Addition of truncated oligosaccharides to influenza virus hemagglutinin results in its temperature-conditional cell-surface expression

In the preceding paper (Hearing, J., E. Hunter, L. Rodgers, M.-J. Gething, and J. Sambrook. 1989. J. Cell Biol. 108:339-353) we described the isolation and initial characterization of seven Chinese hamster ovary cell lines that are temperature conditional for the cell-surface expression of influenza virus hemagglutinin (HA) and other integral membrane glycoproteins. Two of these cell lines appeared to be defective for the synthesis and/or addition of mannose-rich oligosaccharide chains to nascent glycoproteins. In this paper we show that at both 32 and 39 degrees C in two mutant cell lines accumulate a truncated version, Man5GlcNAc2, of the normal lipid-linked precursor oligosaccharide, Glc3Man9GlcNAc2. This is possibly due to a defect in the synthesis of dolichol phosphate because in vitro assays indicate that the mutant cells are not deficient in mannosylphosphoryldolichol synthase at either temperature. A mixture of truncated and complete oligosaccharide chains was transferred to newly synthesized glycoproteins at both the permissive and restrictive temperatures. Both mutant cell lines exhibited altered sensitivity to cytotoxic plant lectins when grown at 32 degrees C, indicating that cellular glycoproteins bearing abnormal oligosaccharide chains were transported to the cell surface at the permissive temperature. Although glycosylation was defective at both 32 and 39 degrees C, the cell lines were temperature conditional for growth, suggesting that cellular glycoproteins were adversely affected by the glycosylation defect at the elevated temperature. The temperature-conditional expression of HA on the cell surface was shown to be due to impairment at 39 degrees C of the folding, trimerization, and stability of HA molecules containing truncated oligosaccharide chains.     

Transport of influenza HA from the trans-Golgi network to the apical surface of MDCK cells permeabilized in their basolateral plasma membranes: energy dependence and involvement of GTP-binding proteins

A procedure employing streptolysin O to effect the selective permeabilization of either the apical or basolateral plasma membrane domains of MDCK cell monolayers grown on a filter support was developed which permeabilizes the entire monolayer, leaves the opposite cell surface domain intact, and does not abolish the integrity of the tight junctions. This procedure renders the cell interior accessible to exogenous macromolecules and impermeant reagents, permitting the examination of their effects on membrane protein transport to the intact surface. The last stages of the transport of the influenza virus hemagglutinin (HA) to the apical surface were studied in pulse-labeled, virus-infected MDCK cells that were incubated at 19.5 degrees C for 90 min to accumulate newly synthesized HA in the trans-Golgi network (TGN), before raising the temperature to 35 degrees C to allow synchronized transport to the plasma membrane. In cells permeabilized immediately after the cold block, 50% of the intracellular HA molecules were subsequently delivered to the apical surface. This transport was dependent on the presence of an exogenous ATP supply and was markedly inhibited by the addition of GTP-gamma-S at the time of permeabilization. On the other hand, the GTP analogue had no effect when it was added to cells that, after the cold block, were incubated for 15 min at 35 degrees C before permeabilization, even though at this time most HA molecules were still intracellular and their appearance at the cell surface was largely dependent on exogenous ATP. These findings indicate that GTP-binding proteins are involved in the constitutive process that effects vesicular transport from the TGN to the plasma membrane and that they are charged early in this process. Transport of HA to the cell surface could be made dependent on the addition of exogenous cytosol when, after permeabilization, cells were washed to remove endogenous cytosolic components. This opens the way towards the identification of cell components that mediate the sorting of apical and basolateral membrane components in the TGN and their polarized delivery to the cell surface.     

Polarized delivery of viral glycoproteins to the apical and basolateral plasma membranes of Madin-Darby canine kidney cells infected with temperature-sensitive viruses

The intracellular route followed by viral envelope glycoproteins in polarized Madin-Darby canine kidney cells was studied by using temperature-sensitive mutants of vesicular stomatitis virus (VSV) and influenza, in which, at the nonpermissive temperature (39.5 degrees C), the newly synthesized glycoproteins (G proteins) and hemagglutinin (HA), respectively, are not transported out of the endoplasmic reticulum. After infection with VSV and incubation at 39.5 degrees C for 4-5 h, synchronous transfer of G protein to the plasma membrane was initiated by shifting to the permissive temperature (32.5 degrees C). Immunoelectron microscopy showed that under these conditions the protein moved to the Golgi apparatus and from there directly to a region of the lateral plasma membrane near this organelle. G protein then seemed to diffuse progressively to basal regions of the cell surface and, only after it had accumulated in the basolateral domain, it began to appear on the apical surface near the intercellular junctions. The results of these experiments indicate that the VSV G protein must be sorted before its arrival at the cell surface, and suggest that passage to the apical domain occurs only late in infection when tight junctions are no longer an effective barrier. In complementary experiments, using the temperature-sensitive mutant of influenza, cultures were first shifted from the nonpermissive temperature (39.5 degrees C) to 18.5 degrees C, to allow entrance of the glycoprotein into the Golgi apparatus (see Matlin, K.S., and K. Simons, 1983, Cell, 34:233-243). Under these conditions HA accumulated in Golgi stacks and vesicles but did not reach the plasma membrane. When the temperature was subsequently shifted to 32.5 degrees C, HA rapidly appeared in discrete regions of the apical surface near, and often directly above, the Golgi elements, and later diffused throughout this surface. To ensure that the anti-HA antibodies had access to lateral domains, monolayers were treated with a hypertonic medium to dilate the intercellular spaces. Some labeling was then observed in the lateral plasma membranes soon after the shift, but this never increased beyond 1.0 gold particle/micron, whereas characteristic densities of labeling in apical surfaces soon became much higher (approximately 10 particles/micron). Our results suggest that the bulk of HA follows a direct pathway leading from the Golgi to regions of the apical surface close to trans-Golgi cisternae.     

Monensin and FCCP inhibit the intracellular transport of alphavirus membrane glycoproteins

Temperature-sensitive mutants of semliki forest virus (SFV) and sindbis virus (SIN) were used to study the intracellular transport of virus membrane glycoproteins in infected chicken embryo fibroblasts. When antisera against purified glycoproteins and (125)I- labeled protein A from staphylococcus aureus were used only small amounts of virus glycoproteins were detected at the surface of SFV ts-1 and SIN Ts-10 infected cells incubated at the restrictive temperature (39 degrees C). When the mutant-infected cells were shifted to the permissive temperature (28 degrees C), in the presence of cycloheximide, increasing amounts of virus glycoproteins appeared at the cell surface from 20 to 80 min after the shift. Both monensin (10muM) and carbonylcyanide-p- trifluoromethoxyphenylhydrazone (FCCP; 10-20 muM) inhibited the appearance of virus membrane glycoproteins at the cell surface. Vinblastine sulfate (10 μg/ml) inhibited the transport by approximately 50 percent, whereas cytochalasin B (1 μg/ml) had only a marginal effect. Intracellular distribution of virus glycoproteins in the mutant-infected cells was visualized in double-fluorescence studies using lectins as markers for endoplasmic reticulum and Golgi apparatus. At 39 degrees C, the virus membrane glycoproteins were located at the endoplasmic reticulum, whereas after shift to 28 degrees C, a bright juxtanuclear reticular fluorescence was seen in the location of the Golgi apparatus. In the presence of monensin, the virus glycoproteins could migrate to the Golgi apparatus, although transport to the cell surface did not take place. When the shift was carried out in the presence of FCCP, negligible fluorescence was seen in the Golgi apparatus and the glycoproteins apparently remained in the rough endoplasmic reticulum. A rapid inhibition in the accumulation of virus glycoproteins at the cell surface was obtained when FCCP was added during the active transport period, whereas with monensin there was a delay of approximately 10 min. These results suggest a similar intracellular pathway in the maturation of both plasma membrane and secretory glycoproteins.     

Role of glycosylation in transport of vesicular stomatitis virus envelope glycoprotein. A new class of mutant defective in glycosylation and transport of G protein

A temperature-sensitive mutant (ts gamma 1) of the Cocal serotype of vesicular stomatitis virus synthesizes at the permissive temperature (32 degrees C) a glycoprotein G whose size is smaller (Mr 68,000) than the wild-type (Mr 71,000) and that renders the virion thermolabile. At the nonpermissive temperature (39 degrees C), reduced amounts of noninfectious virus-like particles deficient in G protein were produced. The size of the intracellular G protein was further decreased (Mr 64,000) at the nonpermissive temperature. Biochemical studies including sugar labeling, tryptic peptide analysis, and NH2-terminal sequence analysis of the various glycoproteins suggest that at 32 degrees C a G protein containing a single glycosidic moiety is synthesized. The G protein containing only 1 oligosaccharide residue is transported to the cell surface and is incorporated in infectious virus particles. In contrast, the G protein synthesized at 39 degrees C is nonglycosylated and fails to reach the cell surface. These results suggest that glycosylation of G protein is essential for its transport to the cell surface, and the presence of a single carbohydrate chain is sufficient for this purpose.     

Characterization of a temperature-sensitive influenza B virus mutant defective in neuraminidase.

ts5, a temperature-sensitive mutant of influenza B virus, belongs to one of seven recombination groups. When the mutant infected MDCK cells at the nonpermissive temperature (37.5 degrees C), infectious virus was produced at very low levels compared with the yield at the permissive temperature (32 degrees C) and hemagglutinating and enzymatic activities were undetectable. However, viral protein synthesis and transport of hemagglutinin (HA) and neuraminidase (NA) to the cell surface were not affected. The NA was found as a monomer within cells even at 32 degrees C, in contrast to wild-type virus NA, existing mostly as an oligomer, but the mutant had oligomeric NA, like the wild-type virus. Its enzymatic activity was more thermolabile than that of wild-type virus. Despite the low yield, large aggregates of progeny virus particles were found to accumulate on the cell surface at the nonpermissive temperature, and these aggregates were broken by treatment with bacterial neuraminidase, with the concomitant appearance of hemagglutinating activity, suggesting that NA prevents the aggregation of progeny virus by removal of neuraminic acid from HA and cell receptor, allowing its release from the cells. Further treatment with trypsin resulted in the recovery of infectivity. When bacterial NA was added to the culture early in infection, many hemagglutinable infectious virus was produced. We also suggest that the removal of neuraminic acid from HA by NA is essential for the subsequent cleavage of HA by cellular protease. Nucleotide sequence analysis of RNA segment 6 revealed that ts5 encoded five amino acid changes in the NA molecule but not in NB.     

An efficient method for introducing defined lipids into the plasma membrane of mammalian cells

An efficient method has been devised to introduce lipid molecules into the plasma membrane of mammalian cells. This method has been applied to fuse lipid vesicles with the apical plasma membrane of Madin-Darby canine kidney cells. The cells were infected with fowl plague or influenza N virus. 4 h after infection, the hemagglutinin (HA) spike glycoprotein of the virus was present in the apical plasma membrane of the cells. Lipid vesicles containing egg phosphatidylcholine, cholesterol, and an HA receptor (ganglioside) were then bound to the cells at 0 degrees C. More than 85% of the vesicles were released by external neuraminidase at 0 degrees C or by simply warming the cells to 37 degrees C for 10 s, probably because of the action of the viral neuraminidase at the cell surface. However, when the cells were warmed to 37 degrees C in a pH 5.3 medium for 30 s, 50% of the bound vesicles could no longer be released by external neuraminidase. This only occurred when the HA protein had been cleaved into its HA1 and HA2 subunits. When we used influenza N virus, whose HA is not cleaved in Madin-Darby canine kidney cells, cleavage with external trypsin was required. The fact that the HA protein has fusogenic properties at low pH only in its cleaved form suggests that fusion of the vesicles with the plasma membrane had taken place. Further confirmation for fusion was obtained using an assay based on the decrease of energy transfer between two fluorescent phospholipids in a vesicle upon fusion of the vesicle with the plasma membrane (Struck, D. K., D. Hoekstra, and R. E. Pagano. 1981. Biochemistry, 20:4093-4099).     

Neuroblastoma Cell Fusion by a Temperature-Sensitive Mutant of Vesicular Stomatitis Virus

A temperature sensitive mutant of vesicular stomatitis virus which does not mature properly when grown at 39 degrees C promoted extensive fusion of murine neuroblastoma cells at this nonpermissive temperature. Polykaryocytes apparently formed as a result of fusion from within the cells that requires low doses of infectious virions for its promotion and is dependent on viral protein synthesis. Although 90% of infected N-18 neuroblastoma cells were fused by 15 h after infection, larger polykaryocytes continued to form, leading to an average of 28 nuclei per polykaryocyte as a result of polykaryocytes fusing to each other. Two neuroblastoma cell lines have been observed to undergo fusion, whereas three other cell lines (BHK-21, CHO, and 3T3) were incapable of forming polykaryocytes, suggesting that nervous system-derived cells are particularly susceptible to vesicular stomatitis virus-induced fusion. Although the normal assembly of the protein components of this virus is deficient at 39 degrees C, the G glycoprotein was inserted into the infected cell membranes at this temperature. Two lines of evidence suggest that the expression of G at the cell surface promotes this polykaryocyte formation: (i) inhibition of glycosylation, which may be involved in the migration of the G protein to the cellular plasma membranes, will inhibit the cell fusion reaction; (ii) addition of antiserum, directed toward the purified G glycoprotein, will also inhibit cell fusion.     

Biosynthesis of plasma membrane components by SV40-virus-transformed 3T3 mouse cells temperature sensitive for expression of some transformed cell properties.

We have studied the plasma membranes of an SV40-transformed 3T3 cell line temperature sensitive for the transformed growth phenotype (ts H6-15 cells), and have found that they vary little as a function of temperature of cultivation. Analysis by polyacrylamide gel electrophoresis was performed on plasma membranes prepared from ts H6-15 cells cultured at the permissive (32 degrees C) and non-permissive (39 degrees C) temperatures and radioactively-labelled in several ways. No significant differences were seen when the electrophoretic patterns of polypeptides of the plasma membranes of ts H6-15 cells, grown through 3-4 generations in medium containing radioactive leucine (32 degrees C and 39 degrees C temperatures) were compared. Plasma membranes derived from cells similarly grown in medium with radioactive glucosamine indicated that extensive alterations in the intrinsic glycopeptides occurred in association with alteration in growth phenotype. A shift towards decreased synthesis of large molecular weight (congruent to 100 000-160 000) glycopeptides occurred in cells grown at the temperature of non-transformed growth (39 degrees C). A decrease in amount of a 120 000 molecular weight glycopeptide at 39 degrees C was the most prominent of these alterations. We have studied the surface exposure of polypeptides and glycopeptides of intact cells grown at 32 and 39 degrees C, using lactoperoxidase-catalyzed iodination, NaBH4 reduction of galactose oxidase-treated cells, and metabolic-labelling with glucosamine of trypsin-sensitive molecules. We found no major qualitative differences between whole cell extracts or between plasma membrane preparations of cells cultivated at the permissive and non-permissive temperatures. Of special interest was the observation that the formation and surface exposure of a trypsin-sensitive, 240 000 molecular weight polypeptide appeared not to be ts in ts H6-15 cells. The significance of these observations will be discussed.     

Temperature-dependent internalization of virus glycoproteins in cells infected with a mutant of Semliki Forest virus.

When the ts-1 mutant of Semliki Forest virus (SFV) was grown in chick embryo or BHK 21 cells at the restrictive temperature (39 degrees C), its membrane glycoproteins were arrested in the endoplasmic reticulum, but started to migrate to the cell surface once the cultures were shifted to the permissive temperature (28 degrees C). If the temperature of infected cells was raised back to 39 degrees C, ts-1 glycoproteins disappeared from the cell surface as evidenced by loss of surface immunofluorescence and by radioimmunoassay based on the binding of 125I-labeled protein A. This phenomenon was specific for ts-1 at 39 degrees C as it was observed neither in cells infected with wild-type SFV at 39 degrees C nor with ts-1 at 28 degrees C. The disappearance of the ts-1 glycoproteins was due to internalization. The internalized proteins were digested, as shown by specific decrease of virus glycoproteins labelled with [35S]methionine at 39 degrees C before shift to 28 degrees C, and by concomitant release of acid soluble 35S-activity into the culture medium. Ts-1 infected cells were treated before shift back to 39 degrees C with Fab' fragments, prepared from IgG against the viral membrane glycoproteins. After shift back to 39 degrees C, the Fab' fragments disappeared from the cell surface. In the presence of chloroquine, they could be visualized in vesicular structures, using an anti-IgG-fluorescein isothiocyanate conjugate. The internalization of ts-1 glycoproteins was not inhibited by carbonylcyanide p-trifluoromethoxy phenylhydrazone, chloroquine, cytochalasin B, vinblastine, colcemid, or monensin.     

Properties of mammalian cells transformed by temperature-sensitive mutants of avian sarcoma virus.

Fibroblasts from European field vole (Microtus agrestis) and from normal rat kidney (NRK) have been infected by avian sarcoma virus mutants which are temperature-sensitive for the maintenance of transformation. These cells are transformed at 33 degrees C, but show normal cell characteristics in morphology, colony formation in agar, saturation density, sugar uptake and membrane proteins at 39 degrees C and 40 degrees C, the nonpermissive temperatures. Ts mutant virus was rescued from most of the ts transformed cell lines. NRK cells infected by avian sarcoma virus ts mutants and kept at the nonpermissive temperature can be transformed by wild-type avian sarcoma virus. The susceptibility of the temperature-sensitive NRK lines to this transformation is higher than the susceptibility of uninfected NRK at either permissive or nonpermissive temperature.     

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.     

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.     

Immunoelectron microscopic studies of the intracellular transport of the membrane glycoprotein (G) of vesicular stomatitis virus in infected Chinese hamster ovary cells

An immunoelectron microscopic study was undertaken to survey the intracellular pathway taken by the integral membrane protein (G-protein) of vesicular stomatitis virus from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane of virus-infected Chinese hamster ovary cells. Intracellular transport of the G-protein was synchronized by using a temperature-sensitive mutant of the virus (0-45). At the nonpermissive temperature (39.8 degrees C), the G-protein is synthesized in the cell infected with 0-45, but does not leave the rough endoplasmic reticulum. Upon shifting the temperature to 32 degrees C, the G-protein moves by stages to the plasma membrane. Ultrathin frozen sections of 0-45-infected cells were prepared and indirectly immunolabeled for the G-protein at different times after the temperature shift. By 3 min, the G-protein was seen at high density in saccules at one face of the Golgi apparatus. No large accumulation of G-protein-containing vesicles were observed near this entry face, but a few 50-70-mm electron-dense vesicular structures labeled for G-protein were observed that might be transfer vesicles between the rough endoplasmic reticulum and the Golgi complex. At blebbed sites on the nuclear envelope at these early times there was a suggestion that the G-protein was concentrated, these sites perhaps serving as some of the transitional elements for subsequent transfer of the G-protein from the rough endoplasmic reticulum to the Golgi complex. By 3 min after its initial asymmetric entry into the Golgi complex, the G-protein was uniformly distributed throughout all the saccules of the complex. At later times, after the G-protein left the Golgi complex and was on its way to the plasma membrane, a new class of G-protein-containing vesicles of approximately 200-nm diameter was observed that are probably involved in this stage of the transport process. These data are discussed, and the further prospects of this experimental approach are assessed.     

Quality control in the endoplasmic reticulum: folding and misfolding of vesicular stomatitis virus G protein in cells and in vitro

Parallel experiments in living cells and in vitro were undertaken to characterize the mechanism by which misfolded and unassembled glycoproteins are retained in the ER. A thermoreversible folding mutant of vesicular stomatitis virus (VSV) G protein called ts045 was analyzed. At 39 degrees C, newly synthesized G failed to fold correctly according to several criteria: intrachain disulfide bonds were incomplete; the B2 epitope was absent; and the protein was associated with immunoglobulin heavy chain binding protein (BiP), a heat shock-related, ER protein. When the temperature was lowered to 32 degrees C, these properties were reversed, and the protein was transported to the cell surface. Upon the shift up from 32 degrees C back to 39 degrees C, G protein in the ER returned to the misfolded form and was retained, while the protein that had reached a pre-Golgi compartment or beyond was thermostable and remained transport competent. The misfolding reaction could be reconstituted in a cell free system using ts045 virus particles and protein extracts from microsomes. Taken together, the results showed that ER is unique among the organelles of the secretory pathway in containing specific factors capable of misfolding G protein at the nonpermissive temperature and thus participating in its retention.     

pH-independent HIV entry into CD4-positive T cells via virus envelope fusion to the plasma membrane

CD4 functions as the cell-surface receptor for human immunodeficiency virus (HIV); however, the mechanism of virus entry into susceptible cells is unknown. To explore this question we used a human T lymphoblastic cell line (VB) expressing high levels of surface CD4. Neutralization of endosomal compartments (pH greater than 6.4) with lysosomotropic agents did not effectively inhibit HIV nucleocapsid entry into the cytoplasm, and virus treated at low pH (5.5) failed to induce rapid cell-to-cell fusion in uninfected cells. Electron microscopy of VB cells acutely exposed to HIV at neutral pH revealed direct fusion of the virus envelope with the plasma membrane within minutes at 4 degrees C. No endocytosed virions were visualized upon rewarming the HIV-exposed cells to 37 degrees C for as long as 60 min. These results indicate that HIV penetrates CD4-positive T cells via pH-independent membrane fusion.     

Apical budding of a recombinant influenza A virus expressing a hemagglutinin protein with a basolateral localization signal

Influenza virions bud preferentially from the apical plasma membrane of infected epithelial cells, by enveloping viral nucleocapsids located in the cytosol with its viral integral membrane proteins, i.e., hemagglutinin (HA), neuraminidase (NA), and M2 proteins, located at the plasma membrane. Because individually expressed HA, NA, and M2 proteins are targeted to the apical surface of the cell, guided by apical sorting signals in their transmembrane or cytoplasmic domains, it has been proposed that the polarized budding of influenza virions depends on the interaction of nucleocapsids and matrix proteins with the cytoplasmic domains of HA, NA, and/or M2 proteins. Since HA is the major protein component of the viral envelope, its polarized surface delivery may be a major force that drives polarized viral budding. We investigated this hypothesis by infecting MDCK cells with a transfectant influenza virus carrying a mutant form of HA (C560Y) with a basolateral sorting signal in its cytoplasmic domain. C560Y HA was expressed nonpolarly on the surface of infected MDCK cells. Interestingly, viral budding remained apical in C560Y virus-infected cells, and so did the location of NP and M1 proteins at late times of infection. These results are consistent with a model in which apical viral budding is a shared function of various viral components rather than a role of the major viral envelope glycoprotein HA.     

Role of neuraminidase in the morphogenesis of influenza B virus.

When ts7, a temperature-sensitive (ts) mutant of influenza B/Kanagawa/73 virus, infected MDCK cells at the nonpermissive temperature (37.5 degrees C), infectious virus was produced at very low levels compared with the yield at the permissive temperature (32 degrees C) and hemagglutinating activity and enzymatic activity of neuraminidase (NA) were negligible. However, viral protein synthesis and transport of hemadsorption-active hemagglutinin to the cell surface were not affected. When the cell lysate was treated with bacterial NA, hemagglutinating activity was recovered but infectivity was not, even after further treatment with trypsin. It was found that ts7 was defective in transport of NA to the cell surface and formation of virus particles. Analysis of the genomes of non-ts recombinants obtained by crossing ts7 and UV-inactivated B/Lee showed that ts7 had the ts mutation only in RNA segment 6 coding for NA and the glycoprotein NB. Nucleotide sequence analysis of the RNA segment revealed that ts7 had four amino acid changes in the NA molecule but not in NB. We suggest that assembly or budding of influenza B virus requires the presence of NA at the plasma membrane, unlike influenza A virus.     

Intracellular transport of influenza virus hemagglutinin to the apical surface of Madin-Darby canine kidney cells

The intracellular pathway followed by the influenza virus hemagglutinin (HA) to the apical surface of Madin-Darby canine kidney cells was studied by radioimmunoassay, immunofluorescence, and immunoelectron microscopy. To synchronize the migration, we used a temperature- sensitive mutant of influenza WSN, ts61, which, at the nonpermissive temperature, 39.5 degrees C, exhibits a defect in the HA that prevents its exit from the endoplasmic reticulum. Upon transfer to permissive temperature, 32 degrees C, the HA appeared in the Golgi apparatus after 10 min, and on the apical surface after 30-40 min. In the presence of cycloheximide, the expression was not inhibited, indicating that the ts defect is reversible; a wave of HA migrated to the cell surface, where it accumulated with a half time of 60 min. After passage through the Golgi apparatus the HA was detected in a population of smooth vesicles, about twice the size of coated vesicles, located in the apical half of the cytoplasm. These HA-containing vesicles did not react with anti- clathrin antibodies. Monensin (10 microM) delayed the surface appearance of HA by 2 h, but not the transport to the Golgi apparatus. Incubation at 20 degrees C retarded the migration to the Golgi apparatus by approximately 30 min and blocked the surface appearance by acting at a late stage in the intracellular pathway, presumably at the level of the post-Golgi vesicles. The initial appearance of HA on the apical surface was in the center; no preference was observed for the tight-junctional regions.     

Cleavage of membrane secretory component to soluble secretory component occurs on the cell surface of rat hepatocyte monolayers

Rat liver secretory component is synthesized as an integral membrane protein (mSC) and cleaved to an 80-kD soluble form (fSC) sometime during transcellular transport from the sinusoidal to the bile canalicular plasma membrane domain of hepatocytes. We have used 24-h monolayer cultures of rat hepatocytes to characterize the conversion of mSC to fSC. Cleavage of mSC in cultured hepatocytes is inhibited by the thiol protease inhibitors leupeptin, antipain, and E-64, but not by other inhibitors, including disopropylfluorophosphate, pepstatin, N-ethylmalemide, p-chloromercuribenzoic acid, and chloroquine. Leupeptin-mediated inhibition of cleavage is concentration dependent and reversible. In the presence or absence of leupeptin, only 10-20% of mSC is accessible at the cell surface. To characterize the behavior of surface as opposed to intracellular mSC, cell surface mSC was labeled with 125I by lactoperoxidase-catalyzed iodination at 4 degrees C. Cell surface 125I-mSC was converted to extracellular fSC at 4 degrees C in the absence of detectable internalization. Cleavage was inhibited by leupeptin and by anti-secretory component antiserum. Cleavage also occurred at 4 degrees C after cell disruption. In contrast, 125I-mSC that had been internalized from the cell surface was not converted to fSC at 4 degrees C in either intact or disrupted cells. Hepatocytes metabolically labeled with [35S]cys also released small quantities of fSC into the medium at 4 degrees C. The properties of fSC production indicate that cleavage occurs on the surface of cultured rat hepatocytes and not intracellularly. Other features of the cleavage reaction suggest that the mSC-cleaving protease is segregated from the majority of cell surface mSC, possibly within a specialized plasma membrane domain.