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S Schlesinger

Publications and source records attributed to S Schlesinger.

At least 19 recordsLinked to original sources
PubMed1995

RNA viruses as vectors for the expression of heterologous proteins.

RNA viruses comprise a wide variety of infectious agents, some of which are the cause of disease in humans, animals, and plants. Recombinant DNA technology is now making it feasible to modify these genomes and engineer them to express heterologous proteins. Several different schemes are being employed that depend on the genome organization of the virus and on the strategy of replication of the particular virus. Several different examples are illustrated and potential uses as well as possible problems are discussed. In the future reverse genetics may convert some of these viruses from agents of disease to agents of cure.

Animals↗
PubMed1994

Interactions between Sindbis virus RNAs and a 68 amino acid derivative of the viral capsid protein further defines the capsid binding site.

In previous studies of encapsidation of Sindbis virus RNA, we identified a 570nt fragment (nt 684-1253) from the 12 kb genome that binds to the viral capsid protein with specificity and is required for packaging of Sindbis virus defective interfering RNAs. We now show that the capsid binding activity resides in a highly structured 132nt fragment (nt 945-1076). We had also demonstrated that a 68 amino acid peptide derived from the capsid protein retained most of the binding activity of the original protein and have now developed an RNA mobility shift assay with this peptide fused to glutathione-S-transferase. We have used this assay in conjunction with the original assay in which the intact capsid protein was immobilized on nitrocellulose to analyze more extensive deletions in the 132-mer. All of the deletions led to a reduction in binding, but the binding of a 5' 67-mer was enhanced by the addition of nonspecific flanking sequences. This result suggests that the stability of a particular structure within the 132nt sequence may be important for capsid recognition.

Base Sequence↗
PubMed1994

Clinical and pathological features of an autosomal dominant, adult-onset leukodystrophy simulating chronic progressive multiple sclerosis.

OBJECTIVE: To study the clinical and pathological features of a kindred with an adult-onset autosomal dominant leukodystrophy. PATIENTS: Five symptomatic and nine asymptomatic at-risk members of the kindred. INTERVENTIONS: Subjects underwent detailed histories and general and neurologic examinations. Further evaluation included electroencephalography, evoked potentials, electromyography, autonomic testing, and analysis of serum, urine, and cerebrospinal fluid. One patient underwent sural nerve biopsy and analysis. Another, previously studied patient, underwent a limited autopsy. RESULTS: Cerebellar and pyramidal dysfunction began in the fourth and fifth decades of life; subtle autonomic symptoms were often present years earlier. Frontal lobe dysfunction and abnormalities of the central visual pathways were mild and of late onset. Sensorineural hearing loss was common. The peripheral nervous system was spared. Autopsy results of one patient revealed severe degeneration of the white matter at all levels of the neuraxis, but most prominent in the frontoparietal and cerebellar regions, with sparing of the subcortical U fibers. Histological and ultrastructural examinations failed to show evidence of a specific pathogenetic mechanism or etiology. CONCLUSION: This disorder seems to be a distinct type of hereditary leukodystrophy, but its exact nature remains unknown.

Chronic Disease↗
PubMed1994

Recombination between Sindbis virus RNAs.

The Sindbis virus RNA genome is divided into two modules--one coding for the nonstructural protein genes and the other coding for the structural protein genes. In our studies of recombination, the two parental RNAs were defective in different modules. Analysis of the recombinant RNAs demonstrated that the parental RNAs each contributed its intact module and that the crossovers occurred within the defective modules. The recombinational events giving rise to infectious virion RNAs could create deletions, rearrangements or insertions as long as they occurred outside of the functional module. These crossovers produced RNA genomes that contained two functional subgenomic RNA promoters.

Crosses, Genetic↗
PubMed1994

Translation of Sindbis virus mRNA: effects of sequences downstream of the initiating codon.

One incentive for developing the alphavirus Sindbis virus as a vector for the expression of heterologous proteins is the very high level of viral structural proteins that accumulates in infected cells. Although replacement of the structural protein genes by a heterologous gene should lead to an equivalent accumulation of the heterologous protein, the Sindbis virus capsid protein is produced at a level 10- to 20-fold higher than that of any foreign protein. Chimeric mRNAs which contain the first 275 nucleotides of the Sindbis virus 26S mRNA fused to the lacZ gene are also translated at the higher level. The enhancing sequences, located downstream of the AUG codon that initiates translation of the capsid protein, have a predicted hairpin-like structure; deletions in this region destroy the activity. These sequences enhance translation in infected cells but have the opposite effect in uninfected cells. Furthermore, translation of this RNA in infected cells is suppressed by a second viral RNA lacking the hairpin-like structure, but translation of the latter RNA is not affected. We propose that the hairpin-like structure presents a barrier to the movement of the ribosomes during translation of mRNA. In infected cells, under conditions in which this mRNA is essentially the only RNA being translated, a slowdown in the transit of the ribosomes gives factors present at low concentrations a chance to bind to the translation complex and permits a high level of functional complexes to be formed. In uninfected cells and in infected cells translating two different viral subgenomic mRNAs, a pause in the movement of the ribosomes along the RNA is no longer an advantage, because the required factors are now usurped by other translation complexes.

Animals↗
PubMed1994

Comparison of the effects of Sindbis virus and Sindbis virus replicons on host cell protein synthesis and cytopathogenicity in BHK cells.

Infection of BHK cells by Sindbis virus leads to rapid inhibition of host cell protein synthesis and cytopathic effects (CPE). We have been studying these events to determine whether the expression of a specific viral gene is required and, in the present study, have focused our attention on the role of the structural proteins--the capsid protein and the two membrane glycoproteins. We tested a variety of Sindbis viruses and Sindbis virus replicons (virus particles containing an RNA that is self-replicating but with some or all of the viral structural protein genes deleted) for their abilities to inhibit host cell protein synthesis and cause CPE in infected BHK cells. Our results show that shutoff of host cell protein synthesis occurred in infected BHK cells when no viral structural proteins were synthesized and also under conditions in which the level of the viral subgenomic RNA was too low to be detected. These results support the conclusion that the early steps in viral gene expression are the ones required for the inhibition of host cell protein synthesis in BHK cells. In contrast, the Sindbis viruses and Sindbis virus replicons were clearly distinguished by the time at which CPE became evident. Viruses that synthesized high levels of the two membrane glycoproteins on the surface of the infected cells caused a rapid (12 to 16 h postinfection) appearance of CPE, and those that did not synthesize the glycoprotein spikes showed delayed (30 to 40 h) CPE.

Animals↗
PubMed1993

Alphaviruses--vectors for the expression of heterologous genes.

DNA viruses and retroviruses are well established as vectors for the expression of heterologous genes, but there is increasing interest in the possibilities of using RNA viruses, which do not replicate through a DNA intermediate, for this purpose. This article summarizes some of the general properties of RNA viruses and concentrates on one class of RNA viruses--the alphaviruses--and their potential as expression vectors.

Alphavirus↗
PubMed1992

Crystallization of Sindbis virus and its nucleocapsid.

Crystals of Sindbis virus, which contains a lipid-bilayer membrane, have been grown using polyethylene glycol. The space group is R32, a = b = 640 A, c = 1520 A. The crystals are highly mosaic, and recorded diffraction is therefore restricted to spacings of about 30 A. The crystals show that the packing of glycoproteins E1 and E2 in the icosahedral outer shell is sufficiently precise that it permits regular and repeated interactions between virus particles in the lattice. Crystals of Sindbis nucleocapsids have also been grown. The limited diffraction data are consistent with close packing of nucleocapsids 404 A in diameter.

Capsid↗
PubMed1992

Expression of the nonstructural proteins of Sindbis virus in insect cells by a baculovirus vector.

The genome of Sindbis virus encodes the polypeptides that are required for the replication and transcription of the virus RNA in infected cells. These polypeptides are translated as a polyprotein that is co- and post-translationally cleaved by an autoproteinase to give rise to four polypeptides designated nsP1, nsP2, nsP3 and nsP4. We have initiated a study of the functions of these proteins by expressing them in the Autographa californica baculovirus polyhedrin expression system. Spodoptera frugiperda cells infected with the recombinant baculovirus synthesized the four Sindbis polypeptides. We used a complementation assay which measures chloramphenicol acetyltransferase (CAT) activity to demonstrate that these proteins were biologically active. The infected cells were transfected with a Sindbis defective RNA that contains the CAT gene downstream of the promoter for the synthesis of the viral subgenomic RNA. CAT activity was found only in cells that had been infected with the recombinant baculovirus, not with wild type baculovirus, indicating that the required Sindbis nsP activities were present. Sindbis virions grew poorly in S. frugiperda cells and self-replicating Sindbis RNAs produced only very low levels of biological activity. Our results suggest that these cells are defective in their ability to replicate Sindbis RNAs and that the block is partially overcome when the Sindbis nsP mRNA is expressed under the control of the baculovirus DNA.

Animals↗
PubMed1991

Complementation between Sindbis viral RNAs produces infectious particles with a bipartite genome.

Sindbis virus, the type member of the alpha-viruses, is an enveloped virus containing a nonsegmented positive-strand RNA genome. We show that the nonstructural and the structural genes can function to produce infectious virus particles when they are expressed on two different RNA segments. The nonstructural genes are translated from an RNA in which the structural genes have been replaced by the chloramphenicol acetyltransferase gene [Xiong, C., Levis, R., Shen, P., Schlesinger, S., Rice, C. M. & Huang, H. V. (1989) Science 243, 1188-1191]. The structural genes are encoded in a defective-interfering RNA but are translated from a subgenomic RNA. Both segments contain the cis-acting sequences required for replication and packaging and are copackaged. This type of genome provides a model for an ancestral intermediate between alphaviruses and the multipartite positive-strand RNA viruses of plants. These different viruses show sequence similarities in their replicative proteins and are thought to have evolved from a common ancestor.

Animals↗
PubMed1991

Recombination between Sindbis virus RNAs.

The genome (49S RNA) of Sindbis virus is a positive-strand RNA of 11.7 kb that consists of two domains. The 5' two-thirds of the RNA codes for the proteins required for replication and transcription of the RNA. The 3' one-third codes for the structural proteins. The latter are translated from a 26S subgenomic RNA identical in sequence to the 3' one-third of the genome. The 26S RNA is transcribed by initiation from an internal promoter that spans the junction between the nonstructural and structural genes. We have used Sindbis virus RNAs transcribed from cloned cDNAs to demonstrate recombination between Sindbis virus RNAs in cultured cells. Several different combinations of deleted or mutationally altered RNAs gave rise to infectious recombinants. In 7 of 10 different crosses, the infectious recombinant RNAs were larger than wild-type 49S RNA. We sequenced the recombinant RNAs in the region spanning the junction between the nonstructural and structural protein genes from five different crosses. In three of the crosses, this is the only region within which recombination could have taken place to produce an infectious 49S RNA. Recombination also occurred in this region in the other two crosses. The recombinant RNAs were distinct from wild-type RNA and from each other. All contained sequence insertions derived from the parental RNAs. One contained a deletion and a rearrangement, and one also contained a stretch of 11 nucleotides not found in the Sindbis virus genome. When each of the parental RNAs contained a functional subgenomic RNA promoter, both promoters were present and functional in the recombinant RNA. Those recombinants with large sequence insertions showed evidence of evolution toward the wild-type single-junction RNA.

Cells, Cultured↗
PubMed1990

Temperature sensitive shut-off of alphavirus minus strand RNA synthesis maps to a nonstructural protein, nsP4.

Minus strand RNA synthesis by the positive strand alphaviruses, Sindbis and Semliki Forest viruses, normally occurs early in infection, is coupled to synthesis of viral nonstructural proteins and to formation of viral replication complexes, and terminates and does not occur late in infection. Previously, ts24 of the A complementation group of Sindbis virus RNA-negative mutants was found to possess, among its other temperature sensitive defects, a temperature sensitivity in the normal cessation of minus strand synthesis which enabled minus strands to be synthesized late in infection at 40 degrees in the absence of protein synthesis. Revertants of ts24 (ts24R1, ts24R2) retained the defect in the shutoff of minus strand synthesis, indicating the lesion was not conditionally lethal and could map outside the A cistron. The studies reported here used an infectious clone of Sindbis virus to identify the mutation responsible for this phenotype. Hybrid viruses were prepared from constructs containing restriction fragments of the cDNA of ts24R1 in place of the corresponding fragments in the infectious SIN HR clone and screened for their ability to synthesize minus strands at 40 degrees in the presence of cycloheximide. A unique base change of an A for a C residue at nt 6339, predicting a change from glutamine to lysine at amino acid 195 in nsP4, was found in genomes of ts24, ts24R1, and ts24R2. Other nucleotide changes present at the 5' and 3' termini did not affect minus strand synthesis. The substitution of the parental Sindbis virus sequence that encompassed nt 6339 in an infectious clone of the ts24R1 revertant eliminated the mutant phenotype. We conclude that the ability to continue minus strand synthesis at 40 degrees exhibited by ts24 and its revertants is caused by an alteration in nsP4, which is the alphavirus replicase or an essential component of the replicase. We hypothesize that this domain of nsP4 functions to fix the minus strand as the stable template of alphavirus replication complexes.

Amino Acid Sequence↗
PubMed1990

Promoter for Sindbis virus RNA-dependent subgenomic RNA transcription.

Sindbis virus is a positive-strand RNA enveloped virus, a member of the Alphavirus genus of the Togaviridae family. Two species of mRNA are synthesized in cells infected with Sindbis virus; one, the 49S RNA, is the genomic RNA; the other, the 26S RNA, is a subgenomic RNA that is identical in sequence to the 3' one-third of the genomic RNA. Ou et al. (J.-H. Ou, C. M. Rice, L. Dalgarno, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 79:5235-5239, 1982) identified a highly conserved region 19 nucleotides upstream and 2 nucleotides downstream from the start of the 26S RNA and proposed that in the negative-strand template, these nucleotides compose the promoter for directing the synthesis of the subgenomic RNA. Defective interfering (DI) RNAs of Sindbis virus were used to test this proposal. A 227-nucleotide sequence encompassing 98 nucleotides upstream and 117 nucleotides downstream from the start site of the Sindbis virus subgenomic RNA was inserted into a DI genome. The DI RNA containing the insert was replicated and packaged in the presence of helper virus, and cells infected with these DI particles produced a subgenomic RNA of the size and sequence expected if the promoter was functional. The initiating nucleotide was identical to that used for Sindbis virus subgenomic mRNA synthesis. Deletion analysis showed that the minimal region required to detect transcription of a subgenomic RNA from the negative-strand template of a DI RNA was 18 or 19 nucleotides upstream and 5 nucleotides downstream from the start of the subgenomic RNA.

Animals↗
PubMed1989

Sindbis virus: an efficient, broad host range vector for gene expression in animal cells.

Sindbis virus, an enveloped virus with a single-stranded RNA genome, was engineered to express a bacterial protein, chloramphenicol acetyltransferase (CAT), in cultured insect, avian, and mammalian cells. The vectors were self-replicating and gene expression was efficient and rapid; up to 10(8) CAT polypeptides were produced per infected cell in 16 to 20 hours. CAT expression could be made temperature-sensitive by means of a derivative that incorporated a temperature-sensitive mutation in viral RNA synthesis. Vector genomic RNAs were packaged into infectious particles when Sindbis helper virus was used to supply virion structural proteins. The vector RNAs were stable to at least seven cycles of infection. The expression of CAT increased about 10(3)-fold, despite a 10(15)-fold dilution during the passaging. Sindbis virus vectors should prove useful for expressing large quantities of gene products in a variety of animal cells.

Aedes↗
PubMed1989

Evidence for specificity in the encapsidation of Sindbis virus RNAs.

We investigated the interaction of the capsid protein of Sindbis virus with Sindbis viral RNAs and defined a region of the genome that is required for binding in vitro and for packaging in vivo. The binding studies were performed with purified capsid protein immobilized on nitrocellulose and 32P-labeled RNAs transcribed in vitro from viral and nonspecific cDNAs. Genomic and defective interfering (DI) RNAs bound capsid protein significantly better than either the subgenomic (26S) RNA or nonspecific RNAs. Transcripts prepared from either truncated or deleted cDNAs were used to define the segment required for binding. This segment, which is represented twice in DI RNA, lies between nucleotides 746 and 1226 of the genomic RNA and is within the coding region of the nonstructural protein nsP1. Insertion of a domain covering these sequences into a nonviral RNA was able to convert it from a background level of binding to an activity that was 80% that of the Sindbis virus DI RNA. We analyzed DI RNA transcripts in detail because they could be studied not only for the ability to bind capsid protein in vitro but also for the ability to be replicated and packaged in vivo in the presence of helper virion RNA. The results obtained with three DI RNAs are reported. One (CTS14), which has one copy of the binding domain, bound efficiently to capsid protein in vitro and was packaged in vivo as measured by amplification on passaging. In contrast, a DI RNA (CTS1) which lacked this region did not bind to capsid protein and was not detected on passaging. By using lipofectin (P. L. Felgner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J.P. Northrop, G. M. Ringold, and M. Danielson, Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987) to enhance RNA uptake, we were able to demonstrate that CTS1 RNA was replicated in the transfected cells. It was replicated to the same level as another DI RNA (CTS253) which has only the 3' 279 nucleotides of the binding domain and these are located near the 3' terminus of the RNA. CTS253 bound capsid protein to an intermediate level but was amplified on passaging. The binding studies and the in vivo packaging data, taken together, provide strong support for the conclusion that there is a specific capsid recognition domain in Sindbis virus RNA that plays a role in nucleocapsid assembly.

Capsid↗