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PubMed1972

Fatty chains of different lipid classes of Semliki forest virus and host cell membranes.

Semliki Forest virus was grown in BHK-21 cells. The major classes of phospho-and glycolipids of the virus were analyzed for the compositions of fatty acids, aldehydes, and sphingosine bases, and the major glycerophospholipids were analyzed for the relative proportions of alkenyl-acyl, alkyl-acyl, and diacyl forms. All viral lipid classes proved to be mixtures of several molecular species. Each class contained a characteristic mixture of fatty chains, which was different in all other classes. All viral lipid classes resembled their counterparts of the host plasma membrane and also those of the endoplasmic reticulum. The gangliosides of the virus and the plasma membrane proved to be similar even at the level of individual molecular species. The number of certain lipid molecules in an average virion was less than the number of the protein molecules.

Aldehydes↗

PubMed1983

Defective viral RNAs in Aedes albopictus C6/36 cells persistently infected with Semliki Forest virus.

A persistent infection of Semliki Forest virus (SFV) has been established in Aedes albopictus C6/36 cells. Only a small number of cells survived the initial infection with this RNA virus and gave rise to a persistently infected culture which produced continuously small amounts of infectious virus. To investigate whether defective viral RNA was analyzed early and late after infection by blot hybridizations. Several defective viral RNAs were detected with a common sequence corresponding to the 3' end of the viral genome during and after the establishment of the persistent infection. These defective viral RNAs resemble the defective interfering RNAs in vertebrate cells generated during serial undiluted passages of standard SFV. The defective viral RNAs are rarely released from cells as virions. The rapid generation of defective viral RNAs may be important for the establishment of a persistent infection in mosquito cells.

Aedes↗

PubMed1984

Infection of rat brain primary cell cultures with an avirulent A7 strain of Semliki Forest virus.

The ability of A7 Semliki Forest Virus (SFV) to infect primary brain cell cultures has been examined using cultures prepared from 1-2-day neonatal rat cerebral hemispheres. These cultures, characterised immunocytochemically using cell-specified markers, contain mainly GFAP+ protoplasmic astrocytes and smaller multiprocessed A2B5+ cells, probably fibrous astrocytes. 10% of the cells are GC+ oligodendrocytes and some neurones are also present. These cultures support virus growth and a cytopathic effect was observed. Using double labelling techniques with the cell-specific markers and anti-SFV antibody A7 has been shown to readily infect cells which carry either the A2B5+ antigen or galactocerebroside marker. Protoplasmic astrocytes (GFAP+/A2B5-) are not readily infected under the conditions used. The protein labelling studies using [35S]methionine show that host cell protein synthesis is not completely shut off and continues in the astrocyte protein region. These results suggest that cells derived from a common A2B5+, GFAP-, GC- progenitor glial cell, i.e. GC+ oligodendrocytes and A2B5+/GFAP+ fibrous astrocytes, are more readily infected than other brain cell types including the protoplasmic astrocytes.

Animals↗

PubMed1996

Role of immune responses in protection and pathogenesis during Semliki Forest virus encephalitis.

The course of Semliki Forest virus (SFV) A7(74) infection in immunocompetent BALB/c, athymic nu/nu and severe combined immunodeficient (SCID) mice was compared. BALB/c mice remained healthy and exhibited transient viraemia and infectious virus in the brain from days 2 to 7. Antibodies were detectable by day 5. In comparison, SCID mice displayed a high incidence of paralysis and died: the average day of death was day 23. From infection until death, virus was present in blood and brain. No antibodies were detectable. Athymic mice were intermediate with a transient viraemia and a persistent (> 210 days) sub-clinical central nervous system (CNS) infection. These mice produced anti-viral IgM but not IgG. The pattern of infection in BALB/c or nu/nu mice could be recreated in infected SCID mice by transfer of immune serum from BALB/c or nu/nu mice with the important exception that although BALB/c immune serum could abolish infectivity titres in the CNS, scattered cells positive for viral RNA remained. Transfer of serum decreased mortality and delayed the onset of paralysis. Transfer to infected SCID mice of a non-neutralizing IgG anti-E2 monoclonal antibody did not affect the viraemia but could also reduce brain virus titres. Irrespective of specific immune responses, virus replication in CNS cells was restricted, was generally non-cytopathic and in the absence of specific immune responses could persist. From day 14 lesions of inflammatory, primary demyelination were observed throughout the CNS of BALB/c mice. In contrast, despite prolonged brain virus titres, no demyelinating lesions were observed in infected nu/nu or SCID mice. Lesions could be initiated in the latter by transfer of spleen cells but not antibody. In summary, the focal restricted infection in the CNS of adult mice infected with SFV A7(74) is independent of specific immune responses. IgM antibodies clear the viraemia. IgG antibodies including non-neutralizing antibodies reduce and clear infectious virus but cells positive for viral RNA remain. These may normally be cleared by T cell responses which are damaging and give rise to lesions of demyelination.

Alphavirus Infections↗

PubMed2000

Biochemical consequences of a mutation that controls the cholesterol dependence of Semliki Forest virus fusion.

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-triggered membrane fusion reaction that requires cholesterol and sphingolipid in the target membrane. Cholesterol-depleted insect cells are highly resistant to alphavirus infection and were used to select srf-3, an SFV mutant that is approximately 100-fold less cholesterol dependent for infection due to a single amino acid change in the E1 spike subunit, proline 226 to serine. Sensitive lipid-mixing assays here demonstrated that the in vitro fusion of srf-3 and wild-type (wt) virus with cholesterol-containing liposomes had comparable kinetics, activation energies, and sphingolipid dependence. In contrast, srf-3 fusion with sterol-free liposomes was significantly more efficient than that of wt virus. Thus, the srf-3 mutation does not affect its general fusion properties with purified lipid bilayers but causes a marked and specific reduction in cholesterol dependence. Upon exposure to low pH, the E1 spike subunit undergoes distinct conformational changes, resulting in the exposure of an acid conformation-specific epitope and formation of an E1 homotrimer. These conformational changes were strongly cholesterol and sphingolipid dependent for wt SFV and strikingly less cholesterol dependent for srf-3. Our results thus demonstrate the functional importance of fusogenic E1 conformational changes in the control of SFV cholesterol dependence.

Animals↗

PubMed1993

Expression of HIV-1 envelope glycoproteins by Semliki Forest virus vectors.

We have used Semliki Forest virus (SFV) vectors to express both the human immunodeficiency virus type 1 (HIV-1) envelope precursor gp160 and the cleaved external portion gp120. Expression of the foreign gene in this system is by transfection of recombinant SFV RNA, or by infection with a recombinant SFV virus that has a wide host range. pSFV1-gp120 or pSFV1-gp160 were expressed in baby hamster kidney (BHK) cells and two human cell lines: HeLa cervical carcinoma and MOLT-4 CD4+ T cells. After SFV1-gp120 infection of HeLa cells, 3.3 micrograms of gp120 was secreted into the media by 1 million cells in a 24-hr period. The secreted envelope glycoprotein was recognized by anti-gp120 monoclonal antibodies directed against both linear and conformation-dependent epitopes in different regions of the molecule. The recombinant gp120 also bound to a soluble form of the CD4 receptor. Syncytium formation was observed when MOLT-4 cells were infected with SFV1-gp160. The gp160 expressed by BHK cells induced syncytia during cocultivation with C8166 CD4+ T cells. These data indicate that SFV vectors can be used to produce the HIV-1 envelope glycoproteins to high levels, and that these proteins are correctly processed, folded, and transported to the cell surface. Furthermore, they exhibit functional activity as indicated by their ability to bind to soluble receptor and induce cell-to-cell fusion.

Animals↗

PubMed1991

Processing of the p62 envelope precursor protein of Semliki Forest virus.

The spike protein of Semliki Forest virus is composed of three subunits, E1, E2, and E3, which mediate the fusion of the virus membrane with that of the host cell. E2 and E3 are synthesized as a precursor, p62, which is cleaved post-translationally after an Arg-His-Arg-Arg sequence. In vitro mutagenesis of a cDNA clone of the spike proteins was used to specifically alter amino acids in this cleavage site. Cleavage of p62 was completely blocked by mutation of the proximal Arg residue to Phe, without affecting transport or surface expression of the spike protein. The cleavage mutation resulted in the loss of spike protein fusion activity within the physiological pH range. Fusion activity was restored by cleavage with exogenous chymotrypsin and showed the same low pH dependence as that of wild type. The cleavage sensitivity of newly synthesized p62 was investigated by pulse-chase analysis and chymotrypsin treatment in detergent solution. p62 was sensitive to cleavage immediately following its synthesis. Protein trapped in the rough endoplasmic reticulum or Golgi apparatus by carbonyl cyanide m-chlorophenylhydrazone, monensin, or Brefeldin A treatment was also fully sensitive to cleavage. These results suggest that p62 does not require an organelle-mediated conformational change for processing. Thus, in vivo, the site of p62 processing is probably controlled by the location or activity of the cleavage enzyme, rather than the sensitivity of the p62 substrate.

Amino Acid Sequence↗

PubMed1975

Polysomes and initiation complexes in vitro induced by Semliki Forest virus 42S and 26S RNA.

Semliki Forest virus 42S and 26S RNA induce the formation of polysomes when translated in vitro in cell-free systems. After analysis on sucrose gradients the polysomal structures, containing prelabelled RNA and nascent peptide chains labelled with [35S]methionine, had sedimentation values from 100 to 200S in the case of 26S RNA and from 150 to over 250S with 42S RNA. After incubation in the presence of inhibitors of elongation 26S RNA was found in initiation complexes sedimenting at about 80S. Under identical conditions 42S RNA had a heterogenous sedimentation pattern, being attached to complexes sedimenting even faster than 250S. These structures, however, fulfilled the criteria of initiation complexes suggesting that there were more than one ribosome attached to each 42S RNA under conditions where 26S RNA bound only one.

Cell-Free System↗

PubMed2003

Semliki Forest virus vectors for gene therapy.

Semliki Forest virus (SFV) vectors transduce a broad range of mammalian and non-mammalian cells, generating high levels of transient expression of heterologous proteins. Generally, they induce apoptosis in mammalian host cells, leading to rapid cell death. These features have made SFV attractive for various gene therapy applications. Recombinant particles, naked RNA and plasmid DNA containing SFV replicons, demonstrate a strong immune response against recombinantly expressed proteins, which has shown protection against tumour challenges. Intratumoural injection of SFV particles has resulted in tumour regression. SFV vectors have been used for production of retrovirus-like particles. Recently, encapsulation of SFV particles into liposomes has generated highly efficient targeting to tumours. Novel SFV vectors based on point mutations in the non-structural genes, and avirulent SFV strains, have further widened the application range.

Animals↗

PubMed1986

Enhanced retinal and optic nerve excitability associated with demyelination in mice infected with Semliki Forest virus.

The physiological effects of Semliki Forest virus (SFV) induced demyelination on the visual system of mice have been examined by recording electroretinograms (ERGs) and the spike activity in the retinal ganglion cell axons in control and SFV-infected mice. The amplitudes of ERGs evoked by a single flash in the dark-adapted eyes of the SFV infected mice were abnormally enhanced, whereas flicker ERGs were slightly depressed. This hyperactivity was also seen in the ganglion cell axonal discharge. Both visually evoked and spontaneous activity recorded from the optic tract fibres of the infected mice were characterized by rhythmic oscillatory bursts of firing. Similar patterns were seen only very rarely in the evoked activity recorded from control mice. We suggest that this oscillatory firing might be a functional correlate of the types of 'positive symptoms' reported by multiple sclerosis patients with optic neuritis.

Animals↗

PubMed1975

Effect of impaired glycosylation on the biosynthesis of Semliki forest virus glycoproteins.

The glycoproteins of Semliki Forest virus, grown in chicken embryo cells, were labeled with radioactive sugars. The data indicate a high mannose content of the nonstructural precursor glycoprotein NSP 63. This protein can also be readily labeled with 2-deoxy-D-glucose. The envelope glycoproteins E1 and E2 are relatively rich in galactose, glucosamine, and fucose. Glycosylation can be impaired by 2-deoxy-D-glucose or D-glucosamine or by omission of sugars in the culture medium. Under these conditions characteristic changes in the electrophoretic profile of the viral polypeptides are observed: in the regions of glycoproteins NSP 97, NSP 63, and E1 and E2 new protein peaks can be detected. These polypeptides seem to be aberrant forms of the glycoproteins. When compared with the normal molecules they have lower molecular weights and contain less carbohydrates, especially mannose. Pulse-chase experiments indicate that the altered glycoproteins are degraded very slowly if at all. If, however, impairment is caused by omission of sugars in the culture medium, the radioactivity is chased after addition of glucose from the region between NSP 63 and E1 + E2 into the E1 + E2 peak. This suggests a completion of the carbohydrate chains under these conditions.

Cells, Cultured↗

PubMed1995

The oligomerization reaction of the Semliki Forest virus membrane protein subunits.

The Semliki Forest virus (SFV) spike is composed of three copies of a membrane protein heterodimer. The two subunits of this heterodimer (p62 and E1) are synthesized sequentially from a common mRNA together with the capsid (C) in the order C-p62-E1. In this work heterodimerization of the spike proteins has been studied in BHK 21 cells. The results indicate that: (a) the polyprotein is cotranslationally cleaved into individual chains; (b) the two membrane protein subunits are initially not associated with each other in the endoplasmic reticulum (ER); (c) heterodimerization occurs predominantly between subunits that originate from the same translation product (heterodimerization in cis); (d) the kinetics of subunit association are very fast (t1/2 = 4 min); and (e) this heterodimerization is highly efficient. To explain the cis-directed heterodimerization reaction we suggest that the p62 protein, which is made before E1 during 26S mRNA translation, is retained at its translocation site until also the E1 chain has been synthesized and translocated at this same site. The mechanism for p62 retention could either be that the p62 anchor sequence cannot diffuse out from an "active" translocation site or that the p62 protein is complexed with a protein folding facilitating machinery that is physically linked to the translocation apparatus.

Animals↗

PubMed1980

The sensitivity to gamma-irradiation of the phases of the virus-host interaction: studies with strains of Semliki forest virus in mice.

A convenient and quantifiable model system, Semliki Forest virus (SFV) in the mouse, has been used to probe the wider problem of the radiation sensitivity of the distinct phases of the virus-host interaction. This has been investigated through the suppression and recovery of the several host defence mechanisms and cellular compartments involved. Direct observations have been made, following whole-body gamma-irradiation at up to 600 R, of the sequential modifications imposed upon the efficiency of primary infection, the stimulation of regulatory immunity (pre-challenge) and the stimulation or boosting of protective immunity (post-challenge). These phases of the virus-host interaction show distinct sensitivities to gamma-radiation which are discussed in terms of the impairment and recovery of the lymphocyte compartments probably involved.

Animals↗

PubMed1982

Temperature-dependent internalization of virus glycoproteins in cells infected with a mutant of Semliki Forest virus.

When the ts-1 mutant of Semliki Forest virus (SFV) was grown in chick embryo or BHK 21 cells at the restrictive temperature (39 degrees C), its membrane glycoproteins were arrested in the endoplasmic reticulum, but started to migrate to the cell surface once the cultures were shifted to the permissive temperature (28 degrees C). If the temperature of infected cells was raised back to 39 degrees C, ts-1 glycoproteins disappeared from the cell surface as evidenced by loss of surface immunofluorescence and by radioimmunoassay based on the binding of 125I-labeled protein A. This phenomenon was specific for ts-1 at 39 degrees C as it was observed neither in cells infected with wild-type SFV at 39 degrees C nor with ts-1 at 28 degrees C. The disappearance of the ts-1 glycoproteins was due to internalization. The internalized proteins were digested, as shown by specific decrease of virus glycoproteins labelled with [35S]methionine at 39 degrees C before shift to 28 degrees C, and by concomitant release of acid soluble 35S-activity into the culture medium. Ts-1 infected cells were treated before shift back to 39 degrees C with Fab' fragments, prepared from IgG against the viral membrane glycoproteins. After shift back to 39 degrees C, the Fab' fragments disappeared from the cell surface. In the presence of chloroquine, they could be visualized in vesicular structures, using an anti-IgG-fluorescein isothiocyanate conjugate. The internalization of ts-1 glycoproteins was not inhibited by carbonylcyanide p-trifluoromethoxy phenylhydrazone, chloroquine, cytochalasin B, vinblastine, colcemid, or monensin.

Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone↗

PubMed1985

On the structure of the acyl linkage and the function of fatty acyl chains in the influenza virus haemagglutinin and the glycoproteins of Semliki Forest virus.

The acylation of the haemagglutinin (HA) of different influenza viruses and of the envelope glycoproteins of Semliki Forest virus (SFV) were analysed. The fatty acid linkage in these acylproteins was found to be resistant to a variety of organic solvents and combinations of these, even after pretreatment with various detergents. Fatty acids are released from influenza virus HA at a pH value between 11.8 and 12.1 at room temperature. Although this mild alkaline cleavage occurs rapidly, the release of fatty acids by treatment with hydroxylamine is time-, temperature- and concentration-dependent. By comparison with model esters the linkage in HA is suggested to be of the oxygenester type rather than a thioester linkage. To assay for possible functions of protein-bound fatty acids the biological activities of influenza virus (A/FPV/Rostock/34) and its solubilized spike glycoproteins were measured after deacylation. While viral haemagglutination activity was not hampered at all, its ability to haemolyse erythrocytes and infectivity were drastically reduced. Likewise, viral spike glycoproteins solubilized with detergents failed to induce haemolysis at low pH when fatty acids had been cleaved off. These results indicate the possible involvement of protein-bound fatty acids in fusion induction through the acylated fusogenic spike glycoproteins.

Esters↗

PubMed2001

Recombinant Semliki Forest virus vector exhibits potential for avian virus vaccine development.

The Semliki Forest virus (SFV) expression system was evaluated as a basis for avian vaccine development. Initial studies indicated that 1-day-old specific pathogen-free (SPF) chicks were susceptible to infection with an infectious strain of SFV, producing SFV-specific antibodies but no clinical disease. One-day-old SPF chicks immunised intramuscularly with recombinant replication-defective SFV (rSFV) particles expressing the Escherichia coli (E. coli) lacZ reporter gene developed high titres of beta-gal- specific antibodies at 4 weeks p.i. after two inoculations. In contrast, significantly lower antibody levels were elicited in chicks immunised with a recombinant SFV-based DNA construct or a conventional CMV promoter-based DNA plasmid. rSFV particles encoding the protective VP2 protein or the VP2/VP4/VP3 polyprotein of infectious bursal disease virus (IBDV) were produced and the expressed antigens were characterised in cell culture. Proteins of the correct size were generated and found to react against a range of IBDV-specific monoclonal antibodies. Immunisation of 1-day-old SPF chicks with rSFV particles encoding the IBDV proteins resulted in specific antibodies being elicited in all birds, neutralising antibodies being induced in some but not all birds.

Animals↗

PubMed1980

Characterization of a small, nonstructural viral polypeptide present late during infection of BHK cells by Semliki Forest virus.

BHK cells, late in infection with Semliki Forest virus, were found to contain a small virus-specific polypeptide not found in the mature virion. This polypeptide had an apparent molecular weight of 6,000 and is referred to here as the 6K protein. No [2-3H]mannose was incorporated into 6K, and hence it does not appear to be a glycoprotein. This protein appears to be a primary translation product of the subgenomic 26S mRNA, which encodes the viral structural proteins. The genes encoding the viral structural proteins are arranged on the message in the order of 5'-C-E3-E2-E1-3'. We have found that the gene coding for 6K is located to the 3' side of the gene encoding E2. Subcellular fractionation of pulse-labeled cells infected with Semliki Forest virus demonstrated that 6K, like the viral glycoproteins p62 and E1, was present predominantly in the rough microsomal membrane fraction. 6K appears to be analogous, therefore, to the nonstructural 4.2K protein present in cells infected with Sindbis virus.

Animals↗

PubMed1986

Use of N-acetylethyleneimine [AEI] for the inactivation of Semliki Forest virus in vitro.

N-Acetylethyleneimine (AEI) was used to inactivate the avirulent Togavirus Semliki Forest virus (A774 strain) grown in chick embryo, Vero, and brain cell cultures. The purity of the virus preparation affected the kinetics of inactivation. The rate of inactivation increased with a rise in temperature from 5 to 40 degrees C and in concentration of AEI from 0.025 to 0.1%. The resultant vaccine was inoculated into adult mice to test its antigenicity and into suckling mice to test for the presence of infective virus. Semliki Forest virus-specific IgG was produced equal to that of mice given live virus, and mice were protected against the lethal SFV L10 strain. No suckling mice died, and the brains of the adult mice showed no pathology.

Animals↗