Mutations that alter the activity of the Rous sarcoma virus protease.

Mutations designed by analysis of the Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV)-1 protease (PR) crystal structures were introduced into 1) the substrate binding pocket, 2) the substrate enclosing "flaps," and 3) surface loops of RSV PR. Each mutant PR was expressed in Escherichia coli. Changes in activity were detected by following cleavage of a truncated (NC-PR) precursor polypeptide in E. coli and cleavage of synthetic peptide substrates representing RSV and HIV-1 PR cleavage sites in vitro. Mutations in the substrate binding pocket exchanged amino acid residues located close to the substrate in the HIV-1 PR for structurally equivalent residues in the RSV PR. Changing histidine 65 to glycine (H65G) gave an inactive enzyme, while a double mutant R105P,G106V, as well as the triple mutant, H65G,R105P,G106V, produced enzymes which showed significant activity toward a substrate that represented a HIV-1 cleavage site. Mutating the catalytic aspartate (D37S) or an adjacent conserved alanine to threonine (A40T), produced inactive enzymes. In contrast, the substitution A40S was active, but showed a reduced rate of catalysis. Mutations in the flaps of conserved glycines (G69L, G70L) produced inactive PRs. Two extended RSV PR surface loops were shortened to the size found in HIV-1 PR and resulted in drastically reduced activity. These results have confirmed some of the basic predictions made from structural models but have also revealed unexpected roles and interactions in the protein.     

Chemical Synthesis of 19F-labeled HIV-1 Protease using Fmoc-Chemistry and ChemMatrix Resin

HIV-1 protease (PR) has been extensively studied due to its importance as a target in AIDS therapy. The enzyme can be obtained via expression of its cloned gene in an appropriate system, or via chemical synthesis. We required a reliable source of fluorine-labeled HIV-1 protease for NMR studies. As our attempts to incorporate a labeling step in overexpression experiments in E. coli failed, we turned to chemical synthesis. Herein is described the first chemical synthesis of an active, 99 amino acid residue HIV-1 encoded protease using Fmoc-chemistry on a total PEG-based resin (CM resin), and labeled with ¹⁹F at the Phe residue. Also reported here are NMR studies of the labeled synthetic protein with a synthetic dimerization inhibitor.     

Chemical synthesis and expression of the HIV-1 protease gene in E. coli

The 297bp HIV-1 protease gene was constructed from five discrete synthetic fragments and expressed in E. coli. A soluble protein product of 11.5 Kd was detected by immunoblotting using protease specific antisera. A quantitative assay system, utilizing a synthetic nonapeptide spanning the cleavage site between p17-p24 in the gag polyprotein, was used to measure the specific protease activity in crude extracts. The protease hydrolyzed tyrosyl-proline bonds with an approximate specific activity of 43 pmoles/min/micrograms of total protein. The chemical synthesis of the protease gene and it's expression provides a feasible method for rapid mutant analysis, important for structure-function studies and rational design of potential inhibitors.     

Expression in Escherichia coli of a synthetic gene coding for horse heart myoglobin

A gene for expression of horse heart myoglobin in Escherichia coli has been constructed in one step from long synthetic oligonucleotides. The synthetic gene contains an efficient translation initiation signal and used codons that are commonly found in E. coli. Unique restriction sites are placed throughout the gene. It has been inserted in a phagemid vector and is expressed from the lac promoter in E. coli at high efficiency, the soluble heme protein representing approximately 10% of soluble protein. Two versions of horse heart myoglobin were produced with aspartic acid or asparagine at residue 122. Comparison of chromatographic mobilities of these two proteins with authentic horse heart myoglobin identified aspartic acid as the correct residue 122. The availability of this gene, which is designed to facilitate oligonucleotide mutagenesis or cassette mutagenesis, will allow systematic structure-function analysis of horse heart myoglobin.     

Amino acid residues 88 and 89 in the central hydrophilic region of human immunodeficiency virus type 1 Vif are critical for viral infectivity by enhancing the steady-state expression of Vif

A hydrophilic region consisting of strikingly clustered charged amino acids is present at the center of human immunodeficiency virus type 1 (HIV-1) Vif. In this study, the role for this central hydrophilic region (E(88)WRKKR(93)) in the virus replication in nonpermissive H9 cells was investigated by extensive deletion and substitution analysis. A total of 31 mutants were constructed. Deletion of the E(88) or W(89) residue alone abolished viral infectivity in H9 cells and impaired virus replication in primary macrophage cultures. Substitution analysis indicated that the hydrophilicity and charge of the central region are insignificant for the function of Vif. Of the 16 substitution mutants, 3 mutants with substitution of E(88) and W(89) with an A residue did not grow in H9 cells. Upon transfection, four mutants (i.e., two mutants with deletion of E(88) or W(89); a mutant with substitution of E(88) and W(89) with A; and a mutant with substitution of E(88), W(89), and R(90) with A) were found to express Vif at a very reduced level relative to that by the wild-type clone. These results have thus demonstrated that amino acid residues 88 and 89 of Vif are critical for the replication of HIV-1 in target cells by enhancing the steady-state expression of Vif. In addition, E(88) and W(89) residues were found to be extremely conserved among the Vif proteins of naturally occurring HIV-1 field isolates as well as those of laboratory HIV-1 strains.     

HIV- 1 Protease Inhibits Cap- and Poly(A)-Dependent Translation upon eIF4GI and PABP Cleavage

A number of viral proteases are able to cleave translation initiation factors leading to the inhibition of cellular translation. This is the case of human immunodeficiency virus type 1 protease (HIV-1 PR), which hydrolyzes eIF4GI and poly(A)-binding protein (PABP). Here, the effect of HIV-1 PR on cellular and viral protein synthesis has been examined using cell-free systems. HIV-1 PR strongly hampers translation of pre-existing capped luc mRNAs, particularly when these mRNAs contain a poly(A) tail. In fact, HIV-1 PR efficiently blocks cap- and poly(A)-dependent translation initiation in HeLa extracts. Addition of exogenous PABP to HIV-1 PR treated extracts partially restores the translation of polyadenylated luc mRNAs, suggesting that PABP cleavage is directly involved in the inhibition of poly(A)-dependent translation. In contrast to these data, PABP cleavage induced by HIV-1 PR has little impact on the translation of polyadenylated encephalomyocarditis virus internal ribosome entry site (IRES)-containing mRNAs. In this case, the loss of poly(A)-dependent translation is compensated by the IRES transactivation provided by eIF4G cleavage. Finally, translation of capped and polyadenylated HIV-1 genomic mRNA takes place in HeLa extracts when eIF4GI and PABP have been cleaved by HIV-1 PR. Together these results suggest that proteolytic cleavage of eIF4GI and PABP by HIV-1 PR blocks cap- and poly(A)-dependent initiation of translation, leading to the inhibition of cellular protein synthesis. However, HIV-1 genomic mRNA can be translated under these conditions, giving rise to the production of Gag polyprotein.     

Placement of leucine zipper motifs at the carboxyl terminus of HIV-1 protease significantly reduces virion production

Natural HIV-1 protease (PR) is homodimeric. Some researchers believe that interactions between HIV-1 Gag-Pol molecules trigger the activation of embedded PR (which mediates Gag and Gag-Pol cleavage), and that Gag-Pol assembly domains outside of PR may contribute to PR activation by influencing PR dimer interaction in a Gag-Pol context. To determine if the enhancement of PR dimer interaction facilitates PR activation, we placed single or tandem repeat leucine zippers (LZ) at the PR C-terminus, and looked for a correlation between enhanced Gag processing efficiency and increased Gag-PR-LZ multimerization capacity. We found significant reductions in virus-like particles (VLPs) produced by HIV-1 mutants, with LZ fused to the end of PR as a result of enhanced Gag cleavage efficiency. Since VLP production can be restored to wt levels following PR activity inhibition, this assembly defect is considered PR activity-dependent. We also found a correlation between the LZ enhancement effect on Gag cleavage and enhanced Gag-PR multimerization. The results suggest that PR dimer interactions facilitated by forced Gag-PR multimerization lead to premature Gag cleavage, likely a result of premature PR activation. Our conclusion is that placement of a heterologous dimerization domain downstream of PR enhances PR-mediated Gag cleavage efficiency, implying that structural conformation, rather than the primary sequence outside of PR, is a major determinant of HIV-1 PR activation.     

Structure-function studies of murine epidermal growth factor: expression and site-directed mutagenesis of epidermal growth factor gene

Wild-type murine epidermal growth factor (mEGF) and mutants with Leu47 replaced by serine and valine, respectively, have been produced by recombinant DNA methodology. A synthetic gene for mEGF was fused to the coding sequence for the signal peptide of the outer membrane protein A (ompA) of Escherichia coli in the secretion vector pIN-III-ompA3, and the recombinant plasmid was used to transform E. coli. Upon induction of gene expression, mEGF and the mutants was expressed and secreted into the periplasmic space. Purification of the wild-type Leu47-mEGF and the mutants was carried out by reversed-phase and anion-exchange high-performance liquid chromatography (HPLC). Amino acid analysis and Western blot analysis further confirmed the identities of the proteins. Specific activities for wild-type and mutant proteins were measured in both mEGF receptor binding and autophosphorylation assays. The recombinant mEGF has specific activities identical with that of mEGF purified from mouse submaxillary glands, while both mutants have reduced specific activities in both bioassays. The data demonstrate the importance of the highly conserved Leu47 residue in mEGF for full biological activity.     

High-level expression in Escherichia coli of a chemically synthesized gene for [Leu-28]echistatin

A gene (Ecs) encoding a platelet aggregation inhibitor, echistatin (Ecs), has been chemically synthesized. Met at position 28 of the native protein was replaced by Leu in the recombinant Ecs. To express this synthetic gene in Escherichia coli, an expression vector, pJC264, was constructed by inserting portions of the E. coli cheB and cheY gene complex into the plasmid pUC13. High-level expression of the synthetic [Leu-28]Ecs was achieved by its fusion with the E. coli cheY gene in the expression vector. Recombinant [Leu-28]Ecs was liberated from the fusion protein by CNBr cleavage at the Met inserted between the CheY protein and [Leu-28]Ecs. The recombinant [Leu-28]Ecs was purified to homogeneity by reverse-phase high-performance liquid chromatography. The refolded [Leu-28]Ecs was identical to native Ecs in inhibiting platelet aggregation, suggesting that Met at position 28 is not essential for the biological activity of this platelet aggregation inhibitor.     

Human immunodeficiency virus type 1 gag-protease fusion proteins are enzymatically active.

We have introduced mutations into the region of the genome of human immunodeficiency virus type 1 (HIV-1) that encodes the cleavage sites between the viral protease (PR) and the adjacent upstream region of the polyprotein precursor. Segments containing these mutations were introduced into plasmids, and the retroviral proteins were expressed in Escherichia coli. The mutations prevented cleavage between the PR and the adjacent polypeptide; however, other PR cleavage sites in the polyprotein were cleaved normally, showing that the release of free PR is not a prerequisite for the appropriate processing of HIV-1 precursors.     

Holliday junction resolvase in Schizosaccharomyces pombe has identical endonuclease activity to the CCE1 homologue YDC2.

A novel Holliday junction resolving activity has been identified in fractionated cell extracts of the fission yeast Schizosaccharomyces pombe . The enzyme catalyses endonucleolytic cleavage of Holliday junction-containing chi DNA and synthetic four-way DNA junctions. The activity cuts with high specificity a synthetic four-way junction containing a 12 bp core of homologous sequences but has no activity on another four-way junction (with a fixed crossover point), a three-way junction, linear duplex DNA or duplex DNA containing six mismatched nucleotides in the centre. The major cleavage sites map as single nicks in the vicinity of the crossover point, 3' of a thymidine residue. These data indicate that the activity has a strong DNA structure selectivity as well as a limited sequence preference; features similar to the Holliday junction resolving enzymes RuvC of Escherichia coli and the mitochondrial CCE1 (cruciform-cuttingenzyme 1) of Saccharomyces cerevisiae. A putative homologue of CCE1 in S.pombe (YDC2_SCHPO) has been identified through a search of the sequence database. The open reading frame of this gene has been cloned and the encoded protein, YDC2, expressed in E.coli . The purified recombinant YDC2 exhibits Holliday junction resolvase activity and is, therefore, a functional S.pombe homologue of CCE1. The resolvase YDC2 shows the same substrate specificity and produces identical cleavage sites as the activity obtained from S. pombe cells. Both YDC2 and the cellular activity cleave Holliday junctions in both orientations to give nicks that can be ligated in vitro. The partially purified Holliday junction resolving enzyme in fission yeast is biochemically indistinguishable from recombinant YDC2 and appears to be the same protein.     

Rational design and PCR-based synthesis of an artificial Schizophyllum commune xylanase gene.

A synthetic gene encoding the Schizophyllum commune xylanase XynA was constructed by a novel PCR-based procedure. Three long oligonucleotides were synthesized and used in combination with flanking PCR primers to generate a 607 base pair gene which contained 31 unique locations for restriction enzyme cleavage. The amino acid sequence was tailored for expression in Escherichia coli by using only those codons found in highly expressed E. coli genes. The availability of the gene will facilitate analysis of the structure and function of this and other beta-(1,4) xylanases.     

High-level production of active HIV-1 protease in Escherichia coli.

High levels of active HIV-1 protease (PR) were produced in Escherichia coli, amounting to 8-10% of total cell protein. High production levels were achieved by altering the following parameters: (1) codon preference of the coding region, (2) A+T-richness at the 5' end of the coding region, and (3) promoter. To circumvent the toxicity of HIV-1 PR in E. coli, the gene was expressed as a fusion protein with two different proteolytic autocleavage sequences. In both the cases, the fusion protein could be cleaved in vivo to give an active molecule with the native sequence at the N terminus.     

A dnaT mutant with phenotypes similar to those of a priA2::kan mutant in Escherichia coli K-12

The ability to repair damaged replication forks and restart them is important for cell survival. DnaT is essential for replication restart in vitro and yet no definite genetic analysis has been done in Escherichia coli K-12. To begin, dnaT822, an in-frame six-codon (87-92) deletion was constructed. DnaT822 mutants show colony size, cell morphology, inability to properly partition nucleoids, UV sensitivity, and basal SOS expression similar to priA2::kan mutants. DnaT822 priA2::kan double mutants had phenotypes similar to those of the single mutants. DnaT822 and dnaT822 priA2::kan mutant phenotypes were fully suppressed by dnaC809. Previously, a dominant temperature-sensitive lethal mutation, dnaT1, had been isolated in E. coli 15T(-). DnaT1 was found to have a base-pair change relative to the E. coli 15T(-) and E. coli K-12 dnaT genes that led to a single amino acid change: R152C. A plasmid-encoded E. coli K-12 mutant dnaT gene with the R152C amino acid substitution did not display a dominant temperature-sensitive lethal phenotype in a dnaT(+) strain of E. coli K-12. Instead, this mutant dnaT gene was found to complement the E. coli K-12 dnaT822 mutant phenotypes. The significance of these results is discussed in terms of models for replication restart.