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Biomedical subjects

D Spehner

Publications and source records attributed to D Spehner.

48 records · Page 3Linked to original sources
PubMed1990

Construction of fowlpox virus vectors with intergenic insertions: expression of the beta-galactosidase gene and the measles virus fusion gene.

A DNA fragment from fowlpox virus cloned on a plasmid vector was modified to contain foreign DNA inserts within an intergenic region. In a first step, a 32-base-pair intergenic region from the fowlpox virus genome corresponding to the position of the thymidine kinase locus in the vaccinia virus genome was enlarged to 55 base pairs by site-directed mutagenesis. A unique restriction endonuclease site introduced upstream of the intergenic region was then used to insert various foreign DNA fragments. The lacZ gene encoding beta-galactosidase and the measles virus gene encoding the fusion protein were positioned downstream of two vaccinia virus p7.5 promoter elements in either a direct repeat or inverted repeat orientation. Foreign DNA inserts contained within the fowlpox virus sequence were transferred to the viral genome by homologous recombination occurring in cells infected with a fowlpox virus temperature-sensitive mutant and transfected with both wild-type viral DNA and plasmid DNA. Recombinant viruses were selected for the expression of beta-galactosidase activity by screening for blue plaques in the presence of a chromogenic substrate. Stable recombinants expressing both the lacZ gene and the unselected measles gene were obtained when the p7.5 promoter was present as an inverted repeat. However, when the p7.5 promoter was in the direct repeat orientation, viral recombinants which initially expressed both gene inserts readily deleted the lacZ gene flanked by the promoter repeat. The methods described enable precise insertion and deletion of foreign genes in the fowlpox virus genome and could be applied to other intergenic regions of the same virus as well as other poxviruses.

Amino Acid Sequence↗
PubMed1989

Antibodies directed against a synthetic peptide enable detection of a protein encoded by a vaccinia virus host range gene that is conserved within the Orthopoxvirus genus.

A vaccinia virus gene required for multiplication in some cell lines but not in others has been previously isolated and sequenced. A synthetic peptide predicted from the nucleotide sequence and corresponding to the carboxy-terminal 18 amino acids was used to raise antibodies in rabbits. The immune serum enabled detection of a 29-kilodalton (kDa) polypeptide by either immunoprecipitation or Western immunoblot assays. Synthesis of the 29-kDa polypeptide occurred immediately after infection and lasted for about 3 h. Shutoff of its synthesis was concomitant with the appearance of a delayed early polypeptide that may be antigenically related to the 29-kDa polypeptide. Analysis of cloned segments of the genomes of other orthopoxviruses by hybridization with the vaccinia virus host range gene demonstrates that it is well conserved within this genus.

Genes, Viral↗
PubMed1988

Protection of mice from fatal measles encephalitis by vaccination with vaccinia virus recombinants encoding either the hemagglutinin or the fusion protein.

Vaccinia virus recombinants encoding the hemagglutinin or fusion protein of measles virus have been constructed. Infection of cell cultures with the recombinants led to the synthesis of authentic measles proteins as judged by their electrophoretic mobility, recognition by antibodies, glycosylation, proteolytic cleavage, and presentation on the cell surface. Mice vaccinated with a single dose of the recombinant encoding the hemagglutinin protein developed antibodies capable of both inhibiting hemagglutination activity and neutralizing measles virus, whereas animals vaccinated with the recombinant encoding the fusion protein developed measles neutralizing antibodies. Mice vaccinated with either of the recombinants resisted a normally lethal intracerebral inoculation of a cell-associated measles virus subacute sclerosing panencephalitis strain.

Animals↗
PubMed1988

A cowpox virus gene required for multiplication in Chinese hamster ovary cells.

Cowpox virus, in contrast to vaccinia virus, can multiply in Chinese hamster ovary cells. To study the genetic basis for this difference in host range, recombinants between vaccinia and cowpox viruses were isolated and their DNA restriction patterns were examined. The ability to multiply in Chinese hamster ovary cells could be correlated with the conservation of cowpox virus sequences mapping at the left end of the genome. This was further demonstrated by marker rescue of the host range phenotype with restricted cowpox virus DNA. Marker rescue with cloned restriction fragments of decreasing size enabled the fine localization of the host range function to a 2.3-kilobase-pair fragment. Nucleotide sequencing revealed that the fragment encoded a single major polypeptide of approximately 77,000 daltons. It is suggested that the role of the host range gene from cowpox virus is to prevent the early and extensive shutoff of protein synthesis that normally occurs in Chinese hamster ovary cells infected by vaccinia virus.

Amino Acid Sequence↗
PubMed1987

Similar genetic organization between a region of fowlpox virus DNA and the vaccinia virus HindIII J fragment despite divergent location of the thymidine kinase gene.

DNA from Fowlpox virus, a member of the Avipoxvirus genus, has been found to hybridize to DNA from vaccinia virus, a member of the Orthopoxvirus genus. The greatest homology detected was around the region containing the vaccinia virus thymidine kinase locus. A 3.1-kbp fowlpox virus fragment that hybridizes to the vaccinia virus HindIII J fragment has been cloned and its sequence determined. Comparison of the nucleotide and deduced amino acid sequence to the cross hybridizing vaccinia fragment revealed extensive conservation of six open reading frames as well as a similar organization along the genome. Nevertheless a fowlpox virus gene corresponding to the vaccinia virus thymidine kinase gene was apparently lacking within the region studied and is probably located elsewhere in the genome. Despite this intriguing divergence, our results indicate that the Avipoxviruses are more closely related to the Orthopoxviruses than previously suspected.

Amino Acid Sequence↗
PubMed1986

Localization and sequence of a vaccinia virus gene required for multiplication in human cells.

A vaccinia virus host range mutant (hr mutant) deleted of 18 kilobase pairs at the left end of the genome has been employed to precisely map a viral gene required for multiplication in human cells. DNA fragments from the wild-type virus were inserted into the thymidine kinase gene of mutant virus by means of in vivo homologous recombination, and the recombinants obtained were screened for their ability to multiply in human cells. A short sequence, 855 base pairs long, overlapping the HindIII M and K fragments was able to restore a normal host range on the mutant virus. A single long open reading frame that could encode a polypeptide of 32.5 kDa was found in the nucleotide sequence of the host range gene. The direction of transcription and the length of the open reading frame are in excellent agreement with previous mapping of mRNAs within this region of the genome. In vitro translation of infected cell early mRNA, selected by hybridization to the host range gene, yielded a prominent polypeptide product whose size (29 kDa) was close to that expected from the predicted amino acid sequence. The phenotype of the hr mutant suggests that the host range gene plays a role in maintaining a high level of protein synthesis in human cells. It may behave positively by complementing the lack of an analogous cellular activity or negatively by antagonizing a cell function that inhibits viral multiplication.

Amino Acid Sequence↗
PubMed1985

Mapping of a vaccinia host range sequence by insertion into the viral thymidine kinase gene.

A vaccinia virus mutant deleted of ca. 18 kilobase pairs at the left-hand end of the genome is unable to multiply on many human cell lines. To determine whether all or some of the deleted sequences were responsible for the host range property, the corresponding region from wild-type DNA was cloned in three pieces into a vaccinia transplacement vector containing the thymidine kinase gene on the HindIII J fragment. The next step was to transfer these pieces to the genome of the host range deletion mutant by in vivo homologous recombination around the thymidine kinase locus. Transfer of one 5.2-kilobase-pair EcoRI fragment was found to restore a wild-type phenotype on the host range mutant, thus demonstrating that only a small portion of the 18-kilobase-pair deletion contains the host range function(s). This result also illustrates that the method initially devised for inserting foreign genes into vaccinia virus DNA is useful for studies of the vaccinia genome.

Cell Line↗
PubMed1983

Physical mapping of vaccinia virus temperature-sensitive mutations.

DNA restriction fragments from the Copenhagen strain of vaccinia virus have been molecularly cloned into the plasmid pAT 153. Eleven fragments obtained with SalI and nine fragments with HindIII together overlap about 90% of the 190 kb genome. The positioning of SalI and XhoI restriction sites demonstrated that vaccinia Copenhagen DNA differs in a number of sites from other vaccinia strains. Vaccinia temperature-sensitive mutants in 17 different complementation groups have been mapped to specific regions on the viral genome by marker rescue with cloned restriction fragments of the wild type strain. As a rule, the physical order of the mutations corresponded to the genetic order previously established from recombination data. The results have thus enabled the proper alignment of the genetic and physical maps and provided a means of comparing intervals expressed on these two scales. All ts mutations that have been mapped so far lie within a central region that spans approximately 60% of the genome. This clustering of mutations confirms and extends previous observations suggesting that the majority of the genes required for viral multiplication under in vitro conditions are located in the highly conserved central region of the orthopoxvirus genome.

Animals↗
PubMed1978

Host range restriction of vaccinia virus in Chinese hamster ovary cells: relationship to shutoff of protein synthesis.

Chinese hamster ovary cells were found to be nonpermissive for vaccinia virus. Although early virus-induced events occurred in these cells (RNA and polypeptide synthesis), subsequent events appeared to be prevented by a very rapid and nonselective shutoff of protein synthesis. Within less than 2 h after infection, both host and viral protein syntheses were arrested. At low multiplicities of infection, inhibition of RNA synthesis with cordycepin resulted in failure of the virus to block protein synthesis. Moreover, infection of the cells in the presence of cycloheximide prevented the immediate onset of shutoff after reversal of cycloheximide. Inactivation of virus particles by UV irradiation also impaired the capacity of the virus to inhibit protein synthesis. These results suggested that an early vaccinia virus-coded product was implicated in the shutoff of protein synthesis. Either the nonpermissive Chinese hamster ovary cells were more sensitive to this inhibition than permissive cells, or a regulatory control of the vaccinia shutoff function was defective.

Cell Line↗
PubMedDate not supplied

Expression of rabies virus glycoprotein from a recombinant vaccinia virus.

Rabies is one of the oldest diseases know to man, but its successful control has remained elusive. Although effective vaccines of tissue culture origin against rabies do exist, such preparations are expensive. Live vaccinia virus (VV) recombinants expressing influenza or hepatitis B antigens have recently been used to immunize against these diseases. We have now used this approach to produce a novel rabies vaccine. We first altered the rabies glycoprotein cDNA by site-directed mutagenesis and removed the poly(dG) tail. We then aligned the modified cDNA with an early VV promoter sequence inserted within a cloned copy of the vaccinia thymidine kinase gene and transfected this plasmid into VV-infected cells. Recombination between the virus and the plasmid resulted in a recombinant virus harbouring the rabies glycoprotein cDNA. Inoculation of rabbits with the live recombinant virus induced high titres of rabies virus-neutralizing antibodies, and scarification with the recombinant VV protected mice against challenge with street rabies virus.

Amino Acid Sequence↗