A vaccinia virus transfer vector using a GUS reporter gene inserted into the I4L locus.

A vaccinia virus (VV) transfer vector is described which enables integration of heterologous sequences into the I4L locus (ribonucleotide reductase-encoding gene) through co-insertion of a GUS selection marker. I4L- VV recombinants formed blue plaques when an agarose overlay containing XGluc (5-bromo-4-chloro-3-indolyl-beta-glucuronide) was added to the infected cell monolayer. Viruses already containing a lacZ reporter gene were also suitable recipients for the selection procedure since infection with a VV lacZ recombinant did not produce any blue plaques with XGluc. The addition of a synthetic early promoter downstream from the GUS cassette initiated the predicted-size transcript during an infection. Insertion of genes with VV p7.5-promoters into the I4L, J2R and K1L loci of the same virus produced viable virus recombinants even though recombination between these loci could be demonstrated. These techniques should be valuable for the further development of VV as a polyvalent vector.

The vaccinia virus J5L open reading frame encodes a polypeptide expressed late during infection and required for viral multiplication.

A number of open reading frames (ORFs) are found in the vaccinia virus (VV) genome whose activities in the viral life cycle have not yet been determined. This report examines one such ORF, designated J5L, which was demonstrated to be essential for viral multiplication. Stable inactivation of the J5L ORF by insertion of a lacZ ORF was impossible unless another copy of the J5L ORF was present in the VV genome. Fusion genes between the J5L ORF and either the lacZ gene or the VV K1L gene were employed to study its temporal expression as well as its protein product. These experiments showed that J5L is transcribed late in infection and gives rise to a protein product which migrates by SDS-PAGE with the expected molecular weight (16 kDa). Numerous unsuccessful attempts to establish a stable cell line expressing J5L suggest that the J5L gene product could be cytotoxic.

A functional measles virus replication and transcription machinery encoded by the vaccinia virus genome.

Measles virus encodes three proteins required for the encapsidation, transcription and replication of viral genomes. The genes for these proteins have been inserted into the vaccinia virus genome together with the gene for the bacteriophage T7 RNA polymerase. Cells infected with this recombinant virus were able to encapsidate, transcribe and replicate a CAT gene positioned in the negative polarity behind a T7 promoter and flanked by measles virus genomic termini. Inhibition of the accumulation of the nucleocapsid proteins by actinomycin D led to an increase in CAT expression. Thus the measles virus polymerase activity, encoded by the vaccinia genome, was regulated by the level of measles proteins just as the authentic polymerase. The recombinant vaccinia described in this study could be useful for the production of measles virus-like particles encoding foreign genes and employed in vaccination or gene therapy strategies.

[Update on smallpox vaccines]. / Actualités sur la vaccination antivariolique.

Smallpox is among the most dangerous pathogens that could be used by bioterrorists. The former vaccines produced by scarification on the flanks of calves or sheep could be used to protect the whole French population when used with bifurcated needles. They should be replaced by a second-generation vaccine grown in cell culture and, eventually later by new and safer third-generation vaccines using non-replicative viral strains.

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.

Ureidosuccinic acid uptake in yeast and some aspects of its regulation.

Ureidosuccinic acid (USA) is an intermediary product in pyrimidine biosynthesis. When proline was the sole nitrogen source, USA uptake occurred; however, when ammonium sulfate or glutamic acid was the nitrogen source, uptake was inhibited. Thus, a ura2 strain which does not synthesize USA would not grow when this substance was supplied on an ammonium sulfate or glutamic acid medium. Mutants are described in which uptake was constitutive on such a medium. Permeaseless mutants for USA have been found, and evidence is presented for permease specificity. It is shown that all constitutive mutants use the same transport system that is missing in the permeaseless mutant. These mutants are constitutive for two permeases: the specific USA permease and the general amino acid permease. The transport system studied here, like the general amino acid transport system, is regulated by nitrogen metabolism. These facts and others suggest that our permease constitutive mutants are impaired in nitrogen metabolism.

Biological properties of recombinant HIV envelope synthesized in CHO glycosylation-mutant cell lines.

N-glycosylation of the human immunodeficiency virus type-1 envelope (Env) glycoprotein precursor (gp160) occurs by transfer of Glc3Man9GlcNAc2 onto the nascent protein. Maturation then occurs via cleavage of the three Glc residues, which starts during translation. These events are considered necessary to create Env functional conformation: treatment with "alpha"-glucosidase inhibitors, but not alpha-mannosidase inhibitors (i) impairs gp160 cleavage into gp120 and gp41, (ii) diminishes the accessibility of gp120 V3 region, (iii) prevents gp120 binding to its CD4 receptor, and (iv) prevents gp41-mediated membrane fusion. These inhibitors are of therapeutic interest. Here, using a collection of parent and mutant CHO cells that possess mutations in different steps of glycosylation, we reassessed the role of glycans in both the processing and the properties of recombinant gp160 expressed from a vaccinia virus vector. Mutant cells were as follows: Lec23 (which lacks alpha-glucosidase I activity) produces a collection of triglucosylated structures (Glc3Man7-9GlcNAc2); LEC10 (which has increased GlcNAc transferase III activity) produces complex glycans with a bisected GlcNAc residue; Lec1 (which lacks GlcNAc transferase I) and Lec3.2.8.1 (which lacks GlcNAc transferase I and has decreased activity of CMP-NeuNAc and UDP-Gal translocases) produce Man5GlcNAc2 glycans at complex or hybrid sites. As expected, glycosylation of Env produced from mutants was affected but, irrespective of the glycosylation phenotype, (i) similar quantities of Env were synthesized, (ii) the immunoreactivity of V3 was similar, (iii) gp160 was efficiently cleaved into gp120 and gp41, (vi) Env was exposed at the cell membrane, (v) secreted gp120 bound CD4, and (vi) membrane gp41 was able to induce membrane fusion with CD4+ cells. Thus, the glycosylation alterations examined are dispensable for Env processing and biological activity in CHO cells. In particular, removal of the three outer Glc residues was not required per se for Env folding in this system because functional Env is obtained from Lec23 cells: it appears therefore that lack of modification is not equivalent to drug inhibition of modification. These data are discussed in the light of previous reports describing the use of glycosidase inhibitors to alter glycosylation.

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.

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.

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.

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.

Effect of cell-mediated immune factors on the replication of an attenuated temperature-sensitive mutant of vaccinia virus.

Studies of the in vivo multiplication of an attenuated temperature-sensitive strain of vaccinia virus (ts2) indicated that temperature sensitivity alone could not account for the attenuation. Immunodepressive treatment of intracerebrally inoculated mice had a dramatic stimulatory effect on the multiplication of the attenuated strain and suggested that establishment of ts2 infection in the mice was hindered by host defense mechanisms mediated by cellular elements. Experiments carried out in vitro with the 51Cr release assay showed that cells infected with the ts2 mutant represented more susceptible targets to the cytotoxic action of immune spleen cells than cells infected with the parent vaccinia strain. Moreover, it appeared that ts2 replication, compared with wild-type replication in vitro, was more inhibited by the immune spleen cells. Although this work did not evaluate the role of specifically sensitized lymphocytes within the lesions, indirect evidence suggests that lymphocytes could cause a more effective halt in ts2 virus multiplication than in wild-type multiplication.

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.

Vaccinia virus-related events and phenotypic changes after infection of dendritic cells derived from human monocytes.

The in vitro interactions between vaccinia virus (VV) and monocyte-derived human dendritic cells (DC) have been studied to gain a better understanding of the mechanisms involved in the induction of an immune response by VV. This work showed that VV binds to DC less efficiently than to HeLa cells (HeLa). Capping of viral antigens on the DC surface and electron microscopic examinations suggested that VV enters into DC mainly by endocytosis instead of fusion as for HeLa. Early viral-encoded proteins were expressed in DC but late viral proteins and viral DNA synthesis did not occur. Nevertheless, when successfully infected, DC expressed a similar amount of a foreign, viral-encoded protein, as HeLa, if the early component of the p7.5 promoter was used. VV infection did not lead to DC maturation as determined by following the level of several cell surface markers associated with maturation, but an inhibition of the expression of the costimulatory molecule CD80 was noticed. The proliferation of allogeneic peripheral blood lymphocytes (PBL) was stimulated by VV-infected DC or inhibited depending on the particular donor lymphocytes employed. PBL from VV-vaccinated individuals with good memory responses to VV antigens proliferated in the presence of infected autologous DC. PBL from individuals with poor memory responses to VV and one unvaccinated individual also proliferated, albeit to a lower level, in the presence of infected autologous DC. These results suggest that VV-infected DC could both stimulate memory cells and prime naive cells in vitro.

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.

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.

Citrate synthaseless glutamic acid auxotroph of Saccharomyces cerevisiae.

Relationship of citrate synthase (EC 4.1.3.7) to the biosynthesis of glutamic acid was investigated by characterizing a new glutamic acid auxotroph FL100-D1 (glu 3) of Saccharomyces cerevisiae. Nutritional requirement of the mutant was satisfied by L-glutamic acid, L-glutamic acid peptide as well as several analogs of glutamic acid, but not by proline, ornithine, arginine, lysine or aspartic acid. The mutant was unable to utilize nonfermentable carbon sources, glycerol, acetate or lactate. Mutant glu3 unlike aconitaseless glutamic acid auxotroph glu 1, failed to accumulate 14C-citric acid in vivo from 1-14C-sodium acetate or U-14C-glutamic acid. Both spectrophotometric and radioactive assay procedures demonstrated a lack of significant citrate synthase activity in the dialysed extract of the mutant compared to the wild type strain. Mutant glu 3 complemented with glu 1 and glu 2 individually in vivo and exhibited a significant aconitase (EC 4.2.1.3) activity in vitro.

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.

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.