA) in the presence of FUTHAN was activated to produce infectious virions by trypsin treatment. Uncleaved HA of WSN virus grown in the presence of FUTHAN was found to be accumulated by protein analysis of WSN virus labeled biosynthetically with [35S]-methionine. It was strongly suggested that FUTHAN inhibited viral replication by preventing proteolytic cleavage of HA.     

Molecular events during the interaction of envelopes of myxo- and RNA-tumor viruses with cell membranes. A 270 MHz 1H nuclear magnetic resonance study

The nuclear magnetic resonance (NMR) spectra of chick embryo cells have been analyzed after exposure to Newcastle disease virus (NDV). Virions that contained the envelope glycoproteins in the cleaved form and, thus, had full biological activity have been compared to virions that had reduced infectivity due to the presence of uncleaved glycoprotein F. After exposure to infectious virus, drastic changes occurred in the signals assigned to choline and the hydrocarbon chains of fatty acids. These observations are interpreted to demonstrate alteration of the fluid lipid bilayer structure of the cell membranes. This is compatible with the concept of membrane fusion as a penetration mechanism for NDV. Virus containing uncleaved F glycoprotein did not alter the NMR spectra. This indicates that infection is blocked at the stage of penetration.Similar, though less pronounced, differences have been observed when the effects of highly infectious influenza virus containing the hemagglutinin in the cleaved form were compared to the effects of virus which had a lower infectivity due to the presence of uncleaved hemagglutinin. Thus, it appears that the hemagglutinin of influenza virus is involved in penetration and that cleavage is necessary for this function.Alterations of the NMR spectra of the membrane lipids have also been observed when susceptible chick embryo cells (C/E) were infected with Rous sarcoma virus of subgroup B. Such alterations did not occur when nonsusceptible cells (C/B) were used. Thus, infection appears to be blocked again at the stage of penetration.     

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.     

Recognition of noninfectious influenza virus by class I-restricted murine cytotoxic T lymphocytes

We have recently shown that murine target cells can be sensitized for lysis by class I-restricted influenza virus-specific cytotoxic T lymphocytes (CTL) using noninfectious influenza virus. Sensitization is dependent on inactivation of viral neuraminidase activity (which can be achieved by heating virus); and requires fusion of viral and cellular membranes. In the present study, we have examined recognition of antigens derived from heat-treated virus by cloned CTL lines induced by immunization with infectious virus. Target cells sensitized with heat-treated virus were recognized by all 11 CTL clones that were specific for internal virion proteins (nucleoprotein and basic polymerase 1), and by one of six clones specific for the major viral glycoprotein (the hemagglutinin). Immunization of mice with heat-treated virus primed their splenocytes for secondary in vitro CTL responses. CTL generated in this manner recognized target cells infected with recombinant vaccinia virus expressing cloned influenza virus gene products. These findings indicate that both integral membrane proteins and internal proteins that comprise virions can be processed by antigen-presenting cells for recognition by class I-restricted CTL. It also appears that not all hemagglutinin determinants recognized on virus-infected cells are presented by cells sensitized with heat-treated virus.     

Formation of mixed hemagglutinin trimers during double infection with influenza virus strains of the same subtype

Formation of hemagglutinin spikes in the course of the mixed infection of cell culture by two influenza virus strains belonging to the same antigenic subtype or to different subtypes was studied by means of immunoprecipitation of [14C]-labelled hemagglutinins from cell lysates. The immunoprecipitates were further analysed by polyacrylamide gel electrophoresis. Lysates of separately infected cells mixed before lysis were used as control samples. The analysis of immunoprecipitates revealed the formation of chimeric hemagglutinin spikes in the cells infected by the strains possessing hemagglutinins of the same subtype but not in the cells infected by the strains of different subtypes (H1 and H3). The results are discussed in connection with the homology of amino-acid sequences of influenza virus hemagglutinins.     

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.     

Staphylococcus aureus adherence to influenza A virus-infected and control cell cultures: evidence for multiple adhesins

During major epidemics with influenza, there is an increased number of pneumonias due to Staphylococcus aureus with a subsequent high mortality rate. We have postulated that influenza A virus infection of host cells promotes the adherence of S. aureus ultimately resulting in bacterial superinfection. In the present study we compared the adherence of seven strains of 3H-labeled S. aureus to Madin-Darby canine kidney (MDCK) cell monolayers, uninfected and infected with influenza A/FM/1/47 virus. Test strains included: Cowan I; a Cowan I protein A-deficient mutant (PA-); EMS, a protein A and clumping factor-deficient mutant; HSmR; 52A5, a teichoic acid-deficient mutant of HSmR; M, an encapsulated strain; and, No. 1071, a clinical isolate. By radioassay, six of the seven strains demonstrated significantly enhanced adherence to virus-infected cell monolayers compared to uninfected controls; only the M strain was adherence negative. Surface hydrophobicity of the staphylococci did not correlate with their ability to adhere. Four strains of labeled staphylococci (Cowan I, PA-, EMS, and No. 1071), untreated or treated with 2.5% trypsin, 1.25% protease, or by autoclaving, were tested in the radioassay. Protease treatment, which was more effective than trypsin treatment, reduced adherence of all four test strains by 74-96%. Results of heat treatment suggested the presence of both thermolabile and thermostable adhesins. Staphylococcal thermal extracts, profiled by anion-exchange HPLC, were used to pretreat monolayers in a blocking radioassay. Adherence was decreased to control cells (9-78%) and to virus-infected cells (56-90%). The data suggest that multiple distinct surface proteins mediate the binding of S. aureus to uninfected and influenza A virus-infected cells.     

Human membrane cofactor protein (CD46) acts as a cellular receptor for measles virus.

A monoclonal antibody (MCI20.6) which inhibited measles virus (MV) binding to host cells was previously used to characterize a 57- to 67-kDa cell surface glycoprotein as a potential MV receptor. In the present work, this glycoprotein (gp57/67) was immunopurified, and N-terminal amino acid sequencing identified it as human membrane cofactor protein (CD46), a member of the regulators of complement activation gene cluster. Transfection of nonpermissive murine cells with a recombinant expression vector containing CD46 cDNA conferred three major properties expected of cells permissive to MV infection. First, expression of CD46 enabled MV to bind to murine cells. Second, the CD46-expressing murine cells were able to undergo cell-cell fusion when both MV hemagglutinin and MV fusion glycoproteins were expressed after infection with a vaccinia virus recombinant encoding both MV glycoproteins. Third, M12.CD46 murine B cells were able to support MV replication, as shown by production of infectious virus and by cell biosynthesis of viral hemagglutinin after metabolic labeling of infected cells with [35S]methionine. These results show that the human CD46 molecule serves as an MV receptor allowing virus-cell binding, fusion, and viral replication and open new perspectives in the study of MV pathogenesis.     

Rabies virus protein synthesis in infected BHK-21 cells.

Rabies virus specific polypeptide synthesis was examined under hypertonic conditions, which selectively inhibit cellular protein synthesis. The rabies virus proteins (L, G, N, M1, M2) were synthesized throughout the course of infection, with little change in their relative rates of synthesis. The rates of synthesis of the G and M1 polypeptides were more sensitive to increasing osmolarity than those of the L, N, and M2 polypeptides. Extrapolation to isotonicity of the results obtained under hypertonic conditions indicated that the molar ratios of the polypeptides synthesized under normal conditions were 0.4 (L), 64 (G), 100 (N), 75 (M1) and 35 (M2). A high-molecular-weight polypeptide (190,000), designated polypeptide L, was repeatedly detected both in infected cells and in extracellular virus. The estimated number of L polypeptide molecules per virion was 33. The synthesis of a viral glycoprotein precursor, designated gp78, , preceded the appearance of the mature viral glycoprotein in infected cells labeled with [3H]glucosamine under isotonic conditions. In cells labeled under hypertonic conditions, little or no mature viral glycoprotein was detected, but a virus-specific glycoprotein with an electrophoretic mobility similar to that of gp78 was observed. This glycoprotein could be chased into mature viral glycoprotein when the hypertonic conditions were made isotonic. These results suggest that a reversible block of viral glycoprotein synthesis occurs under hypertonic conditions.     

Induction and activity of class II-restricted, Lyt-2+ cytolytic T lymphocytes specific for the influenza H5 hemagglutinin

In influenza A virus infections, CTL are a significant component of the host immune response which limits viral replication and promotes recovery. To examine the CTL response to the influenza virus A/Ty/Ont/7732/66[H5N9], particularly the H5 hemagglutinin, a long term CTL line was generated from spleen cells of A/Ty/Ont-immune Balb/c [H-2d] mice secondarily stimulated in vitro with A/Ty/Cal/Hurst-2/71[H5N2]. This CTL line was highly specific for influenza viruses of the H5 subtype. From this line, clones were isolated by limiting dilution and shown to be H5 hemagglutinin-specific based on recognition of an H5 vaccinia virus recombinant (H5 Vac). The clones exhibited the classical CTL surface phenotype Lyt-1-2+L3T4-; however, unlike the typically class I-restricted Lyt-2+ CTL, they were restricted in antigen recognition by class II (I-E) MHC molecules based on target cell recognition and antibody blocking of cytotoxicity. The clones recognized both infectious and non-infectious A/Ty/Ont presented by class II+ target cells. In adoptive transfer studies to assess the biologic role of the clones in vivo, these class II-restricted clones did not appear to alter mortality. However, these cells significantly reduced both morbidity and virus titers in the lungs of infected animals at 5 days post-infection. Thus, in the immune response to this virus, class II-restricted Lyt-2+ CTL specific for the H5 hemagglutinin were readily generated and their biologic role in vivo involved viral clearance.     

Fusion of membrane vesicles bearing only the influenza hemagglutinin with erythrocytes, living cultured cells, and liposomes

Membrane vesicles, bearing only the influenza viral hemagglutinin glycoprotein, were reconstituted following solubilization of intact virions with Triton X-100. The viral hemagglutinin glycoprotein was separated from the neuraminidase glycoprotein by agarose sulfanilic acid column. The hemagglutinin glycoprotein obtained was homogenous in gel electrophoresis and devoid of any neuraminidase activity. A quantitative determination revealed that the hemolytic activity of the hemagglutinin vesicles was comparable to that of intact virions. Incubation of fluorescently labeled hemagglutinin vesicles with human erythrocyte ghosts (HEG) or with liposomes composed of phosphatidylcholine/cholesterol or phosphatidylcholine/cholesterol/gangliosides, at pH 5.0 but not at pH 7.4, resulted in fluorescence dequenching. Very little, if any, fluorescence dequenching was observed upon incubation of fluorescently labeled HA vesicles with neuraminidase or glutaraldehyde-treated HEG or with liposomes composed only of phosphatidylcholine. Hemagglutinin vesicles were rendered non-hemolytic by treatment with NH2OH or glutaraldehyde or by incubation at 85 degrees C or low pH. No fluorescence dequenching was observed following incubation of non-hemolytic hemagglutinin vesicles with HEG or liposomes. These results clearly suggest that the fluorescence dequenching observed is due to fusion between the hemagglutinin vesicles and the recipient membranes. Incubation of hemagglutinin vesicles with living cultured cells, i.e. mouse lymphoma S-49 cells, at pH 5.0 as well as at pH 7.4, also resulted in fluorescence dequenching. The fluorescence dequenching observed at pH 7.4 was inhibited by lysosomotropic agents (methylamine and ammonium chloride) as well as by EDTA and NaN3, indicating that it is due to fusion of hemagglutinin vesicles taken into the cells by endocytosis.     

Purification and cDNA cloning of a novel protease inhibitor secreted into culture supernatant by MDCK cells.

The infectivity of influenza viruses to host cells depends on the activation of the viral glycoprotein hemagglutinin (HA) by proteases. Starting from the observation that influenza virus replication in MDCK (Madin Darby canine kidney) cells was impaired by inactivation of trypsin in the culture fluids, we demonstrated that the inhibitory activity was resolved into two Trypsin-inactivating factors (TF), TF A (15 kDa) and TF B (11 kDa). N-terminal protein sequences of the factors revealed that TF A was a known Submandibular Protease Inhibitor (SPI) secreted in dog saliva, while TF B was a novel protein (renamed CKPI; canine kidney protease inhibitor). Following peptide mapping and protein sequencing of CKPI we obtained a 390 bp cDNA encoding a 130-amino-acid protein from MDCK cell total RNA. Protein sequence comparison showed a 63.8% identity with human secretory leukocyte protease inhibitor (SLPI), the molecule containing two conserved whey acidic protein (WAP) motifs, and we suggest that CKPI is thought to be the canine analogue of human SLPI. These results suggest that the inhibitory factors are secreted from MDCK cells, which are involved in prevention of virus replication, and applicable to the protection of host cells from virus infection.     

Rescue of a synthetic chloramphenicol acetyltransferase RNA into influenza virus-like particles obtained from recombinant plasmids.

We have shown previously that COS-1 cells infected with a vaccinia virus recombinant (vTF7-3) which expresses the T7 RNA polymerase gene and then transfected with four pGEM-derived plasmids encoding the influenza A virus core proteins (nucleoprotein, PB1, PB2, and PA polypeptides) can express a synthetic influenza virus-like chloramphenicol [correction of chloramphenical] acetyltransferase (CAT) RNA (I. Mena, S. de la Luna, C. Albo, J. Martín, A. Nieto, J. Ortín, and A. Portela, J. Gen. Virol. 75:2109-2114, 1994). Here we demonstrate that by supplying the vTF7-3-infected cells with plasmids containing cDNAs of all 10 influenza virus-encoded proteins, the transfected CAT RNA can be expressed and rescued into particles that are budded into the supernatant fluids. The released particles can transfer the enclosed CAT RNA to MDCK cultures and resemble true influenza virions in that they require trypsin treatment to deliver the RNA to fresh cells and are neutralized by a monoclonal antibody specific for the influenza A virus hemagglutinin. Moreover, analysis by electron microscopy showed that the culture medium harvested from the transfected cells contained vesicles that could be labeled with an anti-HA monoclonal antibody and that were similar in size and morphology to authentic influenza virus particles. It is also shown that detection of recombinant particles capable of transmitting the CAT RNA does not require expression of the influenza virus nonstructural protein NS1. All of these data indicate that influenza virus-like particles enclosing a synthetic virus-like RNA can be assembled in cells expressing all viral structural components from recombinant plasmids.     

4-Deoxy-4-fluoro-D-mannose inhibits the glycosylation of the G protein of vesicular stomatitis virus

The effect of 4-deoxy-4-fluoro-D-mannose (4F-Man), a synthetic analog of D-mannose, on the synthesis of the glycoprotein (G) of vesicular stomatitis virus was examined. Nearly confluent monolayers of cultured BHK21 cells infected with vesicular stomatitis virus were incubated for 2 h with 4F-Man (0-10 mM) or for 1 h with tunicamycin (2 micrograms/ml) and then pulse-labeled with [35S]methionine or [3H]glucosamine. After a 90-min chase period, the cells were lysed and the viral proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. The 35S-labeled G protein from cells exposed to greater than or equal to 1 mM 4F-Man migrated more rapidly than G protein isolated from control cells and with the same electrophoretic mobility as the glycoprotein produced by cells treated with tunicamycin. When infected cells were labeled with [3H]glucosamine, little or no radioactivity was associated with G protein synthesized in the presence of greater than or equal to 1 mM 4F-Man. The conclusion that 4F-Man blocks the glycosylation of the G protein was supported by experiments which demonstrated that the fluorosugar inhibits the synthesis of lipid-linked oligosaccharides.     

Identification of Influenza C Virus Phosphoproteins

The HMV-II cells infected with influenza C virus were labeled with inorganic [³²P]phosphate to identify phosphorylated proteins. Analysis by radioimmunoprecipitation with antiviral serum or monoclonal antibodies revealed that three major structural proteins of the virus, hemagglutinin-esterase (HE), nucleoprotein (NP), and matrix protein (M1) are all phosphorylated in both infected cells and virions. It was also observed that, in the presence of trypsin (10 μg/ml), the unphosphorylated form of the HE glycoprotein was cleaved efficiently whereas the phosphorylated form was not, raising the possibility that phosphorylation of HE may influence its susceptibility to degradation by proteolytic enzymes.