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J Leis

Publications and source records attributed to J Leis.

At least 37 records · Page 2Linked to original sources
PubMed1996

Fine mapping and characterization of the Rous sarcoma virus Pr76gag late assembly domain.

The p2 region of the Rous sarcoma virus (RSV) Gag polyprotein contains an assembly domain, which is required late in replication for efficient budding of virus-like particles from cells (J. W. Wills, C. E. Cameron, C. B. Wilson, Y. Xiang, R. P. Bennett, and J. Leis, J. Virol. 68:6605-6618, 1994). This domain, referred to as the L domain, was previously mapped to the 11 amino acids of p2b. Through the analysis of a series of deletion and substitution mutations, the L domain has now been fine mapped to a highly conserved amino acid sequence, PPPPYV of p2b. Sequences flanking PPPPYV motif can be deleted without any effect on budding. Defects caused by L-domain deletions can be rescued by placing a wild-type copy of the sequence at several other positions in RSV Gag. A proline-rich P(S/T)APP motif is found in many retroviral Gag polyproteins; the motif found in the p6 region of human immunodeficiency virus type 1 has been implicated in late functions of the virus. Substitution of the RSV L domain with this motif in a 10-amino-acid sequence derived from visna leukemia virus results in wild-type release of virus particles from cells. In contrast, the slightly different sequences from Gibbon ape leukemia virus, Moloney leukemia virus, PSAPP alone, or a proline-rich SH3 binding sequence do not efficiently rescue RSV L-domain mutations.

Amino Acid Sequence↗
PubMed1994

Mutational analysis of the substrate binding pockets of the Rous sarcoma virus and human immunodeficiency virus-1 proteases.

Mutations, designed by analysis of the crystal structures of Rous sarcoma virus (RSV) and human immunodeficiency virus type 1 (HIV-1) protease (PR), were introduced into the substrate binding pocket of RSV PR. The mutations substituted nonconserved residues of RSV PR, located within 10 A of the substrate, for those in structurally equivalent positions of HIV-1 PR. Changes in the activity of purified mutants were detected in vitro by following cleavage of synthetic peptides representing wild-type and modified RSV and HIV-1 gag and pol polyprotein cleavage sites. Substituting threonine for valine 104 (V104T), S107N, I44V, Q63M or deletion of residues 61-63 produced enzymes that were 2.5-7-fold more active than the wild type RSV PR. Substituting I42D, M73V, and A100L produced enzymes with lower activity, whereas a mutant that included both M73V and A100L was as active as wild type. Several substitutions altered the specificity for substrate. These include I42D and I44V, which contribute to the S2 and S2' subsites. These proteins exhibited HIV-1 PR specificity for P2- or P2'-modified peptide substrates but unchanged specificity with P4-, P3-, P1-, P1'-, and P3'-modified substrates. Changes in specificity in the S4 subsite were detected by deletion of residues 61-63. These results confirm the hypothesis that the subsites of the substrate binding pocket of the retroviral protease are capable of acting independently in the selection of substrate amino acids.

Amino Acid Sequence↗
PubMed1994

An assembly domain of the Rous sarcoma virus Gag protein required late in budding.

The Gag protein of Rous sarcoma virus has the ability to direct particle assembly at the plasma membrane in the absence of all the other virus-encoded components. An extensive deletion analysis has revealed that very large regions of this protein can be deleted without impairing budding and has suggested that the essential functions map to three discrete regions. In the studies reported here, we establish the location of assembly domain 2 (AD2) within the proline-rich p2b sequence of this Gag protein. AD2 mutants lacking the p2b sequence were completely defective for particle release even though their Gag proteins were tightly associated with the membrane fraction and exhibited high levels of protease activity. Mutations that inactivate the viral protease did not restore budding to wild-type levels for these mutants, indicating that the defect is not due simply to a loss of protease regulation. AD2 mutants could be rescued into dense particles in genetic complementation assays, indicating that their defect is not due to a gross alteration of the overall conformation of the protein and that the assembly function is not needed on every Gag molecule in the population. Several mutants with amino acid substitutions in the p2b sequence were found to have an intermediate capacity for budding. Inactivation of the protease of these mutants stabilized the Gag polyprotein within the cells and allowed an increase in particle release; however, the rate of budding remained slow. We favor the idea that AD2 is a dynamic region of movement, perhaps serving as a molecular hinge to allow the particle to emerge from the surface of the cell during budding.

Amino Acid Sequence↗
PubMed1994

A specific orientation of RNA secondary structures is required for initiation of reverse transcription.

The 5' end of avian retrovirus RNA near the primer-binding site (PBS) forms two secondary structures, the U5-inverted repeat (U5-IR) and the U5-leader stems, and contains a 7-nucleotide sequence that anneals to the T psi C loop of the tRNA(Trp) primer. Mutations that disrupt any of these base pair interactions cause defects in initiation of reverse transcription both in vivo and in vitro (D. Cobrinik, A. Aiyar, Z. Ge, M. Katzman, H. Huang, and J. Leis, J. Virol. 65:3864-3872, 1991; A. Aiyar, D. Cobrinik, Z. Ge, H.-J. Kung, and J. Leis, J. Virol. 66:2464-2472, 1992). We have now examined the effect of perturbing the non-base-paired intervening "spacer" sequences between these secondary-structure elements. Small deletions or insertions in these intervening sequences decreased initiation of reverse transcription in vitro. In contrast, base substitutions, which maintain the spacing distances between the structures, had no detectable effect. Additionally, a small deletion at the 3' end of the PBS caused a significant decrease in initiation of reverse transcription whereas substitution mutations again had no effect. Together, these results indicate that reverse transcriptase forms a complex in which the different structural elements are maintained in a specific orientation that is required for efficient initiation of reverse transcription. Specific sequence recognition of the duplex structures by reverse transcriptase is also required since mosaic RNAs that combine the human immunodeficiency virus type 1 PBS with avian sequences is not efficiently utilized for reverse transcription even though the primer used can anneal to the substituted PBS.

Avian Sarcoma Viruses↗
PubMed1993

Comparison of the substrate-binding pockets of the Rous sarcoma virus and human immunodeficiency virus type 1 proteases.

A steady state kinetic analysis of the avian myeloblastosis virus/Rous sarcoma virus (AMV/RSV) and human immunodeficiency virus Type 1 (HIV-1) retroviral proteases (PRs) was carried out using a series of 40 peptide substrates that are derivatives of the AMV/RSV nucleocapsid-PR cleavage site. These peptides contain single amino acid substitutions in each of the seven positions of the minimum length substrate required by the PR for specific and efficient cleavage. These peptide substrates are distinguished by the individual enzyme subsites of the AMV/RSV and HIV-1 PRs. The molecular basis for similarities and differences of the individual subsites for both proteases is discussed using steady state kinetic data and modeling based on crystal structures.

Amino Acid Sequence↗
PubMed1992

Mechanism of inhibition of the retroviral protease by a Rous sarcoma virus peptide substrate representing the cleavage site between the gag p2 and p10 proteins.

The activity of the avian myeloblastosis virus (AMV) or the human immunodeficiency virus type 1 (HIV-1) protease on peptide substrates which represent cleavage sites found in the gag and gag-pol polyproteins of Rous sarcoma virus (RSV) and HIV-1 has been analyzed. Each protease efficiently processed cleavage site substrates found in their cognate polyprotein precursors. Additionally, in some instances heterologous activity was detected. The catalytic efficiency of the RSV protease on cognate substrates varied by as much as 30-fold. The least efficiently processed substrate, p2-p10, represents the cleavage site between the RSV p2 and p10 proteins. This peptide was inhibitory to the AMV as well as the HIV-1 and HIV-2 protease cleavage of other substrate peptides with Ki values in the 5-20 microM range. Molecular modeling of the RSV protease with the p2-p10 peptide docked in the substrate binding pocket and analysis of a series of single-amino acid-substituted p2-p10 peptide analogues suggested that this peptide is inhibitory because of the potential of a serine residue in the P1' position to interact with one of the catalytic aspartic acid residues. To open the binding pocket and allow rotational freedom for the serine in P1', there is a further requirement for either a glycine or a polar residue in P2' and/or a large amino acid residue in P3'. The amino acid residues in P1-P4 provide interactions for tight binding of the peptide in the substrate binding pocket.

Amino Acid Sequence↗
PubMed1992

Requirement for a conserved serine in both processing and joining activities of retroviral integrase.

Retroviruses encode a protein, the integrase (IN), that is required for insertion of the viral DNA into the host cell chromosome. IN alone can carry out the integration reaction in vitro. The reaction involves endonucleolytic cleavage near the 3' ends of both viral DNA strands (the processing step), followed by joining of these new viral DNA ends to host DNA (the joining step). Based on their evolutionary conservation, we have previously identified at least 11 amino acid residues of IN that may be essential for the reaction. Here we report that even conservative replacements of one of these residues, an invariant serine, produce severe reductions in both the processing and joining activities of Rous sarcoma virus IN in vitro. Replacement of the analogous serine of the type 1 human immunodeficiency virus IN had similar effects on processing activity. These results suggest that this single conserved serine is a component of the active site and that one active site is used for both processing and joining. Replacement of this serine with certain amino acids resulted in a loss or reduction in DNA binding activities, while other replacements at this position appeared to affect later steps in catalysis. All of the defective Rous sarcoma virus INs were able to compete with the wild-type protein, which supports a model in which IN functions in a multimeric complex.

Avian Sarcoma Viruses↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1992

Analysis of substrate interactions of the Rous sarcoma virus wild type and mutant proteases and human immunodeficiency virus-1 protease using a set of systematically altered peptide substrates.

In the preceding study, mutant Rous sarcoma virus (RSV) proteases are described in which three amino acids found in the human immunodeficiency virus-1 (HIV-1) protease (PR) were substituted into structurally comparable positions (Grinde, B., Cameron, C.E., Leis, J., Weber, I., Wlodawer, A., Burstein, H., Bizub, D., and Skalka, A. M. (1992) J. Biol. Chem. 267, 9481-9490). In this report, the activity of the wild type and these mutant PRs are compared using a set of RSV NC-PR peptide substrates with single amino acid substitutions in each of the P4 to P3' positions. With most substrates, the relative activities of the two active mutants followed that of the RSV PR. Substitutions in the P1 and P1' positions were an exception; in this case, the mutants behaved more like the HIV-1 PR. These results confirm predictions from structural analyses which indicate that residues 105 and 106 of the RSV PR are important in forming the S1 and S1' binding subsites. These results, further analyzed with the aid of computer modeling of the RSV PR with different substrates, provide an explanation for why only partial HIV-1 PR-like behavior was introduced into the above RSV PR mutants.

Amino Acid Sequence↗
PubMed1992

Interaction between retroviral U5 RNA and the T psi C loop of the tRNA(Trp) primer is required for efficient initiation of reverse transcription.

The 5' end of avian sarcoma and leukosis virus RNA near the primer binding site forms two RNA secondary structures, U5-inverted repeat (U5-IR) and U5-leader stems, which are required for efficient initiation of reverse transcription. Lying between these two secondary structures is a 7-base sequence that can anneal to the T psi C loop of the tRNA(Trp) primer. Base substitutions in U5 RNA which disrupt this potential interaction result in a defect in the initiation of reverse transcription both in vivo and in vitro. The defect can be complemented in vitro by base substitutions in the primer. The U5 RNA-T psi C interaction is also dependent upon the presence of both the U5-IR and the U5-leader structures. These RNA secondary structures and primer interactions are conserved in other type C and D retroviruses, suggesting that there is a common mechanism for the initiation of reverse transcription in all of these retroviruses.

Base Sequence↗
PubMed1991

A covalent complex between retroviral integrase and nicked substrate DNA.

Purified retroviral integrase (IN) from avian sarcoma-leukosis viruses can appropriately process the termini of linear viral DNA, cleave host DNA in a sequence-independent manner, and catalyze integrative recombination; an exogenous source of energy is not required for these reactions. Using DNA substrates containing radioactive phosphate groups, we demonstrate that IN becomes covalently joined to the new 5' phosphate ends of DNA produced at sites of cleavage. Most of the phosphodiester linkages between IN and DNA involve serine, but some involve threonine. Computer-assisted alignment of 80 retroviral and retrotransposon IN sequences identified one serine that is conserved in all of these proteins and three less-conserved threonine residues. These results identify candidate active-site residues and provide support for the participation of a covalent IN-DNA intermediate in retroviral integration.

Amino Acid Sequence↗
PubMed1991

Overlapping retrovirus U5 sequence elements are required for efficient integration and initiation of reverse transcription.

A secondary structure in the 5' noncoding region of avian retrovirus RNA, called the U5-leader stem, was shown previously to have a role in initiation of reverse transcription (D. Cobrinik, L. Soskey, and J. Leis, J. Virol. 62:3622-3630, 1988). We now show that an additional RNA secondary structure near the U5 terminus, called the U5-IR stem, is also important for reverse transcription. Mutations that disrupt the U5-IR stem cause a replication defect associated with both a decrease in synthesis of viral DNA in infected cells and a decrease in initiation of reverse transcription in melittin-permeabilized virions. Structure-compensating base substitutions in the U5-IR restore reverse transcription efficiency. In viral DNA, U5-IR sequences are included in the U5 terminal region that functions as a viral integration donor site. When base substitutions are introduced into these sequences, a reduced efficiency of integration in vitro and in vivo is observed. These observations indicate that U5-IR sequences have a structural role in reverse transcription of viral RNA and a sequence-specific role in the integration of viral DNA.

Avian Sarcoma Viruses↗
PubMed1990

The avian retroviral IN protein is both necessary and sufficient for integrative recombination in vitro.

The integration of viral DNA into the host cell chromosome is an essential feature of the retroviral life cycle. The integration reaction requires cis-acting sequences at the ends of linear viral DNA and a trans-acting product of the pol gene, the integration protein (IN). Previously, we demonstrated that avian sarcoma-leukosis virus (ASLV) IN is able to carry out the first step in the integration process in vitro: nicking of the ends of linear viral DNA. In this paper, using two independent assays, we demonstrate that IN, alone, is sufficient to carry out the second step: cleavage and joining to the target DNA. These results demonstrate that the retroviral IN protein is an integrase.

Alpharetrovirus↗
PubMed1990

Structure of the aspartic protease from Rous sarcoma retrovirus refined at 2-A resolution.

The structure of Rous sarcoma virus protease has been solved by multiple isomorphous replacement in the crystal form belonging to space group P3(1)21, with unit-cell parameters a = 88.95 A and c = 78.90 A. The enzyme belongs to the family of aspartic proteases with two identical subunits composing the active homodimer. The noncrystallographic dyad relating these two subunits was identified after preliminary tracing in the MIR map and was used for phase improvement by electron-density averaging. Structure refinement resulted in a model that included 1772 protein atoms and 252 water molecules, with an R factor of 0.144 for data extending to 2 A. The secondary structure of a retroviral protease molecule closely resembles that of a single domain in pepsin-like aspartic proteases and consists of several beta-strands and of one well-defined and one distorted alpha-helix. The dimer interface is composed of the N- and C-terminal chains from both subunits which are intertwined to form a well-ordered four-stranded antiparallel beta-sheet. In each monomer, the catalytic triad (Asp-Ser-Gly) is located in a loop that forms a part of the psi-structure characteristic to all aspartic proteases. The position of a water molecule between the active-site aspartate residues and the general scheme of H bonding within the active site bear close resemblance to those in pepsin-like aspartic proteases and therefore suggest a similar enzymatic mechanism. The binding cleft over the active site is covered by two flap arms, one from each monomer, which are partially disordered. The retroviral protease dimer has been compared with several enzymes of cellular origin, with chains aligning to an rms deviation of 1.90 A or better.

Amino Acid Sequence↗