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At least 127 records · Page 7Linked to original sources
PubMed1999

Respiratory syncytial virus G glycoprotein expressed using the Semliki Forest virus replicon is biologically active.

The respiratory syncytial virus (RSV) G glycoprotein mediates attachment of RSV to cells via an unknown receptor. To study G glycoprotein function we have cloned two variants of the RSV G gene into a Semliki Forest virus (SFV) expression vector, a full length (rG) and soluble (srG) G glycoprotein variant. By immunofluorescence microscopy, rG was found to be predominantly membrane associated, while srG was mostly cytoplasmic. The rG (80-85 kDa) and srG (75-80 kDa) constructs produced heavily glycosylated proteins, however they were slightly smaller than the G glycoprotein expressed in RSV infected HEp-2 cells (85-90 kDa). The biological activity of purified srG was tested by its ability to bind to RSV permissive cells. Purified srG bound to HEp-2 cells and the amount bound increased linearly with the quantity added. Binding was not saturable with the small quantities of protein available. Binding of srG to HEp-2 cells was inhibited (67-68%) by MAb 30 and neutralising anti-G MAb 29. Nonpermissive SF9 insect cells bound 20-50 times less srG than HEp-2 cells. SFV expressed recombinant RSV G glycoprotein should be useful for studying interactions between the RSV G glycoprotein and cells.

Cell Line↗
PubMed1978

Cleavage defect in the non-structural polyprotein of Semliki Forest virus has two separate effects on virus RNA synthesis.

When Semliki Forest virus ts-4 mutant infected cultures are grown at the permissive temperature (28 degrees C) and shifted to the restrictive temperature (39 degrees C), two different defects in RNA synthesis are manifested: (i) the synthesis of 26S RNA is stopped within 60 min (Saraste et al. 1977) and (ii) the increase in RNA synthesizing activity ceases, in contrast to cultures maintained at 28 degrees C, indicating that no new active RNA polymerase is formed at 39 degrees C. Accumulation of a non-structural precursor protein with an apparent mol. wt. of about 220 000 (ns220) was demonstrated in ts-4 infected cultures shifted to 39 degrees C. NS220 was labelled during short pulses given immediately after release of protein synthesis from hypertonic initiation block, suggesting that genes coding for ns220 are located near the initiation site at the 5'-end of the 42S RNA. The viral specificity of ns220 was shown by its disappearance after a shift to 28 degrees C and by labelling in the presence of sucrose, when no host cell protein synthesis is detectable. The two functional defects can be explained if the polypeptides responsible for the RNA polymerizing activity and that responsible for the synthesis of 26S RNA are components of the same non-structural polyprotein. A mutation in the latter polypeptide which prevents cleavage of the polyprotein would thereby prevent the further formation of active RNA polymerase. If cleavage of the polyprotein has taken place at the permissive temperature, the RNA polymerase would remain active also at 39 degrees C, whereas the polypeptide responsible for 26S RNA synthesis would become inactive due to the mutation.

Cell Line↗
PubMed1998

Assembly of viroplasm and virus-like particles of rotavirus by a Semliki Forest virus replicon.

In this study we have used an expression system based on Semliki Forest virus (SFV) to study assembly and intracellular localization of certain capsid proteins of rotavirus in neurons and mammalian epithelial cells. The complete genes of vp2 (vp2A) and vp6 (vp6A) of group A rotavirus (SA-11) and gene 5 encoding vp6 (vp6C) of porcine group C rotavirus (strain Cowden/AmC-1) were inserted into an SFV expression replicon. Transfection of BHK-21 cells with in vitro-made SFV transcripts resulted in a high level of expression of the heterologous genes. Cotransfection with helper RNA encoding the SFV structural proteins, but lacking the genomic RNA packing signal, resulted in production of recombinant infectious virus. Immunological and biochemical analysis revealed that vp6 was expressed to high levels in primary neurons and mammalian epithelial cells and that vp6 was retained as an authentic homotrimer, stabilized by noncovalent interactions with native antigenic determinants. Thin section electron microscopy analysis revealed that vp6 alone assembled into viroplasm-like structures in the cytoplasm. While coexpression of vp2 and vp6 of group A rotavirus resulted in formation of single-shelled-like particles, no evidence of intracellular assembly was found, suggesting that other viral proteins are required for intracellular formation of single-shelled particles. A notable observation was that the vp6 proteins of group A and C rotaviruses showed different immunofluorescence patterns in BHK-21 cells; vp6C displayed an intense punctate immunofluorescence pattern, while vp6A was characterized by a pronounced filamentous staining in close vicinity to the cytoskeleton.

Animals↗
PubMed1977

The effect of irradiation on demyelination induced by avirulent Semliki Forest virus.

A dose of 500 rad total body irradiation before Semliki Forest virus infection was the most effective in producing multiple foci of demyelination in Swiss mice. Animals receiving this dose had the highest virus titre of Semliki Forest virus persisting in the brain and a delayed antibody response. In spite of extensive demyelination no obvious clinical signs such as paralysis were observed.

Animals↗
PubMed1975

Production of defective interfering virus in the brains of mice by an avirulent, in contrast with a virulent, strain of Semliki forest virus.

An avirulent strain (A7) of Semliki Forest virus formed nearly as much haemagglutinating and complement fixing antigen in the brains of adult mice as a virulent (V13) strain, yet the infectivities of the brain tissues were different by about 100-fold. It appeared therefore that defective virus particles were fomed by A7 but these were not demonstrated by fluorescent antibody studies. In short-term organ cultures of adult mouse brain, A7 derived from mouse brain showed a typical interference pattern in inoculum infectivity response curves. Furthermore, when mixed suspensions of brain-grown V13 and A7 with equal infectivities were inoculated the inoculum infectivity response patterns showed significant depressions of the V13 response at higher inocula. Such interference was not detected if chick cell grown A7 and V13 were substituted for the mouse grown virus. The avirulence of A7 in adult mice and its rapid protective effect against lethal V13 infection could be due to the production of defective interfering virus particles in the brain.

Animals↗
PubMed2002

Novel mutations that control the sphingolipid and cholesterol dependence of the Semliki Forest virus fusion protein.

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a membrane fusion reaction mediated by the E1 membrane protein. Efficient SFV-membrane fusion requires the presence of cholesterol and sphingolipid in the target membrane. Here we report on two mutants, srf-4 and srf-5, selected for growth in cholesterol-depleted cells. Like the previously isolated srf-3 mutant (E1 proline 226 to serine), the phenotypes of the srf-4 and srf-5 mutants were conferred by single-amino-acid changes in the E1 protein: leucine 44 to phenylalanine and valine 178 to alanine, respectively. Like srf-3, srf-4 and srf-5 show striking increases in the cholesterol independence of growth, infection, membrane fusion, and exit. Unexpectedly, and unlike srf-3, srf-4 and srf-5 showed highly efficient fusion with sphingolipid-free membranes in both lipid- and content-mixing assays. Both srf-4 and srf-5 formed E1 homotrimers of decreased stability compared to the homotrimers of the wild type and the srf-3 mutant. All three srf mutations lie in the same domain of E1, but the srf-4 and srf-5 mutations are spatially separated from srf-3. When expressed together, the three mutations could interact to produce increased sterol independence and to cause temperature-sensitive E1 transport. Thus, the srf-4 and srf-5 mutations identify novel regions of E1 that are distinct from the fusion peptide and srf-3 region and modulate the requirements for both sphingolipid and cholesterol in virus-membrane fusion.

Cholesterol↗
PubMed2002

A single amino acid change in the nuclear localization sequence of the nsP2 protein affects the neurovirulence of Semliki Forest virus.

The replicase protein nsP2 of Semliki Forest virus (SFV) has a 648RRR nuclear localization signal and is transported to the nucleus. SFV-RDR has a single amino acid change which disrupts this sequence and nsP2 nuclear transport. In BHK cells, SFV4 and SFV-RDR replicate to high titers, but SFV-RDR is less virulent in mice. We compared the replication of SFV4 and SFV-RDR in adult mouse brain. Both SFV4 and SFV-RDR were neuroinvasive following intraperitoneal inoculation. SFV4 spread rapidly throughout the brain, whereas SFV-RDR infection was confined to small foci of cells. Both viruses infected neurons and oligodendrocytes. Both viruses induced apoptosis in cultured BHK cells but not in the cells of the adult mouse brain. SFV-RDR infection of mice lacking alpha/beta interferon receptors resulted in widespread virus distribution in the brain. Thus, a component of the viral replicase plays an important role in the neuropathogenesis of SFV.

Alphavirus Infections↗
PubMed1974

Virus-induced cytoplasmic membrane structures associated with Semliki Forest virus infection studied by the freeze-etching method.

Intracellular membrane structures associated with the Semliki Forest virus replication process were studied from freeze-etch replicas. Cleaved membrane structures inside the CPV I type vacuoles lacked the typical membrane particles present on most other fractured membranes. CPV II type vacuoles present in thin sections were obscured in the freeze-etch replicas by the cytoplasmic ground substance.

Animals↗
PubMed2006

Deletions in the hypervariable domain of the nsP3 gene attenuate Semliki Forest virus virulence.

Major virulence determinants of Semliki Forest virus (SFV) lie within the non-structural genes that form the replicase complex proteins. Gene exchange between virulent and avirulent viruses has shown that the nsP3 gene, which has essential 5' conserved domains and a non-essential hypervariable 3' domain, is one of the virulence determinants. This protein plays a role in subgenomic 26S and negative-strand RNA synthesis and is thought to function with nsP1 to anchor replication complexes to cell membrane structures. Studies to date have focused on analysing the effect of mutational changes spread over the whole gene on virulence of the virus. The virulent SFV4 virus, derived from an infectious clone, was utilized to analyse the effect on virulence of large deletions in the hypervariable domain of nsP3. Two viruses with different in-frame deletions that spanned this domain showed reduced rates of RNA synthesis and multiplication in cell culture. In adult BALB/c mice, these viruses were avirulent after intramuscular and intraperitoneal inoculation, and brains sampled from infected mice showed minimal or no evidence of pathology. These deleted viruses had greatly reduced virulence when administered by the intranasal route and brains from infected mice showed lesions that were much less severe than those seen in SFV4 infection. Mice surviving infection with the deleted viruses resisted challenge with the virulent L10 strain, indicating induction of protective immunity. This work establishes that deletions in the nsP3 hypervariable domain attenuate virulence after peripheral inoculation and also reduce virulence after intranasal inoculation.

Alphavirus Infections↗
PubMed1988

Immunocytochemical evidence for Semliki Forest virus antigen persistence in mouse brain.

Semliki Forest virus (SFV) is neurotropic in mice. Mature virulent virus (strain L10) can be identified within the CNS by electron microscopy in adult mice. Inspite of high virus titres, avirulent SFV A7(74) cannot be visualised in the brain of adult mice. Immunocytochemical studies using monoclonal antibodies (MAbs) to A7(74) E1 and E2 proteins and viral envelope glycolipids, showed viral E1 to be labelled in the cerebral capillaries, the E2 and the putative envelope glycolipids were labelled in the cytoplasm of neurons, particularly in the hippocampal areas and glia in the cerebellum. By double labelling the presence of viral antigens in astrocytes and oligodendrocytes was demonstrated. Viral antigens were identified in the brain up to 183 days after infection. Paraffin sections from Bouin-fixed tissue were found to be the most suitable material for immunocytochemistry of SFV. The presence of life-long anti SFV antibody in the sera of animals after SFV infection, could be due to the persistence of viral antigens acting as constant stimuli to the immune system.

Animals↗
PubMed1979

Messenger ribonucleoprotein complexes containing in vitro-synthesized 26S and 42S Semliki Forest virus RNA.

An extract derived from Semliki Forest virus (SFV) infected cells is described which catalyzes the synthesis of virus-specific RNAs. The newly-synthesized 26S and 42S RNAs are found complexed with protein as messenger ribonucleoproteins (mRNPs). These mRNPs either are non-membrane bound or are associated with large cytoplasmic lipoprotein membranes, and they are found as free mRNPs as well as mRNPs bound to ribosomal subunits, ribosomes, and polysomes. Following treatment with Tween 40 and deoxycholate, membrane-bound mRNPs containing in vitro-synthesized 26S RNA are dissociated and sediment at 33S. These membrane-dissociated mRNPs contain relatively little protein. In contrast, the free or ribosome-bound mRNPs, which are isolated as 30S to 160S particles, remain heterogeneous after detergent treatment and have a much higher protein content. Addition of purified, native 40S ribosomal subunits to the extract leads to the formation of complexes between the added ribosomal subunits and the newly-synthesized viral mRNA. The in vitro-synthesized 26S and 42S RNAs participate in the assembly of translational initiation and elongation complexes.

Cell Line↗
PubMed1985

Differential effects of defective interfering Semliki Forest virus on cellular and virus polypeptide synthesis.

Defective interfering Semliki Forest virus (DI SFV) inhibited virus RNA and virus polypeptide synthesis in cells coinfected with standard virus but did not delay or alter kinetics of RNA synthesis. Inhibition of polypeptide synthesis was 20-fold greater than that of RNA synthesis which presumably reflected the amplification resulting from cumulative translation of mRNAs. At high concentration, DI virus p12e inhibited the shutoff of host protein synthesis and allowed no synthesis of structural or nonstructural polypeptides. Dilution of DI virus restored the inhibition of host protein synthesis but further dilution was necessary before virus-specified polypeptide synthesis could be demonstrated. Another DI virus (p20a) with the same interference titre as p12e also inhibited shutoff of host protein synthesis but synthesis of virus-induced polypeptides was inhibited differentially: significant amounts of polypeptides comigrating with the structural precursor polypeptide p62 and the nonstructural polypeptide nsp63 were present and the synthesis of nsp90 was little affected at any concentration of DI virus p20a tested. None of the DI viruses tested induced the synthesis of any viral or novel polypeptide. It was concluded that DI SFV preparations have qualitatively different interfering activities in relation to their effects on virus and host cell polypeptide synthesis.

Animals↗
PubMed1975

Properties of Semliki Forest virus nucleocapsid.

The nucleocapsid of the Semliki Forest virus is composed of 34% RNA and 66% protein, or one RNA and about 240 capsid protein molecules. The particle is spherical, with a diameter of 38--39 nm. If the nucleocapsid is exposed to slightly acid pH (6.4--5.6) it undergoes a structural change and is contracted to a 32 nm state. A similar contraction can be effected by RNase treatment, in this case, however, in connection with a loss of RNA. Treatment of the nucleocapsid with 0.2 mM SDS results in dissociation of capsid protein from RNA, an effect which suggests strong RNA-protein interaction. At 0.05 mM SDS the protein remains associated with the RNA, but the S-value is reduced from 150 S to 100 S. Electron micrographs of the 100 S ribonucleoprotein showed irregular and strand-like structures.

Capsid↗
PubMed1994

Modulation of thermoprotection and translational thermotolerance induced by Semliki Forest virus capsid protein.

Low amounts of Semliki Forest virus capsid protein transferred into target cells by electroporation-mediated delivery (10(3)-10(4) molecules incorporated/cell) confer thermal resistance resulting in enhanced survival. Furthermore, when exposed to 43 degrees C, these cells display an enhanced expression of heat-shock protein-70 and a translational thermotolerance. Similarly, low amounts of capsid protein transferred into cells in which transcription is blocked by actinomycin D, also protect the translational machinery at 43 degrees C. In a cell-free translation system, added capsid protein appears to modulate translational efficiency of endogenous mRNAs. At approximately 1 molecule/ribosome, capsid protein is able to enhance translation at 30 degrees C and at 43 degrees C. In contrast, high concentrations of capsid protein are responsible for a marked inhibition of protein synthesis at 30 degrees C, but only hamper translational thermotolerance at 43 degrees C. Our results favor the hypothesis that small amounts of capsid protein trigger a chaperone-like activity that is able to protect the translational machinery from thermal damage.

Adaptation, Biological↗
PubMed1999

Polyethylene glycol-mediated infection of non-permissive mammalian cells with semliki forest virus: application to signal transduction studies.

Semliki Forest Virus (SFV) vectors allow the subcloning of a gene of interest directly in the expression vector, thus avoiding the need to select and purify viral recombinants, making this viral expression system attractive over many others for mammalian protein expression. We now describe a novel and generally applicable method for infection of non-permissive mammalian cells with SFV, that greatly enhances the utility of this expression system. We demonstrate that the hygroscopic polymer poly (ethylene glycol), PEG, promotes the infectivity of cells by SFV under conditions that did not promote cell-cell fusion. We also found that the PEG-induced infection and expression of an exogenous protein (green fluorescent protein, GFP) did not elevate the basal tyrosine kinase activity, induce a stress-activated responses, or result in aberrant cell responses. Expression of GFP tagged-Vav, an activator of stress-activated protein kinase (SAPK/JNK), resulted in the expected induction of JNK activity and in the normal redistribution of Vav in response to engagement of the high affinity receptor for IgE (FcepsilonRI). Thus, our findings that PEG allows the infection of non-permissive cells by SFV makes this system extremely attractive for expression of proteins in mammalian cells, and studies on signal transduction and cellular localization in immune and non-immune cells.

3T3 Cells↗
PubMed1996

Expression of the GB virus C E2 glycoprotein using the Semliki Forest virus vector system and its utility as a serologic marker.

A 336-amino-acid segment of the GB virus C second envelope protein (E2) has been produced in BHK-21 cells using the Semliki Forest virus vector system. Secretion of this protein was facilitated by deletion of a hydrophobic region at the C-terminus that may represent the membrane anchoring domain. The E2 protein recovered from the culture supernatant exhibited a molecular mass of approximately 52 kDa, with the increase in size relative to the polyprotein backbone being contributed by N-linked glycosylation. A radioimmunoprecipitation assay using GBV-C E2 was developed to test for the presence of antibodies against this protein in human sera. The prevalence of antibodies to E2 was high among injection drug users and other individuals at risk for acquiring parenterally transmitted agents. There was a much higher percentage of anti-E2 seropositivity in GBV-C RT-PCR negative compared to GBV-C RT-PCR positive samples from these populations. In addition, serial samples from patients transfused with blood containing GBV-C showed seroconversion to anti-E2 positivity and loss of GBV-C viremia as measured by RT-PCR within 11 months of transfusion in five of seven individuals. Thus, this system provided a rapid means to identify GBV-C E2 as a useful antigen for the study of GBV-C exposure.

Base Sequence↗
PubMed1986

Complete nucleotide sequence of the nonstructural protein genes of Semliki Forest virus.

The nucleotide sequence coding for the nonstructural proteins of Semliki Forest virus has been determined from cDNA clones. The total length of this region is 7381 nucleotides, it contains an open reading frame starting at position 86 and ending at an UAA stop codon at position 7379-7381. This open reading frame codes for a 2431 amino acids long polyprotein, from which the individual nonstructural proteins are formed by proteolytic processing steps, so that nsPl is 537, nsP2 798, nsP3 482 and nsP4 614 amino acids. In the closely related Sindbis and Middelburg viruses there is an opal stop codon (UGA) between the genes for nsP3 and nsP4. Interestingly, no stop codon is found in frame in this region of the Semliki Forest virus 42S RNA. In other aspects the amino acid sequence homology between Sindbis, Middelburg and Semliki Forest virus nonstructural proteins is highly significant.

Amino Acid Sequence↗