Infected MDCK Cells Research Articles

Nonpolarized expression of a secreted murine leukemia virus glycoprotein in polarized epithelial cells

Vaccinia virus recombinants were generated which express the intact gp70/p15E of Friend mink cell focus inducing virus (F-MCFV) or truncated forms of the glycoprotein that lack the transmembrane and cytoplasmic domains. The transport of the intact and truncated envelope glycoproteins to apical or basolateral surfaces was studied in the polarized epithelial MDCK cell line. Infection of MDCK cells with the recombinant expressing the intact F-MCFV envelope glycoprotein resulted in transport exclusively to the basolateral surfaces, whereas the recombinant expressing the truncated glycoprotein was found to be secreted from both the apical and basolateral surfaces. Thus removal of the transmembrane and cytoplasmic domains of the p15E protein results in a loss of directional transport to the basolateral membrane of polarized epithelial cells.

Monoclonal antibodies detect different forms of influenza virus hemagglutinin during viral penetration and biosynthesis

Monoclonal antibodies specific for the influenza virus A/PR/8/34 hemagglutinin (HA) were used to examine the structure of the HA glycoprotein by immunofluorescence techniques during infection of MDCK cells. One antibody (Y8-10C2), shown previously to detect conformational alterations in the HA coinciding with the acid induction of viral fusion activity, bound to internalized virus but not to virus adsorbed to the cell surface. The binding of Y8-10C2 was completely inhibited by ammonium chloride treatment of the cells. These findings are consistent with the idea that Y8-10C2 detects conformational changes in the HA which accompany the acid-induced fusion of viral and endosomal membranes. The same antibody also bound to newly synthesized HA but not to later forms of cytoplasmic HA or to HA incorporated into the cell membrane during virus maturation. A possible common denominator of the antigenic changes detected by antibody Y8-10C2 during virus entry and replication may be alterations in the structural relationship among the three HA monomers which form the mature HA molecule.

Virions and intracellular nucleocapsids produced during mixed heterotypic influenza infection of MDCK cells.

Phenotypically mixed virus yields, obtained by coinfection of MDCK cells with influenza A/WSN/33 and B/Lee/40 viruses, contained both A/WSN/33 and B/Lee/40 NP proteins, as revealed by polyacrylamide gel electrophoresis of the purified 14C-amino acids-labeled virus. Virions were lysed with 0.6 M KCl-Triton X-100 buffer, and nucleocapsids were immunoprecipitated with antibodies against NP protein of influenza A virus. Polyacrylamide gel electrophoresis of the immunoprecipitate revealed NP protein of A/WSN/33 but not of B/Lee/40 virus. However, in similar experiments with the lysates of doubly infected cells, the band of B/Lee/40 NP protein was revealed in the polyacrylamide gel electrophoresis patterns of the immunoprecipitates. In an attempt to analyze the RNA content of the immune complexes, we absorbed the lysates of doubly infected [3H]uridine-labeled cells with protein A-containing Staphylococcus aureus covered with antibodies against the NP protein of influenza A virus. RNA extracted from the immune complexes contained genomic RNA segments of both A/WSN/33 and B/Lee/40 viruses. In control samples containing an artificial mixture of cell lysates separately infected with each virus, the analysis revealed homologous components (i.e., A/WSN/33 NP protein or RNA segments) in the immune complexes. The results suggest the presence of phenotypically mixed nucleocapsids in the cells doubly infected with influenza A and B viruses; in the course of the virion formation, the nucleocapsids lacking the heterologous NP protein are selected.

Mechanism of interference between influenza A/WSN and B/Kanagawa viruses.

Simultaneous infection of MDCK cells with influenza viruses A/WSN and B/Kanagawa resulted in mutual interference with virus protein synthesis and in significant suppression of A/WSN growth. When infection by one virus preceded the other by 1 or 2 h, growth of the superinfecting virus was selectively inhibited at the level of transcription. Interference by the pre-infecting virus was strongly dependent on the expression of the viral genome but not on haemagglutinin activity. When the replication of both virus types was restricted to primary transcription by cycloheximide, the only translation products following removal of the drug were those of the preinfecting virus. This result was not affected by blocking secondary transcription by actinomycin D. These findings suggest that intertypic interference occurs at the level of primary transcription. This concept was supported further by the observation that a ts mutant of A/WSN (ts-65) with a defect in primary transcription interfered only with superinfection by B/Kanagawa at the permissive temperature.

Effects of monensin on the processing and intracellular transport of influenza virus haemagglutinin in infected MDCK cells.

The role of the Golgi complex in the intracellular transport of influenza virus haemagglutinin in infected MDCK cell monolayers has been investigated using monensin, a carboxylic ionophore known to disrupt the functioning of this organelle in other cell types. In untreated cells metabolically labelled 5 h post-infection with [35S]methionine haemagglutinin was first seen in core glycosylated form, which was sensitive to the enzyme endo-beta-N-acetylglucosaminidase H (endo H). After approximately 20 min this form was converted into a terminally glycosylated, endo H-resistant form. In the presence of monensin core glycosylation of haemagglutinin was not affected, but terminal glycosylation was interrupted. Two new forms of haemagglutinin were observed, both of which were smaller than the core glycosylated form. Of these, the larger was endo H-sensitive while the smaller was endo H-resistant. These new (and uncharacterized) forms of haemagglutinin are likely to be intermediates in the normal process of terminal glycosylation, which are revealed as a result of the inhibition by monensin of the transport of haemagglutinin through the stack of Golgi cisternae. In untreated cells 85% of the pulse-labelled haemagglutinin had reached the plasma membrane after 90 min of chase, as revealed by its sensitivity to externally applied trypsin. In monensin-treated cells, on the other hand, only 55% of the haemagglutinin had reached the plasma membrane after 90 min of chase, while 94% had arrived there after 180 min of chase. At 5 h post-infection the density of envelope proteins detected at the apical surface of the monolayer by immunofluorescence microscopy was greatly reduced by monensin treatment. Budding of virions from the apical surface of the monolayer at 4 and 7 h post-infection was also reduced, and the normal Golgi complexes were replaced by distended vacuoles that appeared to contain poorly preserved virions.

The detection of influenza A virus antigens in cultured cells by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was employed to investigate the expression of influenza A/Hong Kong/68 (H3N3) virus structural proteins on the surface of infected MDCK cells, and to detect viral antigens in culture media and cell extracts. Infected cells were fixed with 0.1 per cent glutaraldehyde before being examined for the presence of cell-surface antigens. Viral antigens were first observed on the surface of cells 4 hours after infection and reached a maximum 10-12 hours after infection, when measured by haemadsorption with chicken erythrocytes and by ELISA and immunofluorescence with hyperimmune antiserum to Hong Kong virus. A good correlation was found between the three assay systems. The presence of individual virion structural proteins on the cell surface was determined by ELISA using specific antibodies purified by differential affinity chromatography. Either or both or the internal matrix and nucleoprotein antigens were expressed from 2 to 6 hours after infection, with maximum expression after 2 hours, and the strain-specific and common antigenic determinants of haemagglutinin were observed on the cell surface from 4 hours after infection, and reached a maximum 8 to 10 hours after infection. Low levels of neuraminidase were detected between 4 and 8 hours after infection. Culture media and cell extracts were titrated by infectivity and haemagglutination assays, and by ELISA. Titres obtained from the culture media showed a close correlation between the three assay methods, with peak titres being attained 24 hours after infection. Viral antigens were first observed in cell extracts by ELISA 4 hours after infection, and infectious virions and haemagglutinin 2 hours later, but whereas maximum titres of infectious virus and haemagglutinin were found 10 hours after infection, the ELISA titre continued to rise until 24 hours after infection, which suggested that virus structural proteins were being accumulated in the cells after most of the progeny virions had been released. The results are discussed in terms of the potential use of ELISA in rapid virus diagnosis.

Isolation of influenza C virus recombinants.

Recombinants between two different influenza C viruses were isolated. In MDCK (canine kidney) cells, one strain, C/JJ/50, caused lytic plaques, whereas C/JHG/66 virus did not produce clear plaques. From a mixed infection of MDCK cells with C/JHG/66 virus and UV-inactivated C/JJ/50 virus, clones were isolated which possessed the clear-plaque phenotype. Fingerprint analyses indicated that the RNAs of parent viruses had different oligonucleotide patterns and that one of the clones derived from the mixed infection was formed by reassortment of parental genes. This recombinant clone most likely inherited RNAs 1, 2, 3, 6, and 7 from C/JGH/66 virus and RNAs 4 and 5 from C/JJ/50 virus.

Mechanism and specificity of action of ribavirin.

Ribavirin at a concentration of 30 mug/ml added immediately after infection completely inhibited influenza A/Port Chalmers/1/73 (H(3)N(2)) virus hemagglutinin production in infected MDCK cells. Under these conditions, host cell protein synthesis was inhibited by only 10 to 20%. Polyacrylamide gel electrophoresis of [(35)S]methionine-labeled material from virus-infected cultures confirmed that ribavirin inhibited viral but not host cell protein synthesis. In parallel experiments, actinomycin D also preferentially inhibited viral protein synthesis. The possibility that ribavirin inhibited viral protein synthesis as a result of general inhibition of ribonucleic acid synthesis was therefore examined. In uninfected cells, ribavirin at 30 mug/ml inhibited the incorporation of [(14)C]inosine or [(3)H]uridine into ribonucleic acid but stimulated the incorporation of [(3)H]guanosine. The effects noted are consistent with an inhibition of the host cell enzyme inosine 5'-monophosphate dehydrogenase. This suggestion is supported by the finding that addition of guanosine, but not inosine, to the culture medium substantially reversed the antiviral effect of ribavirin. There was no separation between the concentration of ribavirin causing inhibition of influenza A viral protein synthesis or inhibition of MDCK cell ribonucleic acid synthesis, suggesting that ribavirin is not specifically antiviral in this system but inhibits viral protein synthesis as a result of the general inhibition of ribonucleic acid synthesis.

Influenza virus proteins: identity, synthesis, and modification analyzed by two-dimensional gel electrophoresis.

A modification of the two-dimensional protein electrophoresis system of O'Farrell was used to resolve influenza A virus proteins from each other and from host proteins in infected cells. Viral protein spots corresponding to the hemagglutinin proteins, neuraminidase, nucleocapsid protein, and nonstructural protein, were identified on the two-dimensional electrophoretogram. Use of the two-dimensional separation has allowed us to identify glycoprotein heterogeneity, to demonstrate directly the synthesis of neuraminidase, to analyze some viral proteins early after infection, and to demonstrate that the influenza virus NP and NS proteins are phosphorylated in infected MDCK cells.