HIV-1 protease

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HIV-1 protease is a retroviral aspartyl protease (retropepsin) that is essential for the life-cycle of HIV, the retrovirus that causes AIDS. HIV protease cleaves newly synthesized polyproteins (namely, Gag and Gag-Pol) at nine cleavage sites to create the mature protein components of an infectious HIV virion. Without effective HIV protease, HIV virions remain uninfectious. Thus, mutation of HIV protease's active site or inhibition of its activity disrupts HIV's ability to replicate and infect additional cells, making HIV protease inhibition the subject of considerable pharmaceutical research.

Video HIV-1 protease

Structure

Precursor

The Gag-Pol polyprotein, which contains premature coding proteins, includes the precursor domain of HIV-1 protease (PR). PR is located between the reverse transcriptase (which is at the C-terminus of PR) and the p6^pol (which is at the N-terminus of PR) of the transframe region (TFR).

In order for this precursor to become a functional protein, each monomer must associate with another HIV-1 PR monomer to form a functional catalytic active site by each contributing the Asp25 of their respective catalytic triads.

Mature protease

HIV protease exists as a 22kDa homodimer, with each subunit made up of 99 amino acids. A single active site lies between the identical subunits and has the characteristic Asp-Thr-Gly (Asp25, Thr26 and Gly27) catalytic triad sequence common to aspartic proteases. As HIV-1 PR can only function as a dimer, the mature protease contains two Asp25 amino acids, one from each monomer, that act in conjunction with each other as the catalytic residues. Additionally, HIV protease has two molecular "flaps" which move a distance of up to 7 Å when the enzyme becomes associated with a substrate.

Maps HIV-1 protease

Function

When viral HIV-RNA enters the cell, it is accompanied by a reverse transcriptase, an integrase, and a mature protease. The reverse transcriptase essentially converts viral RNA into DNA, facilitating incorporation of viral genetic information with the host cell DNA, via integrase. The proviral DNA can either remain dormant in the nucleus or be transcribed into mRNA and translated into the Gag-Pol polyprotein by the host cell.

HIV-1 PR serves a dual purpose. Precursor HIV-1 PR is responsible for catalyzing its own production into mature PR enzymes via PR auto-processing. Mature protease is able to hydrolyze peptide bonds on the Gag-Pol polyproteins at nine specific sites, processing the resulting subunits into mature, fully-functional proteins. These cleaved proteins, including reverse transcriptase, integrase, and RNaseH, form the coding (from Gag-Pol) components necessary for viral replication.

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Synthesis

The HIV-1 PR precursor catalyzes its own production by facilitating its cleavage from the Gag-Pol polyprotein in a mechanism known as auto-processing. Auto-processing of HIV-1 PR is characterized by two sequential steps: (1) the intramolecular cleavage of the N-terminus at the p6^pol-protease cleavage site, which serves to finalize PR processing and increase enzymatic activity by producing the PR-reverse transcriptase intermediate, and (2) the intermolecular cleavage of the C-terminus at the protease-reverse transcriptase cleavage site, leading to the assembly of two PR subunits into mature dimers. Dimerization of the two subunits allows for fully-functional, combined active site, characterized by two Asp25 catalytic residues (one from each monomer), to form.

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Mechanism

According to the mechanism for HIV-1 protease cleavage proposed by Mariusz Jaskolski and colleagues, water acts as a nucleophile, which acts in simultaneous conjunction with a well-placed aspartic acid to hydrolyze the scissile peptide bond.

As an aspartic protease, the dimerized HIV-1 PR can activate a water molecule via the aspartyl group complex, in order to perform hydrolysis. Of the two Asp25 residues on the combined catalytic active site of HIV-1 PR, one is deprotonated while the other is protonated, due to pH differences in the micro-environment.

In a general aspartic protease mechanism, once the substrate is properly bound to the active site of the enzyme, the deprotonated Asp25 catalytic amino acid undergoes base catalysis, rendering the incoming water molecule a better nucleophile by deprotonating it and protonating itself. The resulting hydroxyl group attacks the carbonyl carbon of the peptide bond, forming an intermediate with a transient oxyanion. The oxyanion re-forms a double bond, leading to the cleavage of the peptide bond between the two amino acids, while the initially deprotonated Asp25 undergoes acid catalysis to donate its proton to the amino group, rendering the amino group a better leaving group for complete peptide bond cleavage.

While HIV-1 PR shares many of the same characteristics as non-viral aspartic protease, evidence by Ashraf Brik and Chi-Huey Wong has shown that HIV-1 PR catalyzes hydrolysis in a concerted manner; in other words, the nucleophilic water molecule and the protonated Asp25 simultaneously attack the peptide bond during catalysis.

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As a drug target

With its integral role in HIV replication, HIV protease has been a prime target for drug therapy. HIV protease inhibitors work by specifically binding to the active site by mimicking the tetrahedral intermediate of its substrate and essentially becoming "stuck," disabling the enzyme. After assembly and budding, viral particles lacking active protease cannot mature into infectious virions. Several protease inhibitors have been licensed for HIV therapy.

There are ten HIV-1 PR inhibitors that are currently approved by the Food and Drug Administration. These include indinavir, saquinavir, ritonavir, nelfinavir, lopinavir, amprenavir, fosamprenevir, atazanavir, tipranavir, and darunavir. Many of the inhibitors have different molecular components and thus different mechanistic actions, such as blocking the active site. Thus, their functional roles also extend to influencing circulation concentrations of other inhibitor drugs (ritonavir) and being used only for certain circumstances in which the virus exhibits tolerance of other inhibitors (tipranavir).

Evolution and resistance

Due to the high mutation rates of retroviruses, especially due to mutationally sensitive regions (notably the region containing the catalytic triad sequence), and considering that changes to a few amino acids within HIV protease can render it much less visible to an inhibitor, the active site of this enzyme can change rapidly when under the selective pressure of replication-inhibiting drugs.

Two types of mutations are generally associated with increasing drug resistance: "major" mutations and "secondary" mutations. Major mutations involve a mutation on the active site of HIV-1 PR, preventing the selective inhibitors from binding it, while secondary mutations refer to molecular changes on the periphery of the enzyme, by which the virus may exhibit tolerance of an inhibitor due to prolonged exposure of similar chemicals.

One approach to minimizing the development of drug-resistance in HIV is to administer a combination of drugs which inhibit several key aspects of the HIV replication cycle simultaneously, rather than one drug at a time. Other drug therapy targets include reverse transcriptase, virus attachment, membrane fusion, cDNA integration and virion assembly.

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See also

• Management of HIV/AIDS
• Discovery and development of HIV-protease inhibitors

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External links

• The MEROPS online database for peptidases and their inhibitors: A02.001
• Proteopedia HIV-1_protease - the HIV-1 protease structure in interactive 3D
• HIV-1 Protease at the US National Library of Medicine Medical Subject Headings (MeSH)

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References

Source of the article : Wikipedia