Aminoglycoside-mediated suppression of nonsense mutations in late infantile neuronal ceroid lipofuscinosis.

The ability of aminoglycoside antibiotics to promote readthrough of eukaryotic stop codons has attracted interest in these drugs as potential therapeutic agents in human disorders caused by nonsense mutations. One disease for which such a therapeutic strategy may be viable is classical late infantile neuronal ceroid lipofuscinosis (LINCL), a fatal childhood neurodegenerative disorder with currently no effective treatment. Premature stop codon mutations in the gene CLN2 encoding the lysosomal tripeptidyl-peptidase 1 (TPP-I) are associated with disease in approximately half of children diagnosed with LINCL. The aim of this study was to examine the ability of the aminoglycoside gentamicin to restore TPP-I activity in LINCL cell lines. In one patient-derived cell line that was compound heterozygous for a commonly seen nonsense mutation, Arg208Stop and a different rare nonsense mutation, approximately 7% of normal levels of TPP-I were maximally restored with gentamicin treatment. In other cell lines from patients that were compound heterozygous for Arg208Stop and a splice junction mutation, approximately 0.5% of maximal activity was restored. These results suggest that pharmacological suppression of nonsense mutations by aminoglycosides or functionally similar pharmaceuticals may have therapeutic potential in LINCL.

Congenital ovine neuronal ceroid lipofuscinosis--a cathepsin D deficiency with increased levels of the inactive enzyme.

We recently showed that a form of neuronal ceroid lipofuscinosis (NCL) in white Swedish landrace sheep is caused by a missense mutation in the cathepsin D gene resulting in complete inactivation of the enzyme. Despite the lack of cathepsin D activity, the brains of the cathepsin D deficient sheep showed strongly increased staining for cathepsin D in immunohistochemistry. By Western blotting, a 5-10 fold increase in the level of cathepsin D was confirmed. These results indicate that the missense mutation in congenital NCL sheep results in the synthesis of an inactive yet stable cathepsin D.

Niemann-Pick disease type C: spectrum of HE1 mutations and genotype/phenotype correlations in the NPC2 group.

In Niemann-Pick disease type C (NPC), a genetic heterogeneity with two complementation groups--NPC1, comprising > or =95% of the families, and NPC2--has been demonstrated. Mutations in the NPC1 gene have now been well characterized. HE1 was recently identified as the gene underlying the very rare NPC2. Here we report the first comprehensive study of eight unrelated families with NPC2, originating from France, Algeria, Italy, Germany, the Czech Republic, and Turkey. These cases represent essentially all patients with NPC2 who have been reported in the literature, as well as those known to us. All 16 mutant alleles were identified, but only five different mutations, all with a severe impact on the protein, were found; these five mutations were as follows: two nonsense mutations (E20X and E118X), a 1-bp deletion (27delG), a splice mutation (IVS2+5G-->A), and a missense mutation (S67P) resulting in reduced amounts of abnormal HE1 protein. E20X, with an overall allele frequency of 56%, was established as the common mutant allele. Prenatal diagnosis was achieved by mutation analysis of an uncultured chorionic-villus sample. All mutations except 27delG were observed in a homozygous state, allowing genotype/phenotype correlations. In seven families (with E20X, E118X, S67P, and E20X/27delG mutations), patients suffered a severe and rapid disease course, with age at death being 6 mo-4 years. A remarkable feature was the pronounced lung involvement, leading, in six patients, to early death caused by respiratory failure. Two patients also developed a severe neurological disease with onset during infancy. Conversely, the splice mutation corresponded to a very different clinical presentation, with juvenile onset of neurological symptoms and prolonged survival. This mutation generated multiple transcripts, including a minute proportion of normally spliced RNA, which may explain the milder phenotype.

Identification of HE1 as the second gene of Niemann-Pick C disease.

Niemann-Pick type C2 disease (NP-C2) is a fatal hereditary disorder of unknown etiology characterized by defective egress of cholesterol from lysosomes. Here we show that the disease is caused by a deficiency in HE1, a ubiquitously expressed lysosomal protein identified previously as a cholesterol-binding protein. HE1 was undetectable in fibroblasts from NP-C2 patients but present in fibroblasts from unaffected controls and NP-C1 patients. Mutations in the HE1 gene, which maps to chromosome 14q24.3, were found in NP-C2 patients but not in controls. Treatment of NP-C2 fibroblasts with exogenous recombinant HE1 protein ameliorated lysosomal accumulation of low density lipoprotein-derived cholesterol.

A mutation in the ovine cathepsin D gene causes a congenital lysosomal storage disease with profound neurodegeneration.

The neuronal ceroid lipofuscinoses (NCLs) constitute a group of neurodegenerative storage diseases characterized by progressive psychomotor retardation, blindness and premature death. Pathologically, there is accumulation of autofluorescent material in lysosome-derived organelles in a variety of cell types, but neurons in the central nervous system appear to be selectively affected and undergo progressive death. In this report we show that a novel form of NCL, congenital ovine NCL, is caused by a deficiency in the lysosomal aspartyl proteinase cathepsin D. A single nucleotide mutation in the cathepsin D gene results in conversion of an active site aspartate to asparagine, leading to production of an enzymatically inactive but stable protein. This results in severe cerebrocortical atrophy and early death, providing strong evidence for an important role of cathepsin D in neuronal development and/or homeostasis.

Prenatal testing for late infantile neuronal ceroid lipofuscinosis.

Classic late infantile neuronal ceroid lipofuscinosis (LINCL) is a neurodegenerative disease in which autofluorescent "curvilinear" storage bodies accumulate in tissues from affected patients. Recently, the LINCL gene (CLN2) has been found to code for a pepstatin-insensitive lysosomal protease whose activity is deficient in LINCL specimens. We report the first 2 cases of successful prenatal testing for LINCL by using DNA and enzyme-based methods on amniocytes, and describe a new private mutation in one of the families analyzed. These approaches allow definitive prenatal diagnosis and represent a significant improvement over previous pathological methods.

Subcellular localization of mannose 6-phosphate glycoproteins in rat brain.

The intracellular transport of soluble lysosomal enzymes relies on the post-translational modification of N-linked oligosaccharides to generate mannose 6-phosphate (Man 6-P) residues. In most cell types the Man 6-P signal is rapidly removed after targeting of the precursor proteins from the Golgi to lysosomes via interactions with Man 6-phosphate receptors. However, in brain, the steady state proportion of lysosomal enzymes containing Man 6-P is considerably higher than in other tissues. As a first step toward understanding the mechanism and biological significance of this observation, we analyzed the subcellular localization of the rat brain Man 6-P glycoproteins by combining biochemical and morphological approaches. The brain Man 6-P glycoproteins are predominantly localized in neuronal lysosomes with no evidence for a steady state localization in nonlysosomal or prelysosomal compartments. This contrasts with the clear endosome-like localization of the low steady state proportion of mannose-6-phosphorylated lysosomal enzymes in liver. It therefore seems likely that the observed high percentage of phosphorylated species in brain is a consequence of the accumulation of lysosomal enzymes in a neuronal lysosome that does not fully dephosphorylate the Man 6-P moieties.

Biochemical characterization of a lysosomal protease deficient in classical late infantile neuronal ceroid lipofuscinosis (LINCL) and development of an enzyme-based assay for diagnosis and exclusion of LINCL in human specimens and animal models.

Classical late-infantile neuronal ceroid lipofuscinosis (LINCL), a progressive and fatal neurodegenerative disease of childhood, results from mutations in a gene (CLN2) that encodes a protein with significant sequence similarity to prokaryotic pepstatin-insensitive acid proteases. We have developed a sensitive protease activity assay that allows biochemical characterization of the CLN2 gene product in various human biological samples, including solid tissues (brain and chorionic villi), blood (buffy coat leukocytes, platelets, granulocytes, and mononuclear cells), and cultured cells (lymphoblasts, fibroblasts, and amniocytes). The enzyme has a pH optimum of 3.5 and is rapidly inactivated at neutral pH. A survey of fibroblasts and lymphoblasts demonstrated that lack of activity was associated with LINCL arising from mutations in the CLN2 gene but not other neuronal ceroid lipofuscinoses (NCLs), including the CLN6 variant LINCL, classical infantile NCL, classical juvenile NCL, and adult NCL (Kufs' disease). A study conducted using blood samples collected from classical LINCL families whose affliction was confirmed by genetic analysis indicates that the assay can distinguish homozygotes, heterozygotes, and normal controls and thus is useful for diagnosis and carrier testing. Analysis of archival specimens indicates that several specimens previously classified as LINCL have enzyme activity and thus disease is unlikely to arise from mutations in CLN2. Conversely, a specimen previously classified as juvenile NCL lacks pepinase activity and is associated with mutations in CLN2. In addition, several animals with NCL-like neurodegenerative symptoms [mutant strains of mice (nclf and mnd), English setter, border collie, and Tibetan terrier dogs, sheep, and cattle] were found to contain enzyme activity and are thus unlikely to represent models for classical LINCL. Subcellular fractionation experiments indicate that the CLN2 protein is located in lysosomes, which is consistent with its acidic pH optimum for activity and the presence of mannose 6-phosphate. Taken together, these findings indicate that LINCL represents a lysosomal storage disorder that is characterized by the absence of a specific protease activity.

Mutational analysis of the defective protease in classic late-infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disorder.

The late-infantile form of neuronal ceroid lipofuscinosis (LINCL) is a progressive and ultimately fatal neurodegenerative disease of childhood. The defective gene in this hereditary disorder, CLN2, encodes a recently identified lysosomal pepstatin-insensitive acid protease. To better understand the molecular pathology of LINCL, we conducted a genetic survey of CLN2 in 74 LINCL families. In 14 patients, CLN2 protease activities were normal and no mutations were identified, suggesting other forms of NCL. Both pathogenic alleles were identified in 57 of the other 60 LINCL families studied. In total, 24 mutations were associated with LINCL, comprising six splice-junction mutations, 11 missense mutations, 3 nonsense mutations, 3 small deletions, and 1 single-nucleotide insertion. Two mutations were particularly common: an intronic G-->C transversion in the invariant AG of a 3' splice junction, found in 38 of 115 alleles, and a C-->T transition in 32 of 115 alleles, which prematurely terminates translation at amino acid 208 of 563. An Arg-->His substitution was identified, which was associated with a late age at onset and protracted clinical phenotype, in a number of other patients originally diagnosed with juvenile NCL.

Specific alterations in levels of mannose 6-phosphorylated glycoproteins in different neuronal ceroid lipofuscinoses.

Mannose 6-phosphate (Man-6-P) is a carbohydrate modification that is generated on newly synthesized lysosomal proteins. This modification is specifically recognized by two Man-6-P receptors that direct the vesicular transport of the lysosomal enzymes from the Golgi to a prelysosomal compartment. The Man-6-P is rapidly removed in the lysosome of most cell types; however, in neurons the Man-6-P modification persists. In this study we have examined the spectrum of Man-6-P-containing glycoproteins in brain specimens from patients with different neuronal ceroid lipofuscinoses (NCLs), which are progressive neurodegenerative disorders with established links to defects in lysosomal catabolism. We find characteristic alterations in the Man-6-P glycoproteins in specimens from late-infantile (LINCL), juvenile (JNCL) and adult (ANCL) patients. Man-6-P glycoproteins in LINCL patients were similar to controls, with the exception that the band corresponding to CLN2, a recently identified lysosomal enzyme whose deficiency results in this disease, was absent. In an ANCL patient, two Man-6-P glycoproteins were elevated in comparison with normal controls, suggesting that this disease also results from a perturbation in lysosomal hydrolysis. In JNCL, total levels of Man-6-P glycoproteins were 7-fold those of controls. In general this was reflected by increased lysosomal enzyme activities in JNCL but three Man-6-P glycoproteins were elevated to an even greater degree. These are CLN2 and the unidentified proteins that are also highly elevated in the ANCL.

Structural organization and sequence of CLN2, the defective gene in classical late infantile neuronal ceroid lipofuscinosis.

Mutations in the CLN2 gene result in classical late infantile neuronal ceroid lipofuscinosis (LINCL), a fatal childhood neurodegenerative disease. In this report, we present the complete sequence of the human CLN2 gene and define its physical relationship with two other genes that have been previously mapped to chromosome 11p15. The CLN2 gene consists of 13 exons and 12 introns and spans 6.65 kb. By S1 mapping and primer extension, the 5'-terminus of the CLN2 mRNA was mapped to 32 nucleotides upstream of the proposed initiation codon. A number of other elements were found to be located in close proximity to CLN2, including the gene encoding transcription factor TAFII30, the gene encoding intregrin-linked kinase, and an approximately 914-bp fragment that is 82% identical to antithrombin III. In addition, an EST cDNA clone that is transcribed on the strand opposite to CLN2 and that overlaps a portion of the CLN2 gene was identified. Finally, a set of primer pairs are presented for the amplification of the coding sequences, putative promoter, and splice junctions of the CLN2 gene. Taken together, this information will facilitate the molecular analysis of and genetic testing for classical LINCL.

Association of mutations in a lysosomal protein with classical late-infantile neuronal ceroid lipofuscinosis.

Classical late-infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal neurodegenerative disease whose defective gene has remained elusive. A molecular basis for LINCL was determined with an approach applicable to other lysosomal storage diseases. When the mannose 6-phosphate modification of newly synthesized lysosomal enzymes was used as an affinity marker, a single protein was identified that is absent in LINCL. Sequence comparisons suggest that this protein is a pepstatin-insensitive lysosomal peptidase, and a corresponding enzymatic activity was deficient in LINCL autopsy specimens. Mutations in the gene encoding this protein were identified in LINCL patients but not in normal controls.

alpha-Glucosidase and N-acetylglucosamine-6-sulphatase are the major mannose-6-phosphate glycoproteins in human urine.

Most newly synthesized lysosomal enzymes contain a transient carbohydrate modification, mannose 6-phosphate (Man-6-P), which signals their vesicular transport from the Golgi to the lysosome via Man-6-P receptors (MPRs). We have examined Man-6-P glycoproteins in human urine by using a purified soluble fragment of the soluble cation-independent MPR (sCI-MPR) as a preparative and analytical affinity reagent. In a survey of urine samples from seven healthy subjects, the pattern of Man-6-P glycoproteins detected with iodinated sCI-MPR as a probe in a blotting assay was essentially identical in each, regardless of sex or age. Two bands of approx. 100 and 110 kDa were particularly prominent. Man-6-P glycoproteins in human urine were purified by affinity chromatography on immobilized sCI-MPR. Seven distinct bands revealed by SDS/PAGE and Coomassie Blue staining were subjected to N-terminal sequence analysis. The prominent 100 and 110 kDa Man-6-P glycoproteins were identified as N-acetylglucosamine-6-sulphatase and alpha-glucosidase respectively. This identification was confirmed by molecular mass determinations on the two major bands after deglycosylation. Sequence analysis revealed arylsulphatase A and several previously unidentified proteins as minor species. Man-6-P glycoproteins were also purified on an analytical scale to determine the proportion of a number of lysosomal enzyme activities represented by the mannose-6-phosphorylated forms. The lysosomal enzymes in urine containing the highest proportion of mannose-6-phosphorylated form were beta-mannosidase (82%), hexosaminidase (27%) and alpha-glucosidase (24%). The profiles of Man-6-P glycoproteins detected by blotting in urine and plasma were not similar, suggesting that the urinary species are not derived from the bloodstream.

Ligand binding specificities of the two mannose 6-phosphate receptors.

Two mannose 6-phosphate (Man-6-P) receptors (MPRs) direct the vesicular transport of newly synthesized lysosomal enzymes that contain Man-6-P from the Golgi to a prelysosomal compartment. In order to understand the respective roles of the Mr = 46,000 cation-dependent (CD-) MPR and the Mr = 300,000 cation-independent (CI-) MPR in lysosomal targeting, an assay has been developed that simultaneously measures the relative affinity of each MPR for multiple ligands. Glycoproteins containing Man-6-P were affinity-purified from the metabolically labeled secretions of mutant mouse fibroblasts lacking both MPRs. They were incubated with purified MPRs, and the resulting receptor-ligand complexes were immunoprecipitated by anti-MPR monoclonal antibodies coupled to agarose beads. Ligands were eluted with Man-6-P and then quantified following SDS-polyacrylamide gel electrophoresis. Saturating concentrations of CI-MPR resulted in the complete recovery of each Man-6-P glycoprotein in receptor-ligand complexes. Apparent affinity constants ranged between 1 and 5 nM for the individual species. Ligands precipitated by the CD-MPR appeared identical to those bound by the CI-MPR, with apparent affinity constants ranging between 7 and 28 nM. The binding affinities of the two receptors for different ligands were not correlated, indicating that the two MPRs preferentially recognize different subsets of lysosomal enzymes. In addition, saturating levels of CD-MPR resulted in the precipitation of only 50% of the total input ligands, suggesting that the CD-MPR binds a subpopulation of the Man-6-P glycoproteins bound by the CI-MPR. These results provide a biochemical mechanism, which, in part, may explain the interaction of the two MPRs with overlapping yet distinct subsets of ligands in vivo.

Rat brain contains high levels of mannose-6-phosphorylated glycoproteins including lysosomal enzymes and palmitoyl-protein thioesterase, an enzyme implicated in infantile neuronal lipofuscinosis.

Mannose 6-phosphate (Man-6-P) is a posttranslational carbohydrate modification typical of newly synthesized acid hydrolases that signals targeting from the Golgi apparatus to the lysosome via Man-6-P receptors (MPRs). Using iodinated cation independent MPR as a probe in a Western blot assay, we surveyed levels of Man-6-P glycoproteins in a number of different rat tissues. Considerable variation was observed with respect to total amounts and types of Man-6-P glycoproteins in the different tissues. Brain contained 2-8-fold more Man-6-P glycoproteins than other tissues, with relative abundance being brain >> testis approximately heart > lung approximately kidney approximately ovary approximately spleen > skeletal muscle approximately liver approximately serum. Analysis of 16 different lysosomal enzyme activities revealed that brain contains lower activities than other tissues which suggested that decreased removal of Man-6-P results in increased levels of Man-6-P glycoproteins. This was directly demonstrated by comparing activities of phosphorylated lysosomal enzymes, purified by immobilized MPR affinity chromatography, with total activities. The phosphorylated forms accounted for a considerable proportion of the MPR-targeted activities measured in brain (on average, 36.2%) but very little in lung, kidney, and liver (on average, 5.5, 2.3, and 0. 7%, respectively). Man-6-P glycoproteins were also isolated from rat brain by MPR affinity chromatography on a preparative scale. Of the 18 bands resolvable by SDS-polyacrylamide gel electrophoresis, seven bands were NH2-terminally sequenced and identified as the known lysosomal enzymes cathepsin L, cathepsin A, cathepsin D, alpha-galactosidase A, arylsulfatase A, and alpha-iduronidase. One of the major Man-6-P glycoproteins was identified as palmitoyl protein thioesterase, which was not previously thought to be lysosomal. This finding raises important questions about the cellular location and function of palmitoyl protein thioesterase, mutations in which result in the neurodegenerative disorder, infantile neuronal ceroid lipofuscinosis.

Increased levels of glycoproteins containing mannose 6-phosphate in human breast carcinomas.

Newly synthesized enzymes destined for lysosomal localization contain mannose 6-phosphate (Man6-P) residues, allowing interaction with Man6-P receptors (MPRs) and subsequent intracellular targeting to the lysosome. In most cultured cells, lysosomal enzymes are rapidly dephosphorylated after targeting, but in some transformed cell lines, these proteins retain the Man6-P marker. To investigate the significance of this in human malignancy, we examined the persistence of the Man6-P marker in human breast biopsy specimens using MPR derivatives as affinity probes. In one approach, extracts of frozen tissue were standardized to protein content, fractionated by SDS-PAGE, immobilized on nitrocellulose, and probed with iodinated MPR. On average, carcinomas contained 4-fold higher levels of Man6-P glycoproteins than did benign tumors or normal breast samples. In about 15% of the carcinomas, levels of Man6-P glycoproteins were highly elevated (7-10-fold). Multiple Man6-P glycoproteins were detected, suggesting a general alteration in the synthesis or processing of many lysosomal enzymes in carcinomas. In a second approach, sections of formalin-fixed breast biopsy specimens were probed with biotinylated MPR. Malignant cells in 25 of 75 carcinomas exhibited granular cytoplasmic staining in what appears to be intracellular vesicles. Staining was specifically inhibited by Man6-P and was not observed in stromal components or lymphocytes. In addition, Man6-phosphorylated proteins were not detected in the 14 normal or benign biopsy samples examined. Staining appeared to be independent of most prognostic factors examined, including p53, cathepsin D, DNA ploidy, and hormone (estrogen and progesterone) receptor status. However, positive staining was significantly associated with high histological and nuclear grades (P < 0.05) and potentially with c-erbB-2 (P < 0.10), suggesting that elevated levels of Man6-P glycoproteins are associated with the more aggressive tumors.

Mapping determinants within cucumber mosaic virus and its satellite RNA for the induction of necrosis in tomato plants.

A mutant of the WL47 satellite (WL47-sat) RNA of cucumber mosaic virus (CMV), constructed in vitro, induces lethal necrosis in tomato plants when associated with either of two subgroup II strains of CMV: LS- or WL-CMV (D. E. Sleat and P. Palukaitis, Proc. Natl. Acad. Sci. USA 87:2946-2950, 1990). The phenotype of this mutant (WLM2-sat RNA) has been examined on tomato plants, after co-inoculation with a wider range of CMV strains. Necrosis was observed when WLM2-sat RNA was associated with any of the subgroup II CMV strains tested. However, WLM2-sat RNA ameliorated the symptoms induced by subgroup I CMV strains without inducing necrosis. Inoculation with WLM2-sat RNA with pseudorecombinants formed between subgroup I and subgroup II CMV strains showed an association of the necrosis induction phenotype with RNA 2 of the subgroup II strains. In contrast to WLM2-sat RNA, infectious transcripts of the naturally necrogenic D4-sat RNA induced lethal necrosis in tomato with all CMV strains tested. Experiments involving the exchange of sequences between WLM2- and D4-sat RNAs indicated that nucleotides sequences in either of two separate regions could influence the necrosis phenotype. Thus, the subgroup-specific necrosis-inducing phenotype may be due to subtle alterations in secondary and/or tertiary structure in the satellite RNA, as well as the presence or absence of particular nucleotide sequences.

Transcriptional activity and mutational analysis of recombinant vesicular stomatitis virus RNA polymerase.

The 241-kDa large (L) protein of vesicular stomatitis virus (VSV) is the multifunctional catalytic component of the viral RNA polymerase. A protocol has been developed for the synthesis of recombinant L protein that will support viral mRNA synthesis in vitro. COS cells were transfected with a transient expression vector (pSV-VSL1 [M. Schubert, G. G. Harmison, C. D. Richardson, and E. Meier, Proc. Natl. Acad. Sci. USA 82:7984-7988, 1985]) which contains the simian virus 40 late promoter for the transcription of a cDNA copy of the L protein of the Indiana serotype of VSV. Cytoplasmic extracts of these cells efficiently transcribed VSV mRNAs in vitro in conjunction with N protein-RNA template purified from virus and recombinant phosphoprotein synthesized in Escherichia coli. mRNA synthesis was completely dependent upon addition of both bacterial phosphoprotein and extracts from cells transfected with the L gene. Extracts from mock-transfected cells or from cells transfected with the expression vector alone did not support VSV RNA synthesis. RNA synthesis was proportional to the concentration of cell extract used, with an optimum of 0.2 mg/ml. Rhabdoviruses and paramyxoviruses contain a highly conserved GDNQ motif which was mutated in the transfected L gene. All constructs with mutations within the core GDN abrogated transcriptional activity except for the mutant containing GDD, which retained 25% activity. Conserved amino acid changes outside of the core GDN and changes corresponding to other paromyxovirus and rhabdovirus L proteins retained variable transcriptional activity. These findings provide experimental evidence that the GDN of negative-strand, nonsegmented RNA viruses is a variant of the GDD motif of plus-strand RNA viruses and of the XDD motif of DNA viruses and reverse transcriptases.

Restricted replication of vesicular stomatitis virus in T lymphocytes is coincident with a deficiency in a cellular protein kinase required for viral transcription.

Vesicular stomatitis virus (VSV) fails to replicate in mouse T lymphocytes unless the cells have been mitogenically stimulated with concanavalin A (Con A). We have examined the possibility that the failure of VSV to replicate in unstimulated T lymphocytes can be attributed to a deficiency in a host protein kinase which activates the viral P protein by phosphorylation, thus rendering it transcriptionally competent. Soluble extracts were prepared from purified mouse T lymphocytes, with or without prior treatment with Con A. The ability of these extracts to phosphorylate bacterially synthesized P protein of two VSV serotypes was measured in vitro. Activity of the protein kinase on the P proteins of the Indiana or New Jersey serotypes of VSV increased, on average 2.4- and 2.1-fold respectively, after treatment of the cells with 3 micrograms/ml Con A. Higher concentrations of Con A induced proportional increases (up to 10-fold) in the activity of the host protein kinase. Activities of the kinase phosphorylating the P protein in separate populations of CD4- and CD8-containing murine T lymphocytes increased similarly on mitogenic activation. No biochemical or immunological differences were observed between the T cell protein kinase and the previously characterized protein kinase (casein kinase II) from BHK-21 cells. The activity of the kinase that phosphorylates the P protein did not vary in CV-1 cells on treatment with alpha- or gamma-interferon, both of which inhibited VSV replication. Similarly, casein kinase II activities in Raji and SIRC cells, which do not normally support VSV growth, were the same as in BHK-21 cells. Thus restriction of VSV replication in these cells, in contrast to T lymphocytes, was not associated with a deficiency in the host casein kinase II activity.

A single nucleotide change within a plant virus satellite RNA alters the host specificity of disease induction.

Some RNA plant viruses contain satellite RNAs which are dependent upon their associated virus for replication and encapsidation. Some cucumber mosaic virus satellite RNAs induce chlorosis on any of several host plants, including either tobacco or tomato. The exchange of sequence domains between cDNA clones of chlorosis-inducing and non-pathogenic satellite RNAs delimited the chlorosis domain for both tobacco and tomato plants to the same region. Site-directed mutagenesis of one nucleotide (149) within this domain changed the host plant specificity for a chlorotic response to satellite RNA infection from tomato to tobacco. Within the chlorosis domain, three conserved nucleotides are either deleted or altered in all satellite RNAs that do not induce chlorosis. Deletion of one of these nucleotides (153) did not affect satellite RNA replication but rendered it non-pathogenic. Thus, two single nucleotides have been identified which play central roles in those interactions between the virus, its satellite RNA and the host plant, and together result in a specific disease state.