Unpacking the incoming influenza virus.
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Amantadine↗
Biomedical subjects
A Helenius
Publications and source records attributed to A Helenius.
Role of ATP and disulphide bonds during protein folding in the endoplasmic reticulum.
Being topologically equivalent to the extracellular space, the lumen of the endoplasmic reticulum (ER) provides a unique folding environment for newly synthesized proteins. Unlike other compartments in the cell where folding occurs, the ER is oxidizing and therefore can promote the formation of disulphide bonds. The reducing agent dithiothreitol, when added to living cells, inhibits disulphide formation with profound effects on folding. Taking advantage of this effect, we demonstrate here that folding of influenza haemagglutinin is energy dependent. Metabolic energy is required to support the correct folding and disulphide bond formation in this well characterized viral glycoprotein, to rescue misfolded proteins from disulphide-linked aggregates, and to maintain the oxidized protein in its folded and oligomerization-competent state.
Adenosine Triphosphate↗
Manipulating disulfide bond formation and protein folding in the endoplasmic reticulum.
Addition of the reducing agent dithiothreitol (DTT) to the medium of living cells prevented disulfide bond formation in newly synthesized influenza hemagglutinin (HA0) and induced the reduction of already oxidized HA0 inside the ER. The reduced HA0 did not trimerize or leave the ER. When DTT was washed out, HA0 was rapidly oxidized, correctly folded, trimerized and transported to the Golgi complex. We concluded that protein folding and the redox conditions in the ER can be readily manipulated by addition of DTT without affecting most other cellular functions, that the reduced influenza HA0 remains largely unfolded, and that folding events that normally take place on the nascent HA0 chains can be delayed and induced post-translationally without loss in efficiency.
Adenosine Triphosphate↗
Misfolding and aggregation of newly synthesized proteins in the endoplasmic reticulum.
As a part of our studies on the folding of glycoproteins in the ER, we analyzed the fate of viral glycoproteins that have misfolded either spontaneously or through inhibition of N-linked glycosylation. Newly synthesized Semliki Forest virus spike glycoproteins E1 and p62 and influenza hemagglutinin were studied in infected and transfected tissue culture cells. Misfolded proteins aggregated in less than 1 min after release from polysomes and aberrant interchain disulfide bonds were formed immediately. When more than one protein was misfolded, mixed aggregates were generated. This indicated that the formation of complexes was nonspecific, random, and not restricted to products from single polysomes. The size of the aggregates varied from small oligomers to complexes of several million daltons. BiP was associated noncovalently with the aggregates and with some of the nonaggregated products. We conclude that aggregation reflects the poor solubility of incompletely folded polypeptide chains.
Carrier Proteins↗
Role of ribosomes in Semliki Forest virus nucleocapsid uncoating.
The mechanism by which Semliki Forest virus nucleocapsids are uncoated was analyzed in living cells and in vitro. In BHK-21 cells, uncoating occurred with virtually complete efficiency within 1 to 2 min after the nucleocapsids entered the cytoplasm. It was inhibited by monensin, which blocks nucleocapsid penetration from endosomes. As previously shown for Sindbis virus (G. Wengler and G. Wengler, Virology 134:435-442, 1984), the capsid proteins from incoming nucleocapsids became associated with ribosomes. The ribosome-bound capsid proteins were distributed throughout the cytoplasm, while the viral RNA remained associated with vacuolar membranes. Using purified nucleocapsids and ribosomes in vitro, we established that ribosomes alone were sufficient for uncoating. Their role was to release the capsid proteins from nucleocapsids and irreversibly sequester them, in a process independent of energy and translation. The process was stoichiometric rather than catalytic, with a maximum of three to six capsid proteins bound to each ribosome. More than 80% of the capsid proteins could thus be removed from the viral RNA, resulting in the formation of nucleocapsid remnants whose sedimentation coefficients progressively decreased from 140S to 80S as uncoating proceeded.
Animals↗
Nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits import.
Because influenza virus replicates in the nucleus and buds from the plasma membrane, its ribonucleoproteins (RNPs) must undergo bidirectional transport across the nuclear membrane. Export from the nucleus to the cytoplasm was found to depend on the viral matrix protein (M1). M1 associated with newly assembled viral RNPs (vRNPs) in the nucleus and escorted them to the cytoplasm through the nuclear pores. In contrast, during entry of the virus into a new host cell, M1 protein dissociated from the RNPs, allowing them to enter the nucleus. Amantadine, an antiviral agent that induces an early block in influenza A infection, was found to block the dissociation event and thereby to prevent import of incoming RNPs into the nucleus. Together, these results showed that M1 modulates the directionality of vRNP transport into and out of the nucleus.
Amantadine↗
The HA2 subunit of influenza hemagglutinin inserts into the target membrane prior to fusion.
The interaction between influenza virus and target membrane lipids during membrane fusion was studied with hydrophobic photoactivatable probes. Two probes, the newly synthesized bisphospholipid diphosphatidylethanolamine trifluoromethyl [3H]phenyl diazirine and the phospholipid analogue 1-palmitoyl-2(11-[4-[3-(trifluoromethyl)diazirinyl]phenyl]-[2-3H]- undecanoyl]-sn-glycero-3-phosphocholine (Harter, C., Bächi, T., Semenza, G., and Brunner , J. (1988) Biochemistry 27, 1856-1864), were used. Both labeled the HA2 subunit of the virus at low pH. By measuring virus-liposome interactions at 0 degrees C, it could be demonstrated that HA2 was inserted into the target membrane prior to fusion. As we have recently demonstrated, at this temperature, exposure of the fusion peptide of HA2 takes place within 15 s after acidification, but fusion does not start for 4 min (Stegmann, T., White, J. M., and Helenius, A. (1990) EMBO J. 9, 4231-4241). HA2 was labeled at least 2 min before fusion. No labeling of the HA1 subunit was seen. These data indicate that fusion is triggered by a direct interaction of the HA2 subunit of a kinetic intermediate form of HA with the lipids of the target membrane. Most likely, it is the fusion peptide of HA2 that is inserted into the target membrane. Just before fusion, HA is thus an integral membrane protein in both membranes. In contrast, the bromelain-derived ectodomain of HA was labeled by 1-palmitoyl-2(11-[4-[3-(trifluoromethyl)diazirinyl]phenyl]- [2-3H]undecanoyl)-sn-glycerol-3-phosphocholine at low pH but not by diphosphatidylethanolamine trifluoromethyl [3H]phenyl diazirine. This indicates that insertion of the fusion peptide of the bromelain-derived ectodomain of HA into a membrane differs from that of viral HA during fusion.
Affinity Labels↗
Folding of influenza hemagglutinin in the endoplasmic reticulum.
The folding of influenza hemagglutinin (HA0) in the ER was analyzed in tissue culture cells by following the formation of intrachain disulfides after short (1 min) radioactive pulses. While some disulfide bonds were already formed on the nascent chains, the subunits acquired their final disulfide composition and antigenic epitopes posttranslationally. Two posttranslational folding intermediates were identified. In CHO cells constitutively expressing HA0, mature HA0 subunits were formed with a half time of 3 min and their folding reached completion at 22 min. The rate of folding was highly dependent on cell type and expression system, and thus regulated by factors other than the sequence of the protein alone. Exposure of cells to stress conditions increased the level of glucose regulated proteins, including BiP, and decreased the folding rate. The efficiency of folding and subsequent trimerization was not dependent on the rate of translation, nor on temperature between 37 and 15 degrees C; however, the rates of folding and trimerization decreased with decreasing temperature. Whereas the rate of folding was independent of expression level, trimerization was accelerated at higher levels of expression.
Animals↗
Transport of incoming influenza virus nucleocapsids into the nucleus.
Upon penetration of the influenza virus nucleocapsid into the host cell cytoplasm, the viral RNA and associated proteins are transported to the nucleus, where viral transcription and replication occur. By using quantitative confocal microscopy, we have found that over half of cell-associated nucleoprotein (NP) entered the nucleus with a half time of 10 min after penetration into CHO cells. Microinjection and immunoelectron microscopy experiments indicated that the NP entered the nucleus through the nuclear pore as part of an intact ribonucleoprotein (RNP) structure and that its transport was an active process. Transport of the incoming RNPs into the nucleus was not dependent on an intact microfilament, microtubule, or intermediate filament network. Subsequent to penetration, the matrix (M1) protein appeared to dissociate from the RNP structure and to enter the nucleus independently of the RNP. We found that 50% of penetrated M1 entered the nucleus with a half time of 25 min after penetration into CHO cells. Nuclear transport of M1 appeared to occur by passive diffusion. Entry of incoming M1 into the nucleus was not a prerequisite for infection.
Animals↗
Characterization and cloning of lgp110, a lysosomal membrane glycoprotein from mouse and rat cells.
lgp110 is a heavily glycosylated intrinsic protein of lysosomal membranes. Initially defined by monoclonal antibodies against mouse liver lysosomes, it consists of a 45-kilodalton core polypeptide with O-linked and 17 asparagine-linked oligosaccharide side chains in mouse cells. Sialic acid residues make the mature protein extremely acidic, with an isoelectric point of between 2 and 4 in both normal tissues and most cultured cell lines. Partial sequencing of mouse lgp110 allowed oligonucleotide probes to be constructed for the screening of several mouse cDNA libraries. A partial cDNA clone for mouse lgp110 was found and used for additional library screening, generating a cDNA clone covering all of the coding sequence of mature rat lgp110 as well as genomic clones covering most of the mouse gene. These new clones bring to seven the number of lysosomal membrane proteins whose amino acid sequences can be deduced, and two distinct but highly similar groups (designated lgp-A and lgp-B) can now be defined. Sequence comparisons suggest that differences within each group reflect species variations of the same protein and that lgp-A and lgp-B probably diverged from a common ancestor prior to the evolup4f1ary divergence of birds and mammals. Individual cells and individual lysosomes possess both lgp-A and lgp-B, suggesting that these two proteins have different functions. Mouse lgp110 is encoded by at least seven exons; intron positions suggest that the two homologous ectodomains of each lgp arose through gene duplication.
Amino Acid Sequence↗
Heavy chain binding protein recognizes incompletely disulfide-bonded forms of vesicular stomatitis virus G protein.
To investigate the function of heavy chain binding protein (BiP, GRP 78) in the endoplasmic reticulum, we have characterized its interaction with a model plasma membrane glycoprotein, the G protein of vesicular stomatitis virus. We used a panel of well characterized mutant G proteins and immunoprecipitation with anti-BiP antibodies to determine if BiP interacted with newly synthesized G protein and/or mutant G proteins retained in the endoplasmic reticulum. We made three major observations: 1) BiP bound transiently to folding intermediates of wild-type G protein which were incompletely disulfide-bonded; 2) BiP did not bind stably to all mutant G proteins which remain in the endoplasmic reticulum; and 3) BiP bound stably only to mutant G proteins which do not form correct intrachain disulfide bonds.
Animals↗
Intermediates in influenza induced membrane fusion.
Our results show that the mechanism by which influenza virus fuses with target membranes involves sequential complex changes in the hemagglutinin (HA, the viral fusion protein) and in the contact site between virus and target membrane. To render individual steps amenable to study, we worked at 0 degree C which decreased the rate of fusion and increased the efficiency. The mechanism of fusion at 0 degree C and 37 degrees C was similar. The process began with a conformational change in HA which exposed the fusion peptides but did not lead to dissociation of the tops of the ectodomain of the trimer. The change in the protein led to immediate hydrophobic attachment of the virus to the target liposomes. Attachment was followed by a lag period (4-8 min at 0 degree C, 0.6-2 s at 37 degrees C) during which rearrangements occurred in the site of membrane contact between the virus and liposome. After a further series of changes the final bilayer merger took place. This final fusion event was not pH dependent. At 0 degree C efficient fusion occurred without dissociation of the top domains of the HA trimer, suggesting that a transient conformation of HA is responsible for fusion at physiological temperatures. The observations lead to a revised model for HA mediated fusion.
Animals↗
Intracellular transport of soluble and membrane-bound glycoproteins: folding, assembly and secretion of anchor-free influenza hemagglutinin.
The influenza hemagglutinin precursor (HA0) and many other glycoproteins fold and oligomerize in the endoplasmic reticulum (ER). Only correctly folded oligomers are transported to the cell surface. To analyse the rules which determine this type of ER sorting, we have extended our analysis of hemagglutinin transport to two soluble, anchor-free recombinant HA0s derived from X31/A/Aichi/68 and A/Japan/305/57 influenza A. The results showed that individual monomers rapidly acquired a folded structure similar to that of monomeric membrane-anchored HA0. They were efficiently transported and secreted, but oligomerization was not required for secretion. Trimers or higher order complexes were either not formed (X31 HA0), or appeared during passage through the late compartments of the secretory pathway, with no effect on the rate of transport (Japan HA0). However, when initial folding was disturbed by inhibition of N-linked glycosylation, anchor-free X31 HA0 was misfolded and retained in the ER as disulfide-linked complexes associated with binding protein, BiP (GRP78). The complexes were similar to those seen for the nonglycosylated membrane-bound HA0, but instead of forming immediately after synthesis they appeared with a half-time of 6 min. Taken together, the data demonstrate that the structural criteria that makes the anchor-free HA0 transport competent are less stringent than those for the membrane form; they must fold correctly but do not need to oligomerize.
Animals↗
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.
Animals↗
Virus entry into animal cells.
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Animals↗
Endocytosis of simian virus 40 into the endoplasmic reticulum.
The endocytosis of SV-40 into CV-1 cells we studied using biochemical and ultrastructural techniques. The half-time of binding of [35S]methionine-radiolabeled SV-40 to CV-1 cells was 25 min. Most of the incoming virus particles remained undegraded for several hours. Electron microscopy showed that some virus entered the endosomal/lysosomal pathway via coated vesicles, while the majority were endocytosed via small uncoated vesicles. After infection at high multiplicity, one third of total cell-associated virus was observed to enter the ER, starting 1-2 h after virus application. The viruses were present in large, tubular, smooth membrane networks generated as extentions of the ER. The results describe a novel and unique membrane transport pathway that allows endocytosed viral particles to be targeted from the plasma membrane to the ER.
Animals↗
Spike--nucleocapsid interaction in Semliki Forest virus reconstructed using network antibodies.
Using in vitro immunization, we have reconstructed three consecutive steps of an idiotype network to show that the nucleocapsid of Semliki Forest virus contains a specific 'receptor' for the cytoplasmic tail of the E2 spike glycoprotein. This interaction could be the basis for the highly selective inclusion of viral glycoproteins--and exclusion of host cell surface proteins--during virus budding.
Antibodies, Monoclonal↗
Assembly of influenza hemagglutinin trimers and its role in intracellular transport.
The hemagglutinin (HA) of influenza virus is a homotrimeric integral membrane glycoprotein. It is cotranslationally inserted into the endoplasmic reticulum as a precursor called HA0 and transported to the cell surface via the Golgi complex. We have, in this study, investigated the kinetics and cellular location of the assembly reaction that results in HA0 trimerization. Three independent criteria were used for determining the formation of quaternary structure: the appearance of an epitope recognized by trimer-specific monoclonal antibodies; the acquisition of trypsin resistance, a characteristic of trimers; and the formation of stable complexes which cosedimented with the mature HA0 trimer (9S20,w) in sucrose gradients containing Triton X-100. The results showed that oligomer formation is a posttranslational event, occurring with a half time of approximately 7.5 min after completion of synthesis. Assembly occurs in the endoplasmic reticulum, followed almost immediately by transport to the Golgi complex. A stabilization event in trimer structure occurs when HA0 leaves the Golgi complex or reaches the plasma membrane. Approximately 10% of the newly synthesized HA0 formed aberrant trimers which were not transported from the endoplasmic reticulum to the Golgi complex or the plasma membrane. Taken together the results suggested that formation of correctly folded quaternary structure constitutes a key event regulating the transport of the protein out of the endoplasmic reticulum. Further changes in subunit interactions occur as the trimers move along the secretory pathway.
Animals↗