Biomedical subjects

H Garoff

Publications and source records attributed to H Garoff.

At least 55 records · Page 3Linked to original sources

PubMed1992

Membrane fusion of Semliki Forest virus involves homotrimers of the fusion protein.

Infection of cells with enveloped viruses is accomplished through membrane fusion. The binding and fusion processes are mediated by the spike proteins in the envelope of the virus particle and usually involve a series of conformational changes in these proteins. We have studied the low-pH-mediated fusion process of the alphavirus Semliki Forest virus (SFV). The spike protein of SFV is composed of three copies of the protein heterodimer E2E1. This structure is resistant to solubilization in mild detergents such as Nonidet P-40 (NP40). We have recently shown that the spike structure is reorganized during virus entry into acidic endosomes (J. M. Wahlberg and H. Garoff, J. Cell Biol. 116:339-348, 1992). The original NP40-resistant heterodimer is dissociated, and the E1 subunits form new NP40-resistant protein oligomers. Here, we show that the new oligomer is represented by an E1 trimer. From studies that use an in vitro assay for fusion of SFV with liposomes, we show that the E1 trimer is efficiently expressed during virus-mediated membrane fusion. Time course studies show that both E1 trimer formation and fusion are fast processes, occurring in seconds. It was also possible to inhibit virus binding and fusion with a monoclonal antibody directed toward the trimeric E1. These results give support for a model in which the E1 trimeric structure is involved in the SFV-mediated fusion reaction.

Animals↗

PubMed1992

Alphavirus assembly and entry: role of the cytoplasmic tail of the E1 spike subunit.

The alphavirus Semliki Forest virus (SFV) matures by budding at the cell surface. This process is driven by interactions of its membrane protein heterodimer E2-E1 and the nucleocapsid. The virus penetrates into new cells by an E1-mediated membrane fusion event. The E1 subunit has a short, strongly positively charged cytoplasmic tail peptide (Arg-Arg) which is very conserved among different alphavirus E1 proteins. In this work, we have used in vitro mutagenesis of a full-size cDNA clone of SFV to study the role of the tail peptide of the E1 subunit in virus budding and fusion processes in baby hamster kidney cell culture. Our results suggest that the E1 tail plays no major role in SFV multiplication in animal cell culture.

Alphavirus↗

PubMed1992

Spike protein-nucleocapsid interactions drive the budding of alphaviruses.

Semliki Forest virus (SFV) particles are released from infected cells by budding of nucleocapsids through plasma membrane regions that are modified by virus spike proteins. The budding process was studied with recombinant SFV genomes which lacked the nucleocapsid protein gene or, alternatively, the spike genes. No subviral particles were released from cells which expressed only the nucleocapsid protein or the spike proteins. Virus release was found to be strictly dependent on the coexpression of the nucleocapsid and the spike proteins. These results provide direct proof for the hypothesis that the alphavirus budding is driven by nucleocapsid-spike interactions. The importance of the viral 42S RNA for virus assembly and budding was investigated by using the heterologous vaccinia virus-T7 expression system for the synthesis of the SFV structural proteins. The results demonstrate that the viral genome is not absolutely required for formation of budding competent nucleocapsids, since small amounts of viruslike particles were assembled in the absence of 42S RNA.

Animals↗

PubMed1992

Alphavirus spike-nucleocapsid interaction and network antibodies.

Vaux et al. (D. J. T. Vaux, A. Helenius, and I. Mellman, Nature (London) 336:36-42, 1988) recently reported the production of network antibodies that were suggested to have reconstructed a specific interaction between the nucleocapsid of Semliki Forest virus and the cytoplasmic tail of the viral E2 spike protein. The F13 anti-idiotype antibody, which was raised against anti-E2 tail antibodies, was claimed to recognize the virus nucleocapsid. In this report, we have used recombinant SFV viruses to demonstrate that the F13 antibody is not nucleocapsid specific but instead most likely recognizes some component of the viral replication machinery.

Animals↗

PubMed1991

Fate of the 6K membrane protein of Semliki Forest virus during virus assembly.

The Semliki Forest virus directs the synthesis of three virus-specific transmembrane proteins p62, 6K, and E1, which all are made in equimolar amounts from a polyprotein precursor molecule. The p62 and E1 spike proteins form heterodimeric complexes in the endoplasmic reticulum before being transported to the cell surface where virus budding occurs. In this study we show that the 6K protein becomes associated to the p62E1 complex in the endoplasmic reticulum and transported with the complex to the cell surface. During virus budding, E1 and p62 (which has matured into the E2 protein) are incorporated into new virions whereas the 6K is mostly excluded. Virus particles released from infected BHK cells contain only about 3% of 6K in their membrane as compared to the spike protein content. The relevance of these findings for the mechanism of SFV assembly is discussed.

Animals↗

PubMed1991

A new generation of animal cell expression vectors based on the Semliki Forest virus replicon.

We have developed a novel DNA expression system, based on the Semliki Forest virus (SFV) replicon, which combines a wide choice of animal cell hosts, high efficiency and ease of use. DNA of interest is cloned into SFV plasmid vectors that serve as templates for in vitro synthesis of recombinant RNA. The RNA is transfected with virtually 100% efficiency into animal tissue culture cells by means of electroporation. Within the cell, the recombinant RNA drives its own replication and capping and leads to massive production of the heterologous protein while competing out the host protein synthesis. The expression system also includes an in vivo packaging procedure whereby recombinant RNA is packaged into infectious virus particles using cotransfection with packaging-deficient helper RNA molecules. The resulting high titer recombinant virus stock can be used to infect a wide range of animal cells with subsequent high expression of the heterologous gene product, but without expression of any structural proteins of the helper. The infected cells produce protein for up to 75 hours post infection after which the heterologous product can constitute as much as 25% of the total cell protein. The general utility of the system is demonstrated through the expression of human transferrin receptor, mouse dihydrofolate reductase, chick lysozyme and Escherichia coli beta-galactosidase.

Animals↗

PubMed1991

Internally located cleavable signal sequences direct the formation of Semliki Forest virus membrane proteins from a polyprotein precursor.

The proteolytic processes involved in the cotranslational production of the Semliki Forest virus proteins p62, 6K, and E1 from a common precursor polypeptide were analyzed by an in vitro translation-translocation assay. By studying the behavior of wild-type and mutant variants of the polyprotein, we show that the signal sequences responsible for membrane translocation of the 6K and E1 proteins reside in the C-terminal regions of p62 and 6K, respectively. We present evidence suggesting that the polyprotein is processed on the luminal side by signal peptidase at consensus cleavage sites immediately following the signal sequences. Our results also lead us to conclude that the 6K protein is a transmembrane polypeptide with its N terminus on the luminal side of the membrane (type I). Thus, the production of all three membrane proteins is directed by alternating signal and stop-transfer (anchor) sequences that function in translocation and cleavage of the virus precursor polyprotein. This also shows conclusively that internally located signal sequences can be cleaved by signal peptidase.

Amino Acid Sequence↗

PubMed1991

In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the small 6,000-molecular-weight membrane protein modulates virus release.

We report on the construction of a full-length cDNA clone of Semliki Forest virus (SFV). By placing the cDNA under the SP6 promoter, infectious RNA can be produced in vitro and used to transfect cells to initiate virus infection. To achieve efficient transfections, a new protocol for electroporation of RNA was developed. This method gave up to 500-fold improvement over the traditional DEAE-dextran transfection procedure. Since virtually 100% of the cells can be transfected by electroporation, this method is a useful tool for detailed biochemical studies of null mutations of SFV that abolish production of infections virus particles. We used the cDNA clone of SFV to study what effects a deletion of the 6,000-molecular-weight membrane protein (6K membrane protein) had on virus replication. The small 6K protein is part of the structural precursor molecule (C-p62-6K-E1) of the virus. Our results conclusively show that the 6K protein is not needed for the heterodimerization of the p62 and E1 spike membrane proteins in the endoplasmic reticulum, nor is it needed for their transport out to the cell surface. The absence of the 6K protein did, however, result in a dramatic reduction in virus release, suggesting that the protein exerts its function late in the assembly pathway, possibly during virus budding.

Animals↗

PubMed1990

Mannosidase II and the 135-kDa Golgi-specific antigen recognized monoclonal antibody 53FC3 are the same dimeric protein.

Monoclonal antibodies are frequently used as organelle-specific markers without identifying the specific antigen recognized. We have purified the protein recognized by a Golgi-specific monoclonal antibody 53FC3 (Burke, B., Griffiths, G., Reggio, H., Louvard, D., and Warren, G. (1982) EMBO J. 1, 1621-1628). Peptide microsequencing suggested that this antigen is mannosidase II, which was confirmed by cross-immunoprecipitation with an anti-mannosidase II antibody and by precipitation of mannosidase activity with the monoclonal antibody. Mannosidase II was found to exist normally as a disulfide-linked dimer.

Amino Acid Sequence↗

PubMed1990

The signal sequence of the p62 protein of Semliki Forest virus is involved in initiation but not in completing chain translocation.

So far it has been demonstrated that the signal sequence of proteins which are made at the ER functions both at the level of protein targeting to the ER and in initiation of chain translocation across the ER membrane. However, its possible role in completing the process of chain transfer (see Singer, S. J., P. A. Maher, and M. P. Yaffe. Proc. Natl. Acad. Sci. USA. 1987. 84:1015-1019) has remained elusive. In this work we show that the p62 protein of Semliki Forest virus contains an uncleaved signal sequence at its NH2-terminus and that this becomes glycosylated early during synthesis and translocation of the p62 polypeptide. As the glycosylation of the signal sequence most likely occurs after its release from the ER membrane our results suggest that this region has no role in completing the transfer process.

Amino Acid Sequence↗

PubMed1990

Oligomers of the cytoplasmic domain of the p62/E2 membrane protein of Semliki Forest virus bind to the nucleocapsid in vitro.

We analyzed the interaction between the nucleocapsid and synthetic peptides corresponding to the complete or truncated cytoplasmic protein domain of the Semliki Forest virus p62/E2 glycoprotein. We found that the peptide corresponding to the full-length domain efficiently bound nucleocapsids when coupled to a solid matrix via specific antibodies, whereas the shorter one did not. In solution, a substantial fraction of the full-length peptide associated into oligomers. Binding studies showed that it was mostly these oligomers, rather than the monomeric form of the peptide, which were able to interact with the nucleocapsid. Thus, our findings demonstrate a direct interaction between the spike proteins and the viral nucleocapsid. Furthermore, they suggest that this interaction is directed through formation of complexes containing several p62 or E2 subunits.

Amino Acid Sequence↗

PubMed1990

Spike protein oligomerization control of Semliki Forest virus fusion.

We have recently shown, using cleavage-deficient mutants of the p62-E1 membrane protein complex of Semliki Forest virus that p62 cleavage to E2 is necessary for the activation of the fusion function of the complex at pH 5.8 (a pH optimal for virus fusion) (M. Lobigs and H. Garoff, J. Virol. 64:1233-1240, 1990). In this study, we show that the mutant precursor complexes can be induced to activate membrane fusion when treated with more acidic buffers (pH 5.0 and 4.5), which also appear to dissociate most of the p62-E1 complexes and change the conformation of the E1 subunit (the supposed fusion protein of Semliki Forest virus into a form which is resistant to trypsin digestion. These data suggest that p62 cleavage is not essential for membrane fusion per se but that the crucial event activating this process seems to be the apparent dissociation of the heterodimer, which in turn is facilitated by the spike precursor cleavage.

Animals↗

PubMed1990

The E2 signal sequence of rubella virus remains part of the capsid protein and confers membrane association in vitro.

The capsid (C) protein of rubella virus is translated from a 24S subgenomic mRNA as the first part of a polyprotein containing all three structural proteins of the virus. It is separated from the following protein (E2) by signal peptidase, which cleaves after the E2 signal sequence. We raised an antipeptide antiserum directed against the signal sequence and used the antiserum to show that this sequence is still a part of the C protein in the mature virion. Furthermore, we also showed that, when the C protein is synthesized by in vitro transcription and translation, the resultant protein is membrane associated. This association is not seen with a variant C protein which lacks the signal sequence, and a normally soluble protein (dihydrofolate reductase) becomes membrane associated when the signal sequence is placed at its carboxy terminus.

Amino Acid Sequence↗

PubMed1990

Fusion function of the Semliki Forest virus spike is activated by proteolytic cleavage of the envelope glycoprotein precursor p62.

The precursor protein p62 of the prototype alphavirus Semliki Forest virus (SFV) undergoes during transport to the cell surface a proteolytic cleavage to form the mature envelope glycoprotein E2. To investigate the biological significance of this cleavage event, single amino acid substitutions were introduced at the cleavages site through mutagenesis of cDNA corresponding to the structural region of the SFV genome. The phenotypes of the cleavage site mutants were studied in BHK cells by using recombinant vaccinia virus vectors. Nonconservative substitutions completely abolished p62 cleavage. Uncleaved p62 was transported with normal kinetics to the cell surface, where it became accessible to low concentrations of exogenous trypsin. The proteolytic cleavage of envelope glycoprotein precursors has been shown to activate the membrane fusion potential of viral spikes in several virus families. Here we demonstrate that the fusion function of the SFV spike is activated by the cleavage of p62. Cleavage-deficient p62 expressed at the cell surface did not function in low-pH-triggered (pH 5.5) cell-cell membrane fusion; however, cleavage of the mutated p62 with exogenous trypsin restored the fusion function. We discuss a model for SFV assembly and fusion where p62 cleavage plays a crucial role in the stability of the multimeric association of the viral envelope glycoproteins.

Animals↗

PubMed1990

Function of Semliki Forest virus E3 peptide in virus assembly: replacement of E3 with an artificial signal peptide abolishes spike heterodimerization and surface expression of E1.

The Semliki Forest virus spike glycoproteins E1 and p62 form a heterodimeric complex in the endoplasmic reticulum (ER) and are transported as such to the cell surface. In the mature virus particle, the heterodimeric association of E1 and E2 (the cleavage product of p62) is maintained, but as a more labile and acid-sensitive oligomer than the E1-p62 complex. The E3 peptide forms the N-terminal part of the p62 precursor and carries the signal for the translocation of p62 into the lumen of the ER. The question of whether E3 is also important in the formation and stabilization of the E1-p62 heterodimer has been addressed here with the aid of an E3 deletion mutant cDNA. In this construct, the entire E3 was replaced with a cleavable, artificial signal sequence which preserved the membrane topology of an authentic E2. The E3 deletion, when expressed via a recombinant vaccinia virus, abolished heterodimerization of the spike proteins. It also resulted in the complete retention of E1 in the ER and almost total inhibition of E2 transport to the plasma membrane. The oligomerization and transport defect of E1 expressed from the E3 deletion mutant could be complemented with a wild-type p62 provided from a separate coding unit in double infections. These results point to a central role of E3 in complex formation and transport of the viral structural components to the site of budding. In conjunction with earlier work (M. Lobigs and H. Garoff, J. Virol. 64:1233-1240, 1990; J. Wahlberg, W. A. M. Boere, and H. Garoff, J. Virol. 63:4991-4997, 1989), the data support a model of spike protein oligomerization control of Semliki Forest virus assembly and disassembly which may be mediated by the presence of E3 in the uncleaved p62 precursor and release of E3 after cleavage.

Animals↗

PubMed1989

The heterodimeric association between the membrane proteins of Semliki Forest virus changes its sensitivity to low pH during virus maturation.

The budding and the fusion processes of the enveloped animal virus Semliki Forest virus serve the purpose of transporting its nucleocapsid, containing its genome, from the cytoplasm of an infected cell into that of an uninfected one. We show here that, in the infected cell, the viral membrane (spike) proteins p62 and E1 are organized as heterodimers which are very resistant to dissociation in acidic conditions. In contrast, the mature form of the heterodimer, E2E1, which is found in the virus particle and which is generated by proteolytic processing of p62, is very prone to dissociate upon treatment with mildly acidic buffers. We discuss the possibility that this difference in behavior of the intracellular precursor form and the mature form of the spike protein complex represents an important regulatory mechanism for the processes involving membrane binding around the nucleocapsid during budding and membrane release from the nucleocapsid at the stage of virus fusion.

Animals↗

PubMed1989

Role of heterologous and homologous glycoproteins in phenotypic mixing between Sendai virus and vesicular stomatitis virus.

Phenotypic mixing between Sendai virus and vesicular stomatitis virus (VSV) or the mutant VSV ts045 was studied. Conditions were optimized for double infection, as shown by immunofluorescence microscopy. Virions from double-infected cells were separated by sequential velocity and isopycnic gradient centrifugations. Two types of particles with mixed protein compositions were found. One type was VSV particles with Sendai virus spikes, i.e., phenotypically mixed particles. A second type was Sendai virus-VSV associations, which in plaque assays also behaved as phenotypically mixed particles. The ratio of VSV G protein to Sendai virus glycoproteins on the cell surface was varied, using the VSV mutant ts045 in double infections. Thus, different amounts of the VSV G protein were allowed to reach the cell surface at 32, 38, and 39 degrees C in Sendai virus-infected cells. However, a fixed number of Sendai virus spikes was always found in the ts045 virions. This represented 12 to 16% of the number of G proteins present in normal VSV. Furthermore, the yield of ts045 virions was radically reduced during double infection when the temperature was raised to block G-protein transport to the cell surface, suggesting that the Sendai virus glycoproteins were not able to compensate for G protein in budding. These results emphasize the role of the G protein in VSV assembly.

Animals↗

PubMed1988

Expression, tyrosine sulfation, and secretion of yolk protein 2 of Drosophila melanogaster in mouse fibroblasts.

Tyrosine sulfation is a post-translational modification in the trans Golgi that has been found in all animal species studied. In the preceding paper (Baeuerle, P. A., Lottspeich, F., and Huttner, W. B. (1988) J. Biol. Chem. 263, 14925-14929), we have identified the site of tyrosine sulfation in an insect secretory protein, yolk protein 2 (YP2) of Drosophila melanogaster. In the present report, tyrosine sulfation of this protein was examined after expression in a heterologous mammalian cell system. Mouse fibroblasts, transfected with Drosophila YP2 genomic DNA inserted into the eucaryotic expression vector pSV2, secreted the fly protein in sulfated form. Analyses of Drosophila YP2 produced by the mouse cells showed that the features of sulfation of this protein were identical to those previously determined for YP2 isolated from flies. YP2 secreted from mouse fibroblasts was found to be exclusively sulfated on tyrosine residues. The stoichiometry of tyrosine sulfation was approximately 1 mol of sulfate/mol of YP2. Sulfate was linked to the same tyrosine residue as in YP2 isolated from flies, tyrosine 172. These results show that essential parameters of the tyrosine sulfation reaction are very similar in insects and mammals and thus highly conserved in evolution.

Animals↗