Tài liệu Báo cáo khoa học: Effect of sequence polymorphism and drug resistance on two HIV-1 Gag

Effect of sequence polymorphism and drug resistance on two HIV-1

Gag processing sites

Anita Fehe´r

1

, Irene T. Weber2

, Pe´ter Bagossi1

, Pe´ter Boross1

, Bhuvaneshwari Mahalingam2

,

John M. Louis3

, Terry D. Copeland4

, Ivan Y. Torshin5

, Robert W. Harrison5 and Jo´ zsef To¨ zse´r

1

1

Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Hungary; 2

Department of

Biology, Georgia State University, Atlanta, GA, USA; 3

Laboratory of Chemical Physics, National Institute of Diabetes,

Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA; 4

NCI-Frederick, Frederick, MD, USA; 5

Department of Computer Science, Georgia State University, Atlanta, GA, USA

The HIV-1 proteinase (PR) has proved to be a good target

for antiretroviral therapy of AIDS, and various PR inhibi￾tors are now in clinical use. However, there is a rapid selec￾tion of viral variants bearing mutations in the proteinase that

are resistant to clinical inhibitors. Drug resistance also

involves mutations of the nucleocapsid/p1 and p1/p6 clea￾vage sites of Gag, both in vitro and in vivo. Cleavages at these

sites have been shown to be rate limiting steps for polypro￾tein processing and viral maturation. Furthermore, these

sites show significant sequence polymorphism, which also

may have an impact on virion infectivity. We have studied

the hydrolysis of oligopeptides representing these cleavage

sites with representative mutations found as natural varia￾tions or that arise as resistant mutations. Wild-type and five

drug resistant PRs with mutations within or outside the

substrate binding site were tested. While the natural varia￾tions showed either increased or decreased susceptibility of

peptides toward the proteinases, the resistant mutations

always had a beneficial effect on catalytic efficiency. Com￾parison of the specificity changes obtained for the various

substrates suggested that the maximization of the van der

Waals contacts between substrate and PR is the major

determinant of specificity: the same effect is crucial for

inhibitor potency. The natural nucleocapsid/p1 and p1/p6

sites do not appear to be optimized for rapid hydrolysis.

Hence, mutation of these rate limiting cleavage sites can

partly compensate for the reduced catalytic activity of drug

resistant mutant HIV-1 proteinases.

Keywords: HIV-1 proteinase; Gag processing sites; oligo￾peptide substrates; substrate specificity; molecular modeling.

All replication competent retroviruses code for an aspartic

proteinase (PR) whose function is critical for virion

replication (reviewed in [1]). The HIV-1 PR has proved to

be an excellent target for antiretroviral therapy of AIDS,

and various PR inhibitors are now in clinical use (reviewed

in [2]). However, as observed with reverse transcriptase

inhibitors, resistant viruses rapidly emerge in PR inhibitor

therapy. Moderate to high level of resistance (2- to 100-fold)

to PR inhibitors has been observed both in vitro and in vivo,

and has been attributed to the appearance of mutations in

the PR gene. Many of these mutations are located in the

substrate binding site of the PR, and these mutations

have considerable impact on PR activity and specificity.

Other resistant mutations alter residues outside of the

substrate binding site. The compromised catalytic capability

of the multiple drug resistant HIV-1 mutants is reflected by

impaired processing of Gag precursors in PR-mutated

virions [3,4] and by decreased in vitro catalytic efficiency of

the PR towards peptides representing natural cleavage sites

[5–8]. The development of high levels of resistance to PR

inhibitors, possibly requiring multiple mutations in the PR,

was therefore expected to be limited by the functional

constraints of the enzyme, which must cleave all precursor

cleavage sites during viral replication. Subsequently, a

second locus was found to be involved in drug resistance

to HIV PR inhibitors, both in vitro and in vivo, at the

nucleocapsid (NC)/p1 and p1/p6 cleavage sites [9–14].

Evolution of PR cleavage sites other than NC/p1 and

p1/p6 in the internal (P2-P2¢) positions is limited, and

mutations are rarely observed even upon drug treatment

[12]. Cleavage at these sites appears to be a rate limiting step

in polyprotein processing [9,11]. Peptides representing these

sites have the lowest specificity constants (kcat/Km) among

all HIV-1 cleavage sites [15,16]. Furthermore, there is a

significant sequence polymorphism at these sites, which also

may have an impact on virion infectivity [17–19]. Natural

polymorphism and resistant mutations occurring at these

sites are shown in Fig. 1. Some of these amino acid

substitutions are frequently detected, others have been

found only in one clone including the P1 Asp, Ile and Lys

substituted NC/p1 sites, which are not expected to be

cleaved by the PR, based on previous extensive specificity

studies [20]. If cleavage at this site is important for virus

replication, as indicated by the mutations seen in resistance,

Correspondence to J. To¨zse´r, Department of Biochemistry and

Molecular Biology, Faculty of Medicine, University of Debrecen,

H-4012 Debrecen, PO Box 6, Hungary.

Fax: + 36 52 314989, Tel.: + 36 52 416432,

E-mail: [email protected]

Abbreviations: MA , matrix protein; CA , capsid protein; NC, nucleo￾capsid protein; PR, proteinase.

Enzyme: retropepsin (EC 3.4.23.16).

Note: nomenclature of viral proteins is according to Leis et al. [50].

(Received 9 May 2002, revised 8 July 2002, accepted 11 July 2002)

Eur. J. Biochem. 269, 4114–4120 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.03105.x