Expression of exogenous proteins in mammalian cells with the Semliki Forest virus vector.
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Animals↗
Biomedical subjects
V M Olkkonen
Publications and source records attributed to V M Olkkonen.
Cloning and subcellular localization of novel rab proteins reveals polarized and cell type-specific expression.
Small GTPases of the rab subfamily are specific regulators of vesicular transport. The intracellular localization of these proteins has been mostly investigated in cultured cells where they have been found associated with distinct compartments of the exocytic and endocytic pathways. Using a PCR-based cloning approach we have recently identified several novel rab proteins, extending the total number of this family to more than 30 members. Here, we have investigated the mRNA expression in different tissues and the intracellular localization in organ cryosections of two rab proteins, rab18 and rab20. Both northern blot analysis and confocal immunofluorescence microscopy demonstrated that these proteins are expressed in a tissue- and cell type-dependent manner. Despite their presence in non-polarized cells and polarized cells, both proteins are highly expressed on the apical side of kidney tubule epithelial cells. Electron microscopic studies revealed that rab18 and rab20 are located in apical dense tubules, endocytic structures underlying the apical plasma membrane, suggesting that they play a role in apical endocytosis/recycling. In intestinal epithelial cells as well, both proteins were localized apically, but, in addition, rab18 was found associated with the basolateral domain, suggesting that this protein is not restricted to the apical transport machinery of polarized epithelial cells. The results demonstrate that, depending on the epithelial cell type, rab proteins that are also expressed in non-polarized cells may be enriched in one or both surface domains. Together with the observed tissue- and cell type-dependent variation in the expression of the rab proteins, this suggests that the large number of mammalian rab proteins might reflect the specific requirements in the organization of membrane traffic encountered by different cell types.
Amino Acid Sequence↗
Membrane insertion and intracellular transport of yeast syntaxin Sso2p in mammalian cells.
Proteins of the syntaxin family are suggested to play a key role in determining the specificity of intracellular membrane fusion events. They belong to the class of membrane proteins which are devoid of N-terminal signal sequence and have a C-terminal membrane anchor. Sso2p is a syntaxin homologue involved in the Golgi to plasma membrane vesicular transport in yeast. The protein was transiently expressed in BHK-21 cells using the Semliki Forest virus vector, and its localization and mode of membrane insertion were studied. By immunofluorescence and immuno-EM we show that Sso2p is transported to its final location, the plasma membrane, along the biosynthetic pathway. Experiments with synchronized Sso2p synthesis or expression of the protein in the presence of brefeldin A indicate endoplasmic reticulum as the initial membrane insertion site. During a 20 degrees C temperature block Sso2p accumulated in the Golgi complex and was chased to the plasma membrane by a subsequent 37 degrees C incubation in the presence of cycloheximide. The in vitro translated protein was able to associate with dog pancreatic microsomes post-translationally. A truncated form of Sso2p lacking the putative membrane anchor was used to show that this sequence is necessary for the membrane insertion in vivo and in vitro. The results show that this syntaxin-like protein does not directly associate with its target membrane but uses the secretory pathway to reach its cellular location, raising interesting questions concerning regulation of SNARE-type protein function.
Animals↗
Semliki Forest virus as a tool for protein expression in cultured rat hippocampal neurons.
We use the Semliki Forest Virus (SFV) as a tool for protein expression in primary cultures of rat hippocampal neurons. These cells develop in vitro into polarized neurons that can be infected with recombinant SFV with an efficiency of 60 - 80%. SFV-driven protein expression is detectable within 3-4 hours postinfection, at which time the newly-synthesized proteins are mainly present in the cell soma. By 6 to 8 hours postinfection foreign proteins are detectable in the neurites. Protein expression can continue for up to 48 hours. However, after 8 - 10 hours infected neurons start to suffer from cytopathic effects as evidenced by a change in morphology and detachment from the coverslip. The infection does not seem to affect the polarized distribution of proteins. Upon overexpression of rab8, a somatodendritic distribution is observed, similar to that of the endogenous protein. Therefore, the SFV expression system is suitable for short-term expression of proteins and can be used successfully to study the polarized distribution of heterologous proteins expressed in cultured hippocampal neurons.
Animals↗
Expression of heterologous proteins in cultured rat hippocampal neurons using the Semliki Forest virus vector.
The Semliki Forest virus expression vector (Liljeström and Garoff: Bio/Technology 9:1356-1361, 1991) was tested in cultured rat hippocampal neurons using two Madin-Darby canine kidney (MDCK) cell membrane-associated proteins as reporters: rab8, a small GTPase involved in post-Golgi vesicle transport, and VIP21, an integral membrane protein of caveolae, trans-Golgi network, and post-Golgi vesicles. Expression of the c-myc epitope-tagged proteins was visualized by immunofluorescence microscopy. The proteins were first detected in neurons after 3-4 hr infection by the recombinant viruses. The infection efficiency on neurons was high: after 6 hr infection at a multiplicity of one, 50-60% of the cells expressed the reporter proteins. The neurons tolerated the infection well up to 8 hr. Their polarized organization was not disturbed, as judged from morphology and from distribution of the dendritic MAP2 and axonal synaptophysin marker proteins. The Semliki Forest virus vector thus seems suitable for short-term expression of proteins in cultured neurons.
Amino Acid Sequence↗
Overproduction, purification, and characterization of DNA-binding protein P19 of bacteriophage PRD1.
The early protein, P19, of bacteriophage PRD1 was purified after overexpression of the cloned gene, XIX, in Escherichia coli DH5 alpha cells. The purified protein binds as multimers to single-stranded DNA (ssDNA), and with a lower affinity to double-stranded DNA (dsDNA), without sequence-specificity. Two distinct P19-ssDNA complexes were discovered in gel- mobility-shift assays at different protein:DNA ratios. P19 was capable of fully protecting ssDNA against nuclease P1. Electron microscopy of protein P19-ssDNA complexes showed DNA molecules which were extensively coated with protein and whose contour length was clearly reduced by P19 binding. The results suggest that P19 binds to ssDNA with moderate cooperativity and are consistent with the DNA being wrapped around the P19 multimers.
Bacteriophages↗
Topology of the major capsid protein P3 of bacteriophage PRD1: analysis using monoclonal antibodies and C-terminally truncated proteins.
Trimeric capsomeres of protein P3 (395 aa) are the main component of the phage PRD1 capsid, which encloses a lipid-protein vesicle containing the viral dsDNA genome. In this study we characterize a panel of monoclonal antibodies (MAb) against P3. The epitopes recognized by the MAbs are analyzed by immunoprecipitation of intact virions or of released P3 trimers, and by Western blotting using a series of C-terminally truncated P3 molecules. Nine of the MAbs recognize epitopes on the virion surface, whereas five require unmasking of epitopes by disruption of the virions. Several of the MAbs are capable of neutralizing the virus; this is most probably due to virus aggregation by the antibodies. Analysis of the C-terminal truncations (the 6 Western blot-positive MAbs were used) delineates three major antigenic regions of the protein. The epitope of MAb 3T74 is included in the 66 N-terminal amino acids, and is not accessible on the virion surface, suggesting that the N-terminus is internally located in the capsid. MAbs 3N81 and 3R2 recognize epitopes in the region of amino acids 159-168, which is part of the first predicted beta-barrel structure of P3. The third antigenic region is in the second predicted beta-barrel, between amino acids 217-242, where the epitopes of 3N180, 3P4, and 3T5 map. The trimerization of P3 was found to be independent of the non-structural assembly factor proteins P10 and P17. Functional studies of the truncated proteins reveal that molecules comprising of 294 or more residues from the P3 N-terminus are capable of trimer formation.
Antibodies, Monoclonal↗
Molecular cloning and subcellular localization of three GTP-binding proteins of the rab subfamily.
Small GTPases of the rab subfamily are involved in regulation of intracellular membrane transport events. We recently used a PCR approach to isolate short cDNA fragments of a number of novel rab sequences. These PCR fragments have not been used with cDNA library screening and PCR-based techniques to clone the cDNAs encoding three of these proteins, rab12, rab22, and rab24. By northern blot analysis, the messages were found to be present in a wide variety of mouse tissues. However, quantitative differences in the mRNA levels between the tissues were detected. We determined the subcellular localization of the GTPases by expressing the c-myc epitope-tagged proteins with the Semliki Forest virus and the vaccinia T7 vector systems. Transiently expressed rab12 was localized to the Golgi complex. This localization was confirmed using a polyclonal anti-peptide antibody detecting the endogenous protein in BHK cells. rab22 expressed from the cDNA was localized to endosomal compartments and to the plasma membrane. After longer periods of expression, the protein was found on abnormally large perinuclear endosomal structures, suggesting that it is a potent regulator of events in the endocytic pathway. Finally, rab24 was found in the endoplasmic reticulum/cis-Golgi region and on late endosomal structures. The localization of rab24 may indicate its involvement in autophagy-related processes.
Amino Acid Sequence↗
Sequence of a canine cDNA clone encoding a Ran/TC4 GTP-binding protein.
We report the isolation and characterization of a canine cDNA encoding a 216-amino acid GTP-binding protein of the Ras superfamily. The protein is almost identical to the human TC4 [Drivas et al., Mol. Cell. Biol. 10 (1990) 1793-1798] and Ran [Bischoff and Ponstingl, Proc. Natl. Acad. Sci. USA 88 (1991) 10830-10834; Nature 354 (1991) 80-82] proteins, the latter of which has been found to be involved in cell cycle control. Furthermore, the protein is highly similar to the fission yeast spi1 gene product [Matsumoto and Beach, Cell 66 (1991) 347-360]. The high degree of evolutionary conservation in this protein suggests that it plays a vital role in the eukaryotic cell.
Amino Acid Sequence↗
Construction of a transducing virus from double-stranded RNA bacteriophage phi6: establishment of carrier states in host cells.
Bacteriophage phi 6 contains three double-stranded RNA (dsRNA) genomic segments. We have constructed a plasmid that contains a cDNA copy of the middle (M) segment, with a gene for kanamycin resistance (kan) inserted into the PstI site. A transcript of this cDNA was incorporated in vitro into procapsids along with natural transcripts of the S and L segments. The procapsids were coated with nucleocapsid surface protein P8 and transfected into Pseudomonas syringae pv. phaseolicola. The resulting infectious virus, phi 6 K1, was found to contain an M segment that was 1.2 kbp larger than the normal 4.1 kbp. K1 formed small, turbid plaques, and its genome was unstable. Preparations of K1 contained from about 0.1 to 10% large, clear-plaque forms of the virus which were usually missing the kan gene, and in some cases, the resulting segment M was smaller than its normal size. Cells picked from lawns of host cells infected with K1 yielded colonies that were resistant to kanamycin (Kan). These colonies could be passaged on kanamycin-containing medium. The cells were found to contain large amounts of dsRNA corresponding to the viral genomic segments. Some strains continued to produce viable phage, while others lost this ability. One strain completely lost the small genomic segment S. Approximately 1 in 10,000 infected cells acquired the carrier state with the original phage isolate K1. However, we isolated a viral mutant that was able to induce the carrier state in 10 to 20% of the infected cells. The ability to use drug resistance as a test for the carrier state makes this system very useful for the study of the mechanisms of induction of persistent infections.
Bacteriophages↗
Generation of infectious nucleocapsids by in vitro assembly of the shell protein on to the polymerase complex of the dsRNA bacteriophage phi 6.
A method for the in vitro uncoating of the phi 6 nucleocapsid (NC) was developed. The resulting particle, designated as the NC core, containing the genomic double-stranded (ds) RNA segments and the proteins P1, P2, P4 and P7, was not infectious but had a highly enhanced in vitro transcriptase activity compared to that of the intact NC. The NC shell protein P8 was purified by immunoaffinity chromatography, and it was shown to self-assemble to shell-like structures upon addition of calcium ions. The conditions for the self-assembly of the shell were optimized. Shell reassembly on to the NC cores restored the infectivity but resulted in a decrease of transcriptase activity. No reassembly of the shell on to RNA-less cores (procapsids) produced from a cDNA construction in Escherichia coli was observed. Our results suggest that the intracellular uncoating of the NC is the event activating the phi 6 dsRNA transcriptase and that the NC shell is necessary for infectivity, probably for the passage of the NC through the host cytoplasmic membrane. Packaging of the dsRNA segments into the procapsid appears to be a prerequisite for NC shell assembly.
Bacteriophages↗
In vitro assembly of infectious nucleocapsids of bacteriophage phi 6: formation of a recombinant double-stranded RNA virus.
A system is described for assembling infectious bacteriophage phi 6 nucleocapsids in vitro. Procapsids encoded by cDNA copies of genomic segment L in Escherichia coli were used to package and replicate viral RNA segments. The resulting filled particles were shown to be capable of infecting host cell spheroplasts after incubation with purified nucleocapsid shell protein P8. The infected spheroplasts yielded infectious virions. A modified cDNA-derived RNA segment was inserted into virions by this method. The resulting infectious virions contained the same 4-base-pair deletion as the modified cDNA. These findings support the contention that the preformed procapsids are the "machine" that replicates the phi 6 genome, by showing that the cDNA-derived procapsids are competent to package and replicate RNA properly.
Bacteriophages↗
Quantitation of the adsorption and penetration stages of bacteriophage phi 6 infection.
The enveloped dsRNA bacteriophage phi 6 uses the pilus of Pseudomonas syringae as its receptor. It enters the host cell by fusion of the virus envelope with the host outer membrane, followed by penetration of the cytoplasmic membrane by the phage nucleocapsid. In this investigation we quantitated the adsorption and penetration of phi 6wt and a host range mutant, phi 6h 1s, to five bacterial strains. Adsorption rate constants were measured for the different phage-host combinations, the constant for phi 6wt with the standard host was 3.3 X 10(10) ml/min. Infections with 14C-labeled phage at different phage/cell ratios were used to measure the numbers of adsorbing and entering virions/sensitive cell. At high phage/cell ratios (200-250) the standard host adsorbed on the average 35-40 wild-type virions/cell, the saturation level being somewhat higher. It was shown that at phage/host cell ratios of 0.1-1 practically every virion produces an infectious center. The average number of entering phage particles per infectious center reached saturation around the phage/cell ratio of 50 and did not exceed 3 for the standard host. The phi 6 preparations used in this study had a specific infectivity of 0.7-0.9.
Adsorption↗
The nucleocapsid of bacteriophage phi 6 penetrates the host cytoplasmic membrane.
Bacteriophage phi 6 infects its host, the Gram-negative bacterium Pseudomonas syringae, by a protein-targeted fusion of the virus envelope with the host outer membrane. In this investigation we present results suggesting that the phage nucleocapsid penetrates the host cytoplasmic membrane via a membrane invagination and an intracellular vesicle. This indicates that the prokaryotic plasma membrane might be more dynamic and have more common features with eukaryotic membrane systems than previously expected. Most of the nucleocapsid surface lattice protein is degraded in the cell, and the nucleocapsid core particle containing the viral dsRNA segments and the proteins necessary for the viral RNA polymerase activity can be isolated from the infected cells. The penetration is dependent on the energized state of the host cytoplasmic membrane. About 25% of the entering core particles are re-used in the progeny viruses.
Bacteriophages↗
Monoclonal antibodies to the major structural proteins of bacteriophage phi 6.
A panel of 38 monoclonal antibodies to the five major structural proteins of phi 6 was generated and characterized. The panel includes antibodies recognizing the receptor recognition protein P3, the major hydrophobic envelope protein P9, the nucleocapsid surface protein P8, and the nucleocapsid proteins P1 and P4, which are involved in the viral RNA polymerase activity and form the internal protein skeleton of the nucleocapsid. Six out of the fourteen antibodies to the receptor recognition protein, P3, showed neutralizing activity, interfering with the adsorption of phi 6 to host cells.
Antibodies, Monoclonal↗
The nucleocapsid of the lipid-containing double-stranded RNA bacteriophage phi 6 contains a protein skeleton consisting of a single polypeptide species.
The nucleocapsid of the enveloped double-stranded RNA bacteriophage phi 6 was isolated by extraction with the nonionic detergent Triton X-114 and subjected to disruption analysis with chelating and protein-denaturing agents. The subnucleocapsid particles were separated in rate-zonal sucrose gradients, and their ultrastructure and protein composition were analyzed. The role of divalent cations in the nucleocapsid structure was studied by using a precipitation assay of the isolated nucleocapsid proteins. The phi 6 nucleocapsid had a cagelike skeleton consisting of a single polypeptide species (P1). Two other proteins (P2 and P4) were associated with the P1 cage. These three early proteins were previously known to be involved in the RNA synthesis machinery of the virus. The stability of the nucleocapsid surface lattice consisting of protein P8 was dependent on Ca2+ ions.
Bacteriophages↗