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J Leis

Publications and source records attributed to J Leis.

At least 55 records · Page 3Linked to original sources
PubMed1989

Avian retroviral protease and cellular aspartic proteases are distinguished by activities on peptide substrates.

The avian sarcoma/leukemia virus protease (PR), purified from avian myeloblastosis virus has a native molecular mass of 26 kDa, suggesting a dimer structure. The enzymatic activity of PR has been characterized using synthetic peptide substrates. PR is most active at pH 5.5, 35 degrees C and 2-3 M NaCl. Under these conditions PR cleaves decapeptides which are resistant in low ionic strength. This high, nonphysiological, salt concentration also increases the proteolytic activity of a cellular aspartic protease, pepsin. PR and pepsin show additional similarities: they both cleave a synthetic decapeptide at the same Tyr-Pro bond in low and high salt, while the cleavage site preferences of human renin and cathepsin-D in this substrate are altered at high salt concentrations. In addition, iodination of the tyrosine residue in this decapeptide causes an increase in the rates of hydrolysis by both PR and pepsin. However, Km values are too high to be estimated accurately for PR using Tyr-Pro and Tyr(I)-Pro decapeptides as substrates. Comparison of the digestion products of two additional decapeptides, altered in a single amino acid residue, shows that PR cleaves at fewer sites than all three cellular enzymes. Furthermore pepstatin, a strong inhibitor of pepsin, renin, and cathepsin-D has little effect on PR.

Avian Leukosis Virus↗
PubMed1989

Molecular modeling of the HIV-1 protease and its substrate binding site.

The human immunodeficiency virus (HIV-1) encodes a protease that is essential for viral replication and is a member of the aspartic protease family. The recently determined three-dimensional structure of the related protease from Rous sarcoma virus has been used to model the smaller HIV-1 dimer. The active site has been analyzed by comparison to the structure of the aspartic protease, rhizopuspepsin, complexed with a peptide inhibitor. The HIV-1 protease is predicted to interact with seven residues of the protein substrate. This information can be used to design protease inhibitors and possible antiviral drugs.

Amino Acid Sequence↗
PubMed1989

Crystal structure of a retroviral protease proves relationship to aspartic protease family.

Retroviral gag, pol and env gene products are translated as precursor polyproteins, which are cleaved by virus-encoded proteases to produce the mature proteins found in virions. On the basis of the conserved Asp-Thr/Ser-Gly sequence at the putative protease active sites, and other biochemical evidence, retroviral proteases have been predicted to be in the family of pepsin-like aspartic proteases. It has been suggested that aspartic proteases evolved from a smaller, dimeric ancestral protein, and a recent model of the human immunodeficiency virus (HIV) protease postulated that a symmetric dimer of this enzyme is equivalent to a pepsin-like aspartic protease. We have now determined the crystal structure of Rous sarcoma virus (RSV) protease at 3-A resolution and find it is dimeric and has a structure similar to aspartic proteases. This structure should provide a useful basis for the modelling of the structures of other retroviral proteases, such as that of HIV, and also for the rational design of protease inhibitors as potential antiviral drugs.

Amino Acid Sequence↗
PubMed1989

Phosphorylation of avian retrovirus matrix protein by Ca2+/phospholipid-dependent protein kinase.

The matrix protein from avian myeloblastosis virus and the Rous sarcoma virus, Prague C strain, is a phosphoprotein. A comparison of the amino acid sequences shows these phosphoproteins are very similar. The sites of phosphorylation of the matrix protein purified from virions are identified as serine residues 68 and 106. Treatment with purified rabbit skeletal-muscle protein phosphatase 1 or 2A, selectively releases phosphate from serine 68, while alkali treatment releases phosphate from both sites. When analyzed as a substrate for six different protein kinases, only the Ca2+/phospholipid-dependent protein kinase modifies the matrix protein. The serine residues phosphorylated in vivo are identical to those phosphorylated in vitro by this protein kinase. The role of these phosphorylation events in viral production is discussed.

Amino Acid Sequence↗
PubMed1989

The avian retroviral integration protein cleaves the terminal sequences of linear viral DNA at the in vivo sites of integration.

The purified integration protein (IN) of avian myeloblastosis virus is shown to nick double-stranded oligodeoxynucleotide substrates that mimic the ends of the linear form of viral DNA. In the presence of Mg2+, nicks are created 2 nucleotides from the 3' OH ends of both the U5 plus strand and the U3 minus strand. Similar cleavage is observed in the presence of Mn2+ but only when the extent of the reaction is limited. Neither the complementary strands nor sequences representing the termini of human immunodeficiency virus type 1 DNA were cleaved at analogous positions. Analysis of a series of substrates containing U5 base substitutions has defined the sequence requirements for site-selective nicking; nucleotides near the cleavage site are most critical for activity. The minimum substrate size required to demonstrate significant activity corresponds to the nearly perfect 15-base terminal inverted repeat. This in vitro activity of IN thus produces viral DNA ends that are joined to host DNA in vivo and corresponds to an expected early step in the integrative recombination reaction. These results provide the first enzymatic support using purified retroviral proteins for a linear DNA precursor to the integrated provirus.

Avian Leukosis Virus↗
PubMed1988

Site-directed mutagenesis of the avian retrovirus nucleocapsid protein, pp 12, at Serine 40, the primary site of phosphorylation in vivo.

The major nucleocapsid protein of avian retroviruses, pp 12, binds to single-stranded viral RNA with high affinity. Phosphorylation at Ser-40 is necessary for this binding. In order to examine the role of phosphorylation of serine 40 in the biological function of pp 12, we have introduced a series of amino acid substitutions at this position in the Rous sarcoma virus (Pr-C) protein. Substitution of threonine, alanine, or three other amino acids for Ser-40 had very little or no detectable effect on viral replication, nor did the control substitution of glycine for Ser-43, a nonphosphorylated residue. In vivo and in vitro, the Ala-40 and probably the Thr-40 substituted p 12 proteins are phosphorylated at alternative sites which are phosphorylated to a minor extent in vivo in the wild type protein. A study of the RNA binding properties of Ala-40 substituted p 12 has indicated that the protein has been stabilized in a high affinity RNA binding state which is independent of phosphorylation. The viability of the Ala-40 mutant virus indicates that this high binding affinity may be required for biological activity.

Amino Acid Sequence↗
PubMed1988

Site-directed mutagenesis of the avian retrovirus nucleocapsid protein, pp 12. Mutation which affects RNA binding in vitro blocks viral replication.

Site-directed mutagenesis was used to replace the lysine residues of the avian retrovirus nucleocapsid protein pp 12 that have previously been shown to be important for RNA binding. Single amino acid substitutions at Lys-36, -37, and -39 of the protein were not sufficient to affect virus production as measured by reverse transcriptase activity in virus particles released from transfected cells. However, when Lys-36 and Lys-37 were simultaneously replaced by isoleucine residues, there was a complete block of viral replication. As expected from the single mutant analyses, the wild type phenotype could be restored by reverting either or both of the isoleucine residues to lysine residues. Analysis of a bacterially produced rp 12 protein containing Ile-36 and Ile-37 indicated that the protein has a low affinity binding for RNA, as compared to wild type protein. Unlike wild type, the binding is independent of phosphorylation at Ser-40, the major site of phosphorylation of the protein in vivo. A quail cell line was established that expresses virus particles containing the doubly mutated pp 12. Analysis of these particles indicated that they lack viral RNA. Thus, the binding defect in pp 12 is correlated with the inability to package viral RNA.

Animals↗
PubMed1988

Synthetic peptides as substrates and inhibitors of a retroviral protease.

Processing of the gag and pol gene precursor proteins of retroviruses is essential for infectivity and is directed by a viral protease that is itself included in one of these precursors. We demonstrate here that small synthetic peptides can be used as both model substrates and inhibitors to investigate the specificity and molecular parameters of the reaction. The results indicate that a peptide that extends five amino acids but not three amino acids in both directions from a known cleavage site is accurately hydrolyzed by the protease of avian sarcoma-leukosis virus. Substitutions of the amino acids to either side of the peptide bond to be cleaved affect the ability of the peptide (as well as a larger precursor protein) to serve as a substrate. The specificity is more stringent for the amino acid that will become the carboxyl end after cleavage. Some substitutions produced peptides that were not cleaved but could act as inhibitors of cleavage of a susceptible peptide. Thus, small model substrates may be used to explore both the binding and catalytic properties of these important proteases.

Alpharetrovirus↗
PubMed1988

Conserved cysteine and histidine residues of the avian myeloblastosis virus nucleocapsid protein are essential for viral replication but are not "zinc-binding fingers".

The nucleocapsid protein from the Rous sarcoma virus has two regions of sequence with the motif Cys-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Gly-His-Xaa-Xaa-Xaa-Cys. All retrovirus nucleocapsid proteins contain one or two of these motifs, and they represent the only conserved sequences among these proteins. Sequence analysis of nucleocapsid from avian myeloblastosis virus shows that it also contains two Cys-His sequences and, in fact, differs from the Rous sarcoma nucleocapsid protein only in three residues near the carboxyl terminus. The hypothesized role of the conserved cysteines and histidines as zinc ligands was tested experimentally. No tightly bound metal ions were detected for avian myeloblastosis nucleocapsid protein, and the molar amount of zinc in virions was less by a factor of 50 than that of the nucleocapsid protein. Added Zn2+ did not significantly affect nucleocapsid binding to poly(ethenoadenylic acid) or its secondary structure, as determined from circular dichroism. Nevertheless, the conserved cysteine and histidine residues of the Rous sarcoma (Prague-C strain) nucleocapsid protein are essential for fully functional virus, as shown by the fact that single-site substitutions of five of the six conserved cysteines and either of the two histidine residues blocked viral replication.

Avian Leukosis Virus↗
PubMed1988

A retroviral RNA secondary structure required for efficient initiation of reverse transcription.

Genetic evidence is presented which suggests the existence of an important structural element in the 5' noncoding region of avian retrovirus RNA. The proposed structure, which we term the U5-leader stem, is composed of sequences in the middle of U5 and in the leader, flanking the primer-binding site. U5 and leader mutations which would disrupt this structure caused a partial replication defect. However, nucleotide substitutions in the leader, which would structurally compensate for a U5 deletion mutation, restored normal replication. Analysis of replication intermediates of viruses with the above mutations suggests that the U5-leader stem is required for efficient DNA synthesis in vivo and for initiation of DNA synthesis from the tRNA(Trp) primer in melittin-activated virions. However, this structure does not appear to be required for binding of the tRNA(Trp) primer to viral RNA. These results support a role for the U5-leader stem structure, independent of its primary sequence, in the initiation of retroviral replication.

Animals↗
PubMed1988

Properties of avian sarcoma-leukosis virus pp32-related pol-endonucleases produced in Escherichia coli.

The gag-pol precursor protein of the avian sarcoma-leukosis virus is processed into three known pol-encoded mature polypeptides; the 95- and 63-kilodalton (kDa) beta and alpha subunits, respectively, of reverse transcriptase and the 32-kDa pp32 protein. The pp32 protein possesses DNA endonuclease activity and is produced from the precursor by two proteolytic cleavage events, one of which removes 4.1 kDa of protein from the C terminus. A 36-kDa protein (p36pol) which retains this C-terminal segment is detectable in small quantities in virions. We have constructed Escherichia coli plasmid clones that express the C-terminal domains of pol corresponding to pp32 and p36. These proteins have been purified by column chromatographic methods to near homogeneity. No significant differences could be detected in the enzymatic properties of the bacterially produced p32pol and p36pol proteins. Both possess DNA endonuclease activity and, like the pp32 protein isolated from virions, can cleave near the junction of two tandem avian sarcoma-leukosis virus long terminal repeats in double-stranded supercoiled DNA substrates. In the presence of Mg2+, both p32pol and viral pp32 cleave either strand of DNA 2 nucleotides 5' to the junction.

Amino Acid Sequence↗
PubMed1987

Proteolytic processing of avian sarcoma and leukosis viruses pol-endo recombinant proteins reveals another pol gene domain.

Three pol gene products have been identified in avian retroviral particles: the full-length 95-kilodalton (kDa) beta chain of reverse transcriptase and two proteolytic cleavage products of beta, a 63-kDa reverse transcriptase alpha chain derived from the amino terminus of beta and a 32-kDa (pp32) endonuclease from its carboxy terminus. By using molecularly cloned retroviral DNA and synthetic oligonucleotides to introduce initiator ATGs and codons corresponding to the authentic N termini, we constructed two bacterial-expression clones; one clone contains the entire pol gene, and the other contains the region encoding the pp32 domain. A 99-kDa protein was synthesized in Escherichia coli by the full-length clone, and a 36-kDa protein was synthesized by the endonuclease domain clone. The recombinant proteins exceeded the size of both the mature viral beta chain and the pp32, respectively, by approximately 4 kDa. These larger sizes, however, are consistent with predictions from the DNA sequence of the pol gene. Processing of the recombinant pol proteins was examined by using p15 protease purified from virus particles and antisera directed against synthetic peptides corresponding to three domains in pol. Proteolytic digestion of the 99-kDa product with p15 produced a 63-kDa protein that comigrated on polyacrylamide gels with the alpha chain of reverse transciptase and a 36-kDa fragment that comigrated with the endonuclease domain product. Further digestion of the 36-kDa protein yielded a 32-kDa protein that comigrated with viral pp32 endonuclease. Thus, we concluded that two p15-sensitive sites exist in pol. Cleavage at the previously identified site produces alpha, and cleavage at the newly discovered site removes approximately 4 kDa from the C terminus of the primary protein product. Since the 36-kDa protein was also detected in protein isolated from virus particles, it seems probable that processing at the C-terminal site is a normal step in the production of mature beta and pp32 endonuclease products.

Amino Acid Sequence↗
PubMed1987

Avian sarcoma and leukosis virus pol-endonuclease recognition of the tandem long terminal repeat junction: minimum site required for cleavage is also required for viral growth.

Integration of retroviral DNA is a site-specific reaction involving an endonuclease encoded by the viral pol gene (pol-endo). In vitro the pol-endo from avian sarcoma and leukosis viruses (ASLVs) cleaves both DNA strands near the U5-U3 junction of tandem long terminal repeats (LTR-LTR junction) in single-stranded and replicative form (RF)-I substrates. We have reported previously that the sequences that are required for cleavage of single-stranded substrates by the alpha beta form of the pol-endo differ for the plus and minus strands (G. Duyk, M. Longiaru, D. Cobrinik, R. Kowal, P. deHaseth, A. M. Skalka, and J. Leis, J. Virol. 56:589-599, 1985). This is not the case with RF-I substrates, in which a maximum of 22 base pairs of U5 and 8 base pairs of U3 were required for alpha beta pol-endo cleavage in each strand. Insertion of a palindromic octanucleotide (CATCGATG) at the LTR-LTR junction abolished cleavage in RF-I but not in single-stranded DNA substrates. Deletion of the four nucleotides (TTAA) at the junction prevented cleavage in the plus strand of RF-I DNA, but did not affect cleavage of single-stranded DNA. Furthermore, the alpha beta form of ASLV pol-endo did not recognize heterologous LTR-LTR junction sequences from the reticuloendotheliosis virus or Moloney murine leukemia virus in either substrate form, despite their sequence and structural similarities to the ASLV junction. These results support a role for a sequence-specific interaction between the ASLV pol-endo and the LTR-LTR junction domains that are required for cleavage. By using the infectious Rous sarcoma virus clone pATV8-K, we introduced a set of deletions into the U5 region that would be incorporated into the LTR-LTR junction on viral replication. In the unintegrated provirus, the deletions started 43 base pairs from the LTR-LTR junction and extended various lengths toward the junction. Results of transfection studies with these clones indicated that the U5 sequences that are required for virus production in vivo correspond to those that are required for cleavage of RF-I DNA in vitro.

Alpharetrovirus↗
PubMed1986

Avian retrovirus nucleocapsid protein, pp12, produced in Escherichia coli has biochemical properties identical to unphosphorylated viral protein.

The avian sarcoma and leukosis viruses contain an RNA binding nucleocapsid protein, pp12, in the phosphorylated form. An Escherichia coli expression vector carrying the Rous sarcoma virus Prague C strain gene coding for this protein has been constructed using a site-directed deletion mutagenesis method to place start and stop codons into translational reading frame with the N- and C-terminal coding sequences of the protein, respectively. The protein is produced efficiently in bacteria (rp12), is soluble and readily purified. It is also indistinguishable from the unphosphorylated viral pp12 protein in its migration on SDS-polyacrylamide gels, reactivity with rabbit antisera directed against purified viral pp12, low RNA-binding affinity, and ability to serve as a substrate for in vitro phosphorylation at serine-40 by protease-activated kinase I. The ability to analyze the biochemical activities of the normal, modified, and mutant forms of this protein is an essential step in elucidating its role in the retroviral life cycle. This expression clone will be especially useful in testing for the effects of mutations before they are reconstructed into retroviral genomes for analyses.

Avian Sarcoma Viruses↗
PubMed1985

Site-specific phosphorylation of avian retrovirus nucleocapsid protein pp12 regulates binding to viral RNA. Evidence for different protein conformations.

Phosphorylation of serine 40 of the major nucleocapsid protein of avian retroviruses, pp12, regulates binding to viral RNA (Leis, J., Johnson, S., Collins, L. S., and Traugh, J. A. (1984) J. Biol. Chem. 259, 7726-7732). The phosphorylation state of the protein can be altered in vitro, resulting in the interconversion of the protein between a state of high affinity for single-stranded RNA and low affinity for single- or double-stranded RNA. The reversible phosphorylation of serine 40 is accompanied by a change in the conformation of the protein as demonstrated by quenching of intrinsic tryptophan fluorescence and chemical modification studies. Quenching of fluorescence of the sole tryptophan residue, Trp 80, by poly(U), KI, and CsCl indicates that the microenvironment of this residue is more positive in pp12 than in p12. Chemical modification studies indicate that the 3 lysine residues at positions 36, 37, and 39 of pp12 react with 2,4,6-trinitrobenzenesulfonic acid, while only 1 of these residues reacts in p12. The addition of single-stranded, but not double-stranded RNA, to pp12 protects 2 of the 3 lysine residues from chemical modification, suggesting that the two protected lysyl groups are required for binding to single-stranded viral RNA. In contrast to the phosphorylation of serine 40, phosphorylation of serine 43, catalyzed by protease-activated kinase II in vitro, does not induce changes in the protein conformation nor does it alter the RNA binding properties of the protein.

Amino Acid Sequence↗
PubMed1985

Circles with two tandem long terminal repeats are specifically cleaved by pol gene-associated endonuclease from avian sarcoma and leukosis viruses: nucleotide sequences required for site-specific cleavage.

The avian retroviral pol gene-encoded DNA endonuclease (pol-endo) has been shown to selectively cleave the viral long terminal repeat sequences (LTRs) in single-stranded DNA substrates in a region known to be joined to host DNA during integration (G. Duyk, J. Leis, M. Longiaru, and A.M. Skalka, Proc. Natl. Acad. Sci. USA 80:6745-6749, 1983). The preferred sites of cleavage were mapped to the unique U5/U3 junctions found only in covalently closed circular DNA molecules containing two tandem LTRs. The cuts occurred three nucleotides 5' to the axis of symmetry of the 12-of-15-base-pair nearly perfect inverted repeat which marks the LTR junction. Experiments with double-stranded supercoiled DNA substrates revealed a similar specificity for nicking. Also, the endonuclease associated with the pol cleavage product, pp32, has the same specificity as the alpha beta form. The limits of sequence required for site-selective cleavage near the U5/U3 junction were established with single-stranded DNA substrates. A domain no larger than 44 base pairs allowed site-selective cleavage in each strand in vitro. Recognition of either strand appeared to be independent of the other, and in each case, the critical sequence was asymmetrically distributed with respect to the U5/U3 junction. The predominant contribution was from the U5 domain; this is consistent with its conservation in the LTR sequences of a number of avian sarcoma and leukosis viruses.

Alpharetrovirus↗