Intracellular expression of human immunodeficiency virus type 1 (HIV-1) protease variants inhibits replication of wild-type and protease inhibitor-resistant HIV-1 strains in human T-cell lines

Junker, Uwe; Escaich, Sonia; Plavec, Ivan; Baker, Joni; McPhee, Fiona; Rose, Jim; Craik, Charles S.; Böhnlein, Ernst

doi:10.1128/jvi.70.11.7765-7772.1996

Cited by 21 publications

(10 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The next considered position was 49, which is located near the tip of the flap at the top of the substrate-binding site. Gly49 could be found in the wt monomer and it could be changed even to the largest residue (Trp) without any detrimental effect to the desired inhibition (Babe et al, 1995;Junker et al, 1996;McPhee et al, 1996;Rozzelle et al, 2000;Todd et al, 2000a;2000b). Our calculations also showed that large hydrophobic residues (Phe, Tyr) and positively charged residues (Lys, Arg) gave good intermolecular interaction energy values ( Table I).…”

Section: Resultssupporting

confidence: 54%

“…Furthermore, the PR may also be targeted by macromolecular inhibitors that could provide higher interaction surface and hence a lower chance for the development of resistance (Babe et al, 1995;McPhee et al, 1996;Rozzelle et al, 2000;Todd et al, 2000a). Designing defective PR monomers and expressing them in the infected cells has been demonstrated to be an effective method (Babe et al, 1995;Junker et al, 1996;McPhee et al, 1996;Rozzelle et al, 2000;Todd et al, 2000a;2000b). Similar strategy worked for Gag (Trono et al, 1989;Shimano et al, 1999), Tat (Pearson et al, 1990;Modesti et al, 1991;Fraisier et al, 1998), Rev (Bevec et al, 1992;Liu et al, 1994), Env (Buchschacher et al, 1992;Steffy and Wong-Staal, 1993;Buchschacher et al, 1995;Chen et al, 1996) and Vpr (Sawaya et al, 2000) proteins of HIV-1.…”

Section: Introductionmentioning

confidence: 99%

“…Studies on trans-dominant negative HIV-1 PR inhibitors were pioneered by Craik and co-workers (Babe et al, 1995;Junker et al, 1996;McPhee et al, 1996;Rozzelle et al, 2000;Todd et al, 2000a;2000b). They showed that a mutant form of the PR carrying Asp25Lys, Gly49Trp and Ile50Trp mutations (PR KWW ) was the most potent inhibitor for the wild-type (wt) PR in vitro and in cell culture experiments.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Novel macromolecular inhibitors of human immunodeficiency virus-1 protease

Miklóssy

Tőzsér

Kádas

et al. 2008

Protein Engineering Design and Selection

View full text Add to dashboard Cite

An intracellularly expressed defective human immunodeficiency virus type-1 (HIV-1) protease (PR) monomer could function as a dominant-negative inhibitor of the enzyme that requires dimerization for activity. Based on in silico studies, two mutant PRs harboring hydrophilic mutations, capable of forming favorable inter- and intra-subunit interactions, were selected: PR(RE) containing Asp25Arg and Gly49Glu mutations, and PR(RER) containing an additional Ile50Arg mutation. The mutants were expressed and tested by PR assays, nuclear magnetic resonance (NMR) and cell culture experiments. The mutant PRs showed dose-dependent inhibition of the wild-type PR in a fluorescent microtiter plate PR assay. Furthermore, both mutants were retained by hexahistidine-tagged wild-type HIV-1 PR immobilized on nickel-chelate affinity resin. For the first time, heterodimerization between wild-type and dominant-negative mutant PRs were also demonstrated by NMR spectroscopy. (1)H-(15)N Heteronuclear Single Quantum Coherence NMR spectra showed that although PR(RE) has a high tendency to aggregate, PR(RER) exists mainly as a folded monomer at 25-35 microM concentration, but in the presence of wild-type PR in a ratio of 1:1, heterodimerization occurs with both mutants. While the recombinant virus containing the PR(RE) sequence showed only very low level of expression, expression of the viral proteins of the virus with the PR(RER) sequence was comparable with that of the wild-type. In cell culture experiments, infectivity of viral particles containing PR(RER) protein was reduced by 82%, at mutant to wild-type infective DNA ratio of 2:1.

show abstract

Section: Resultssupporting

confidence: 54%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Novel macromolecular inhibitors of human immunodeficiency virus-1 protease

Miklóssy

Tőzsér

Kádas

et al. 2008

Protein Engineering Design and Selection

View full text Add to dashboard Cite

show abstract

“…This likely feature of the KSHV protease provides the opportunity to develop KSHV-specific or more broadly active antiherpesviral agents, through disruption of Pr or Pr/AP homodimer formation. This strategy, using small-molecule or dominantnegative inhibitors of dimerization, has proven to be effective in inhibiting the HIV Pr (4,35,42). Such inhibitors can be used to dissect the role of the Pr in the KSHV viral life cycle and to examine the impact of antiviral therapy on the natural history of KSHV infection.…”

Section: Discussionmentioning

confidence: 99%

The protease and the assembly protein of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8)

Ünal

Pray

Lagunoff

et al. 1997

J Virol

Self Cite

View full text Add to dashboard Cite

A genomic clone encoding the protease (Pr) and the assembly protein (AP) of Kaposi's sarcoma-associated herpesvirus (KSHV) (also called human herpesvirus 8) has been isolated and sequenced. As with other herpesviruses, the Pr and AP coding regions are present within a single long open reading frame. The mature KSHV Pr and AP polypeptides are predicted to contain 230 and 283 residues, respectively. The amino acid sequence of KSHV Pr has 56% identity with that of herpesvirus saimiri, the most similar virus by phylogenetic comparison. Pr is expressed in infected human cells as a late viral gene product, as suggested by RNA analysis of KSHV-infected BCBL-1 cells. Expression of the Pr domain in Escherichia coli yields an enzymatically active species, as determined by cleavage of synthetic peptide substrates, while an active-site mutant of this same domain yields minimal proteolytic activity. Sequence comparisons with human cytomegalovirus (HCMV) Pr permitted the identification of the catalytic residues, Ser114, His46, and His134, based on the known structure of the HCMV enzyme. The amino acid sequences of the release site of KSHV Pr (Tyr-Leu-Lys-Ala*Ser-Leu-Ile-Pro) and the maturation site (Arg-Leu-Glu-Ala*Ser-Ser-Arg-Ser) show that the extended substrate binding pocket differs from that of other members of the family. The conservation of amino acids known to be involved in the dimer interface region of HCMV Pr suggests that KSHV Pr assembles in a similar fashion. These features of the viral protease provide opportunities to develop specific inhibitors of its enzymatic activity.

show abstract

“…Trans-dominant mutant protease has been constructed and shown to prevent protease activation and virion maturation (148). Junker and co-workers showed that T-cell lines which constitutively expressed the mutant protein had dramatically reduced HIV replication when compared to cells expressing the wild-type protein and this approach was also shown to be effective against protease-resistant isolates (149). Another approach to inhibit the protease through competitive inhibition has been taken by Serio and colleagues who overexpressed a chimeric Vpr molecule which contained a protease cleavage site (150) (see 4.7, Vpr) .…”

Section: Proteasementioning

confidence: 99%

Human immunodeficiency virus type I as a target for gene therapy

Gottfreðsson¹

1997

Front Biosci

View full text Add to dashboard Cite

Recent progress in our understanding of the human immunodeficiency virus type 1 (HIV-1) life cycle has lead to the identification and characterization of viral genes or gene products that have been evaluated as targets for gene therapy. Virtually every stage in the viral life cycle and every viral gene product is a potential target. Gene therapy approaches directed at several of these viral targets have been successful at inhibiting HIV-1 replication in cultured human cells, but clinical trials involving gene therapy directed at HIV-1 are still in their infancy. This manuscript begins with a brief review of the viral life cycle with an emphasis on the function of viral gene products and then summarizes the gene therapy approaches that have targeted these viral genes or gene products to inhibit HIV-1 replication.

show abstract

Intracellular expression of human immunodeficiency virus type 1 (HIV-1) protease variants inhibits replication of wild-type and protease inhibitor-resistant HIV-1 strains in human T-cell lines

Cited by 21 publications

References 38 publications

Novel macromolecular inhibitors of human immunodeficiency virus-1 protease

Novel macromolecular inhibitors of human immunodeficiency virus-1 protease

The protease and the assembly protein of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8)

Human immunodeficiency virus type I as a target for gene therapy

Contact Info

Product

Resources

About