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At least 73 records · Page 4Linked to original sources
PubMed2003

Expression of the murine CB2 cannabinoid receptor using a recombinant Semliki Forest virus.

A recombinant Semliki Forest virus (SFV) RNA construct, SFV1-mCB(2) RNA, was employed for the high-level expression of the murine CB(2) (mCB(2)) cannabinoid receptor in baby hamster kidney cells. Biosynthetic radiolabel incorporation studies in concert with urea-sodium dodecylsulfate-polyacrylamide gel electrophoresis (urea-SDS-PAGE) and western immunoblotting revealed that two major proteins of approximately 26 and 40kDa were produced by the construct. The 40kDa product, but not the 26kDa product, was glycosylated as determined by 2-deoxy-D-glucose incorporation and peptide-N-glycosidase F digestion analysis. Assessment of [3H]CP55940 ([3H]-(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol) binding data for membranes of cells transfected with SFV1-mCB(2) RNA indicated a K(d) of 0.35+/-0.04nM and a B(max) of 24.4+/-2.7pmol/mg. A rank order of binding affinities for cannabinoids, which paralleled that reported for native mCB(2) receptors, was observed. The CB(2) receptor-specific antagonist SR144528 (N-[(1S)-endo-1,3,3-trimethyl bicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) blocked binding of [3H]CP55940, while the CB(1) receptor-specific antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] had a minimal effect. These results indicate that the recombinant receptor expressed from SFV1-mCB(2) RNA exhibits properties, including ligand binding features, that are consistent with those for the native mCB(2) receptor. However, the presence of both 26 and 40kDa receptor species is consistent with alternative translation from two AUG start sites using the SFV1-mCB(2) RNA expression system.

Amidohydrolases↗
PubMed1997

Infectious particles derived from Semliki Forest virus vectors encoding murine leukemia virus envelopes.

Semliki Forest virus vectors encoding murine leukemia virus (MLV) envelope protein with a truncated cytoplasmic tail generate submicrometer, cell-associated, membranous particles that transmit replication-competent vector RNA specifically to cells bearing the MLV receptor. Such "minimal" viruses could have applications as retroviral vaccines or in the study of virus evolution.

3T3 Cells↗
PubMed1981

Shutoff of neuroblastoma cell protein synthesis by Semliki Forest virus: loss of ability of crude initiation factors to recognize early Semliki Forest virus and host mRNA's.

A crude ribosomal wash containing the initiation factors of protein synthesis was isolated from mouse neuroblastoma cells 8 h after infection with Semliki Forest virus (SFV). The activity of this wash was compared with that of a wash from control cells in a cell-free protein-synthesizing "pH5" system, with early SFV mRNA (42S), late SFV mRNA (26S), encephalomyocarditis virus (EMC) mRNA, or neuroblastoma polyadenylated mRNA templates. A pronounced loss of activity (+/-80%) of the crude ribosomal wash from infected cells was observed with host mRNA (neuroblastoma polyadenylated mRNA) and early SFV mRNA, messengers which contain a cap structure at the 5' terminus. However, these washes were only slightly less active in systems programmed with (noncapped) EMC mRNA and late SFV mRNA. Although late SFV mRNA (26S) is capped, the synthesis of late (= structural) proteins in infected lysates was insensitive to inhibition by cap analogs. Purified initiation factors eIF-4B (M(r), 80,000) and cap-binding protein (M(r), 24,000) from reticulocytes (but none of the others) were able to restore the activity of infected factors to about 90% of control levels in systems programmed with early SFV mRNA and host mRNA. These observations indicate that infection-exposed crude initiation factors have a decreased level of eIF-4B and cap-binding protein activity. However, after partial purification of these and other initiation factors from infected and control cells, we found no significant difference in activity when model assay systems were used. Furthermore, both eIF-4B and cap-binding protein from infected cells were able to restore the activity of these infection-exposed factors to the same level obtained when these factors isolated from control cells or reticulocytes were added. A possible mechanism for the shutoff of host cell protein synthesis is discussed.

Animals↗
PubMed1998

Human MxA protein confers resistance to Semliki Forest virus and inhibits the amplification of a Semliki Forest virus-based replicon in the absence of viral structural proteins.

Mx proteins form a small family of interferon (IFN)-induced GTPases with potent antiviral activity against various negative-strand RNA viruses. To examine the antiviral spectrum of human MxA in homologous cells, we stably transfected HEp-2 cells with a plasmid directing the expression of MxA cDNA. HEp-2 cells are permissive for many viruses and are unable to express endogenous MxA in response to IFN. Experimental infection with various RNA and DNA viruses revealed that MxA-expressing HEp-2 cells were protected not only against influenza virus and vesicular stomatitis virus (VSV) but also against Semliki Forest virus (SFV), a togavirus with a single-stranded RNA genome of positive polarity. In MxA-transfected cells, viral yields were reduced up to 1,700-fold, and the degree of inhibition correlated well with the expression level of MxA. Furthermore, expression of MxA prevented the accumulation of 49S RNA and 26S RNA, indicating that SFV was inhibited early in its replication cycle. Very similar results were obtained with MxA-transfected cells of the human monocytic cell line U937. The results demonstrate that the antiviral spectrum of MxA is not restricted to negative-strand RNA viruses but also includes SFV, which contains an RNA genome of positive polarity. To test whether MxA protein exerts its inhibitory activity against SFV in the absence of viral structural proteins, we took advantage of a recombinant vector based on the SFV replicon. The vector contains only the coding sequence for the viral nonstructural proteins and the bacterial LacZ gene, which was cloned in place of the viral structural genes. Upon transfection of vector-derived recombinant RNA, expression of the beta-galactosidase reporter gene was strongly reduced in the presence of MxA. This finding indicates that viral components other than the structural proteins are the target of MxA action.

3T3 Cells↗
PubMed1990

Neuropeptide immunoreactivity and choline acetyltransferase activity in the mouse urinary bladder following inoculation with Semliki Forest Virus.

The effect of Semliki Forest Virus, a known central demyelinating agent and a proposed model for multiple sclerosis, on the innervation of the mouse urinary bladder has been examined 3, 6, 9 and 12 weeks after inoculation. Three weeks after Semliki Forest Virus inoculation, vasoactive intestinal polypeptide content of the bladder was reduced and the density of vasoactive intestinal polypeptide-immunoreactive nerves was decreased in the smooth muscle, but not in the mucosa. Choline acetyltransferase activity and neuropeptide Y and substance P content was normal, as was the pattern of innervation by acetylcholinesterase-containing and neuropeptide Y- and substance P-immunoreactive nerve fibres. Six weeks after Semliki Forest Virus inoculation, the choline acetyltransferase activity was significantly reduced. Between 6 and 9 weeks the level of vasoactive intestinal polypeptide in the bladder of Semliki Forest Virus-infected mice significantly increased, so that at 9 weeks it was higher than the control value. However, by 12 weeks both choline acetyltransferase activity and vasoactive intestinal polypeptide content were normal. At this time, the substantial age-related increase in substance P content of the bladder was more pronounced in the Semliki Forest Virus-treated animals. Thus there are transitory changes in the innervation of the mouse bladder by vasoactive intestinal polypeptide-containing and cholinergic nerve fibres after exposure to a central demyelinating agent which may reflect changes in bladder dysfunction seen in multiple sclerosis patients.

Animals↗
PubMed1991

Fate of the 6K membrane protein of Semliki Forest virus during virus assembly.

The Semliki Forest virus directs the synthesis of three virus-specific transmembrane proteins p62, 6K, and E1, which all are made in equimolar amounts from a polyprotein precursor molecule. The p62 and E1 spike proteins form heterodimeric complexes in the endoplasmic reticulum before being transported to the cell surface where virus budding occurs. In this study we show that the 6K protein becomes associated to the p62E1 complex in the endoplasmic reticulum and transported with the complex to the cell surface. During virus budding, E1 and p62 (which has matured into the E2 protein) are incorporated into new virions whereas the 6K is mostly excluded. Virus particles released from infected BHK cells contain only about 3% of 6K in their membrane as compared to the spike protein content. The relevance of these findings for the mechanism of SFV assembly is discussed.

Animals↗
PubMed1994

Nuclear targeting of Semliki Forest virus nsP2.

The Semliki Forest virus-specific nonstructural protein nsP2 is transported into the nuclei of both infected and transfected BHK cells. The pentapeptide sequence P 648R RRV is an essential part of the nuclear localization signal (NLS) of nsP2, the middle arginine being the most critical residue for nuclear targeting. Host DNA and RNA syntheses are rapidly inhibited in virus-infected cells, and nsP2 could be involved in these processes. It has been postulated that the inhibition of cellular replication could be due to viral NTPase activity. We have expressed and purified nsP2 in E. coli using the highly efficient T7 based expression system. Purified nsP2 was shown to have ATPase and GTPase activities, and these specific activities were increased in the presence of single-stranded RNA, a typical feature of RNA helicases. The role of nsP2 in the nucleus was studied by creating a mutant virus SFV-RDR, which contained an altered NLS (PRDRV). The mutation affected neither the processing nor the stability of nsP2, but it did render nsP2 completely cytoplasmic. SFV-RDR was shown to be fully infectious, and no difference could be seen in the expression of viral proteins. In addition, the inhibition of host DNA synthesis was almost equally efficient in both wild-type and mutant-infected cells. The pathogenic properties of the mutant will be further studied.

Animals↗
PubMed2006

Role of the amphipathic peptide of Semliki forest virus replicase protein nsP1 in membrane association and virus replication.

Semliki Forest virus RNA replication takes place in association with specific cytoplasmic vacuoles, derived from the endosomal apparatus. Of the four virus-encoded replicase proteins, nsP1 serves as the membrane anchor of the replication complex. An amphipathic peptide segment, G245STLYTESRKLLRSWHLPSV264, has been implicated in the membrane binding of nsP1. nsP1 variants with changes within the peptide were studied after protein expression and in the context of virus infection. Proteins with mutations R253E and W259A accumulated in the cytoplasm and were very poorly palmitoylated. The same mutations also drastically affected the localization of the precursor polyprotein P123, and they were lethal when introduced into the virus genome. Mutations R253A and L255A+L256A partially changed the localization of nsP1, and the respective viruses acquired compensatory changes. L255A+L256A only yielded virus encoding L255A+L256V, indicating the importance of a hydrophobic residue in the central 256 position. When fused to green fluorescent protein, the peptide was required in at least two tandem copies to effect a change in localization, but even then the fusion protein was associated with membranes in a nonspecific manner. Thus, the amphipathic peptide is a crucial element for the membrane association of nsP1 and the replication complex. It provides essential affinity for membranes, and other regions of nsP1 also appear to contribute to the localization of the protein.

Amino Acid Sequence↗
PubMed1986

Immunological relationship between a demyelinating RNA enveloped budding virus (Semliki Forest) and brain glycolipids.

A monoclonal antibody (MAb) raised against central nervous system (CNS) myelin (212) and a MAb (308) raised against brain with Semliki Forest virus (SFV) were both found to react against the same CNS glycolipids. Both these MAbs were also found to react strongly with SFV and against certain brain glycolipid fractions in an immunosorbent assay (ELISA). This demonstrates the presence of common glycolipid antigens in the viral envelope and CNS myelin. MAb 212 had no SFV neutralising capacity and that of MAb 308 was not significant. However, MAb 212 inhibited the neutralisation of the virus by the MAbs (302, 307) specific to SFV proteins. The implications of these findings in relation to the viral induced CNS autoimmunity and persistence of virus in the CNS is discussed.

Antibodies, Monoclonal↗
PubMed1980

pH-dependent fusion between the Semliki Forest virus membrane and liposomes.

Semliki Forest virus was mixed with liposomes containing phosphatidylcholine,phosphatidylethanolamine, sphingomyelin, and cholesterol. When the pH of the mixture was dropped to 6 or below, rapid fusion between the membranes of the virus and the liposomes occurred, resulting in the transfer of viral nucleocapsids into the liposomes. Fusion was demonstrated biochemically by trapping RNase or trypsin within the liposomes. Trapped RNase digested the viral RNA into acid-soluble form, providing a simple quantitative assay for fusion. Trapped trypsin digested the viral capsid protein. Fusion was also demonstrated by electron microscopy as the formation of large vesicles containing viral glycoproteins on the surface and nucleocapsids inside. The efficiency of fusion was 91 +/- 6%. In addition to low pH, it required that the viral glycoproteins be intact. In the target liposomes, cholesterol (but none of the individual phospholipids) was essential. Divalent cations were not required. Our previous studies with tissue culture cells indicated that the final step in the penetration of the Semliki Forest virus genome into host cells might involve a fusion event between the membrane of lysosomally trapped viruses and the lysosomal membrane [Helenius, A., Kartenbeck, J., Simons, K. & Fries, E. (1980) J. Cell Biol, 84, 404--420]. The data presented here are fully compatible with this hypothesis.

Capsid↗
PubMed1994

Assembly and entry mechanisms of Semliki Forest virus.

The alphavirus Semliki Forest (SFV) is an enveloped virus with a positive single-stranded RNA genome. The genome is complexed with 240 copies of a capsid protein into a nucleocapsid structure. In the membrane the virus carries an equal number of copies of a membrane protein heterodimer. The latter oligomers are grouped into clusters of three. These structures form the spikes of the virus and carry its entry functions, that is receptor binding and membrane fusion activity. The membrane protein heterodimer is synthesized as a p62E1 precursor protein which upon transport to the cell surface is cleaved into the mature E2E1 form. Recent studies have given much new information on the assembly and entry mechanism of this simple RNA virus. Much of this work has been possible through the construction of a complete cDNA clone of the SFV genome which can be used for in vitro transcription of infectious RNA. One important finding has been to show that a spike deletion variant and a capsid protein deletion variant are budding-negative when expressed separately but can easily complement each other when transfected into the same cell. This shows clearly that enveloped viruses use different budding strategies: one which depends on a nucleocapsid-spike interaction as exemplified by SFV and another one which is based on a direct core-lipid bilayer interaction as shown before to be the case with retroviruses. Another important finding concerns the activation process of the presumed fusion protein of SFV, the E1 subunit. In the original p62E1 heterodimer E1 is completely inactive. Activation proceeds in several steps. First p62 cleavage activates the potential for low pH inducible fusion. Next the low pH which surrounds incoming virus in endosomes induces dissociation of the heterodimeric structure. This is followed by a rearrangement of E1 subunits into homotrimers which are fusion active.

Animals↗
PubMed1976

Inactivation of Semliki Forest Virus in aerosols.

Purified Semliki forest virus in aerosols is inactivated rapidly at 40% and above 70% relative humidity. At all humidities tested the decay of virus infectivity runs parallel with the decrease in hemagglutination activity, whereas the biological integrity of the virus ribonucleic acid is preserved. Also, free infectious ribonucleic acid is stable after spraying at all relative humidities. Evidence is presented for the hypothesis that above 20% relative humidity, virus inactivation in aersols is mainly due to surface-dependent factors, damaging the virus coat.

Aerosols↗
PubMed2003

Expression of the nucleoprotein of the Puumala virus from the recombinant Semliki Forest virus replicon: characterization and use as a potential diagnostic tool.

Puumala virus (Bunyaviridae family, Hantavirus genus) causes a mild form of hemorrhagic fever with renal syndrome (HFRS) called nephropathia epidemica in northern and central Europe. Serological tests are used for diagnosis, but antigen production is difficult because the virus grows poorly in tissue culture. We expressed the N protein (nucleoprotein) of Puumala virus via the Semliki Forest virus (SFV) replicon in mammalian cells and compared its antigenic properties with those of the native antigen derived from Puumala virus-infected cells. Detection of immunoglobulin G or immunoglobulin M by enzyme-linked immunosorbent assay (ELISA), micro -capture ELISA, and indirect immunofluorescence assay was (at least) as effective with the recombinant antigen as with the native antigen when HFRS patient sera or organ washes from wild rodents were tested. No nonspecific reaction was observed. Thus, the SFV-expressed N protein of Puumala virus appears as a valid antigen, specific and sensitive for serological investigations.

Animals↗
PubMed1993

Cholesterol is required in the exit pathway of Semliki Forest virus.

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a membrane fusion reaction triggered by low pH. For fusion to occur cholesterol is required in the target membrane, as demonstrated both in in vitro fusion assays and in vivo for virus infection of a host cell. In this paper we examine the role of cholesterol in postfusion events in the SFV life cycle. Cholesterol-depleted insect cells were transfected with SFV RNA or infected at very high multiplicities to circumvent the fusion block caused by the absence of cholesterol. Under these conditions, the viral spike proteins were synthesized and transported to the site of p62 cleavage with normal kinetics. Surprisingly, the subsequent exit of virus particles was dramatically slowed compared to cholesterol-containing cells. The inhibition of virus production could be reversed by the addition of cholesterol to depleted cells. In contrast to results with SFV, no cholesterol requirement for virus exit was observed for the production of either the unrelated vesicular stomatitis virus or a cholesterol-independent SFV fusion mutant. Thus, cholesterol was only critical in the exit pathway of viruses that also require cholesterol for fusion. These results demonstrate a specific and unexpected lipid requirement in virus exit, and suggest that in addition to its role in fusion, cholesterol is involved in the assembly or budding of SFV.

Aedes↗
PubMed1990

Biosynthesis, maturation, and acid activation of the Semliki Forest virus fusion protein.

The Semliki Forest virus spike protein has a potent membrane fusion activity which is activated in vivo by the low pH of endocytic vacuoles. The spike protein is composed of two transmembrane subunits, E1 and E2, plus E3, a peripheral polypeptide. Acid-induced conformational changes in the E1 or E2 subunits were analyzed by using monoclonal antibodies specific for the acid-treated spike protein. E1 and E2 reacted with the antibodies after treatment of wild-type or mutant virus at the pH of fusion. The E1 conformational change resembled fusion in its requirement for both low pH and cholesterol. Pulse-chase analysis and intracellular pH treatment were then used to determine the ability of the newly synthesized spike to undergo acid-induced conformational changes. p62, the precursor to E2 and E3, was shown to undergo a pH-dependent conformational change similar to that of E2 and was sensitive to acid very soon after biosynthesis. In contrast, a posttranslational maturation event was required for the conversion of E1 to the pH-sensitive form. E1 maturation occurred fairly late in the exocytic pathway, after the virus spike had passed the medial Golgi but before incorporation of the spike into a new virus particle.

Acetylglucosaminidase↗
PubMed1999

Growth and stability of a cholesterol-independent Semliki Forest virus mutant in mosquitoes.

Semliki Forest virus (SFV) is an enveloped alphavirus that is transmitted in the wild by mosquito vectors. In tissue culture cells, SFV requires cholesterol in the cell membrane both for virus membrane fusion and for the efficient exit of progeny virus from the cell. A previously isolated SFV mutant, srf-3, is strikingly less cholesterol-dependent for virus fusion, exit, and growth due to a single amino acid change in the E1 spike protein subunit, proline 226 to serine. Here we show that when mosquitoes were infected by intrathoracic injection at a range of virus multiplicities, the growth of srf-3 was significantly more rapid than that of wild-type virus, particularly at low multiplicity infection. The differential cholesterol requirements for wild-type and srf-3 infection were maintained during virus passage through mosquitoes. The presence or absence of cholesterol in the srf-3 virus membrane did not affect its infection properties in mosquitoes. Thus the srf-3 mutation causes a growth advantage in the tissues of the mosquito host.

Alphavirus Infections↗
PubMed2005

Rab7 associates with early endosomes to mediate sorting and transport of Semliki forest virus to late endosomes.

Semliki forest virus (SFV) is internalized by clathrin-mediated endocytosis, and transported via early endosomes to late endosomes and lysosomes. The intracellular pathway taken by individual fluorescently labeled SFV particles was followed using immunofluorescence in untransfected cells, and by video-enhanced, triple-color fluorescence microscopy in live cells transfected with GFP- and RFP-tagged Rab5, Rab7, Rab4, and Arf1. The viruses progressed from Rab5-positive early endosomes to a population of early endosomes (about 10% of total) that contained both Rab5 and Rab7. SFV were sequestered in the Rab7 domains, and they were sorted away from the early endosomes when these domains detached as separate transport carriers devoid of Rab5, Rab4, EEA1, Arf1, and transferrin. The process was independent of Arf1 and the acidic pH in early endosomes. Nocodazole treatment showed that the release of transport carriers was assisted by microtubules. Expression of constitutively inactive Rab7T22N resulted in accumulation of SFV in early endosomes. We concluded that Rab7 is recruited to early endosomes, where it forms distinct domains that mediate cargo sorting as well as the formation of late-endosome-targeted transport vesicles.

ADP-Ribosylation Factor 1↗