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Results for “SEMLIKI FOREST VIRUS”

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PubMed1988

Impaired interleukin 1 and interleukin 2 production following in vitro abortive infection of murine spleen mononuclear cells by Semliki Forest virus.

The effect of Semliki Forest virus (SFV) infection of murine spleen mononuclear cells was investigated in vitro. A small percentage of spleen macrophages expressed viral antigens, but no infectious virus particles were released, indicating an abortive-type infection. Wild-type SFV infected a higher percentage of macrophages than the attenuated, demyelinating mutant A7. The proliferation of spleen mononuclear cells under Con A stimulation was inhibited by the viral infection. The supernatant (SN) harvested from infected and Con A-stimulated spleen adherent cells could not stimulate thymocytes in an interleukin 1 (IL-1) assay and indomethacin treatment of infected cultures had no effect. The stimulatory effect of SN from noninfected cultures in the IL-1 assay was reduced when SN from infected cultures was added, suggesting the presence of an IL-1 inhibitor. Interleukin 2 (IL-2) production by splenocytes also decreased after viral infection, but exogenous IL-2 restored the response to Con A stimulation of infected spleen cells. This study demonstrates that abortive SFV infection of spleen macrophages has an immunosuppressive effect which may lead to an aberrant immune regulation.

Animals↗

PubMed2000

Replicase complex genes of Semliki Forest virus confer lethal neurovirulence.

Semliki Forest virus (SFV) is a mosquito-transmitted pathogen of small rodents, and infection of adult mice with SFV4, a neurovirulent strain of SFV, leads to lethal encephalitis in a few days, whereas mice infected with the avirulent A7(74) strain remain asymptomatic. In adult neurons, A7(74) is unable to form virions and hence does not reach a critical threshold of neuronal damage. To elucidate the molecular mechanisms of neurovirulence, we have cloned and sequenced the entire 11,758-nucleotide genome of A7(74) and compared it to the highly neurovirulent SFV4 virus. We found several sequence differences and sought to localize determinants conferring the neuropathogenicity by using a panel of chimeras between SFV4 and a cloned recombinant, rA774. We first localized virulence determinants in the nonstructural region by showing that rA774 structural genes combined with the SFV4 nonstructural genome produced a highly virulent virus, while a reciprocal recombinant was asymptomatic. In addition to several amino acid mutations in the nonstructural region, the nsp3 gene of rA774 displayed an opal termination codon and an in-frame 21-nucleotide deletion close to the nsp4 junction. Replacement in rA774 of the entire nsp3 gene with that of SFV4 reconstituted the virulent phenotype, whereas an arginine at the opal position significantly increased virulence, leading to clinical symptoms in mice. Completion of the nsp3 deletion in rA774 did not increase virulence. We conclude that the opal codon and amino acid mutations other than the deleted residues are mainly responsible for the attenuation of A7(74) and that the attenuating determinants reside entirely in the nonstructural region.

Alphavirus Infections↗

PubMed1999

RNA helicase activity of Semliki Forest virus replicase protein NSP2.

Semliki Forest virus replicase protein nsP2 shares sequence homology with several putative NTPases and RNA helicases. NsP2 has RNA-dependent NTPase activity. Here we expressed polyhistidine-tagged nsP2 in Escherichia coli, purified it by metal-affinity chromatography, and used it in RNA helicase assays. RNA helicase CI of plum pox potyvirus was used as a positive control. Unwinding of alpha-32P-labelled partially double-stranded RNA required nsP2, Mg2+ and NTPs. NsP2 with a mutation, K192N, in the NTP-binding sequence GVPGSGK192SA could not unwind dsRNA and had no NTPase activity. This is the first demonstration of RNA helicase activity within the large alphavirus superfamily.

Cysteine Endopeptidases↗

PubMed2001

Induction of therapeutic antitumor antiangiogenesis by intratumoral injection of genetically engineered endostatin-producing Semliki Forest virus.

Antiangiogenic therapy using Semliki Forest virus (SFV) carrying Endostatin gene for malignant brain tumor was investigated to improve the therapeutic efficacy. The efficiency of SFV-mediated gene delivery was first evaluated for B 16 cells and compared with the efficiency in cells of endothelial origin (HMVECs). HMVECs are more susceptible to SFV infection than B 16 cells. For the in vivo treatment model, phosphate-buffered saline, SFV-LacZ, retrovirus vector GCsap-Endostatin, and SFV-Endostatin were injected to mice bearing B 16 brain tumors. A very significant inhibition of tumor growth was observed in the group that had been treated with SFV-Endostatin. A marked reduction of intratumoral vascularization was seen in the tumor sections from the SFV-Endostatin group compared with tumor sections from the SFV-LacZ or GCsap-Endostatin groups. Moreover, at day 7 after intravenous administration of SFV-Endostatin, the serum level of endostatin was augmented more than 3-fold compared to that after intravenous administration of GCsap-Endostatin. The results indicated that treatment with SFV-Endostatin inhibited the angiogenesis with established tumors. Gene therapy with Endostatin delivered via SFV may be a candidate for the development of new therapy for brain tumors.

Animals↗

PubMed1992

Nuclear and nucleolar targeting signals of Semliki Forest virus nonstructural protein nsP2.

Semliki Forest virus nonstructural protein nsP2, when expressed in the absence of other viral proteins, is transported into the nucleus, with a specific enrichment in nucleoli. This implies that information for its nuclear targeting must be in nsP2 itself. To define the nuclear and nucleolar targeting signals of nsP2, a series of in-frame deletions were made in its coding sequence. We have identified one amino-terminal (residues 25-110) and another carboxy-terminal (residues 634-661) deletion, both of which rendered nsP2 cytoplasmic. The carboxy-terminal region had a pentapeptide sequence P 648R RRV, which resembles some of the known nuclear localization signals. When the three arginines were substituted with aspartic acids, the mutant nsP2 was localized in the cytoplasm. Especially arginines 648-649 were shown to be critical. The carboxy-terminal 232 amino acid residues of nsP2 were able to translocate beta-galactosidase to the nucleus as a fusion protein, whereas the amino-terminal 110 residues failed to do so. Studies with different beta-galactosidase-nsP2 fusions showed that the pentapeptide is necessary for nuclear transport but that its activity is strongly affected by the context. The deletion analysis indicated also that the nuclear localization signal was not sufficient for nucleolar targeting of nsP2. The nucleolar targeting sequence of nsP2 was tentatively mapped between residues 470 and 539, and at least residues 470-489 are an essential part of it.

Animals↗

PubMed1996

Functional significance of the nuclear-targeting and NTP-binding motifs of Semliki Forest virus nonstructural protein nsP2.

Semliki Forest virus-specific polypeptide nsP2 is a nonstructural protein involved in multiple steps during viral RNA replication. It was recently shown to possess single-stranded RNA-stimulated ATPase and GTPase activities. Replacement of the highly conserved lysine (Lys-192) within the classical nucleotide-binding motif A/GXXGXGKS/T with asparagine abolished its NTP-hydrolyzing activity. Also, about half of nsP2 is transported into the nucleus during viral infection. Substitution of the second arginine in its nuclear localization signal (P648RRRV) with aspartic acid rendered nsP2 totally cytoplasmic. To assess the functional importance of these sequence motifs, the same mutations were introduced into a cDNA clone of Semliki Forest virus, from which infectious RNA can be produced in vitro. Transfection of an RNA encoding Lys-192 --> Asn mutation into BHK cells did not promote viral infection. However, revertants encoding the wild-type amino acid were obtained. Cells transfected with RNA coding for Arg-649 --> Asp mutation gave rise to infectious virus termed SFV-RDR. Indirect immunofluorescence and subcellular fractionation of SFV-RDR-infected cells confirmed the cytoplasmic localization of nsP2. Measurement of host DNA synthesis late in infection revealed that infection with the parental virus inhibited DNA synthesis to 10% of control cells. In contrast, infection with SFV-RDR led only to a partial shutoff of cellular DNA synthesis. Mice experiments indicated that the pathogenicity of SFV-RDR was attenuated.

Adenosine Triphosphate↗

PubMed1985

pH-induced alterations in the fusogenic spike protein of Semliki Forest virus.

The spike glycoproteins of Semliki Forest virus mediate membrane fusion between the viral envelope and cholesterol-containing target membranes under conditions of mildly acidic pH (pH less than 6.2). The fusion reaction is critical for the infectious cycle, catalyzing virus penetration from the acidic endosome compartment. To define the role of the viral spike glycoproteins in the fusion reaction, conformational changes in the spikes at acid pH were studied using protease digestion and binding assays to liposomes and nonionic detergent. A method was also developed to prepare fragments of both transmembrane subunit glycopolypeptides of the spike, E1 and E2, which lacked the hydrophobic anchor peptides. Unlike the intact spikes the fragments were monomeric and therefore useful for obtaining information on conformational changes in individual subunits. The results showed that both E1 and E2 undergo irreversible conformational changes at the pH of fusion, that the conformational change of E1 depends, in addition to acidic pH, on the presence of cholesterol, and that no major changes in the solubility properties of the spikes takes place. On the basis of these findings it was concluded that fusion involves both subunits of the spike and that E1 confers the stereo-specific sterol requirement. The results indicated, moreover, that acid-induced fusion of Semliki Forest virus differs in important respects from that of influenza virus, another well-defined model system for protein-mediated membrane fusion.

Cholesterol↗

PubMed1984

The effect of liposomal charge on the neutralizing antibody response against inactivated encephalomyocarditis and Semliki Forest viruses.

The primary neutralizing antibody response to encephalomyocarditis (EMC) virus and Semliki Forest virus (SFV) is enhanced by addition of either negatively or positively charged liposomes. The purified u.v. light inactivated viruses were merely mixed with liposomes and injected intraperitoneally (i.p.) into mice. In contrast, neutral liposomes were unable to enhance the primary response to these viruses. Furthermore primary immunization with inactivated SFV mixed with either neutral, positively or negatively charged liposomes was associated with an enhancement of the secondary humoral response after i.p. booster injection of mice with inactivated virus alone. But neutral liposomes seemed to be less effective in this respect than either positively or negatively charged liposomes.

Animals↗

PubMed1953

An antiviral substance from Penicillium funiculosum. II. Effect of helenine upon infection in mice with Semliki Forest virus.

Helenine exerts a therapeutic effect against Semliki Forest virus infections of mice. Cures, that is to say the survival of treated animals, were more frequently observed in Semliki Forest virus infections than they were in SK virus infections. It is believed that this difference in end-result probably represented only a quantitative difference in the therapeutic effect of helenine against these two viruses and not a qualitative difference in its mechanism of therapeutic action. The findings reported in this paper with regard to the treatment of Semliki Forest virus infections with helenine parallel very closely those described in an accompanying paper which deals with the action of helenine on SK. virus infections.

Animals↗

PubMed1999

An epitope of the Semliki Forest virus fusion protein exposed during virus-membrane fusion.

Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells via a membrane fusion reaction triggered by acidic pH in the endocytic pathway. Fusion is mediated by the spike protein E1 subunit, an integral membrane protein that contains the viral fusion peptide and forms a stable homotrimer during fusion. We have characterized four monoclonal antibodies (MAbs) specific for the acid conformation of E1. These MAbs did not inhibit fusion, suggesting that they bind to an E1 region different from the fusion peptide. Competition analyses demonstrated that all four MAbs bound to spatially related sites on acid-treated virions or isolated spike proteins. To map the binding site, we selected for virus mutants resistant to one of the MAbs, E1a-1. One virus isolate, SFV 4-2, showed reduced binding of three acid-specific MAbs including E1a-1, while its binding of one acid-specific MAb as well as non-acid-specific MAbs to E1 and E2 was unchanged. The SFV 4-2 mutant was fully infectious, formed the E1 homotrimer, and had the wild-type pH dependence of infection. Sequence analysis demonstrated that the relevant mutation in SFV 4-2 was a change of E1 glycine 157 to arginine (G157R). Decreased binding of MAb E1a-1 was observed under a wide range of assay conditions, strongly suggesting that the E1 G157R mutation directly affects the MAb binding site. These data thus localize an E1 region that is normally hidden in the neutral pH structure and becomes exposed as part of the reorganization of the spike protein to its fusion-active conformation.

Animals↗

PubMed1979

Comparison of the structural properties of Sindbis and Semliki forest virus nucleocapsids.

The envelope spikes of Sindbis and Semliki Forest virus are arranged in a T = 4 icosahedral surface lattice and, by deduction, it has been suggested that the nucleocapsid proteins are similarly arranged. After treatment of the virions with a non-ionic detergent the released nucleocapsids sediment in sucrose gradients at about 160S and 150S and have densities in CsCl of 1.42 g/ml and 1.425 g/ml, respectively, for Sindbis and Semliki Forest virus. At pH 6.0 Sindbis nucleocapsids do not contract like those of Semliki Forest virus. Nucleocapsids of both viruses are sensitive to the action of ribonuclease but only those of Semliki Forest virus undergo a drastic structural rearrangement due to the treatment. EDTA treatment in hypotonic conditions results in a decrease in the S-value for both particles. Electron micrographs show that the SFV nucleocapsids are partly 'unfolded' while those of Sindbis appear slightly contracted after exposure to EDTA.

Capsid↗

PubMed1979

Functional defects of RNA-negative temperature-sensitive mutants of Sindbis and Semliki Forest viruses.

Defects in RNA and protein synthesis of seven Sindbis virus and seven Semliki Forest virus RNA-negative, temperature-sensitive mutants were studied after shift to the restrictive temperature (39 degrees C) in the middle of the growth cycle. Only one of the mutants, Ts-6 of Sindbis virus, a representative of complementation group F, was clearly unable to continue RNA synthesis at 39 degrees C, apparently due to temperature-sensitive polymerase. The defect was reversible and affected the synthesis of both 42S and 26S RNA equally, suggesting that the same polymerase component(s) is required for the synthesis of both RNA species. One of the three Sindbis virus mutants of complementation group A, Ts-4, and one RNA +/- mutant of Semliki Forest virus, ts-10, showed a polymerase defect even at the permissive temperature. Seven of the 14 RNA-negative mutants showed a preferential reduction in 26S RNA synthesis. The 26S RNA-defective mutants of Sindbis virus were from two different complementation groups, A and G, indicating that functions of two viral nonstructural proteins ("A" and "G") are required in the regulation of the synthesis of 26S RNA. Since the synthesis of 42S RNA continued, these functions of proteins A and G are not needed for the polymerization of RNA late in infection. The RNA-negative phenotype of 26S RNA-deficient mutants implies that proteins regulating the synthesis of this subgenomic RNA must have another function vital for RNA synthesis early in infection or in the assembly of functional polymerase. Several of the mutants having a specific defect in the synthesis of 26S RNA showed an accumulation of a large nonstructural precursor protein with a molecular weight of about 200,000. One even larger protein was demonstrated in both Semliki Forest virus- and Sindbis virus-infected cells which probably represents the entire nonstructural polyprotein.

DNA-Directed RNA Polymerases↗

PubMed1997

Efficient multiplication of a Semliki Forest virus chimera containing Sindbis virus spikes.

Using the Semliki Forest virus (SFV) and Sindbis virus (SIN) cDNAs we have constructed recombinants in which the spike genes were exchanged. Analyses of expression showed that the SFV/SIN(spike) RNA directed efficient assembly of infectious virus, whereas the reciprocal SIN/SFV(spike) RNA was completely unable to assemble virus. This was apparently due to a defective capsid-spike interaction.

Amino Acid Sequence↗

PubMed1975

Mouse encephalitis induced by avirulent Semliki forest virus.

Encephalitis caused by avirulent Semliki Forest virus was studied at regular intervals for 6 weeks in experimentally infected mice. The most obvious lesion was perivascular cuffing. Microglia were predominant near the vessels early in the infection. The cells of the cuffs were mostly lymphocytes. Although the virus produced acute encephalitis, which gradually resolved by 6 weeks, at no time were there abnormal neurological clinical signs. The cells of the brain and the immune response reacted to eliminate the virus.

Animals↗

PubMed1984

Variation in homotypic and heterotypic interference by defective interfering viruses derived from different strains of Semliki Forest virus and from Sindbis virus.

There was strong interference between various virulent and avirulent strains of Semliki Forest virus (SFV) and their respective defective interfering (DI) viruses but in other combinations interference was variable: it could be equally strong, weak or could not be demonstrated. On passage, this spectrum of interfering activity changed, some combinations showing greater interference than before and others less. Heterotypic interference between DI SFV, DI Sindbis virus and standard viruses was clearly demonstrated although this was strongest between DI SFV preparations and Sindbis standard virus than in the reciprocal combinations. Variation in interference between DI SFVs and different SFV strains was similar in magnitude to that between DI SFVs and Sindbis virus, suggesting that a similar DI RNA sequence is recognized by both viruses.

Animals↗

PubMed1993

Fusion of enveloped viruses with sperm cells: interaction of Sendai, influenza, and Semliki Forest viruses with bull spermatozoa.

Incubation of fluorescently labeled Sendai, influenza, as well as Semliki Forest viruses with bull sperm cells resulted in fluorescence dequenching. Fluorescence dequenching was observed with Sendai virus at pH 7.4 while with influenza and Semliki Forest viruses at pH 5.0, a pH value which is required for triggering their fusogenic activity. Control experiments performed with nonfusogenic Sendai and influenza viruses, or with bull sperm cells from which the viral receptors have been removed by treatment with neuraminidase, showed little fluorescence dequenching. These results clearly indicate that animal enveloped viruses are able to interact and to fuse with bull sperm cells. The possibility that following virus-sperm fusion spermatozoa can serve as a carrier of the virus genome and introduce it into recipient eggs during fertilization is discussed.

Animals↗

PubMed2002

Immobilisation of Semliki forest virus for atomic force microscopy.

Semliki Forest virus (SFV), an alphavirus, is a single-stranded positive-sense RNA virus. The RNA genome is surrounded by a protein shell known as the capsid which itself is surrounded by a lipid envelope of host cell origin. In this study, SFV strain L10 enveloped virus and its capsid were immobilised onto silicon wafer supports which had been pre-coated with a monolayer of the relevant anti-viral antibody. After drying, the samples were imaged in air, using non-contact mode atomic force microscopy (AFM). Quantification of the AFM images has revealed that both the strain L10 enveloped virus and capsid collapse when immobilised in this manner. The capsid undergoes more significant collapse compared to the enveloped virus. The dimensions of the immobilised enveloped virus and capsid have been compared to a model where the free spherical particles collapse into ellipsoids during immobilisation. For the immobilised capsid the dimensions are consistent with this model whereas for the enveloped virus the model is less effective. The dimensions of the enveloped virus appear to be affected by the antibody used for immobilisation.

Animals↗

PubMed1986

Kinetics of endosome acidification detected by mutant and wild-type Semliki Forest virus.

The fusogenic properties of Semliki Forest virus (SFV) and its mutants were used to follow the kinetics of acidification during the endocytic uptake of virus by BHK-21 cells. It has previously been shown that the low pH of endocytic vacuoles triggers a conformational change in the SFV spike glycoprotein, activating membrane fusion and initiating virus infection. This conformational alteration was here shown to occur in endosomes and to follow the same time course as the intracellular fusion reaction, demonstrating that fusion occurs rapidly after virus exposure to endosome acidity. The kinetics of endosome acidification were monitored using wild type (wt) SFV and fus-1, an SFV mutant with a lower fusion pH threshold. The results presented here demonstrated that wt and mutant virus were internalized with a t1/2 of 10 min, and that endosomes were acidified to the wt threshold of pH 6.2 with a t1/2 of 15 min. In contrast, endosome pH reached the fus-1 threshold of 5.3 with a much longer t1/2 of 45 min. The subsequent degradation of SFV in lysosomes had a t1/2 of 90 min. It was found that after the initial uptake of virus from the plasma membrane, its transit through the endocytic pathway, exposure to endosome acidity and eventual delivery to lysosomes were markedly asynchronous.

Animals↗