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Post-transplantation lymphoproliferative disease (PTLD) is associated with Epstein-Barr virus (EBV). Quantitative and qualitative differences in EBV in peripheral blood mononuclear cells (PBMCs) of PTLD patients and healthy controls were characterized. A quantitative competitive polymerase chain reaction (QC-PCR) technique confirmed previous reports that EBV load in PBMCs is increased in patients with PTLD in comparison with healthy seropositive controls (18 539 vs 335 per 10(6) PBMCs, P =.0002). The average frequency of EBV-infected cells was also increased (271 vs 9 per 10(6) PBMCs, P =.008). The distribution in numbers of viral genome copies per cell was assessed by means of QC-PCR at dilutions of PBMCs. There was no difference between PTLD patients and healthy controls. Similarly, no differences in the patterns of viral gene expression were detected between patients and controls. Finally, the impact of therapy on viral load was analyzed. Patients with a past history of PTLD who were disease-free (after chemotherapy or withdrawal of immunosuppression) at the time of testing showed viral loads that overlapped with those of healthy seropositive controls. Patients treated with rituximab showed an almost immediate and dramatic decline in viral loads. This decline occurred even in patients whose PTLD progressed during therapy. These results suggest that the increased EBV load in PBMCs of PTLD patients can be accounted for by an increase in the number of infected B cells in the blood. However, in terms of viral copy number per cell and pattern of viral gene expression, these B cells are similar to those found in healthy controls. Disappearance of viral load with rituximab therapy confirms the localization of viral genomes in PBMCs to B cells. However, the lack of relationship between the change in viral load and clinical response highlights the difference between EBV-infected PBMCs and neoplastic cells in PTLD.

Three conserved histidine residues, His-243, His-781, and His-788, located within the large subunit of carbamoyl phosphate synthetase from Escherichia coli were identified by sequence identity comparisons. These three histidine residues were individually mutated to asparagine residues. The H243N mutant enzyme was found to be critical for carbamoyl phosphate synthesis as the mutant protein was unable to synthesize carbamoyl phosphate at a significant rate (< 1/1500). By analysis of the effects of this mutation on the partial reactions catalyzed by CPS, it was determined that this mutation blocked the formation of the carbamate intermediate from carboxyphosphate and ammonia. The H781N mutant enzyme had an order of magnitude reduction for both the rate of carbamoyl phosphate formation and ATP synthesis which is consistent with the proposal that the carboxyl-terminal half of the large subunit is primarily involved in the phosphorylation of the putative carbamate intermediate. This mutation also reduced the effects of the allosteric activator ornithine on the Km parameters for ATP in the overall biosynthetic reaction and ADP in the ATP synthesis reaction. The H788N mutant enzyme is a functional protein which maintains the ability to synthesize carbamoyl phosphate at a rate comparable to that of the wild-type enzyme. The effects of this mutation are 10-fold reductions of the ATP synthetase and the bicarbonate-dependent ATPase activities with substantial increases in the Km values for ATP in the full biosynthetic reaction and for ADP in the ATP synthesis reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

A homologue to the glycoprotein H (gH) gene of herpes simplex virus (HSV) has been identified in the genome of infectious bovine rhinotracheitis virus (IBR, BHV-1). The gene is located immediately downstream from the thymidine kinase gene, and codes for an open reading frame (orf) of 842 amino acids. The orf has the characteristics of a membrane glycoprotein, including an N-terminal hydrophobic region resembling a signal sequence, a C-terminal region which is probably a transmembrane domain, and six potential sites for N-linked glycosylation. This orf shows significant homology to the gH sequences of both HSV and pseudorabies virus (PRV). We conclude that this gene encodes BHV-1 gH.

Sodium butyrate was used to enhance expression levels and thereby facilitate the generation of analytical quantities of nine different tissue plasminogen activator (tPA) analogues expressed under the control of the cytomegalovirus immediate early (CMV IE) promoter by the Chinese hamster ovary (CHO) mammalian expression system. Production involved growth in roller bottles, using serum free or low serum media formulations, together with repetitive, sodium butyrate inductions. Average inductions in the presence of sodium butyrate ranged from 2 to 9-fold relative to uninduced controls, using cell lines with no previous butyrate exposure. Retardation of growth rate by butyrate minimized the need to split cells during the production runs, extending longevity of roller bottles containing cells secreting at induced levels. SDS-PAGE analyses indicate a consistently high percentage of single-chain material. Measurements of specific activity and fibrinogen fragment enhancement for one of the analogues demonstrate that neither of these two critical parameters are affected by production in the presence of butyrate. Induction kinetic data and growth curves for the expression of sCD4 under control of the SV40 early promoter demonstrate that the benefits of butyrate can be realized with different promoters and heterologous genes, and are additive when used in conjunction with an amplified cell line constitutively expressing at elevated levels. This work demonstrates the practical application of sodium butyrate in the production of analytical quantities of protein from the CHO expression system, and suggests a role for sodium butyrate in commercial scale processes as well.

Two Aujeszky's disease virus glycoprotein genes, gX and g1, have been used to produce deletion mutants which have then been developed into vaccines. These deletions then allow differentiation between pigs infected with wild type virus and those given the vaccine. It is not clear whether the glycoproteins encoded for by these genes are needed to induce a full protective immune response, in which case deletion mutants would suffer from lack of potency. To test this, commercially available Aujeszky's virus vaccines which lacked either gX or g1 were compared and isogenic constructs were made which differed only in the absence or presence of gX and, or, g1. These constructs and vaccines were used to vaccinate the natural host of Aujeszky's disease, the pig, and potency was measured using challenge with wild type virus. In all cases vaccines which lacked g1 performed significantly less well than those in which g1 was present, whereas deletions of gX had no significant effect on vaccine performance.

The O-glycosidically linked oligosaccharides on the pseudorabies virus (PRV) glycoprotein gp50 synthesized by three different cell lines were studied. The intact membrane protein (gp50) was expressed in Vero cells and in the insect cell line Sf9. In addition, a truncated, secreted form lacking the transmembrane and cytoplasmic domains (gp50T), was expressed in CHO and Sf9 cells. The protein, both in intact and truncated form, synthesized by the two mammalian cells contained only the disaccharide Gal beta 1-3GalNAc, either unsubstituted or substituted with one or two sialic acid residues. By contrast, the major O-linked structure on gp50 and gp50T synthesized by Sf9 cells was the monosaccharide GalNAc. The Sf9 cells also linked lower amounts of Gal beta 1-3GalNAc to gp50 (12%) and gp50T (26%). None of the structures synthesized by Sf9 cells contained sialic acid. Measurements of the two relevant glycosyltransferases revealed that while all three cell lines contain comparable levels of UDP-GalNAc:polypeptide, N-acetylgalactosaminyltransferase activity, there is a greater variation in the levels of UDP-Gal:N-acetylgalactosamine, beta 1-3 galactosyltransferase, with the Sf9 cells containing the lowest level.

Pseudorabies virus (PRV) is a herpesvirus of pigs. Homologous recombination with plasmids offers a method to engineer precise changes in the PRV genome to produce advantageous live vaccines. Safety can be ensured by using a non-reverting deletion to inactivate the thymidine kinase gene. One particularly important feature of new PRV vaccines is deletion of an antigen, so that vaccinated pigs are serologically distinguishable from infected pigs. We have constructed a live vaccine strain with deletions in the thymidine kinase gene and in the gene for a glycoprotein, gX. Molecular engineering techniques made it possible to choose deletion of gX, which has no known immunological significance, over deletion of other glycoproteins that contribute to protective immunity. Extensive experiments in pigs with isogenic virus pairs show that deletion of gX does not compromise efficacy of a vaccine as gI deletions do. Deletion of gX also suggests a site for replacement with antigens from other pathogens. In addition to molecular engineering of a live vaccine strain, research on PRV glycoproteins has led to the discovery that expression of the glycoprotein gp50 makes cells resistant to PRV infection. Perhaps this observation could be extrapolated to the level of a whole animal to allow engineering of pigs to become an alternative to engineered vaccines.

We have developed a technique for the insertion of any DNA fragment into the herpes simplex virus (HSV) genome at specific sites. This technique was used to resolve a specific problem concerning the isomerization of the HSV genome. Briefly, HSV DNA consists of four isomers differing in the orientation of two covalently linked components, L and S, relative to each other. Each component consists of unique sequences flanked by inverted repeats. To determine whether the isomerization of HSV DNA is the result of generalized recombinatin between homologous reiterated sequences in the inverted repeats or the result of site-specific recombination, we constructed plasmids in which DNA fragments derived from various regions of the viral genome were inserted in both orientations into the thymidine kinase gene, rendering it nonfunctional. The HSV DNA sequences in the plasmids were then recombined into the viral genome, and viral recombinants were selected for their thymidine kinase-deficient phenotype. The insertion of these fragments by homologous recombination was highly efficient in that all the viral clones isolated contained the inserted fragment at the expected location. The only fragments that promoted additional inversions of the viral genome were those spanning the junction between the L and S components. Furthermore, analysis of isomers formed by these recombinants indicates that the inversions occur only when sequences in the inserted fragment are in inverted orientation in relation to homologous sequences at the termini or at the authentic junction.

Experience with cloning pseudorabies virus (PRV) DNA in the lambda gt11 phage vector has shown that there are special requirements for the antisera used in screening the libraries, in addition to the requirement that the antisera recognize proteins on a Western blot. Initial screening of a lambda gt11 library of sheared PRV DNA fragments in Escherichia coli for expression of PRV antigens using PRV hyperimmune antisera was unsuccessful. It was only after screening the library with antisera raised against PRV proteins eluted from sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels that positive results were obtained. These "gel-slice" antisera (GSA) were equivalent in potency to hyperimmune antisera in standard immunoassays (including ELISA, immunoprecipitation, Western blots, and neutralization of virus), but only the GSA could recognize PRV fusion proteins expressed by recombinant lambda gt11 phage. This difference was seen despite the fact that hyperimmune antisera performed satisfactorily on Western blots of denatured PRV-infected cell extracts. These results show that the efficiency of screening expression libraries in E. coli can be improved if antibodies are raised against denatured proteins.

A pseudorabies virus (PRV) mutant with deletions in genes for glycoprotein X (gX) and thymidine kinase, designated delta GX delta TK, was constructed and evaluated as a vaccine for protecting swine against PRV-induced mortality. Doses greater than or equal to 10(3) plaque-forming units (PFU) of this strain given to mice provided protection from challenge exposure with virulent PRV. Sera tested from mice inoculated with delta GX delta TK had high titers of neutralizing antibody to PRV, but reactivity in the same sera was not significantly different from that in sera from noninoculated mice (controls) when sera from both groups were evaluated by use of an ELISA with gX antigen produced in Escherichia coli. Compared with noninoculated pigs (controls), those given delta GX delta TK (greater than or equal to 10(2) PFU) were protected completely from lethal challenge exposure, without experiencing adverse effects on weight gain and with reduction of shedding of virulent challenge virus. Serotest results indicated that, although inoculated pigs responded with strong neutralizing antibody titers, the response of delta GX delta TK-inoculated pigs to gX, as determined by ELISA before challenge exposure, was not significantly greater than the ELISA values obtained from control pigs. The ELISA values from a group of pigs inoculated with a commercially available vaccine were significantly (P less than 0.05) higher than those of control pigs. The experimental vaccine, delta GX delta TK, was avirulent for mice, swine, and sheep, but was mildly virulent for calves (mortality, 1 of 12) and more virulent for dogs (mortality, 3 of 6) and cats (mortality, 2 of 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Previous studies showed that the gene for RNA polymerase subunit alpha at 72 min on the Escherichia coli chromosome is co-transcribed with genes for ribosomal proteins (r-proteins) S11, S4, and L17, and probably S13. DNA sequence analysis of a deletion mutant has now established that the S13 gene is a part of the alpha operon and the gene order is promoter (P alpha), rpsM (S13), rpsK (S11), rpsD(S4), rpoA(alpha), and rplQ(L17). The DNA sequence extending 650 bases before S13 gene was determined. In vitro transcription experiments establish the probable location of the alpha promoter (P alpha) within this sequence. The start site is 94 nucleotides upstream from the initiation codon (GUG) of the S13 gene. This promoter has features previously noted as common to E. coli promoters. However, comparison with four other sequenced promoters of r-protein operons shows no unique common features that might account for the common regulation of synthesis of r-proteins. This lack of sequence similarity in promoters of r-protein operons may be because r-protein synthesis is regulated at least partially at a post-transcriptional level.

The lambda fus3 transducing phage carries several operons for ribosomal proteins of E. coli, including the str operon. A mutant transducing phage with a promoter mutation in this operon has been isolated. This mutant shows reduced stimulation of synthesis of proteins encoded by the operon, S12, S7, and elongation factors G and Tu, in ultraviolet-irradiated cells. This mutation also abolishes in vitro transcription from the str promoter. The DNA sequence of the mutant promoter shows that it is a point mutation 6 bases upstream from the in vitro transcription start site, changing the "Pribnow box" sequence from TAAAATT to TAAAACT. These results indicate that the site altered by the mutation, which is in the region just preceding the transcription start site, is important for the expression of the str operon.

The gene for RNA polymerase subunit alpha is a co-transcribed with several ribosomal protein genes (Jaskunas, S.R., Burgess, R.R., and Nomura, M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 5036-5040). The DNA sequence whicch codes for the COOH-terminal amino acids of S4 and the NH2-terminal amino acids of alpha, and the 25 nucleotide intercistronic sequence have been determined. This short distance supports the idea that some post-transcriptional regulation determines the differential synthesis of alpha and ribosomal proteins.

The cDNA coding for human tissue plasminogen activator (tPA) was cloned downstream from the promoter for pseudorabies virus (PRV) glycoprotein and flanked by downstream PRV DNA. After co-transfection with PRV DNA, this plasmid recombined to insert the tPA cDNA into the viral genome. In cells infected by this recombinant virus, tPA was detected by immunoprecipitation analysis and by enzymatic activity. Since it has a wide host range but does not infect humans, PRV is a possible vaccine vector for genes from animal pathogens.

A baculovirus expression vector was constructed with the tissue plasminogen activator (TPA) cDNA under the control of the viral polyhedrin promoter. After infection of insect cells with the recombinant baculovirus, active TPA was secreted into the medium in which these cells were grown. TPA was isolated from the conditioned media using metal chelate affinity chromatography followed by immunoaffinity purification using mouse monoclonal anti-human TPA coupled to Sepharose. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions and sequence analysis of recombinant human TPA have revealed a two-chain form of the enzyme. The N-terminal amino acid was identified to be serine, indicating that it was processed at its N-terminus by the insect cell culture in a manner similar to that observed for mammalian cells. The relative specific activity of recombinant TPA from insect cells is comparable to that of Bowes melanoma TPA standard. Its activity is stimulated in the presence of fibrinogen fragments, but by a factor about 2.3-fold lower than the Bowes melanoma TPA. The apparent molecular weight of recombinant TPA from insect cells was about 60K by fibrin agar activity gels, suggesting less complex glycosylation than recombinant TPA from mammalian cells.

Pseudorabies virus (PRV) produces a glycoprotein, gX, that accumulates in the medium of infected cells. The gX gene was expressed in Chinese hamster ovary cells (CHOgX cells) using the cytomegalovirus Towne major immediate early promoter. Like PRV-infected cells, CHOgX cells produced gX and exported it into the medium. Tunicamycin reduced the molecular weight of the gX in the medium to 89 kDa, compared with 99 kDa for gX made in the absence of drug. In the presence of tunicamycin gX produced by both PRV-infected cells and CHOgX cells was still glycosylated, as indicated by incorporation of [14C]glucosamine. The most likely form of this glycosylation is O-linked. In a pulse-chase experiment, gX first appeared in a 90-kDa form, then a 115-kDa form. This 115-kDa form is probably cleaved to give the 99-kDa form of gX that is released into the medium. The 115-kDa form was much more persistent in the PRV-infected Vero cells than in the CHOgX cells. In both cell types, gX was labeled by [35S]sulfate in the presence and absence of tunicamycin.

We describe a general method for inactivation and deletion of genes at specific sites in large DNA genomes. In the first step of the procedure, the herpes simplex virus thymidine kinase is inserted into the genome at a specific site. In the second step, the thymidine kinase gene is desired sequences flanking the insertion site are deleted. Both steps involve recombination of the genomes with cloned chimeric fragments and utilize the available selection for or against thymidine kinase to select the desired genomes. We have applied the procedure to inactivate and to delete portions of an alpha gene of herpes simplex virus 1 specifying protein 22. The recombinant virus carrying the thymidine kinase inserted into the gene 22 and viruses exhibiting 0.1 kb and 0.7 kb deletions in the gene 22 specify new alpha polypeptides with molecular weights approximately 30% of the wild-type gene 22 product and grown normally in Vero cell cultures.

We report a system for investigating promoters of eucaryotic cell and virus genes based on analyses of the regulation of herpes simplex virus 1 (HSV-1) thymidine kinases whose structural gene sequences have been fused to the promoter of the gene under study. In infected cells, the polypeptides specified by HSV-1 form at least three groups, alpha, beta and gamma, whose synthesis is coordinately regulated and sequentially ordered at the transcriptional level. To identify the DNA sequence responsible for the regulation of transcription of alpha genes, we fused the sequence encoding the 5' end of an alpha gene to the structural gene sequence of the thymidine kinase, a beta gene. The resultant recombinant DNA was inserted into the viral genome and was also used to convert Ltk- cells to tk+ phenotype. In cells infected with recombinant virus, the thymidine kinase gene was regulated and expressed as an alpha gene-that is, it was transcribed and processed in the absence of prior infected cell protein synthesis. Moreover, mRNA selected by hybridization to sequences encoding the thymidine kinase contains at its 5' terminus sequences homologous to the donor sequence encoding the t'terminus of the alpha mRNA. In converted tk+ cells, the fused thymidine kinase gene, like the wild-type gene, is stimulated by superinfection with the tk- virus. However, the stimulation is many times greater and is due to non-alpha-gene products, whereas in cells converted by the wild-type gene, the stimulation is by alpha gene products. We conclude that the alpha genes are identified for transcription by sequences at or near those encoding the 5' terminus of the mRNA, and transposition of these sequences to a beta gene is all that is required to convert it to an alpha gene. Transcription of alpha genes appears to be regulated by non-alpha-gene products, which could be contained within the structure of the virion. In converted Ltk+ cells, the thymidine kinase gene uses its own promoter.

Hybridomas were selected for secretion of monoclonal antibodies directed against pseudorabies virus (PRV) glycoproteins. Each monoclonal antibody was capable of neutralizing PRV in vitro in the presence of complement. This panel of antibodies was used in passive immunization studies to protect mice and swine from PRV-induced mortality. The most protective antibody in mice was 3A4, specific for PRV glycoprotein gp50, which afforded as high as 100% protection. Although antibody 3A4 was partially protective in swine, antibody 3D11, which is specific for PRV glycoprotein glll, afforded greater protection--83% protection when ascitic fluid was used and 100% protection when immunoglobulin concentrated from cell cultures was used at a dose of 150 mg/pig. These studies demonstrated that monoclonal antibodies may be useful for short-term prophylaxis against PRV-induced disease and that antibody directed against either PRV glycoprotein glll or gp50 is sufficient to protect animals from PRV-induced mortality.

The DNA sequences have been determined for promoter regions of two ribosomal protein operons in E. coli, the str operon and the spc operon. The site of in vitro transcription initiation within each of these promoter regions has been determined. The start site of the str operon occurs 69 bases upstream from the initiation codon of the S12 gene. The start site of the spc operon occurs 72 bases upstream from the L14 gene, and only 91 bases downstream from the termination codon of the S17 gene (which is in the preceding S10 operon). Both promoters are similar to other sequenced promoters in that they each have an identifiable "Pribnow box" sequence 5 bases upstream from the transcription start site. The spc promoter has a long sequence of 2 fold symmetry centered within the Pribnow box; the str promoter has a shorter but similar symmetry. At positions -69 through -40 in the spc operon, another long region of symmetry is present which may be the termination signal of the preceding S10 operon. Extensive sequence similarity between the str and spc promoter regions is found downstream from the Pribnow box-that is, in a transcribed region preceding the translation start sites.

The str operon at 72 min on the Escherichia coli chromosome contains genes for ribosomal proteins (r-proteins) S12 (str or rpsL) and S7 (rpsG) and elongation factors G (fus) and Tu (tufA). The sequence of the entire S12 gene, the S12-S7 intercistronic region, and the beginning of the S7 gene is reported. Also, the sequence of the end of the S7 gene, the S7-G intercistronic region, and the beginning of the elongation fractor G gene is reported. The S12-S7 intercistronic region is 96 base pairs long, in contrast to other intercistronic regions in r-protein operons which have been found to vary from 3 to 66 base pairs. The S7-G intercistronic region is only 27 bases long, supporting the previous conclusion that r-protein and elongation factor genes are co-transcribed. A comparison of translation initiation sites of the S12 and S7 genes, and other examples of co-transcribed r-protein genes, reveals no obvious features that could account for equimolar synthesis of all r-proteins. The codon usage in the S12 and S7 genes follows the pattern observed in other r-protein genes; that is, there is a highly preferential usage of codons recognized by the most abundant of isoaccepting tRNA species. This pattern could reflect the cell's need for efficient translation or minimal errors, or both, in r-protein synthesis.

Transfection of Chinese hamster ovary (CHO) cells with a plasmid containing the cDNA for human preprorenin has provided cell lines that secrete 15 mg of native prorenin per liter of culture medium. Tryptic activation of the prorenin occurs by selective cleavage of the Arg66-Leu67 bond (numbering as in preprorenin). The renin product, purified in a single step and in high yield by affinity chromatography, is fully stable for as long as 8 months when stored in solution at 4 degrees C and pH 6.5. Purity of the renin was judged to be greater than 95% by gel electrophoresis, compositional and N-terminal sequence analyses, and specific enzyme activity. An important aspect of the present work is the development of a direct assay for renin which permits accurate and reproducible evaluation of enzyme units and kinetic parameters. Application of methods described herein, combined with appropriate scale-up fermentation capabilities, provides the means for generating gram quantities of human renin and its zymogen.

An open reading frame coding for an 11-kDa protein was located downstream from the gI gene of pseudorabies virus (PRV). This open reading frame is homologous to an open reading frame (US9) in an analogous position in herpes simplex virus and to an open reading frame (US1) in a different position in varicella zoster virus. The open reading frame encoding the 11-kDa protein is in a region known to be deleted in live attenuated vaccine strains of PRV.

The promoters from the str and spc operons of ribosomal proteins from E. coli were inserted into the Hind II cleavage site of mini-Col E1 (pVH51) plasmid. For both promoters, strains with the hybrid plasmid accumulated a small RNA species not present in strains carrying the vector. These RNAs were analyzed by RNA sequencing techniques and compared to DNA sequences. In both cases, synthesis of the new RNA species is initiated by the cloned r protein promoter at the site predicted by previous in vitro experiments. The RNAs extend across the Hind II site used for cloning and terminate specifically in the vector sequences. The termination site was localized to six consecutive thymine nucleotides preceded by a sequence with dyad symmetry. We found that the RNA from the str promoter was 205 (+/- 3) nucleotides long and that from the spc promoter was 177 (+/- 3) nucleotides long. These "hybrid mRNAs" are much more stable than ordinary mRNA. The str hybrid mRNA has a half-life of about 8 min, and the spc hybrid mRNA has a half-life of about 18 min at 37 degrees C. These hybrid mRNAs provide an in vivo system with which to examine directly the discrete transcription products from ribosomal protein promoters, and to study promoter function and mRNA metabolism in vivo.