Human immunodeficiency virus protease expressed in Escherichia coli exhibits autoprocessing and specific maturation of the gag precursor.

Debouck, Christine; Gorniak, Joselina G.; Strickler, J. Rudi; Meek, Thomas D.; Metcalf, Brian W.; Rosenberg, Martin

doi:10.1073/pnas.84.24.8903

Cited by 260 publications

(172 citation statements)

References 22 publications

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“…This shift occurs only four codons after the initiation of the pol frame. Therefore, the primary products of FS mutants are expected to resemble closely the products generated by normal FS in HIVinfected cells, which are large gag-pol fusion polyproteins consisting of a truncated gag region (p48 minus four amino acids) and the pol proteins (Debouck et al, 1987;Gendelman et al, 1987;Jacks et al, 1988). Proteolytic cleavage of the fusion proteins generates gag products, a p41 intermediate and fully processed gag proteins.…”

Section: Discussionmentioning

confidence: 99%

Role of the gag and pol genes of human immunodeficiency virus in the morphogenesis and maturation of retrovirus-like particles expressed by recombinant vaccinia virus: an ultrastructural study

Hoshikawa¹,

Kojima²,

Yasuda³

et al. 1991

Journal of General Virology

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Section: Discussionmentioning

confidence: 99%

Role of the gag and pol genes of human immunodeficiency virus in the morphogenesis and maturation of retrovirus-like particles expressed by recombinant vaccinia virus: an ultrastructural study

Hoshikawa¹,

Kojima²,

Yasuda³

et al. 1991

Journal of General Virology

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“…drug target ͉ neutron diffraction ͉ reaction mechanism ͉ transition-state analog T he HIV-1 protease (EC 3.4.23.16) is a dimeric aspartic protease that cleaves the nascent polyproteins of HIV-1 and plays an essential role in viral replication (1)(2)(3). Currently, the development of HIV-1 protease inhibitors is regarded as a major success of structure-based drug design (4), and the inhibitors of HIV-1 protease are important compounds for establishing highly-active antiretroviral therapy for AIDS (5).…”

mentioning

confidence: 99%

Structure of HIV-1 protease in complex with potent inhibitor KNI-272 determined by high-resolution X-ray and neutron crystallography

Adachi

Ohhara

Kurihara

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

136

115

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HIV-1 protease is a dimeric aspartic protease that plays an essential role in viral replication. To further understand the catalytic mechanism and inhibitor recognition of HIV-1 protease, we need to determine the locations of key hydrogen atoms in the catalytic aspartates Asp-25 and Asp-125. The structure of HIV-1 protease in complex with transition-state analog KNI-272 was determined by combined neutron crystallography at 1.9-Å resolution and X-ray crystallography at 1.4-Å resolution. The resulting structural data show that the catalytic residue Asp-25 is protonated and that Asp-125 (the catalytic residue from the corresponding diad-related molecule) is deprotonated. The proton on Asp-25 makes a hydrogen bond with the carbonyl group of the allophenylnorstatine (Apns) group in KNI-272. The deprotonated Asp-125 bonds to the hydroxyl proton of Apns. The results provide direct experimental evidence for proposed aspects of the catalytic mechanism of HIV-1 protease and can therefore contribute substantially to the development of specific inhibitors for therapeutic application.drug target ͉ neutron diffraction ͉ reaction mechanism ͉ transition-state analog

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“…Of the 63 secondary structural assignments made for the crystallographic monomer, 46 (73%) were conserved in each monomer of the unbound structure, and 54 (86%) were conserved in each monomer of the Zn2+-bound structure. 2 crystallographic structure are maintained in solution both in the unbound and Zn 2-bound simulations. These include interactions at the amino-and carboxy-terminal g strands (residues 1-A, 95-99), which form a four-stranded antiparallel B sheet, and in the region of the active site triads (residues [25][26][27], which interlock in the "fireman's grip" characteristic of aspartyl proteases (33).…”

Section: Methodsmentioning

confidence: 99%

“…Human immunodeficiency virus type 1 protease (HIV-1 PR) is a 99-amino acid, virally encoded protein responsible for proteolytic cleavage of viral gag and gag-pol fusion polyproteins into functional products (1)(2)(3). The activity of the protease is required for viral infectivity in vitro (4).…”

mentioning

confidence: 99%

Molecular Modeling Studies Suggest That Zinc Ions Inhibit HIV-1 Protease by Binding at Catalytic Aspartates

York¹,

Darden²,

Pedersen³

et al. 1993

Environmental Health Perspectives

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Human immunodeficiency virus type 1 protease (HIV-1 PR) is a 99-amino acid, virally encoded protein responsible for proteolytic cleavage of viral gag and gag-pol fusion polyproteins into functional products (1-3). The activity of the protease is required for viral infectivity in vitro (4). Consequently, HIV-1 PR is an attractive therapeutic target for rational drug design (5,6), and the focus of a tremendous amount of research.HIV-1 PR has been characterized as an aspartic acid protease based on sequence homology to related enzymes (7, catalytic pH studies (8), and inhibition by wellknown aspartyl protease inhibitors (9,10).The active form of the enzyme behaves as a dimer (11,12). These findings have been confirmed by the elucidation of X-ray crystallographic structures of the enzyme both unbound (13-15) and bound to synthetic inhibitors (16)(17)(18)(19)(20).One feature that distinguishes the HIV-1 protease from cellular aspartyl proteases is the pH dependence of its activity. The optimum pH range for activity of the HIV-1 protease (4.5-6.0) (8) is considerably higher than for cellular enzymes in vitro (typically 2.0-4.0) (21). A notable exception is human renin, which has a pH optimum in the range of 5.5-7.5 (22). The pH optima of these enzymes presumably reflect the catalytic mechanism generally accepted for aspartyl proteases which requires one of the catalytic aspartate residues to be protonated. Recently, Zhang et al. (23) reported that zinc ions are inhibitors of both renin and HIV-1 PR at neutral pH. Although the zinc binding site(s) that lead to inactivation have not been determined, evidence suggests that one site may occur at or near the catalytic aspartate residues. In this study, molecular dynamics methodology was used to explore the possibility of zinc binding in the active site of HIV-1 PR and predict the effects of this binding on the structure. Our results are consistent with the requirements of zinc binding reported by Zhang et al. (23) and give supporting evidence that this is the site that leads to inhibition of the protease. MethodsWe performed molecular mechanics and dynamics calculations using a modified version ofAMBER3.0 (Revision A) (24,25). The all-atom force field (26) was used for all standard residues, and solvent was treated explicitly using the TIP3P model (27). Parameters for chloride ions and zinc ions were taken from Lybrand et al. (28) and Bartolotti et al. (29), respectively. Electrostatic and van der Waals interactions were treated using a "twin range" (9/18 A) residue-based cutoff, updated every 20 steps. We performed simulations under constant temperature (300 K) and pressure (1 bar) conditions using a 1-fsec integration time step, carried out to 200 psec.We obtained the starting positions of the heavy atoms for the unbound and Zn2-bound dimers from the crystallographic structure of the synthetic (Aba6795) HIV-1 protease at pH 7.0 (14).The net charge of the unbound dimer was assumed to be +4, consistent with the normal protonation states of the component amino acids at ...

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Human immunodeficiency virus protease expressed in Escherichia coli exhibits autoprocessing and specific maturation of the gag precursor.

Abstract: The mature gag and pol proteins of human immunodeficiency virus (HIV) and all retroviruses derive from large gag and gag-pol polyprotein precursors by posttranslational cleavage.

Cited by 260 publications

References 22 publications

Role of the gag and pol genes of human immunodeficiency virus in the morphogenesis and maturation of retrovirus-like particles expressed by recombinant vaccinia virus: an ultrastructural study

Role of the gag and pol genes of human immunodeficiency virus in the morphogenesis and maturation of retrovirus-like particles expressed by recombinant vaccinia virus: an ultrastructural study

Structure of HIV-1 protease in complex with potent inhibitor KNI-272 determined by high-resolution X-ray and neutron crystallography

Molecular Modeling Studies Suggest That Zinc Ions Inhibit HIV-1 Protease by Binding at Catalytic Aspartates

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