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R Drillien

Publications and source records attributed to R Drillien.

At least 37 records · Page 2Linked to original sources
PubMed1993

Detection of a protein encoded by the vaccinia virus C7L open reading frame and study of its effect on virus multiplication in different cell lines.

Vaccinia virus encodes several proteins, the activity of which is essential for multiplication in different cell types. Both the C7L and K1L open reading frames (ORFs) have been characterized as viral determinants for multiplication in human cells. To confirm and extend these findings we inserted the C7L ORF into the genome of a mutant virus unable to multiply in human cells and showed that this virus recovered its ability to replicate. Deletion of C7L from a wild-type viral genome did not adversely affect virus multiplication in human cells but it did reduce replication in hamster Dede cells. When both C7L and K1L were deleted from the vaccinia virus genome only poor or no viral yields were obtained from various human cell lines. Recombinant viruses were also constructed to facilitate the study of C7L protein synthesis during infection. One virus in which the lacZ ORF was fused downstream and in-frame with the C7L ORF enabled us to characterize the C7L protein as an early gene product. Another recombinant virus was constructed so that the carboxy terminus of the C7L ORF product contained an additional 28 amino acids from the carboxy terminus of K1L. Tagging of C7L in this way allowed us to detect the fusion protein by immunoprecipitation with antibodies against the K1L protein. Furthermore, the hybrid protein retained its biological properties. The recombinant viruses constructed in this work should be useful for studies of the molecular basis of the activity of viral host range proteins.

Animals↗
PubMed1993

The conserved N-terminal region of Sendai virus nucleocapsid protein NP is required for nucleocapsid assembly.

Sendai virus nucleocapsid protein NP synthesized in the absence of other viral components assembled into nucleocapsid-like particles. They were identical in density and morphology to authentic nucleocapsids but were smaller in size. The reduction in size was probably due to the fact that they contained RNA only 0.5 to 2 kb in length. Nucleocapsid assembly requires NP-NP and NP-RNA interactions. To identify domains on NP protein involved in nucleocapsid formation, 29 NP protein mutants were tested for the ability to assemble. Any deletion between amino acid residues 1 and 399 abolished formation of nucleocapsid-like particles, but mutants within this region exhibited two different phenotypes. Deletions between positions 83 and 384 completely abolished all interactions. Deletions between residues 1 and 82 and between residues 385 and 399, at the N- and C-terminal ends of the region from 1 to 399, resulted in unstructured aggregates of NP protein, indicating only a partial loss of function. Deletions within the C-terminal 124 amino acids were the only ones that did not affect assembly. The results suggest that NP protein can be divided into at least two separate domains which function independently of each other. Domain I (residues 1 to 399) seems to contain all of the structural information necessary for assembly, while domain II (residues 400 to 524) is not involved in nucleocapsid formation.

Animals↗
PubMed1992

Restricted replication of ectromelia virus in cell culture correlates with mutations in virus-encoded host range gene.

Ectromelia virus (strain Moscow) was shown to replicate poorly or not at all in cell lines derived from the rabbit or hamster. The failure of ectromelia virus to replicate in cell lines derived from the hamster suggested that the virus lacked a functional CHO host range (hr) gene required for multiplication in these cells. A DNA fragment which hybridized to the CHO hr gene was cloned from the ectromelia virus genome and shown by sequence analysis to be deleted of 506 bp within the ectromelia virus CHO hr homologue. Two additional ectromelia viruses (Hampstead and Mill Hill strains) were also shown to lack an intact CHO hr gene. Insertion of the CHO gene from cowpox virus into the ectromelia virus genome extended the host range of ectromelia virus in tissue culture. These results demonstrate that an intact CHO hr gene is not required for maintenance of ectromelia virus in nature and provide a partial explanation for ectromelia virus' narrow host range, as opposed to the broad host range of cowpox virus, which has a functional CHO hr gene.

Animals↗
PubMed1992

Construction of vaccinia virus recombinants expressing several measles virus proteins and analysis of their efficacy in vaccination of mice.

Measles virus genes encoding the haemagglutinin (HA), fusion protein (F) or nucleoprotein (NP) have been inserted into the vaccinia virus genome either alone or in various combinations. In each case the measles virus genes were expressed from the 7.5K promoter and were incorporated into the thymidine kinase (tk) or K1L loci of the Copenhagen strain of vaccinia virus. Cells infected by the recombinants synthesized measles virus proteins indistinguishable from those induced in measles virus-infected cells. However, in some instances the level of expression in cells infected by recombinants expressing more than one measles virus gene was reduced when compared to those encoding a single gene. The sera from mice immunized with recombinants containing either HA, HA.F, HA.NP or HA.F.NP had similar levels of measles virus neutralizing antibodies which remained constant throughout a 7 month period. Analysis of these sera by immunoprecipitation of radiolabelled measles virus confirmed the presence of specific antibody to each of the antigens where appropriate. The introduction of the measles virus genes into the K1L and the tk sites despite attenuating the virus for mice by 10-fold and 1000-fold respectively did not affect the vaccination efficiency, i.e. ability to induce measles virus antibody and protect mice. Vaccination of BALB/c (H2d) mice with HA and F, but not NP, recombinants completely protected the animals against a lethal measles virus challenge. In contrast, although the HA recombinant protected CBA (H2k) mice, the F recombinant did so poorly. However, by immunizing CBA mice with a recombinant expressing both F and NP, protection was increased to more than 75%. Our findings demonstrate the ability of three measles virus antigens expressed from the vaccinia virus genome alone or in combination to contribute to protective immunity against measles virus infection of mice. They also suggest that the association of measles virus antigens in a single recombinant DNA vaccine could be beneficial to overcome host-related restriction of the immune response to particular antigens.

Animals↗
PubMed1991

Assembly of nucleocapsidlike structures in animal cells infected with a vaccinia virus recombinant encoding the measles virus nucleoprotein.

A vaccinia virus recombinant containing the measles virus nucleoprotein gene was shown to induce the synthesis of a 60 kDa phosphorylated nucleoprotein similar to authentic measles virus nucleoprotein. Mammalian or avian cells infected with the recombinant virus displayed tubular structures reminiscent of viral nucleocapsids both in the cytoplasm and in the nucleus. Such structures could be labelled in situ by using an immunogold detection method specific for measles virus proteins. Electron microscopic examination of tubular structures purified from cells infected with the vaccinia virus recombinant indicated that they displayed most of the features of measles virus nucleocapsids, although their length was on the average shorter. These results demonstrate the spontaneous assembly of measles virus nucleocapsids in the absence of viral leader RNA and provide a means for a detailed molecular analysis of the requirements for nucleocapsid assembly. Furthermore, these findings raise the possibility of achieving complete assembly of measles virus particles, devoid of infectious RNA, by using a vaccinia virus vector.

Animals↗
PubMed1990

Fowlpox virus recombinant encoding the measles virus fusion protein: protection of mice against fatal measles encephalitis.

A recombinant Fowlpox virus engineered to encode the measles virus fusion protein was shown to protect mice against a challenge measles infection. A vaccine dose of about 10(6) p.f.u. was needed to protect nearly 100% of the animals. Mice failed to develop a significant level of antibodies directed against measles virus suggesting that other components of the immune system may be involved.

Animals↗
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

One hundred base pairs of 5' flanking sequence of a vaccinia virus late gene are sufficient to temporally regulate late transcription.

A vaccinia virus late gene coding for a major structural polypeptide of 11 kDa was sequenced. Although the 5' flanking gene region is very A+T rich, it shows little homology either to the corresponding region of vaccinia early genes or to consensus sequences characteristic of most eukaryotic genes. Three DNA fragments (100, 200, and 500 base pairs, respectively), derived from the flanking region and including the late gene mRNA start site, were inserted into the coding sequence of the vaccinia virus thymidine kinase (TK) early gene by homologous in vivo recombination. Recombinants were selected on the basis of their TK- phenotype. Cells were infected with the recombinant viruses and RNA was isolated at 1-hr intervals. Transcripts initiating either from the TK early promoter, or from the late gene promoter at its authentic position, or from the translocated late gene promoters within the early gene were detected by nuclease S1 mapping. Early after infection, only transcripts from the TK early promoter were detected. Later in infection, however, transcripts were also initiated from the translocated late promoters. This RNA appeared at the same time and in similar quantities as the RNA from the late promoter at its authentic position. No quantitative differences in promoter efficiency between the 100-, 200-, and 500-base-pair insertions were observed. We conclude that all necessary signals for correct regulation of late-gene expression reside within only 100 base pairs of 5' flanking sequence.

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↗