PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “SEMLIKI FOREST VIRUS”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3Linked to original sources
PubMed1982

The effect of defective-interfering Semliki Forest virus on the histopathology of infection with virulent Semliki Forest virus in mice.

The majority of mice inoculated with a mixture of a lethal dose of virulent Semliki Forest virus (SFV) strain ts+ and defective-interfering (DI) SFV remained completely health, with virus infectivity levels in brain tissue reduced by 99.9%. The results of previous studies had suggested that these effects were primarily the result of the intrinsic interfering capacity of DI virus rather than of host defense responses. Because SFV strain ts+ and an avirulent strain of SFV have clearly distinguishable histopathologic effects in brain tissue, the capability of DI virus to change the virulent into the avirulent form of the disease was examined. Modulation of strain ts+ virus infection by DI virus was accompanied by a complete absence of histopathologic changes despite significant levels of infectious virus and thus differed qualitatively from infection with avirulent SFV. These results provide further evidence that the interference is not mediated through stimulation of an immune cell infiltration.

Animals↗
PubMed1998

A single point mutation controls the cholesterol dependence of Semliki Forest virus entry and exit.

Membrane fusion and budding are key steps in the life cycle of all enveloped viruses. Semliki Forest virus (SFV) is an enveloped alphavirus that requires cellular membrane cholesterol for both membrane fusion and efficient exit of progeny virus from infected cells. We selected an SFV mutant, srf-3, that was strikingly independent of cholesterol for growth. This phenotype was conferred by a single amino acid change in the E1 spike protein subunit, proline 226 to serine, that increased the cholesterol independence of both srf-3 fusion and exit. The srf-3 mutant emphasizes the relationship between the role of cholesterol in membrane fusion and virus exit, and most significantly, identifies a novel spike protein region involved in the virus cholesterol requirement.

Amino Acid Sequence↗
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↗
PubMed1994

Membrane and protein interactions of a soluble form of the Semliki Forest virus fusion protein.

Semliki Forest virus is an enveloped alphavirus that infects cells by a membrane fusion reaction triggered by the low pH present in endocytic vacuoles. Fusion is mediated by the E1 spike protein subunit. During fusion, several conformational changes occur in E1 and E2, the two transmembrane subunits of the spike protein. These changes include dissociation of the E1-E2 dimer, alteration of the trypsin sensitivity and monoclonal antibody binding patterns of E1, and formation of a sodium dodecyl sulfate (SDS)-resistant E1 homotrimer. A critical characteristic of Semliki Forest virus fusion is also its dependence on the presence of both cholesterol and sphingomyelin in the target membrane. We have here examined the conformational changes induced by low pH treatment of E1*, the water-soluble, proteolytically truncated ectodomain of the E1 subunit. Following low pH treatment, E1* was shown to bind efficiently to artificial liposomes. Similar to virus fusion, optimal E1*-liposome binding required low pH, cholesterol, and sphingomyelin. The E1 ectodomain, although monomeric in its neutral pH form, assembled into an SDS-resistant oligomer following treatment at low pH. This low pH-induced oligomerization required target membranes containing both cholesterol and sphingomyelin. Our results demonstrate that the E1 ectodomain responds to low pH similarly to the full-length E1 subunit. The ectodomain facilitates the characterization of conformational changes and membrane binding in the absence of virus fusion or other virus components.

Animals↗
PubMed1993

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↗
PubMed1975

Replication of Semliki Forest virus.

Replication of Semliki Forest virus, a typical alphavirus, takes place in the cytoplasm of many eukaryotic cells. The virus genome, the 42 S RNA, directs the synthesis of at least two RNA-dependent RNA polymerases. By the aid of these enzymes complementary 45 S RNA is synthesized; it serves as a template for the synthesis of positive RNA strands with sedimentation values of 45 S and 26 S. In BHK cells close to 200,000 molecules of each RNA species are produced per cell. Both 26 S and 42 S RNAs are associated with polysomes synthesizing viral structural proteins. The 26 S RNA is a duplication of the nucleotide sequences coding for the virion proteins. These are translated as a polyprotein with the capsid protein at the N-terminal end followed by the envelope proteins E2 and E1. Usually only small amounts of nonstructural proteins are synthesized at the exponential phase of virus growth, indicating that a translational control operates in Semliki Forest virus-infected cells. One of our temperature-sensitive mutants, ts-1, directs, however, the synthesis of two nonstructural proteins with MWs of 78,000 and 86,000 when grown at the nonpermissive temperature. The assembly of the viral nucleocapsid begins by association of the capsid protein with the 42 S RNA, which is still serving as a messenger. In this process a cytoplasmic structure sedimenting at about 65 S is presumably one of the capsid protein donors. The 140 S nucleocapsid buds through the host cell plasma membrane whereby the capsid protein interacts with the envelope proteins creating a specific viral envelope devoid of host proteins. Altogether 5,000 to 20,000 virus particles are released from each cell by the end of the growth cycle, representing about 10% of the 42 S RNA molecules synthesized during the infection.

Animals↗
PubMed2003

Molecular dynamics simulations of the E1/E2 transmembrane domain of the Semliki Forest virus.

Transmembrane (TM) helix-helix interactions are important for virus budding and fusion. We have developed a simulation strategy that reveals the main features of the helical packing between the TM domains of the two glycoproteins E1 and E2 of the alpha-virus Semliki Forest virus and that can be extrapolated to sketch TM helical packing in other alpha-viruses. Molecular dynamics simulations were performed in wild-type and mutant peptides, both isolated and forming E1/E2 complexes. The simulations revealed that the isolated wild-type E1 peptide formed a more flexible helix than the rest of peptides and that the wild-type E1/E2 complex consists of two helices that intimately pack their N-terminals. The residues located at the interhelical interface displayed the typical motif of the left-handed coiled-coils. These were small and medium residues as Gly, Ala, Ser, and Leu, which also had the possibility to form interhelical Calpha-H...O hydrogen bonds. Results from the mutant complexes suggested that correct packing is a compromise between these residues at both E1 and E2 interhelical interfaces. This compromise allowed prediction of E1-E2 contact residues in the TM spanning domain of other alphaviruses even though the sequence identity of E2 peptides is low in this domain.

Amino Acid Motifs↗
PubMed2006

The dynamic envelope of a fusion class II virus. Prefusion stages of semliki forest virus revealed by electron cryomicroscopy.

Semliki Forest virus is among the prototypes for Class II virus fusion and targets the endosomal membrane. Fusion protein E1 and its envelope companion E2 are both anchored in the viral membrane and form an external shell with protruding spikes. In acid environments, mimicking the early endosomal milieu, surface epitopes in the virus rearrange along with exposure of the fusion loop. To visualize this transformation into a fusogenic stage, we determined the structure of the virus at gradually lower pH values. The results show that while the fusion loop is available for external interaction and the shell and stalk domains of the spike begin to deteriorate, the E1 and E2 remain in close contact in the spike head. This unexpected observation points to E1 and E2 cooperation beyond the fusion loop exposure stage and implies a more prominent role for E2 in guiding membrane close encounter than has been earlier anticipated.

Cryoelectron Microscopy↗
PubMed1986

Adoptive transfer of immunity against virulent Semliki Forest virus with immune spleen cells from mice infected with avirulent Semliki Forest virus.

This paper describes the minimal requirements to protect mice adoptively against challenge with virulent Semliki Forest virus (SFV). Early immune serum, from donor mice infected with an avirulent strain of SFV, contained mainly neutralizing IgM immunoglobulins. More of these antibodies (1.80 PND50 vs 0.06 PND50) were needed to protect recipient mice against intraperitoneal challenge (10 LD50 of SFV) than against subcutaneous challenge (7 LD50). Adoptive transfer experiments indicated that a minimum of 3 X 10(7) six-day immune spleen cells were also able to protect recipient mice against intraperitoneal challenge with 10 LD50 of SFV. Treatment of these donor cells with cytotoxic antisera and complement revealed both T- and B-lymphocytes were required for optimum adoptive immunity. Surviving recipients of either immune serum or immune spleen cells developed significantly less neutralizing antibodies than control mice. The lower antibody titres in protected mice might be related to either immune serum or immune spleen cell mediated restriction of virus replication; meaning a reduced antigenic stimulus in these mice compared to control mice.

Animals↗
PubMed2003

Furin processing and proteolytic activation of Semliki Forest virus.

The alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-dependent membrane fusion reaction mediated by the E1 envelope protein. Fusion is regulated by the interaction of E1 with the receptor-binding protein E2. E2 is synthesized as a precursor termed "p62," which forms a stable heterodimer with E1 and is processed late in the secretory pathway by a cellular furin-like protease. Once processing to E2 occurs, the E1/E2 heterodimer is destabilized so that it is more readily dissociated by exposure to low pH, allowing fusion and infection. We have used FD11 cells, a furin-deficient CHO cell line, to characterize the processing of p62 and its role in the control of virus fusion and infection. p62 was not cleaved in FD11 cells and cleavage was restored in FD11 cell transfectants expressing human furin. Studies of unprocessed virus produced in FD11 cells (wt/p62) demonstrated that the p62 protein was efficiently cleaved by purified furin in vitro, without requiring prior exposure to low pH. wt/p62 virus particles were also processed during their endocytic uptake in furin-containing cells, resulting in more efficient virus infection. wt/p62 virus was compared with mutant L, in which p62 cleavage was blocked by mutation of the furin-recognition motif. wt/p62 and mutant L had similar fusion properties, requiring a much lower pH than control virus to trigger fusion and fusogenic E1 conformational changes. However, the in vivo infectivity of mutant L was more strongly inhibited than that of wt/p62, due to additional effects of the mutation on virus-cell binding.

Animals↗
PubMed1991

Mutagenesis of the putative fusion domain of the Semliki Forest virus spike protein.

Semliki Forest virus (SFV), an alphavirus, infects cells via a low pH-triggered membrane fusion reaction that takes place within the cellular endocytic pathway. Fusion is mediated by the heterotrimeric virus spike protein, which undergoes conformational changes upon exposure to low pH. The SFV E1 spike subunit contains a hydrophobic domain of 23 amino acids that is highly conserved among alphaviruses. This region is also homologous to a domain of the rotavirus outer capsid protein VP4. Mutagenesis of an SFV spike protein cDNA was used to evaluate the role of the E1 domain in membrane fusion. Mutant spike proteins were expressed in COS cells and assayed for cell-cell fusion activity. Four mutant phenotypes were identified: (i) substitution of Gln for Lys-79 or Leu for Met-88 had no effect on spike protein fusion activity; (ii) substitution of Ala for Asp-75, Ala for Gly-83, or Ala for Gly-91 shifted the pH threshold of fusion to a more acidic range; (iii) mutation of Pro-86 to Asp, Gly-91 to Pro, or deletion of amino acids 83 to 92 resulted in retention of the E1 subunit within the endoplasmic reticulum; and (iv) substitution of Asp for Gly-91 completely blocked cell-cell fusion activity without affecting spike protein assembly or transport. These results argue that the conserved hydrophobic domain of SFV E1 is closely involved in membrane fusion and suggest that the homologous region in rotavirus VP4 may be involved in the entry pathway of this nonenveloped virus.

Amino Acid Sequence↗
PubMed1990

Blocking by anti-idiotypic antibodies of monoclonal antibody-mediated protection against lethal Semliki Forest virus in mice.

Semliki Forest virus-(SEV) neutralizing monoclonal antibodies (MoAbs), produced after fusion of spleen cells from BALB/c mice and myeloma cell line P3-X63-AG8. 653 or SP2/0, were used for anti-idiotypic immunization of female BALB/c mice. Two intracutaneous immunizations (2 x 40 micrograms per animal), 3 weeks apart, with keyhole limpet haemocyanin-conjugated MoAbs mixed with the saponin Quil A were sufficient to induce high levels of anti-idiotypic antibodies in the circulation of these mice with the capacity to block specifically in vitro MoAb-mediated virus neutralization. Anti-idiotypic antibodies against SFV-neutralizing MoAbs, either passively transferred or actively acquired by immunization, are also able to abrogate (specifically) passive immunity, mediated by critical protective doses of MoAb, in mice against infection with a lethal strain of SFV. Furthermore we confirmed by intervention with anti-idiotypic serum in vivo that an SFV-neutralizing MoAb exerts its greatest protective effect during the first 2 days of infection.

Adjuvants, Immunologic↗
PubMed1993

Role of spike protein conformational changes in fusion of Semliki Forest virus.

The alphavirus Semliki Forest virus (SFV) and a number of other enveloped animal viruses infect cells via a membrane fusion reaction triggered by the low pH within endocytic vesicles. In addition to having a low pH requirement, SFV fusion and infection are also strictly dependent on the presence of cholesterol in the host cell membrane. A number of conformational changes in the SFV spike protein occur following low-pH treatment, including dissociation of the E1-E2 dimer, conformational changes in the E1 and E2 subunits, and oligomerization of E1 to a homotrimer. To allow the ordering of these events, we have compared the kinetics of these conformational changes with those of fusion, using pH treatment near the fusion threshold and low-temperature incubation to slow the fusion reaction. Dimer dissociation, the E1 conformational change, and E1 trimerization all occur prior to the mixing of virus and cell membranes. Studies of cells incubated at 20 degrees C showed that as with virus fusion, E1 trimerization occurred in the endosome before transport to lysosomes. However, unlike the strictly cholesterol-dependent membrane fusion reaction, the E1 homotrimer was produced in vivo during virus uptake by cholesterol-depleted cells or in vitro by low-pH treatment of virus in the presence of artificial liposomes with or without cholesterol. Purified, lipid-free spike protein rosettes were assayed to determine the requirement for virus membrane cholesterol in E1 homotrimer formation. Spike protein rosettes were found to undergo E1 oligomerization upon exposure to low pH and target liposomes and showed an enhancement of oligomerization with cholesterol-containing membranes. The E1 homotrimer may represent a perfusion complex that requires cholesterol to carry out the final coalescence of the viral and target membranes.

Animals↗
PubMedDate not supplied

Expression of the alpha 1b-adrenergic receptor and G protein subunits in mammalian cell lines using the Semliki Forest virus expression system.

Semliki Forest virus (SFV) vectors have been efficiently used for rapid high level expression of several G protein-coupled receptors. Here we describe the use of SFV vectors to express the alpha 1b-adrenergic receptor (AR) alone or in the presence of the G protein alpha q and/or beta 2 and gamma 2 subunits. Infection of baby hamster kidney (BHK) cells with recombinant SFV-alpha 1b-AR particles resulted in high specific binding activity of the alpha 1b-AR (24 pmol receptor/mg protein). Time-course studies indicated that the highest level of receptor expression was obtained 30 hours post-infection. The stimulation of BHK cells, with epinephrine led to a 5-fold increase in inositol phosphate (IP) accumulation, confirming the functional coupling of the receptor to G protein-mediated activation of phospholipase C. The SFV expression system represents a rapid and reproducible system to study the pharmacological properties and interactions of G protein coupled receptors and of G protein subunits.

Animals↗
PubMed1990

Karyophilic properties of Semliki Forest virus nucleocapsid protein.

Semliki Forest virus capsid (C) protein molecules (Mr, 33,000) can be introduced efficiently into the cytoplasm of various target cells by electroporation, liposome, and erythrocyte ghost-mediated delivery (M. Elgizoli, Y. Dai, C. Kempf, H. Koblet, and M.R. Michel, J. Virol. 63:2921-2928, 1989). Here, we show that the transferred C protein molecules partition rapidly from the cytosolic compartment into the nucleus. Transport of the C protein molecules into the nucleus was reversibly arrested by metabolic inhibitors, indicating that the transfer process is energy dependent. Fractionation of isolated nuclei revealed that the delivered C protein preferentially associates with the nucleoli. This finding was confirmed by morphological studies, showing that in an in vitro system containing ATP isolated nuclei rapidly accumulated rhodamine-labeled C protein in their nucleoli. Furthermore, in this assay system, the lectin wheat germ agglutinin prevented transfer of C protein through nuclear pores. These results are in agreement with our observation that nucleoli contain measurable amounts of newly synthesized C protein as early as 5 h after infection of cells with SFV. Thereafter, nucleolar-associated C protein increased progressively during the course of infection.

Adenosine Triphosphate↗
PubMed2001

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.

Animals↗