Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein
To explore the sequence context-dependent nature of the human immunodeficiency virus type 1 (HIV-1) protease’s specificity and to provide a rationale for viral mutagenesis to study the potential role of the nucleocapsid (NC) processing in HIV-1 replication, synthetic oligopeptide substrates represen...
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MDPI AG
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/13/6/1092 |
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| author | János András Mótyán Márió Miczi Stephen Oroszlan József Tőzsér |
| author_facet | János András Mótyán Márió Miczi Stephen Oroszlan József Tőzsér |
| author_sort | János András Mótyán |
| collection | DOAJ |
| description | To explore the sequence context-dependent nature of the human immunodeficiency virus type 1 (HIV-1) protease’s specificity and to provide a rationale for viral mutagenesis to study the potential role of the nucleocapsid (NC) processing in HIV-1 replication, synthetic oligopeptide substrates representing the wild-type and modified versions of the proximal cleavage site of HIV-1 NC were assayed as substrates of the HIV-1 protease (PR). The S1′ substrate binding site of HIV-1 PR was studied by an in vitro assay using KIVKCF↓NCGK decapeptides having amino acid substitutions of N17 residue of the cleavage site of the first zinc-finger domain, and in silico calculations were also performed to investigate amino acid preferences of S1′ site. Second site substitutions have also been designed to produce “revertant” substrates and convert a non-hydrolysable sequence (having glycine in place of N17) to a substrate. The specificity constants obtained for peptides containing non-charged P1′ substitutions correlated well with the residue volume, while the correlation with the calculated interaction energies showed the importance of hydrophobicity: interaction energies with polar residues were related to substantially lower specificity constants. Cleavable “revertants” showed one residue shift of cleavage position due to an alternative productive binding mode, and surprisingly, a double cleavage of a substrate was also observed. The results revealed the importance of alternative binding possibilities of substrates into the HIV-1 PR. The introduction of the “revertant” mutations into infectious virus clones may provide further insights into the potential role of NC processing in the early phase of the viral life-cycle. |
| first_indexed | 2024-03-10T10:37:02Z |
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| id | doaj.art-50c54b9aefd64f39a10ae947da8a5340 |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-10T10:37:02Z |
| publishDate | 2021-06-01 |
| publisher | MDPI AG |
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| series | Viruses |
| spelling | doaj.art-50c54b9aefd64f39a10ae947da8a53402023-11-21T23:13:47ZengMDPI AGViruses1999-49152021-06-01136109210.3390/v13061092Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid ProteinJános András Mótyán0Márió Miczi1Stephen Oroszlan2József Tőzsér3Laboratory of Retroviral Biochemistry, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, HungaryLaboratory of Retroviral Biochemistry, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, HungaryHIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD 21702, USALaboratory of Retroviral Biochemistry, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, HungaryTo explore the sequence context-dependent nature of the human immunodeficiency virus type 1 (HIV-1) protease’s specificity and to provide a rationale for viral mutagenesis to study the potential role of the nucleocapsid (NC) processing in HIV-1 replication, synthetic oligopeptide substrates representing the wild-type and modified versions of the proximal cleavage site of HIV-1 NC were assayed as substrates of the HIV-1 protease (PR). The S1′ substrate binding site of HIV-1 PR was studied by an in vitro assay using KIVKCF↓NCGK decapeptides having amino acid substitutions of N17 residue of the cleavage site of the first zinc-finger domain, and in silico calculations were also performed to investigate amino acid preferences of S1′ site. Second site substitutions have also been designed to produce “revertant” substrates and convert a non-hydrolysable sequence (having glycine in place of N17) to a substrate. The specificity constants obtained for peptides containing non-charged P1′ substitutions correlated well with the residue volume, while the correlation with the calculated interaction energies showed the importance of hydrophobicity: interaction energies with polar residues were related to substantially lower specificity constants. Cleavable “revertants” showed one residue shift of cleavage position due to an alternative productive binding mode, and surprisingly, a double cleavage of a substrate was also observed. The results revealed the importance of alternative binding possibilities of substrates into the HIV-1 PR. The introduction of the “revertant” mutations into infectious virus clones may provide further insights into the potential role of NC processing in the early phase of the viral life-cycle.https://www.mdpi.com/1999-4915/13/6/1092human immunodeficiency virusHIV-1proteaseviral proteasesspecificityviral proteins |
| spellingShingle | János András Mótyán Márió Miczi Stephen Oroszlan József Tőzsér Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein Viruses human immunodeficiency virus HIV-1 protease viral proteases specificity viral proteins |
| title | Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein |
| title_full | Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein |
| title_fullStr | Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein |
| title_full_unstemmed | Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein |
| title_short | Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein |
| title_sort | specificity of the hiv 1 protease on substrates representing the cleavage site in the proximal zinc finger of hiv 1 nucleocapsid protein |
| topic | human immunodeficiency virus HIV-1 protease viral proteases specificity viral proteins |
| url | https://www.mdpi.com/1999-4915/13/6/1092 |
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