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A Salminen

Publications and source records attributed to A Salminen.

At least 55 records · Page 3Linked to original sources
PubMed1997

Glutamate-induced changes in the DNA-binding complexes of transcription factor YY1 in cultured hippocampal and cerebellar granule cells.

Transcription factor YY1 is a zinc finger protein which can interact and form complexes with several proteins. Depending on its binding partners, YY1 can either activate or repress the transcription of several genes. We have studied whether the regulation of YY1 is affected by glutamate-induced excitotoxic insult in cultured primary hippocampal and cerebellar granule cells. Using electrophoretic mobility shift (EMSA) technique, we observed that glutamate treatment induced a dramatic increase in the YY1-binding activity of the smallest of three complexes both in the hippocampal and cerebellar granule cells. Two larger complexes disappeared after glutamate treatment in cerebellar granule cells. Supershift assays with specific YY1 antibody showed that all three complexes contained YY1 protein. Western blot assays did not show any changes in the nuclear levels of YY1 protein. Our results show that excitotoxic treatment affects the regulation of YY1 transcription factor and suggest that YY1 is a significant nuclear target for stress-related signaling pathways in neuronal degeneration.

Animals↗
PubMed1997

Down-regulation of Ku autoantigen, DNA-dependent protein kinase, and poly(ADP-ribose) polymerase during cellular senescence.

During aging and cellular senescence mutations accumulate in genomic and mitochondrial DNA. Ku autoantigens, DNA-dependent protein kinase, and poly (ADP-ribose) polymerase have an essential role in DNA damage recognition. Our purpose was to find out whether cellular senescence of fibroblasts affects the protein components that recognize DNA damage and induce the repair process. We compared presenescent and replicatively senescent human WI-38 fibroblasts with each other and with SV-40 immortalized and serum-deficient quiescent WI-38 cells. Our results showed that replicative senescence significantly decreased the nuclear level of both p70 and p86 components of Ku autoantigen. SV-40 immortalization and cellular quiescence did not affect the level of the p86 component but slightly increased that of p70. Both replicative senescence and cellular quiescence decreased the activity of DNA-dependent protein kinase in WI-38 fibroblasts. On the other hand, SV-40 immortalization increased the activity of DNA-dependent protein kinase. The protein level of poly(ADP-ribose) polymerase (PARP) was strongly decreased in replicatively senescent fibroblasts. Quiescence of early-passage fibroblasts also slightly reduced the protein level of PARP. Apoptosis was not observed in replicatively senescent fibroblasts. Our results show that replicative senescence and to some extent cellular quiescence down-regulate the recognition system of DNA damage involving Ku autoantigens, DNA-dependent protein kinase, and PARP and hence could enhance the accumulation of DNA damage during aging.

Amino Acid Sequence↗
PubMed1997

Analysis of a yeast SNARE complex reveals remarkable similarity to the neuronal SNARE complex and a novel function for the C terminus of the SNAP-25 homolog, Sec9.

SNARE proteins represent a family of related proteins that are thought to have a central role in vesicle targeting and fusion in all eukaryotic cells. The binding properties of the neuronal proteins synaptobrevin 1 (VAMP1), syntaxin 1, SNAP-25, and soluble N-ethylmaleimide-sensitive factor attachment protein (alpha-SNAP), have been extensively studied. We report here the first biochemical characterization of a nonneuronal SNARE complex using recombinant forms of the yeast exocytic SNARE proteins Snc1, Sso1, and Sec9 and the yeast alpha-SNAP homolog, Sec17. Despite the low level of sequence identity, the association properties of the yeast and neuronal complexes are remarkably similar. The most striking difference we have found between the yeast and neuronal proteins is that individually neither of the target membrane SNAREs (t-SNAREs), Sso1 nor Sec9, show any detectable binding to the synaptobrevin homolog, Snc1. However, as a hetero-oligomeric complex, Sec9 and Sso1 show strong binding to Snc1. The clear dependence on the Sso1-Sec9 complex for t-SNARE function suggests that regulating the formation of this complex may be a key step in determining the site of vesicle fusion. In addition, we have used this in vitro assay to examine the biochemical effects of several mutations in Sec9 that result in pronounced growth defects in vivo. As expected, a temperature-sensitive mutation in the region most highly conserved between Sec9 and SNAP-25 is severely diminished in its ability to bind Sso1 and Snc1 in vitro. In contrast, a temperature-sensitive mutation near the C terminus of Sec9 shows no defect in SNARE binding in vitro. Similarly, a deletion of the C-terminal 17 residues, which is lethal in vivo, also binds Sso1 and Snc1 normally in vitro. Interestingly, we find that these same two C-terminal mutants, but not mutants that show SNARE assembly defects in vitro, act as potent dominant negative alleles when expressed behind a strong regulated promoter. Taken together these results suggest that the C-terminal domain of Sec9 is specifically required for a novel interaction that is required at a step following SNARE assembly.

Amino Acid Sequence↗
PubMed1997

Age-related changes in the regulation of transcription factor NF-kappa B in rat brain.

Aging process involves an increase in stress at cellular level. We studied whether aging affects the regulation of stress responsive transcription factor NF-kappa B in brain samples of Wistar rats. Hippocampus, cerebellum, and temporal and frontal lobes of cortex were studied. We observed a significant up-regulation in the constitutive, nucleus-located NF-kappa B binding activity in 30-month-old Wistar rats compared to young and 18-month-old rats. The increase was most prominent in cerebellum and in frontal cortex, but age-related changes did not occur in hippocampus. Inducible, cytoplasmic NF-kappa B binding activity was not affected by aging in any of the samples studied. Western blot assays did not show any age-related changes in the nuclear level of p50, p52, and p65 protein components of NF-kappa B complex. Cytoplasmic level of inhibitory I kappa B-alpha was also unaffected. The increase in nuclear constitutive NF-kappa B binding activity during aging may be related to the NF-kappa B driven cellular response to adapt neurons against apoptotic pressure, as observed recently in several apoptotic conditions.

Aging↗
PubMed1996

Down-regulation of transcription factors AP-1, Sp-1, and NF-kappa B precedes myocyte differentiation.

Terminal differentiation of myocytes involves withdrawal from the cell cycle, induction of myogenin expression, and finally formation of myotubes. To study the factors that regulate the initial phase of muscle differentiation, we analyzed the binding activities of transcription factors AP-1, Sp-1, and NF-kappa B in L6, C2C12, and rhabdomyosarcoma BA-Han-1C cells. Temporal changes in transcription factor binding activities were compared to the activation of myogenin promoter-driven CAT reporter gene and the expression level of myogenin, a master gene of myogenic differentiation. We observed a prominent decrease in the nuclear binding activities of AP-1, Sp-1, and NF-kappa B already 12 to 24 h after the transfer of cells to differentiation medium. The response was very similar in L6 and C2C12 myocytes and in BA-Han-1C rhabdomyosarcoma cells. The down-regulation clearly preceded the activation of myogenin promoter and the induction of myogenin and retinoblastoma expression, as well as the initiation of myocyte fusion. Cholera toxin and okadaic acid, established inhibitors of myogenin expression and muscle differentiation, strongly up-regulated the binding activities of AP-1, Sp-1, and NF-kappa B in differentiation medium. Myogenin expression and myocyte fusion were also inhibited. Levels of nuclear c-Fos and c-Jun proteins, components of the AP-1 complex, showed a prominent decrease already after 12 h in differentiation medium. These results show that the down-regulation of the proliferation-promoting transcription factors is a prerequisite to the initiation of myocyte differentiation.

Animals↗
PubMed1996

Down-regulation of nuclear binding activities of OXBOX-REBOX transcription factors during cellular senescence.

Functional capacity of mitochondria declines during aging and this impairment may have a major role in aging process. Several observations indicate that transcriptional efficiency is reduced during aging. Our purpose was to find out whether aging and cellular senescence affect the nuclear binding activities of transcription factors which bind to OXBOX-REBOX sequence present in promoter regions of numerous nuclear genes encoding mitochondrial proteins. These factors regulate and coordinate the expression of mitochondrial proteins. We observed a strong down-regulation in the nuclear binding activities of OXBOX-REBOX factors in replicatively senesced human WI-38 and IMR-90 fibroblasts. On the contrary, SV-40 immortalization highly increased the nuclear binding activities. A considerable down-regulation of OXBOX-REBOX factors was also observed in UVB-irradiated WI-38 fibroblasts. Irradiation induced photoaging in fibroblasts which involved cell cycle arrest and senescent morphology. Interestingly, the nuclear binding activities of OXBOX-REBOX factors were also prominently decreased in the liver of Wistar rats at the age of 30 months but not yet at the age of 18 months. Our results suggest that the down-regulation of OXBOX-REBOX factors could affect the expression level of mitochondrial proteins encoded in nucleus and hence induce disturbances in mitochondrial function and promote the cellular aging process.

Animals↗
PubMed1996

Changes associated with aging and replicative senescence in the regulation of transcription factor nuclear factor-kappa B.

Both the aging of animals and the senescence of cultured cells involve an altered pattern of gene expression, suggesting changes in transcription factor regulation. We studied age-related changes in transcription factors nuclear factor (NF)-kappa B, activator protein factor-1 (AP-1) and Sp-1 by using electrophoretic mobility shift binding assays; we also analysed changes in the protein components of NF-kappa B complex with Western blot assays. Nuclear and cytoplasmic extracts were prepared from heart, liver, kidney and brain of young adult and old NMRI mice and Wistar rats as well as from presenescent, senescent and simian virus 40-immortalized human WI-38 fibroblasts. Aging of both mice and rats induced a strong and consistent increase in the nuclear binding activity of NF-kappa B factor in all tissues studied, whereas those of AP-1 and Sp-1 decreased, e.g. in liver. Protein levels of p50, p52 and p65 components of the NF-kappa B complex did not show any age-associated changes in the cytoplasmic fraction but in the nuclear fraction the level of p52 strongly increased in heart and liver during aging. The protein levels of inhibitory I kappa B-alpha and Bcl-3 components were not affected by aging in any of the tissues studied. Replicative cellular senescence of human WI-38 fibroblasts induced a strong decrease in nuclear NF-kappa B, AP-1 and Sp-1 binding activities. Protein levels of p50 and p52 components of NF-kappa B complex were decreased in the nuclear fraction of senescent WI-38 fibroblasts but in the cytoplasm of senescent fibroblasts the level of p65 protein was increased. Cellular senescence also slightly decreased the protein levels of I kappa B-alpha and Bcl-3. Transfection assays with NF-kappa B-enhancer-driven chloramphenicol acetyltransferase reporter gene showed a significant down-regulation of NF-kappa B promoter activity in senescent WI-38 fibroblasts. Results suggest that the aging process might be regulated differently in tissues and cultured fibroblasts, perhaps reflecting differences between mitotic and post-mitotic cells. In tissues, aging seems to involve specific changes in the regulation of NF-kappa B components and perhaps also changes in the DNA-binding affinities of the NF-kappa B complex.

Aging↗
PubMed1996

Aging-induced up-regulation of nuclear binding activities of oxidative stress responsive NF-kB transcription factor in mouse cardiac muscle.

The accumulation of lipofuscin to cardiomyocytes is a classical parameter of aging and is believed to reflect oxidative stress. NF-kB transcription factor complex is one of the cellular sensors which responds to oxidative stress and regulates gene expression. Our purpose was to study whether aging affects the level and distribution of DNA binding activities of NF-kB transcription factors both in cardiac sarcoplasm and nuclear extracts. We used electrophoretic mobility shift assays (EMSA) to characterize the DNA binding activities of NF-kB and two other transcription factors. AP-1 and Sp-1, in the myocardium of 4 months and 24 months old male and female NMRI-mice. The protein levels of p50, p52, and p65 components of NF-kB-complex and an inhibitory IkB-alpha/MAD-3 were assayed with Western blots. Surprisingly, aging upregulated by 123% the nuclear NF-kB binding activity in the male and female mice. The sarcoplasmic NF-kB activity, activated by deoxycholate, did not show any change during aging. Aging-induced increase in nuclear NF-kB protein-DNA binding activity was observed both by gel retardation and UV-crosslinking assays. In immunoblotting, the level of p52 component but not those of p50 and p65 components of NF-kB-complex was slightly increased in nuclear fractions. Aging did not affect the sarcoplasmic levels of p50, p52, and p65 proteins. Supershift EMSA assays showed that the nuclear NF-kB complex contained p50, p52, and p65 components. The level of inhibitory IkB-alpha/MAD-3 protein was unaffected by aging both in nuclear and sarcoplasmic fractions. Aging down-regulated the nuclear Sp-1 binding activities but did not affect AP-1 binding activities. Statistically significant sex-related differences did not appear in the aging responses of transcription factors. These results indicate that NF-kB transcription factor pathway is activated during aging in cardiac muscle and could be the signaling route regulating gene expression. However, the activation mechanism of NF-kB during aging whether oxidative stress responsive or not in vivo needs further studies.

Aging↗
PubMed1995

Alteration of transcription factor binding activities in the ischemic rat brain.

Transient focal ischemia-reperfusion in the cerebral cortex caused regional alteration of DNA-binding activities of transcription factors AP-1, CREB, Sp-1, and NF-kB. The changes were time-dependent. During the first 24 hr of reperfusion after 90 min ischemia, there was an increase in the binding activity of AP-1 only in the region surrounding the ischemic cortex. Five days after ischemia, an increase in the binding activities of CREB, Sp-1, and NF-kB, but not AP-1, was noted in the ischemic cortex, and to a lesser extent, Sp-1 and NF-kB, in the surrounding region. The binding activities of these transcription factors were reduced by hydrogen peroxide but could be restored by dithiothreitol and 2-mercaptoethanol. These results are the first demonstration of ischemia-induced differential regulation of transcription factor binding activities which are time-, region-, and redox state dependent.

Animals↗
PubMed1994

Increased expression of c-fos mRNA and AP-1 transcription factors after cortical impact injury in rats.

Levels of c-fos mRNA and AP-1 transcription factors co-expression were measured in a controlled lateral cortical impact model of traumatic brain injury (TBI) in rats. Ipsilateral cerebral cortex and bilateral hippocampal c-fos mRNA increases were revealed by in situ hybridization after lateral cortical impact injury. Based on regional in situ hybridization data, we employed semi-quantitative RT-PCR methods to study the temporal profile of changes in the ipsilateral cortex at the site of injury. We found that TBI produces transient increases of c-fos mRNA expression in the ipsilateral cerebral cortex at 5 min postinjury, which peaks at 1 h postinjury and subsides by 1 day postinjury. Gel shift nuclear protein binding assays showed that AP-1 transcription factor binding was robustly increased in injured cerebral cortex at 1 h, 3 h, 5 h and 1 day after injury. These data indicate that TBI can produce significant increases in c-fos expression and subsequent upregulation of the AP-1 transcription factors. Thus, AP-1 transcription factors modulation of downstream gene expression may be an important component of pathophysiological responses to TBI.

Animals↗
PubMed1994

Suppression of ischemia-induced fos expression and AP-1 activity by an antisense oligodeoxynucleotide to c-fos mRNA.

Activation of c-fos, an immediate early gene, and the subsequent expression of the Fos protein have been noted following focal cerebral ischemia. Fos and Jun form a heterodimer as activator protein 1 (AP-1), which transregulates the expression of several genes. To study the postischemic events related to c-fos expression, we suppressed the expression of c-fos by intraventricular infusion of an antisense oligodeoxynucleotide (anti-rncfosr115) of c-fos mRNA. The effectiveness of anti-rncfosr115 was confirmed first by its capability to block in vitro c-fos mRNA translation. In vivo, after intraventricular infusion of 32P-labeled anti-rncfosr115, the oligodeoxynucleotide was internalized within 6 hours and detectable also in the nucleic acids fraction up to 41 hours. Treatment of the recovered nucleic acids with RNase H separated the labeled oligodeoxynucleotide from the nucleic acid fraction, indicating an association of the antisense oligodeoxynucleotide and cellular RNA after uptake. When focal cerebral ischemia was induced 16 hours after the infusion of anti-rncfosr115, the postischemic increase in Fos expression and AP-1 binding activity were suppressed. Specificity of the effect of anti-rncfosr115 was suggested by its failure to suppress the DNA binding activity of nuclear cyclic AMP response elements. These results support the hypothesis that increased AP-1 binding activity following focal cerebral ischemia is dependent on Fos expression and can be inhibited in vivo by antisense c-fos oligodeoxynucleotides.

Animals↗
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↗
PubMed1993

Differentiation of BA-HAN-1C rhabdomyosarcoma cells is controlled by a pertussis toxin sensitive signaling pathway.

BA-Han-1C rat rhabdomyosarcoma cells grow with a transformed phenotype and do not differentiate efficiently. Here, we report that these cells can be induced with pertussis toxin (PTX) to rapidly express the myogenin gene and form terminally differentiated myotubes. Potential targets for the effect mediated by PTX are G alpha i-2 and G alpha i-3 proteins, the only inhibitor GTP-binding proteins expressed in these cells. While G alpha i-2 is found at the plasma membrane, G alpha i-3 is predominantly associated with Golgi vesicles and endoplasmic reticulum, suggesting that it may regulate protein trafficking. Differentiation of BA-Han-1C cells can also be induced by suramin, heparin, and other polyanions. As these compounds bind certain peptide growth factors, we assume that differentiation of BA-Han-1C cells is blocked by pathways involving autocrine or paracrine acting growth stimulating peptides. We present evidence that bFGF and cAMP inhibit induced differentiation in BA-Han-1C cells similar to normal myogenic cell lines, suggesting that signaling pathways mediated by these compounds are unaltered.

Adenosine Diphosphate↗
PubMed1992

Membrane fusion process of Semliki Forest virus. II: Cleavage-dependent reorganization of the spike protein complex controls virus entry.

The envelope of the Semliki Forest virus (SFV) contains two transmembrane proteins, E2 and E1, in a heterodimeric complex. The E2 subunit is initially synthesized as a precursor protein p62, which is proteolytically processed to the mature E2 form before virus budding at the plasma membrane. The p62 (E2) protein mediates binding of the heterodimer to the nucleocapsid during virus budding, whereas E1 carries the entry functions of the virus, that is, cell binding and low pH-mediated membrane fusion activity. We have investigated the significance of the cleavage event for the maturation and entry of the virus. To express SFV with an uncleaved p62 phenotype, BHK-21 cells were transfected by electroporation with infectious viral RNA transcribed from a full-length SFV cDNA clone in which the p62 cleavage site had been changed. The uncleaved p62E1 heterodimer was found to be used for the formation of virus particles with an efficiency comparable to the wild type E2E1 form. However, in contrast to the wild type virus, the mutant virus was virtually noninfectious. Noninfectivity resulted from impaired uptake into cells, as well as from the inability of the virus to promote membrane fusion in the mildly acidic conditions of the endosome. This inability could be reversed by mild trypsin treatment, which converted the viral p62E1 form into the mature E2E1 form, or by treating the virus with a pH 4.5 wash, which in contrast to the more mild pH conditions of endosomes, effectively disrupted the p62E1 subunit association. We conclude that the p62 cleavage is not needed for virus budding, but regulates entry functions of the E1 subunit by controlling the heterodimer stability in acidic conditions.

Animals↗
PubMed1992

Retinoic acid induces myogenin synthesis and myogenic differentiation in the rat rhabdomyosarcoma cell line BA-Han-1C.

Two clonal rat rhabdomyosarcoma cell lines BA-Han-1B and BA-Han-1C with different capacities for myogenic differentiation have been examined for the expression of muscle regulatory basic helix-loop-helix (bHLH) proteins of the MyoD family. Whereas cells of the BA-Han-1C subpopulation constitutively expressed MyoD1 and could be induced to differentiate with retinoic acid (RA), BA-Han-1B cells did not express any of the myogenic control factors and appeared to be largely differentiation-defective. Upon induction with RA, BA-Han-1C cells expressed also myogenin, in contrast to BA-Han-1B cells which never activated any of the genes encoding muscle bHLH factors. The onset of myogenin transcription in BA-Han-1C cells required de novo protein synthesis and DNA replication suggesting that RA probably did not act directly on the myogenin gene. Although MyoD1 was expressed in proliferating BA-Han-1C myoblasts, muscle-specific reporter genes were not activated indicating that MyoD was biologically inactive. However, transfections with plasmid expressing additional MyoD1 protein resulted in the transactivation of muscle genes even in the absence of RA. mRNA encoding the negative regulatory HLH protein Id was expressed in proliferating BA-Han-1C cells and disappeared later after RA induction which suggested that it may be involved in the regulation of MyoD1 activity. The myogenic differentiation of malignant rhabdomyosarcoma cells strictly correlated with the activation of the myogenin gene. In fact, stable transfections of BA-Han-1C cells with myogenin expressing plasmids resulted in spontaneous differentiation. Together, our results suggest that the transformed and undifferentiated phenotype of BA-Han-1C rhabdomyosarcoma cells is associated with the inactivation of the myogenic factor MyoD1 as well as lack of myogenin expression. RA alleviates the inhibition of myogenic differentiation, probably by activating MyoD protein and myogenin gene transcription. BA-Han-1B cells did not respond to RA and the differentiated phenotype could not be restored by overexpression of MyoD1 or myogenin.

Animals↗
PubMed1992

Regulation of myogenin expression in normal and transformed myogenic cell lines.

The control of myogenin (Myf-4), one of the muscle-specific regulatory proteins, is particularly interesting since its expression appears obligatory in myoblasts at the onset of differentiation. We isolated the human Myf-4 (myogenin) gene and determined promoter elements which direct cell type-specific expression and are subject to transactivation by the muscle transcription factors Myf-5 and MyoD1 in fibroblasts. Extrinsic signals such as serum components and purified growth factors or potential intracellular signals such as cAMP down-regulate transcription of the myogenin gene. Constitutive expression of the catalytic subunit of PKA completely suppresses transactivation of the myogenin promoter by Myf-5 or MyoD1 suggesting that cAMP may act via phosphorylation by PKA. In contrast to normal myogenic cell lines in which differentiation and myogenin expression can be induced by the removal of serum components, retinoic acid (RA) is required for differentiation in the rat rhabdomyosarcoma cell line BA-Han-1C. This model system was utilized to investigate factors which influence the balance between the transformed state and differentiation. Administration of retinoic acid to BA-Han-1C cells leads to the accumulation of myogenin mRNA approximately 48 h after the addition of RA. This late induction requires ongoing protein- and DNA-synthesis suggesting that trans- and cis-acting factors may be involved in the control. The critical involvement of myogenin in the process of terminal muscle differentiation was also demonstrated in the rat L6 muscle cell line which has been blocked for differentiation by the transforming protein E1a of Ad5 adenovirus. In cells which stably express E1a, myogenin expression is completely suppressed while Myf-5 continues to be synthesized normally. However, E1a inhibits the transactivator function of Myf-5, as demonstrated on GAL4-Myf5 chimeric proteins. A possible interpretation of this result is that Myf-5 or factors activated by Myf-5 are required for the expression of myogenin and myogenin itself is necessary for the terminal differentiation of myoblasts.

Blood↗
PubMed1991

Two mutations in the envelope glycoprotein E2 of Semliki Forest virus affecting the maturation and entry patterns of the virus alter pathogenicity for mice.

The prototype strain of Semliki Forest virus (SFV) of known sequence and virus produced by the cDNA clone derived from it were lethal following intranasal (i.n.) infection of 40-day-old and intraperitoneal (i.p.) infection of pregnant BALB/c mice; this lethality was related to neuronal necrosis in the central nervous system (CNS). We conclude that the virulence of the prototype strain, and virus from the cDNA clone derived from it, is similar to that of L10 (the original SFV isolate). The effects of two mutations in the p62 envelope protein region of the clone were determined. Substitution of Glu for Lys at position 162 (mut64) extended the mean time of death following i.n. inoculation of 40-day-old mice. Pregnant mice infected with this virus survived but lethal infection of some fetuses did occur. Substitution of Leu for Arg at position 66 (mL), the cleavage site of the E2 and E3 proteins, results in the production of particles containing uncleaved p62. These particles were less virulent than the prototype strain when inoculated i.n. and induced immunity to virulent SFV challenge. The virus also induced the formation of multifocal glial nodules in the CNS of surviving mice. The differences in pathogenicity between the two mutants and the virulent parental virus are probably related to differences in the efficiency of virus multiplication in infected mice. The mut64 mutation attenuated the virus and allowed survival of pregnant mice infected i.p. so that the effects of fetal infection could be detected. The mL mutation allowed survival of i.n.-infected mice so that the later effects of virus multiplication in the CNS could be assessed. In the former case, this is probably a result of reduced virus release, whereas in the latter case it is due to inefficient entry of host cells. The results are consistent with our previous suggestion that lethality for virulent SFV infection results from a lethal threshold of damage to neurons in the CNS and that attenuating mutations may reduce neuronal damage below this threshold level.

Animals↗