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A Functional Interplay between Human Immunodeficiency Virus Type 1 Protease Residues 77 and 93 Involved in Differential Regulation of Precursor Autoprocessing and Mature Protease Activity.

Counts CJ, Ho PS, Donlin MJ, Tavis JE, Chen C - PLoS ONE (2015)

Bottom Line: The free mature PR is liberated as a result of precursor autoprocessing upon virion release.Furthermore, the 77I93V covariant enhanced precursor autoprocessing and Gag polyprotein processing but decreased the mature PR activity.Our data also suggests that these covariance pairs differentially regulate precursor autoprocessing and the mature protease activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, United States of America.

ABSTRACT
HIV-1 protease (PR) is a viral enzyme vital to the production of infectious virions. It is initially synthesized as part of the Gag-Pol polyprotein precursor in the infected cell. The free mature PR is liberated as a result of precursor autoprocessing upon virion release. We previously described a model system to examine autoprocessing in transfected mammalian cells. Here, we report that a covariance analysis of miniprecursor (p6*-PR) sequences derived from drug naïve patients identified a series of amino acid pairs that vary together across independent viral isolates. These covariance pairs were used to build the first topology map of the miniprecursor that suggests high levels of interaction between the p6* peptide and the mature PR. Additionally, several PR-PR covariance pairs are located far from each other (>12 Å Cα to Cα) relative to their positions in the mature PR structure. Biochemical characterization of one such covariance pair (77-93) revealed that each residue shows distinct preference for one of three alkyl amino acids (V, I, and L) and that a polar or charged amino acid at either of these two positions abolishes precursor autoprocessing. The most commonly observed 77V is preferred by the most commonly observed 93I, but the 77I variant is preferred by other 93 variances (L, V, or M) in supporting precursor autoprocessing. Furthermore, the 77I93V covariant enhanced precursor autoprocessing and Gag polyprotein processing but decreased the mature PR activity. Therefore, both covariance and biochemical analyses support a functional association between residues 77 and 93, which are spatially distant from each other in the mature PR structure. Our data also suggests that these covariance pairs differentially regulate precursor autoprocessing and the mature protease activity.

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Related in: MedlinePlus

Mutations of residue 93 to non-covariance amino acids abolish fusion precursor autoprocessing.(A) Miniprecursor fusion schematic showing the distal and proximal cleavage sites and residue 93 mutants that were tested here. (B) Autoprocessing of the L-MBP fusion precursors carrying the indicated mutations. Post-nuclear total lysates were resolved on SDS-PAGE and analyzed via western blotting. Two sets of lysate samples were resolved and probed separately with mouse Flag (top) or HA (bottom) antibodies. Both were probed with IR800 goat anti-mouse secondary antibody and scanned using a LI-COR Odyssey scanner. The bracket to the left indicates the autoprocessing products resulted from the proximal site cleavage. (C) Precursor fusion schematic showing the p6* sequence and the truncated sequence of M4, in which the distal cleavage site is deleted. X corresponds to either C2-MBP or GST. Autoprocessing of the indicated constructs is shown in the context of C2-MBP (D) or GST (E) with the wild type or M4 p6* peptide. The corresponding full length precursors were denoted with triangles.
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pone.0123561.g003: Mutations of residue 93 to non-covariance amino acids abolish fusion precursor autoprocessing.(A) Miniprecursor fusion schematic showing the distal and proximal cleavage sites and residue 93 mutants that were tested here. (B) Autoprocessing of the L-MBP fusion precursors carrying the indicated mutations. Post-nuclear total lysates were resolved on SDS-PAGE and analyzed via western blotting. Two sets of lysate samples were resolved and probed separately with mouse Flag (top) or HA (bottom) antibodies. Both were probed with IR800 goat anti-mouse secondary antibody and scanned using a LI-COR Odyssey scanner. The bracket to the left indicates the autoprocessing products resulted from the proximal site cleavage. (C) Precursor fusion schematic showing the p6* sequence and the truncated sequence of M4, in which the distal cleavage site is deleted. X corresponds to either C2-MBP or GST. Autoprocessing of the indicated constructs is shown in the context of C2-MBP (D) or GST (E) with the wild type or M4 p6* peptide. The corresponding full length precursors were denoted with triangles.

Mentions: DNA plasmids encoding for the fusion precursors used in this study were constructed by standard mutagenesis and molecular cloning techniques as described previously [18,30]. The GST-Flag-p6*-PR-HA encoding plasmid was used as the parental expression vector [18,24]. The GST coding sequence was then replaced with the L-MBP or C2-MBP coding sequence to express fusion precursors carrying different N-terminal tags. The L-MBP tag contains the full-length maltose binding protein sequence plus an additional peptide (MATSSHHHHHHSSGLVPRGSH) fused to its N-terminus. The C2-MBP sequence is derived from the full-length MBP but lacking the N-terminal signal peptide (MKIKTGARILALSALTTMMFSASALA). Therefore, the L-MBP and C2-MBP differ only at their N-terminal sequences (C2-MBP is 46 aa shorter) and have the same C-terminal sequences (366 aa). Unless noted elsewhere, these fusion constructs contain the entire p6* sequence carrying two natural cleavage sites, one between p6* and PR (the proximal site, also referred to as the N-terminal cleavage site) and the other at the N-terminal region of the p6* peptide (the distal site) (Fig 3A). The majority of the p6* peptide was truncated in M4 construct (Fig 3C) [18]. The pNL-PR proviral construct was previously described [30]. The individual point mutations described herein were introduced to the indicated plasmids by standard PCR-mediated mutagenesis and cloning procedures. All plasmids were sequenced to confirm the presence of the desired mutations and detailed sequence information is available upon request.


A Functional Interplay between Human Immunodeficiency Virus Type 1 Protease Residues 77 and 93 Involved in Differential Regulation of Precursor Autoprocessing and Mature Protease Activity.

Counts CJ, Ho PS, Donlin MJ, Tavis JE, Chen C - PLoS ONE (2015)

Mutations of residue 93 to non-covariance amino acids abolish fusion precursor autoprocessing.(A) Miniprecursor fusion schematic showing the distal and proximal cleavage sites and residue 93 mutants that were tested here. (B) Autoprocessing of the L-MBP fusion precursors carrying the indicated mutations. Post-nuclear total lysates were resolved on SDS-PAGE and analyzed via western blotting. Two sets of lysate samples were resolved and probed separately with mouse Flag (top) or HA (bottom) antibodies. Both were probed with IR800 goat anti-mouse secondary antibody and scanned using a LI-COR Odyssey scanner. The bracket to the left indicates the autoprocessing products resulted from the proximal site cleavage. (C) Precursor fusion schematic showing the p6* sequence and the truncated sequence of M4, in which the distal cleavage site is deleted. X corresponds to either C2-MBP or GST. Autoprocessing of the indicated constructs is shown in the context of C2-MBP (D) or GST (E) with the wild type or M4 p6* peptide. The corresponding full length precursors were denoted with triangles.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4404164&req=5

pone.0123561.g003: Mutations of residue 93 to non-covariance amino acids abolish fusion precursor autoprocessing.(A) Miniprecursor fusion schematic showing the distal and proximal cleavage sites and residue 93 mutants that were tested here. (B) Autoprocessing of the L-MBP fusion precursors carrying the indicated mutations. Post-nuclear total lysates were resolved on SDS-PAGE and analyzed via western blotting. Two sets of lysate samples were resolved and probed separately with mouse Flag (top) or HA (bottom) antibodies. Both were probed with IR800 goat anti-mouse secondary antibody and scanned using a LI-COR Odyssey scanner. The bracket to the left indicates the autoprocessing products resulted from the proximal site cleavage. (C) Precursor fusion schematic showing the p6* sequence and the truncated sequence of M4, in which the distal cleavage site is deleted. X corresponds to either C2-MBP or GST. Autoprocessing of the indicated constructs is shown in the context of C2-MBP (D) or GST (E) with the wild type or M4 p6* peptide. The corresponding full length precursors were denoted with triangles.
Mentions: DNA plasmids encoding for the fusion precursors used in this study were constructed by standard mutagenesis and molecular cloning techniques as described previously [18,30]. The GST-Flag-p6*-PR-HA encoding plasmid was used as the parental expression vector [18,24]. The GST coding sequence was then replaced with the L-MBP or C2-MBP coding sequence to express fusion precursors carrying different N-terminal tags. The L-MBP tag contains the full-length maltose binding protein sequence plus an additional peptide (MATSSHHHHHHSSGLVPRGSH) fused to its N-terminus. The C2-MBP sequence is derived from the full-length MBP but lacking the N-terminal signal peptide (MKIKTGARILALSALTTMMFSASALA). Therefore, the L-MBP and C2-MBP differ only at their N-terminal sequences (C2-MBP is 46 aa shorter) and have the same C-terminal sequences (366 aa). Unless noted elsewhere, these fusion constructs contain the entire p6* sequence carrying two natural cleavage sites, one between p6* and PR (the proximal site, also referred to as the N-terminal cleavage site) and the other at the N-terminal region of the p6* peptide (the distal site) (Fig 3A). The majority of the p6* peptide was truncated in M4 construct (Fig 3C) [18]. The pNL-PR proviral construct was previously described [30]. The individual point mutations described herein were introduced to the indicated plasmids by standard PCR-mediated mutagenesis and cloning procedures. All plasmids were sequenced to confirm the presence of the desired mutations and detailed sequence information is available upon request.

Bottom Line: The free mature PR is liberated as a result of precursor autoprocessing upon virion release.Furthermore, the 77I93V covariant enhanced precursor autoprocessing and Gag polyprotein processing but decreased the mature PR activity.Our data also suggests that these covariance pairs differentially regulate precursor autoprocessing and the mature protease activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, United States of America.

ABSTRACT
HIV-1 protease (PR) is a viral enzyme vital to the production of infectious virions. It is initially synthesized as part of the Gag-Pol polyprotein precursor in the infected cell. The free mature PR is liberated as a result of precursor autoprocessing upon virion release. We previously described a model system to examine autoprocessing in transfected mammalian cells. Here, we report that a covariance analysis of miniprecursor (p6*-PR) sequences derived from drug naïve patients identified a series of amino acid pairs that vary together across independent viral isolates. These covariance pairs were used to build the first topology map of the miniprecursor that suggests high levels of interaction between the p6* peptide and the mature PR. Additionally, several PR-PR covariance pairs are located far from each other (>12 Å Cα to Cα) relative to their positions in the mature PR structure. Biochemical characterization of one such covariance pair (77-93) revealed that each residue shows distinct preference for one of three alkyl amino acids (V, I, and L) and that a polar or charged amino acid at either of these two positions abolishes precursor autoprocessing. The most commonly observed 77V is preferred by the most commonly observed 93I, but the 77I variant is preferred by other 93 variances (L, V, or M) in supporting precursor autoprocessing. Furthermore, the 77I93V covariant enhanced precursor autoprocessing and Gag polyprotein processing but decreased the mature PR activity. Therefore, both covariance and biochemical analyses support a functional association between residues 77 and 93, which are spatially distant from each other in the mature PR structure. Our data also suggests that these covariance pairs differentially regulate precursor autoprocessing and the mature protease activity.

Show MeSH
Related in: MedlinePlus