Recombinant Semliki Forest Virus Particles Research Articles

Semliki Forest virus biodistribution in tumor-free and 4T1 mammary tumor-bearing mice: a comparison of transgene delivery by recombinant virus particles and naked RNA replicon

Semliki Forest virus (SFV) vectors are promising tools for cancer gene therapy because they ensure a high level of transgene expression and a rapid and strong cytopathic effect. However, broad tissue tropism and transient expression make it more difficult to develop an optimal cancer treatment strategy. In this study, we have compared the distribution of recombinant SFV particles (recSFV) and naked viral RNA replicon (recRNA) in tumor-free and 4T1 mammary tumor-bearing mice as a consequence of different vector administration strategies. The high potential of SFV recRNA as a biosafe approach for the development of therapeutic treatment was demonstrated. Intravenous (i.v.) inoculation of recRNA provided primary brain targeting in both tumor-free and 4T1 tumor mouse models, but local intratumoral inoculation revealed a high expression level in tumors. Moreover, we observed the predominant tumor targeting of recSFV at a reduced viral dose on i.v. and intraperitoneal (i.p.) virus inoculation, whereas the dose increase led to a broad virus distribution in mice. To prolong transgene expression, we have tested several i.v. and i.p. reinoculation strategies. A detailed evaluation of vector distribution and readministration properties could have an impact on cancer gene therapy clinical trial safety and efficacy.

Characterization of specific immune responses in cattle immunized with plasmid DNA construct and recombinant Semliki Forest virus particles encoding Cu,Zn superoxide dismutase (SOD) of Brucella abortus

Characterization of specific immune responses in cattle immunized with plasmid DNA construct and recombinant Semliki Forest virus particles encoding Cu,Zn superoxide dismutase (SOD) of Brucella abortus

Vaccination with recombinant Semliki Forest virus particles expressing translation initiation factor 3 of Brucella abortus induces protective immunity in BALB/c mice

Recombinant replicons of Semliki Forest virus (SFV) can be used to induce high-level, transient expression of heterologous proteins in vivo. We constructed infectious but replication-deficient SFV particles carrying recombinant RNA encoding the Brucella abortus translation initiation factor 3 (IF3). The recombinant SFV particles (SFV-IF3 particles) were then evaluated for their ability to induce immune responses and to protect BALB/c mice against a challenge with B. abortus 2308 following vaccination. Animals inoculated with SFV-IF3 developed IF3-specific IgM antibodies at day 14 post-immunization. In vitro stimulation of splenocytes from vaccinated mice with either recombinant IF3 (rIF3) or crude Brucella protein extracts resulted in a T-cell proliferative response and induction of interferon gamma secretion, but not interleukin-4. In addition, mice immunized with SFV-IF3 exhibited a significant level of resistance against challenge with the virulent B. abortus strain 2308 ( P<0.01). These findings indicate that an SFV-based vector carrying RNA encoding Brucella IF3 has potential for use as a vaccine to induce protection against B. abortus infections.

Evaluation of Brucella abortus DNA and RNA vaccines expressing Cu–Zn superoxide dismutase (SOD) gene in cattle

This study was conducted to evaluate the immunogenicity of a DNA or RNA vaccines encoding Brucella abortus Cu–Zn superoxide dismutase (SOD) in cattle. Intramuscular injection of plasmid DNA carrying Brucella SOD gene (pcDNA-SOD) into animals elicited both humoral and cellular immune responses. Animals injected with pcDNA-SOD developed SOD IgG antibody with predominance of immunoglobulin G1 (IgG1) isotype over IgG2. In addition, the DNA vaccine elicited a specific T-cell-proliferative response. Furthermore, intraperitoneal injection of cattle with recombinant Semliki Forest virus particles carrying recombinant RNA encoding SOD (SFV-SOD) did not lead to the induction of SOD IgG 1 or 2 antibody, but induced specific T-cell activation. Both vaccines were able to induce a non-significant secretion of gamma interferon and did not induce the secretion of IL-4 or tumor necrosis factor (TNF)-α. These results suggest that SOD gene in a genetic vaccine formulation (DNA or RNA) might be of potential us as a vaccine to induce cell-mediated immunity in cattle. To our knowledge, this is the first study to evaluate a genetic vaccine against Brucella in cattle.

Efficient expansion of HIV-1-specific T cell responses by homologous immunization with recombinant Semliki Forest virus particles

Vaccines based on recombinant viruses represent a promising strategy for the development of a prophylactic vaccine against HIV-1. However, despite a proven capacity to stimulate potent HIV-1-specific immune responses, viral systems have limited utility in homologous prime-boost regimens due to the generation of anti-vector immune responses. It is therefore important to develop a diverse set of vaccine candidates that can be combined in different heterologous prime-boost regimens and/or to identify a vaccine candidate that is less sensitive to anti-vector mediated immunity. In this report, we describe the design and pre-clinical immunogenicity of a Semliki Forest virus-based vaccine, VREP-C, encoding Indian origin HIV-1 clade C antigens. We show that a single immunization with VREP-C stimulates HIV-1-specific IFNγ ELISPOT responses, which were efficiently boosted by a second and a third homologous VREP-C immunization resulting in highly potent cytotoxic T cell responses. These results suggest that VREP-C may be a valuable component of a future prophylactic vaccine against HIV-1.

An RNA Vaccine Based on Recombinant Semliki Forest Virus Particles Expressing the Cu,Zn Superoxide Dismutase Protein of Brucella abortus Induces Protective Immunity in BALB/c Mice

We constructed infectious but replication-deficient Semliki Forest virus (SFV) particles carrying recombinant RNA encoding Brucella abortus Cu,Zn superoxide dismutase (SOD). The recombinant SFV particles (SFV-SOD particles) were then evaluated for their ability to induce a T-cell immune response and to protect BALB/c mice against a challenge with B. abortus 2308. Intraperitoneal injection of mice with recombinant SFV-SOD particles did not lead to the induction of SOD-specific antibodies, at least until week 6 after immunization (the end of the experiment). In vitro stimulation of splenocytes from the vaccinated mice with either recombinant Cu,Zn SOD (rSOD) or crude Brucella protein resulted in a T-cell proliferative response and the induction of gamma interferon secretion but not interleukin-4. In addition, the splenocytes exhibited significant levels of cytotoxic T-lymphocyte activity against Brucella-infected cells. The SFV-SOD particles, but not the control virus particles, induced a significant level of protection in BALB/c mice against challenge with B. abortus virulent strain 2308. These findings indicated that an SFV-based vector carrying the SOD gene has potential for use as a vaccine to induce resistance against B. abortus infections.

Vesicular stomatitis virus glycoprotein: a transducing coat for SFV-based RNA vectors.

Semliki Forest virus (SFV) vectors have a great potential for the induction of protective immunity in a large number of clinical conditions including cancer. Such a potential accounts for the huge efforts made to improve the in vivo expression from SFV vectors. It is noteworthy that efficient in vivo expression strongly relies on the ability to deliver high-titre vectors. To achieve this, the generation of recombinant SFV particles, using independent expression systems for structural SFV genes, has been proposed. However, despite several modifications in the production process, a risk of contamination with replication-competent, or partially recombined, virus has remained. Here, we exploit the ability of the vesicular stomatitis virus glycoprotein (VSV-G), expressed in trans, to hijack full-length genomic SFV RNA into secreted virus-like particles (VLPs). To allow SFV vector mobilisation, we designed a CMV driven SFV vector in which the internal 26S promoter has been extensively mutated. With this vector, mobilisation events were monitored using the Green Fluorescent Protein (GFP). The production procedure involves a sequential transfection protocol, of plasmids expressing the VSV-G and the SFV vector respectively. We show that the VLPs are effective for cellular delivery of SFV vectors in a broad range of human and non-human cellular targets. Furthermore, production of VLPs is easy and allows, through concentration, the harvest of high-titre vector. The present paper describes a convenient process aimed at mobilising full length SFV vectors. A major issue to consider, while developing clinically relevant gene transfer vectors, is the risk of undesirable generation of replication competent by-products. Importantly, as the VSV-G gene shares no homology with the SFV genome, our VLPs offer a strong guarantee of biosafety.

Effect of intranasal administration of semliki forest virus recombinant particles expressing reporter and cytokine genes on the progression of experimental autoimmune encephalomyelitis

We have initiated studies to determine the feasibility of employing the Semliki Forest virus (SFV) expression system as a central nervous system (CNS) vector. We investigated the effects of infecting Balb/c mice intranasally (i.n.) with recombinant SFV particles expressing the enhanced green fluorescent protein (EGFP) reporter gene. EGFP expression was detected by fluorescence microscopy in the olfactory bulb as early as 1 day postinfection. No pathological changes were associated with infection. Viral RNA could be detected in the olfactory mucosa only, whereas fluorescence was detected in axons in the olfactory bulb, indicating that only the expressed protein was present. A vector expressing interleukin 10 (IL-10) was constructed and shown to induce good cytokine expression in cultured cells. IL-10 expression in the nasal passage and olfactory bulb of infected mice was enhanced following i.n. administration of such particles. Mice induced for experimental autoimmune encephalomyelitis (EAE) were treated i.n. with vectors expressing EGFP and IL-10 and with empty vector. The EGFP-expressing and empty vectors were found to exacerbate EAE, whereas that expressing IL-10 ameliorated EAE. It is concluded that the mice showed a significant biological response when treated i.n. with recombinant SFV particles and that such particles administered by the i.n. route have potential as a noninvasive vector for protein delivery to the CNS.

Eradication of established HPV16-transformed tumours after immunisation with recombinant Semliki Forest virus expressing a fusion protein of E6 and E7

Previously, we described the efficacy of immunisation with recombinant Semliki Forest virus (SFV), expressing the human papillomavirus 16 (HPV) oncoproteins E6 and E7, in inducing HPV-specific CTLs and anti-tumour responses. Recently, we developed a novel recombinant SFV construct encoding a relatively stable fusion protein of HPV16 E6 and E7 under control of a translational enhancer derived from the SFV capsid protein. In the present study we demonstrate that immunisation of tumour-bearing mice with this improved vector results in the regression and complete elimination of established tumours. We furthermore demonstrate that a long-term high level of CTL activity, up to 340 days, accompanies the anti-tumour response. Thus, immunisation with recombinant SFV particles encoding increased levels of a fusion protein of HPV16 E6 and E7 efficiently induces CTL activity and CTL memory resulting in a potent therapeutic anti-tumour effect.

Hepatitis C virus non-structural protein 3-specific cellular immune responses following single or combined immunization with DNA or recombinant Semliki Forest virus particles.

The capacity of recombinant Semliki Forest virus particles (rSFV) expressing the hepatitis C virus non-structural protein 3 (NS3) to induce, in comparison or in combination with an NS3-expressing plasmid, specific cellular and humoral immune responses in murine models was evaluated. In vitro studies indicated that both types of vaccine expressed the expected size protein, albeit with different efficacies. The use of mice transgenic for the human HLA-A2.1 molecule indicated that the rSFV-expressed NS3 protein induces, as shown previously for an NS3 DNA vaccine, NS3-specific cytotoxic lymphocytes (CTLs) targeted at one dominant HLA-A2 epitope described in infected patients. All DNA/rSFV vaccine combinations evaluated induced specific CTLs, which were detectable for up to 31 weeks after the first injection. Overall, less than 1 log difference was observed in terms of the vigour of the bulk CTL response induced and the CTL precursor frequency between all vaccines (ranging from 1:2.6x10(5) to 1:1x10(6)). Anti-NS3 antibodies could only be detected following a combined vaccine regimen in non-transgenic BALB/c mice. In conclusion, rSFV particles expressing NS3 are capable of inducing NS3-specific cellular immune responses targeted at a major HLA-A2 epitope. Such responses were comparable to those obtained with a DNA-based NS3 vaccine, whether in the context of single or combined regimens.

Induction of apoptosis in BCL-2-expressing rat prostate cancer cells using the Semliki Forest virus vector.

The Semliki Forest virus (SFV) vector is a transient RNA expression vector that has an inherent p53-independent apoptosis-inducing property. It is administered as recombinant SFV particles (rSFV) that undergo 1 round of replication only and express a gene cloned into the multicloning site. In our study we have investigated the ability of the SFV vector to induce apoptosis and inhibit tumour growth in rat prostate cancer (AT3-Neo) cells expressing the Bcl-2 oncogene (AT3-Bcl-2 cells), which normally inhibits apoptosis. rSFV expressing the enhanced green fluorescent protein (EGFP) gene (rSFV-EGFP), or recombinant RNA transfected into cells by electroporation, induced delayed apoptosis in AT3-Bcl-2 cells. SFV-mediated expression of a cloned pro-apoptotic Bax gene by the vector, however, enhanced apoptosis induction both in AT3-Bcl-2 cells and standard BHK-21 cells. Such Bax-expressing particles could be produced only at low titers compared to EGFP-expressing particles under standard conditions for particle production, but lowering the incubation temperature for particle production to 33 degrees C partially alleviated this effect. Bax-expressing particles were shown to inhibit the growth of AT3-Neo and AT3-Bcl-2 tumours in nude mice, as did high titre EGFP-expressing particles. It is concluded that SFV recombinant particles have potential as anti-tumour agents to treat apoptosis-resistant tumours.

Activation of peritoneal cells upon in vivo transfection with a recombinant alphavirus expressing GM-CSF

In this study we determined the in vivo localization of recombinant proteins expressed by intraperitoneally (i.p.) injected recombinant Semliki Forest virus (SFV) particles. Subsequently, we investigated the influence of i.p. administered SFV particles encoding recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) on intraperitoneal recruitment and activation of cells. Finally, the therapeutic effect of SFV-GM-CSF treatment on an i.p. growing ovarian tumor was determined. Intraperitoneal injections of recombinant SFV particles encoding the reporter protein luciferase resulted in a high level of luciferase activity in cells of the peritoneal lining and tumor cells in the peritoneal cavity. Low levels of luciferase activity were found in liver, spleen and lungs. Injection of SFV-GM-CSF particles resulted in a slight increase in the number of peritoneal macrophages and in a significant increase in the number of neutrophils. In contrast to multiple i.p. injections with commercially available recombinant GM-CSF, i.p. injected SFV-GM-CSF particles activated the macrophages to tumor cytotoxicity. Although treatment of tumor-bearing mice with SFV-GM-CSF particles did not result in prolonged survival, tumor growth was inhibited for 2 weeks. Our findings indicate that macrophage-activating cytokines expressed by the efficient and safe recombinant SFV system when administered i.p. may provide an immunotherapeutic treatment modality additional to current chemotherapeutic treatment of intraperitoneally growing cancers.

Antigenic Properties of Recombinant Envelope Glycoproteins Derived from T-Cell-Line-Adapted Isolates or Primary Human Immunodeficiency Virus Isolates and Their Relationship to Immunogenicity

The native envelope glycoproteins of primary HIV-1 virions have weaker antigenicity than do T-cell laboratory-adapted (TCLA) viruses. These antigenic properties require further evaluation if recombinant envelope glycoproteins are produced as part of a vaccine strategy. In this study, we compared the antigenicity of recombinant envelope glycoproteins derived from three primary isolates (PI) (HIV-1BX08, HIV-1CHA, and HIV-1133) and two TCLA viruses (HIV-1HXB2 and HIV-1MN) produced using the Semliki Forest virus (SFV) system. This analysis was performed by radioimmunoprecipitation assays and flow cytometry. The results suggest that the SFV produces envelope glycoproteins with features in common with the envelopes found in naturally occurring virions. In particular, the PI envelopes had weak heterogeneous antigenic properties. However, the cytometric analysis also showed that there was less envelope glycoprotein on the cell surface for the PI envelopes than for those of TCLA viruses, suggesting differences in their intracellular trafficking. The immunogenic properties of the various envelope glycoproteins were evaluated in mice using recombinant SFV particles as vaccine vectors. The PI envelopes were less immunogenic than the TCLA envelopes, probably due to both their low antigenicity and cell surface expression level. Thus, it may be difficult to design an effective vaccine based on native recombinant PI envelopes.

Induction of P815 tumor immunity by recombinant Semliki Forest virus expressing the P1A gene.

The methylcholantrene-induced P815 mastocytoma tumor is derived from DBA/2 mice and expresses a weak tumor rejection antigen, P815A. The P1A gene, which encodes for the P815A antigen, is silent in most normal tissues with the exception of testis and placenta. These characteristics make P815 an interesting mouse model for the human MAGE-type tumor antigens. Recombinant Semliki Forest virus particles (rSFV) were constructed that expressed variants of the P815 antigen. Such particles, when used for vaccination, express the antigen only transiently since the viral vector is incapable of productive replication. Nevertheless, mice vaccinated with rSFV generated strong CTL responses and were protected against P815 tumor challenge.

Alphaviruses to the rescue?

The ability to genetically engineer animal viruses enables their application to medical research. The development of non-retroviral RNA viruses as vectors to deliver foreign genes for vaccination and gene therapy is particularly interesting, especially the domestication of the alphavirus group. Two papers in this field have recently been published1xTwo-helper RNA system for production of recombinant Semliki Forest virus particles. Smerdou, C. and Liljestrom, P. J. Virol. 1999; 73: 1092–1098PubMedSee all References, 2xNoncytopathic Sindbis virus RNA vectors for heterologous gene expression. Agapov, E.V. et al. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12689–12994Crossref | PubMed | Scopus (155)See all References.Alphaviruses offer several advantages as expression vectors: they have a broad host range, high levels of expression and a small genome that is easy to manipulate. Vectors based on Semliki Forest virus (SFV), Sindbis virus and Venezuelan equine encephalitis have been constructed and deliver and express their cargo efficiently. However, the generation of wild-type virus, owing to recombination between the artificial genome and a helper genome, remains a problem in many recombinant viral systems. This biosafety issue is a concern, especially if vectors are to be used in vaccine delivery, and methods have been developed to reduce this risk. In the SFV system, Smerdou and Liljestrom1xTwo-helper RNA system for production of recombinant Semliki Forest virus particles. Smerdou, C. and Liljestrom, P. J. Virol. 1999; 73: 1092–1098PubMedSee all References1 have prevented the production of wild-type virus by using two, independent helper RNAs; this system also circumvents the requirement of a viral protease and this portion of the genome has been mutated, conferring additional biosafety. Thus, at least two recombination events and one reversion are required to generate replication-competent virus particles. Extensive analysis has failed to demonstrate the presence of wild-type viruses, emphasizing the biosafety of the two-helper system.The expression strategy of alphaviruses imposes a severe burden on normal cellular biochemistry and infected cells die quickly, restricting the use of these vectors when long-term gene expression is required. However, Agapov et al.2xNoncytopathic Sindbis virus RNA vectors for heterologous gene expression. Agapov, E.V. et al. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12689–12994Crossref | PubMed | Scopus (155)See all References2 have developed noncytotoxic Sindbis virus vectors, which produce large amounts of foreign protein, by transfecting Sindbis virus replicons that express a puromycin N-acetyltransferase into cells grown in the presence of puromycin. Cells that survive in the presence of the drug carry replicons that are noncytotoxic and express the puromycin-inactivating enzyme. Such replicons can then be engineered to express other foreign genes for long periods in a noncytotoxic manner, which could be useful in both vaccinology and in the development of gene therapy.

Specific interactions between retrovirus Env and Gag proteins in rat neurons.

In this work we have studied the intracellular localization properties of the Gag and Env proteins of Moloney murine leukemia virus (MLV) and human immunodeficiency virus (HIV) in dorsal root ganglion (DRG) neurons of rat. These neurons form thick bundles of axons, which facilitates protein localization studies by immunofluorescence analyses. When such neuron cultures were infected with recombinant Semliki Forest virus particles carrying the gag genes of either retrovirus, the expressed Gag proteins were localized to both the somatic and the axonal regions of the DRG neurons. In contrast, the Env proteins were confined only to the somatic region. When the Gag and Env proteins were coexpressed, the Gag proteins were also excluded from the axons. This effect of the Env proteins was shown to be dependent on the concentration of the Gag proteins in the neuron and also to be specific for homologous pairs of retrovirus proteins. Therefore, the results suggest that there are specific interactions between the Env and the Gag proteins of MLV and HIV in the DRG neurons.

Effect of temperature on recombinant protein expression in Semliki Forest virus infected mammalian cell lines growing in serum- free suspension cultures.

The firefly luciferase gene was introduced into the Semliki Forest virus (SFV) vector and high titer recombinant SFV particles generated. The broad host range of SFV allowed efficient infection and high level expression of four mammalian cell lines growing in serum-free suspension cultures. The incubation temperature had dramatic effects on the level and duration of recombinant protein expression. For example, the luciferase activity was significantly higher in the rodent BHK and CHO cell lines incubated at 33 degrees C compared to 37 degrees C when harvested 19 h post-infection. At 33 degrees C the specific expression levels increased 10-20 fold during prolongation of the post-infection time up to 50 h. In contrast, a significant decrease in luciferase activity was observed from 26 h post-infection for cell cultures incubated at 37 degrees C. Only a slight temperature effect on luciferase expression was seen in the human cell line HEK293 and no effect was observed for the subclone293(EBNA).

Generation of Monoclonal Antibodies against Human Prion Proteins in PrP0/0 Mice

Prion diseases belong to a group of neurodegenerative disorders affecting humans and animals. The human diseases include kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). The pathogenic mechanisms of the prion diseases are not yet understood. Monoclonal antibodies provide valuable tools in the diagnosis, as well as in the basic research, of several diseases; however, monospecific antisera or monoclonal antibodies (mAbs) against human prion proteins were, until now, not available. We have developed an immunization protocol based on nucleic acid injection into nontolerant PrP0/0 mice. DNA or RNA coding for different human prion proteins including the mutated sequences associated with CJD, GSS, and FFI were injected into muscle tissue. Mice were primarily inoculated with DNA plasmids encoding the prion protein (PRNP) gene and boosted either with DNA, RNA, or recombinant Semliki Forest Virus particles expressing PRNP. Hybridomas were then prepared. Different mAbs against human prion proteins were obtained, and their binding behavior was analyzed by peptide enzyme-linked immunosorbent assay, Western blot, immunofluorescence, and immunoprecipitation. Their cross-reactivity with prion protein from other species was also determined. Our mAbs are directed against four different linear epitopes and may also recognize discontinuous regions of the native prion protein. These antibodies should allow us to address questions concerning the nature of the prion protein as well as the initiation and progression of prion diseases. Moreover, these mAbs can now be used for the diagnosis of prion diseases of humans and animals.

Proposed Schizophrenia-Related Gene Polymorphism: Expression of the Ser9Gly Mutant Human Dopamine D3Receptor with the Semliki Forest Virus System

Homozygotes for aBalI polymorphism resulting in the Ser9Gly mutation in the human dopamine D3receptor gene are suggested to display a twofold higher risk of schizophrenia. The cDNAs for this mutant and the wildtype receptor were introduced into the pSFV1C vector and recombinant Semliki Forest virus particles generated. CHO cells infected with SFV-D3virus resulted in high level expression of recombinant receptor. Double infections with wildtype and Ser9Gly dopamine D3SFV stocks generated an artificial heterozygote in CHO cells. Receptor binding analysis of several structurally different dopamine D3ligands showed similar pharmacological properties for all compounds tested except for dopamine and the D3-selective ligand GR99841. A significantly higher dopamine binding afffinity for the Ser9Gly homozygote was detected. The heterozygote binding did not differ from the wildtype. However, both the homo- and heterozygote for Ser9Gly showed significantly higher binding activity for GR99841 compared to the D3wildtype.

Transient Expression of a Mitochondrial Precursor Protein

In order to study mitochondrial protein import in the context of whole cell metabolism, we have used the transfection technique based on Semliki Forest virus (SFV) to express a mitochondrial precursor protein within BHK21 cells and human fibroblasts. Recombinant SFV particles mediate a highly efficient, transient transfection of higher eukaryotic cells. The mitochondrial precursor protein used is a fusion protein consisting of the mitochondrial targeting sequence of Neurospora crassa ATPase subunit 9 and mouse dihydrofolate (H2folate) reductase. Transfected BHK21 cells synthesized substantial amounts of subunit-9-H2folate-reductase. Immunofluorescence staining revealed that the protein colocalized with the mitochondria. The precursor protein was processed to the intermediate and mature form, implying that is was successfully imported into the mitochondrial matrix. Import was dependent on a proton gradient across the mitochondrial membranes since uncoupling of oxidative phosphorylation inhibited the process. The mature-sized protein was folded into a protease-resistant conformation. These results indicate that, in mammalian cells, transport of the precursor subunit-9-H2folate-reductase into mitochondria and its subsequent maturation occurs in a similar way as in lower eukaryotes. Import and processing of the fusion protein proceeded very rapidly in BHK21 cells but were substantially slower in human fibroblasts. SFV-mediated transfection proved to be excellently suited to study protein import into mitochondria of living cells and is probably applicable to transport studies with other organelles as well. The approach could also be helpful in the diagnosis of hereditary disorders or organelle protein import.