Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants.

The antibiotic G418 was shown to be an effective inhibitor of vaccinia virus replication when an appropriate concentration of it was added to cell monolayers 48 h before infection. Genetic engineering techniques were used in concert with DNA transfection protocols to construct vaccinia virus recombinants containing the neomycin resistance gene (neo) from transposon Tn5. These recombinants contained the neo gene linked in either the correct or incorrect orientation relative to the vaccinia virus 7.5-kilodalton gene promoter which is expressed constitutively throughout the course of infection. The vaccinia virus recombinant containing the chimeric neo gene in the proper orientation was able to grow and form plaques in the presence of G418, whereas both the wild-type and the recombinant virus with the neo gene in the opposite polarity were inhibited by more than 98%. The effect of G418 on virus growth may be mediated at least in part by selective inhibition of the synthesis of a subset of late viral proteins. These results are discussed with reference to using this system, the conferral of resistance to G418 with neo as a positive selectable marker, to facilitate constructing vaccinia virus recombinants which contain foreign genes of interest.

Viral acylproteins: greasing the wheels of assembly.

Viruses take advantage of the host's protein modification and targeting pathways to modify their own proteins and to ensure that they assume active configurations and locate appropriately for assembly. In many viruses, one recurrent theme in such processes is exploitation of cellular protein acylation pathways for the addition of myristic and palmitic acid to capsid or envelope proteins.

Expression vector pT7:TKII for the synthesis of authentic biologically active RNA encoding vaccinia virus thymidine kinase.

A transcription vector, pT7: TKII, was constructed by a novel application of the polymerase chain reaction. Chimeric oligodeoxynucleotides were used to direct the synthesis of a DNA fragment which consisted of a truncated bacteriophage T7 promoter element fused to the vaccinia virus (VV) thymidine kinase gene (tk). This fragment was cloned into a pUC118 plasmid and sequenced to ensure no mutations had occurred during its synthesis. When linearized at the 3' end of the VV tk gene at the BamHI site located in the polylinker region of the vector, pT7:TKII was efficiently transcribed by T7 RNA polymerase into a 595 nucleotide transcript whose 5' end was identical to that found on authentic nascent VV tk mRNA. When translated in a rabbit reticulocyte lysate system, the synthetic VV tk RNA was shown to be biologically active in that it directed the synthesis of a 20-kDa protein which assembled into an enzymatically active 80-kDa tetrameric complex which was indistinguishable from VV thymidine kinase (TK) enzyme isolated from VV-infected cells. The pT7:TKII vector provides a powerful approach with which: (i) to investigate the translational and posttranslational regulation of the VV tk gene; (ii) to use directed genetics to identify potential cis-acting regulatory sequences or structures present within the VV tk RNA; and (iii) to apply protein engineering procedures to identify the catalytic, allosteric and subunit interactive domains of the VV TK enzyme. As an example, the translational effects of adding a m7G cap structure to the pT7:TKII-derived VV tk RNA are presented.

Phosphonates and phosphinates: novel leaving groups for benzisothiazolone inhibitors of human leukocyte elastase.

A novel class of alkyl and aryl phosphonate and phosphinate acid-based leaving groups has been developed for utilization in the synthesis of benzoisothiazolone (BIT) inhibitors of human leukocyte elastase (HLE). A number of BITs were synthesized with phosphonate and phosphinate acid-based leaving groups and were found to be potent inhibitors of HLE. Compound 3c with a diethyl phosphonate leaving group is the most potent inhibitor synthesized in this series with Ki* = 0.035 nM and ED50 = 2.0 mg/kg.

A novel class of cyclic beta-dicarbonyl leaving groups and their use in the design of benzisothiazolone human leukocyte elastase inhibitors.

Human leukocyte elastase (HLE) has been proposed to be a primary mediator of pulmonary emphysema, and inhibitors of this enzyme should be effective in the treatment of emphysema and other pulmonary diseases. We have discovered a novel class of alicyclic and heterocyclic leaving groups which share one common structural feature, a cyclic beta-dicarbonyl. This design concept for leaving groups has not been previously reported. A structure-activity relationship has been developed and the concept extended to several types of alicyclic and heterocyclic beta-dicarbonyl systems. This work led to the identification of a potent (K*i of 0.066 nM) and tissue stable (in vitro: blood t1/2 = 160 min, liver t1/2 > 240 min) benzisothiazolone HLE inhibitor, WIN 65936 (13b).

Inhibition of vaccinia virus replication by nicotinamide: evidence for ADP-ribosylation of viral proteins.

Replication of vaccinia virus (VV) in monolayers of BSC40 cells was inhibited 99.9% in the presence of 60 mM nicotinamide (NIC), a competitive inhibitor of ADP-ribosylation reactions. Dot-blot hybridization analysis of infected cell extracts utilizing a VV DNA-specific probe indicated that the drug had only minimal effects on viral DNA synthesis. SDS-polyacrylamide gel electrophoresis of newly synthesized VV proteins pulse-labeled at early (2 h) or late (8 h) times post-infection revealed that although the full spectrum of expected viral polypeptides was evident, quantitative differences in the levels of expression of a distinct subset of viral proteins were observed in the presence of the drug. Velocity sedimentation of virus-infected cell lysates established that no mature particles were assembled in drug treated cells. Additional evidence suggesting that VV morphogenesis was abortive in the presence of NIC was obtained by pulse-chase labeling experiments that demonstrated that the two VV major late core polypeptide precursors P94 and P65, whose proteolytic processing to VP62 and VP60 is intimately associated with viral assembly, were not cleaved in the presence of NIC. Interestingly, growth of VV in the presence of [3H]adenosine resulted in the metabolic labeling of eight proteins that were associated with purified virions. These proteins co-migrated with proteins labeled with [3H]adenosine that were present in extracts of VV-infected, but not uninfected, cells. These analyses also revealed that the [3H]adenosine-labeling of a subset of cellular proteins (MW 18-20 kDa, possibly histones) was increased 4-fold by VV infection. The observed induction of either increased synthesis or hyper-modification of these 18-20 kDa proteins was inhibited by NIC. These results are discussed with respect to whether one or more VV polypeptides are subject to obligatory ADP-ribosylation modification reactions in order to attain their active configuration, and if so, whether the enzymes catalyzing these reactions are specified by the virus or host cell.

Expression and regulation of the vaccinia virus thymidine kinase gene in non-permissive cells.

The expression and regulation of the vaccinia virus (VV) thymidine kinase (tk) gene was examined in two non-permissive cell lines, CHO and MDBK, which restrict VV development at different stages of the viral replication cycle. The VV tk gene was expressed in these two cell lines with kinetics similar to a fully permissive cell line BSC40. These results are consistent with the hypothesis that inhibition of tk mRNA translation by another viral early gene product is a normal component of the overall strategy employed to express and regulate the VV tk gene during a productive infection.

Novel acylation of poxvirus A-type inclusion proteins.

Myristylation is one of several post-translational modifications that occur on vaccinia virus (VV) proteins. Previously, time course labeling of VV-infected cells with myristic acid had indicated that five late proteins (17, 25, 36, 38 and 92 kDa) are myristylated. Four of these proteins were mapped to the E7R, L1R, AI6L and G9R open-reading frames, respectively, because of the predicted presence of the N-myristyltransferase recognition sequence (M-G-X-X-X-S/T/A) at their amino termini. In contrast, computer analyses of large (80-100 kDa) VV open reading frames did not reveal any predicted species with this N-terminal motif. By immunoprecipitation with monospecific sera and transient expression of cloned gene products, the myristylated 92-kDa protein has been demonstrated to be the A-type inclusion protein encoded by the Western Reserve (WR) strain of VV. Labeling of cowpox virus (CPV) infected cells with myristic acid indicated that the 160-kDa A-type inclusion protein appears to be myristylated as well. Both the VV 92-kDa and the CPV 160-kDa A-type inclusion proteins labeled with myristic acid were stable to hydroxylamine treatment, suggesting an amide linkage between the fatty acid and the acceptor protein. HPLC analysis confirmed that the 92-kDa protein was in fact myristylated. This data suggests that poxvirus ATI proteins may be subject to a novel type of internal myristylation modification, and the roles such modifications may play in the replication cycles of these viruses is discussed.

Quantitative assay of recombinant vaccinia virus-encoded neomycin phosphotransferase in infected eukaryotic cell lysates.

A method for the detection and quantitation of neomycin phosphotransferase (NPT II) activity in recombinant vaccinia virus (VV)-infected eukaryotic cell lysates is described. The assay is linear with respect to both protein concentration and time of incubation. Cytoplasmic extracts of cells infected with a recombinant VV expressing the bacterial neo gene exhibited NPT II levels more than 50-fold higher than those detected in extracts from either uninfected or VV-infected cells. These results indicate that interference from cellular or viral-induced ATPase activities is sufficiently low that NPT II enzyme activity can be measured in crude cell lysates without employing additional protein purification procedures.

Immunisation of cattle with a recombinant togavirus-vaccinia virus strain.

Genetic engineering techniques have been used to construct a vaccinia virus recombinant which contains and expresses togavirus (Sindbis) genetic information. Intradermal inoculation of this recombinant strain into calves caused a transient pock-type lesion at the site of inoculation and elicited the production of substantial levels of anti-Sindbis virus neutralising antibodies. These results suggest that recombinant vaccinia virus vaccines may have potential for use in veterinary medicine.

Reasons for family refusal of ocular tissue donation.

INTRODUCTION: Corneal donations do not fill the transplant demand. The waiting list had 5512 individuals in Brazil and 143 in Rio Grande do Sul in December 2012. The aim of this study was to identify the reasons for family refusal of ocular tissues donation. METHODS: This retrospective study analyzed interview records for ocular tissue procurement performed in a general, public university hospital located in Southern Brazil between January 2008 and December 2012. It identified the reasons of family refusal for ocular tissue donation. RESULTS: A total of 1010 interviews for ocular tissues procurement were performed. From these, 513 (50.79%) refused donation with the following reasons: 60 (11.69%) family members were unaware of the desire of the potential donor, 153 (29.82%) of potential donors spoke against donation in life, 113 (22.02%) family members were undecided about the donation, 156 (30.40%) family members were against donation, 3 (0.58%) family members were unhappy with the service, 11 (2.14%) family members were afraid of body release delay, 6 (1.16%) families expressed religious convictions against donation, and 11 (2.14%) family members wanted to keep the body intact. CONCLUSION: There are many reasons for ocular tissues donation refusal, and the knowledge provides better strategies for family interviews. In this study, most of the reasons, around 90%, can be related to lack of information or communication about the subject. Greater awareness of the population about the subject can be a good way to increase ocular tissue procurement indexes.

Expression of recombinant vaccinia virus-derived alphavirus proteins in mosquito cells.

A recombinant vaccinia virus strain which contains and expresses a 26S cDNA insert encoding Sindbis virus structural proteins (VV:3S) was used to infect a continuous line of Aedes albopictus mosquito cells. There were not visible cytopathic effects due to the virus infection and the cells continued to grow normally. However, examination of the proteins present in the cytoplasm of the infected cells with Sindbis virus-specific antisera revealed that Sindbis virus proteins were being synthesized and processed. These results are discussed with respect to vaccinia virus as a non-lethal expression vector to deliver and express eukaryotic genetic information in insect cell systems and using this system (VV:3S) to dissect various facets of togavirus-insect cell interactions.

Association of non-viral proteins with recombinant vaccinia virus virions.

Evidence is presented which suggests that recombinant vaccinia virus particles (VV:CAT), containing the bacterial chloramphenicol acetyl transferase gene, are capable of encapsidating both the foreign protein which they encode (CAT) as well as cellular enzymes such as thymidine kinase. These results are discussed with respect to using VV to passively introduce biologically-active proteins into cells or organisms.

Expression and single-step purification of enzymatically active vaccinia virus thymidine kinase containing an engineered oligohistidine domain by immobilized metal affinity chromatography.

A method has been developed for the controlled expression in Escherichia coli and rapid purification of an enzymatically active vaccinia virus (VV) thymidine kinase protein containing an engineered oligohistidine domain. The nucleotide sequence that encodes the VV thymidine kinase open reading frame was inserted into a plasmid expression vector (pET-16b, Novagen Inc., Madison, WI) under the control of a strongly repressed bacteriophage T7 promoter and high efficiency translational signals. The construct (pET-16b:TK) directs the synthesis of a fusion protein (His-TK) with an N-terminal histidine decapeptide fused to the VV thymidine kinase polypeptide. Upon induction of E. coli strain BL21(DE3)pLysS with isopropyl beta-D-thiogalactoside, accumulation of large quantities of a 22-kDa protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein reacted with polyclonal antiserum raised against a TrpE-VVTK fusion protein. The predominantly soluble fusion protein (approximately 13% of the total soluble bacterial protein) was purified to homogeneity from crude bacterial extracts in a single-step by immobilized metal chelate affinity chromatography (Ni(2+)-nitrilotriacetic acid-agarose) under nondenaturing conditions and was shown to have thymidine kinase activity. The yield of the purification scheme was about 15 mg recombinant protein/liter of bacterial culture. The availability of purified VV TK protein should greatly facilitate biochemical studies on its enzymatic activity, as well as analyses of its structural and functional domains.

Use of a cell-free system to identify the vaccinia virus L1R gene product as the major late myristylated virion protein M25.

A 25-kDa vaccinia virus (VV) virion protein, designated M25, is modified in vivo by covalent addition of myristic acid. The predicted amino acid sequences of all VV open reading frames which have been reported were searched for the sequence M-G-X-X-X-(S/T/A), which has been proposed to be the consensus recognition signal for cotranslational modification of proteins by N-myristyltransferase. This conserved signal was found at the amino terminus of a single locus, which corresponded to the leftmost rightward-reading open reading frame (L1R) initiating within the VV HindIII L DNA fragment. By using synthetic oligonucleotides in concert with polymerase chain reaction techniques, a chimeric gene consisting of open reading fram L1R fused to a bacteriophage T7 promoter was constructed and cloned into a plasmid vector. Transcripts derived from the wild-type expression plasmid (designated pL1G1) were translated in vitro in a wheat germ extract to yield a polypeptide with an apparent molecular mass of 25 kDa. This polypeptide was labeled with either [35S]methionine or [3H]myristic acid and comigrated with in vivo-labeled protein M25 on sodium dodecyl sulfate-polyacrylamide gels. Polyclonal antiserum generated in rabbits against a trpE:L1R fusion protein immunoprecipitated a 25-kDa protein labeled either in vitro (the L1R gene product, designated protein L1) or in vivo (from purified VV, protein M25), identifying the M25 protein as the gene product of open reading frame L1R. Chromatographic analysis of the protein L1-bound fatty acid moieties liberated after acid methanolysis resulted in recovery of greater than 99% of the fatty acid as myristate-associated label. Cell-free translation of proteins derived from a set of deletions from the carboxy terminus of the open reading frame L1R suggested that the site of myristylation maps near the amino terminus of protein L1. This hypothesis was supported by cell-free translation of mutant L1R transcripts in which the penultimate glycine codon had been altered by site-directed mutagenesis to encode either an aspartic acid (pL1D1) or alanine (pL1A1) residue. In both cases, the mutant transcripts were translated into a 25-kDa protein which could be labeled in vitro with [35S]methionine but not with [3H]myristic acid. These data demonstrate that VV open reading frame L1R encodes a myristylated protein and provide evidence that the site of modification of protein L1 is the amino-terminal glycine residue.

Construction of a recombinant vaccinia virus which expresses immunoreactive plant virus proteins.

A chimeric transcription unit was assembled consisting of a vaccinia virus promoter linked to a 2400 bp(3) double-stranded complementary DNA (cDNA) made to the 3' end of the genomic RNA of the plant pathogen, tobacco etch virus (TEV). Marker rescue techniques were used to introduce virus recombinant genes into the vaccinia virus genome. The recombinant virus (designated WNAT) was isolated, purified, and subjected to molecular genetic analyses. The ability of WNAT to direct the expression of plant virus genetic information in a mammalian system was assessed by infecting a line of monkey kidney cells. The plant virus cDNA insert was transcribed into RNA molecules that were correctly processed, and subsequently translated into proteins that were recognized by monospecific antisera directed against either the capsid protein or nuclear inclusion protein of TEV. These results are discussed in relation to using vaccinia virus to investigate other aspects of the plant virus replicative cycle.

Studies on the genomic organization of recombinant Streptococcus gordonii and the development of a novel intergenic integration site for foreign gene expression.

The methods currently employed to produce recombinant Streptococcus gordonii strains for use as vaccines and/or protein expression vectors result in the insertion of foreign genes into an unknown integration site with no information on the transcriptional context or potential phenotypic consequences. Therefore, the genomic organization surrounding the insertion site of a recombinant strain of S. gordonii (GP1223) containing a portion of the emm6 gene of Streptococcus pyogenes was determined. The nucleotide sequence of chromosomal walks in both directions from the insertion site revealed that the insert was flanked by a duplicated 3061-bp ClaI fragment. A consensus gram-positive promoter and a factor-independent RNA polymerase terminator sequence could be deduced in the fragment immediately upstream of the insertion site. The ClaI fragment also encoded open reading frames (ORFs) with high homology and parallel structural organization to the leucine biosynthesis operon of Lactococcus lactis subsp. lactis. Chromosomal walks downstream of the identified promoter region on the non-recombinant parental strain, GP204, yielded the sequence of two ORFs which would be normal targets of the transcription derived from this promoter. Northern analyses detected a highly expressed M6-specific transcript in recombinant strain GP1223 consistent in size with the proposed transcription unit. Transcripts analogous in length to those observed for the leucine biosynthesis operon of L. lactis subsp. lactis were also detected encompassing the homologous ORFs of S. gordonii. This information has enabled the construction of a recombinant S. gordonii strain in which the emm6 gene from S. pyogenes was targeted to a distinct intergenic locus within the S. gordonii genome. This new recombination site allows for expression of foreign gene products with minimal perturbation of the genomic organization of the wild-type S. gordonii strain and has provided information essential for further optimization of foreign gene expression levels.

Fatty acid acylation of vaccinia virus proteins.

Labeling of vaccinia virus-infected cells with [3H]myristic acid resulted in the incorporation of label into two viral proteins with apparent molecular weights of 35,000 and 25,000 (designated M35 and M25, respectively). M35 and M25 were expressed in infected cells after the onset of viral DNA replication, and both proteins were present in purified intracellular virus particles. Virion localization experiments determined M25 to be a constituent of the virion envelope, while M35 appeared to be peripherally associated with the virion core. M35 and M25 labeled by [3H]myristic acid were stable to treatment with neutral hydroxylamine, suggesting an amide-linked acylation of the proteins. Chromatographic identification of the protein-bound fatty acid moieties liberated after acid methanolysis of M25, isolated from infected cells labeled during a 4-h pulse, resulted in the recovery of 25% of the protein-bound fatty acid as myristate-associated label and 75% as palmitate, indicating that interconversion of myristate to palmitate had occurred during the labeling period. Similar analyses of M25 and M35, isolated from infected cells labeled during a 0.5-h pulse, determined that 46 and 43%, respectively, of the protein-bound label had been elongated to palmitate even during this brief labeling period. In contrast, M25 and M35 isolated from purified intracellular virions labeled continuously during 24 h of growth contained 75 and 70%, respectively, myristate-associated label, suggesting greater stability of these proteins or a favored interaction of the proteins containing myristate with the maturing or intracellular virion.

Proteolytic maturation of vaccinia virus core proteins: identification of a conserved motif at the N termini of the 4b and 25K virion proteins.

Three structural proteins (4a, 4b and 25K) located within the virion core of vaccinia virus are cleavage products of precursor polypeptides (P4a, P4b and P25K) synthesized late in viral infection. Pulse-chase labelling experiments revealed that cleavage of the core proteins lags considerably behind precursor synthesis and that processing requires continuous protein synthesis. The N-terminal sequences of 4b and 25K, but not 4a, were determined by microsequencing core proteins isolated from purified virions. Comparison of these data with the predicted amino acid sequence of P4b and P25K revealed a conserved Ala-Gly-Ala motif flanking the apparent N termini of both proteins, as well as several additional sequence similarities between the P4b and P25K precursors both upstream and downstream of the putative cleavage site. The Ala-Gly-Ala tripeptide signal was also found in the same region of the amino acid sequences of the homologous proteins of fowlpox virus.