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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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Gag processing in the presence of various C-terminal tags (A) and 77–93 covariants (B) in transfected HEK293T cells and the released virus like particles.The blots were probed with a mouse monoclonal anti-p24 antibody and visualized by a LI-COR Odyssey scanner.
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pone.0123561.g007: Gag processing in the presence of various C-terminal tags (A) and 77–93 covariants (B) in transfected HEK293T cells and the released virus like particles.The blots were probed with a mouse monoclonal anti-p24 antibody and visualized by a LI-COR Odyssey scanner.

Mentions: We next sought to examine whether the observed functional correlation is reproducible in a proviral context. Because the fusion precursor system utilizes a tag at the C-terminus of the precursor to facilitate mature PR detection, we first examined whether various C-terminal tags would have any impact on Gag processing as an indirect readout of precursor autoprocessing and mature PR activity. A previously reported pNL-PR construct [18,30] was used to serve as a positive control, to which we introduced Flag, HA, Myc, and V5 tags individually fused to the C-terminus of PR. The resulting constructs were then expressed in transfected HEK293T cells. A mouse monoclonal p24 antibody was used to detect the full-length Gag precursor (p55) and the other p24-containing products associated with the total cell lysates and released virus-like particles (VLPs). We also tested two PR sequences, i.e., the pseudo wild type PR [15,24] and NL4-3 derived PR, which differ by six amino acids, to see if there is any difference in Gag processing by different PRs. Both wild type PRpse and PRNL4-3 produced p24 as the dominant band along with some p25 plus minimal amounts of full length Gag and other p24-containing intermediates in the released VLPs (Fig 7A, lanes 2 and 5), confirming the presence of the mature PR as a result of effective precursor autoprocessing. As a negative control, a D25N mutant completely abolished PR activity, leading to detection of the full length Gag polyprotein (p55) as the predominant product (Fig 7A, lane 4). The fusion of Flag, HA, Myc, and V5 tags all resulted in greater levels of p25 and the detection of several p24-containing intermediates plus full length Gag in transfected cells and VLPs (Fig 7A, lanes 3, 6–8). Our data suggested that all C-terminal fusions have moderate impact on Gag polyprotein processing in the provirus context. It is not discernable whether the impact is due to effects on the mature PR, the precursor, or a combination of the two. Nonetheless, this is consistent with our previous publication [20] and previous reports showing that C-terminal fusions to RT or GFP remain replication competent but with reduced infectivity [12–14]. Interestingly, each fusion exhibited a slightly different Gag processing pattern in that certain intermediate(s) are more predominant than others. This suggests that PR autoprocessing and/or proteolysis activities can be modulated by various flanking sequences as well. In light of this information, we decided to evaluate 77–93 covariants using the tag-free pNL-PR construct (Fig 7B).


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)

Gag processing in the presence of various C-terminal tags (A) and 77–93 covariants (B) in transfected HEK293T cells and the released virus like particles.The blots were probed with a mouse monoclonal anti-p24 antibody and visualized by a LI-COR Odyssey scanner.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123561.g007: Gag processing in the presence of various C-terminal tags (A) and 77–93 covariants (B) in transfected HEK293T cells and the released virus like particles.The blots were probed with a mouse monoclonal anti-p24 antibody and visualized by a LI-COR Odyssey scanner.
Mentions: We next sought to examine whether the observed functional correlation is reproducible in a proviral context. Because the fusion precursor system utilizes a tag at the C-terminus of the precursor to facilitate mature PR detection, we first examined whether various C-terminal tags would have any impact on Gag processing as an indirect readout of precursor autoprocessing and mature PR activity. A previously reported pNL-PR construct [18,30] was used to serve as a positive control, to which we introduced Flag, HA, Myc, and V5 tags individually fused to the C-terminus of PR. The resulting constructs were then expressed in transfected HEK293T cells. A mouse monoclonal p24 antibody was used to detect the full-length Gag precursor (p55) and the other p24-containing products associated with the total cell lysates and released virus-like particles (VLPs). We also tested two PR sequences, i.e., the pseudo wild type PR [15,24] and NL4-3 derived PR, which differ by six amino acids, to see if there is any difference in Gag processing by different PRs. Both wild type PRpse and PRNL4-3 produced p24 as the dominant band along with some p25 plus minimal amounts of full length Gag and other p24-containing intermediates in the released VLPs (Fig 7A, lanes 2 and 5), confirming the presence of the mature PR as a result of effective precursor autoprocessing. As a negative control, a D25N mutant completely abolished PR activity, leading to detection of the full length Gag polyprotein (p55) as the predominant product (Fig 7A, lane 4). The fusion of Flag, HA, Myc, and V5 tags all resulted in greater levels of p25 and the detection of several p24-containing intermediates plus full length Gag in transfected cells and VLPs (Fig 7A, lanes 3, 6–8). Our data suggested that all C-terminal fusions have moderate impact on Gag polyprotein processing in the provirus context. It is not discernable whether the impact is due to effects on the mature PR, the precursor, or a combination of the two. Nonetheless, this is consistent with our previous publication [20] and previous reports showing that C-terminal fusions to RT or GFP remain replication competent but with reduced infectivity [12–14]. Interestingly, each fusion exhibited a slightly different Gag processing pattern in that certain intermediate(s) are more predominant than others. This suggests that PR autoprocessing and/or proteolysis activities can be modulated by various flanking sequences as well. In light of this information, we decided to evaluate 77–93 covariants using the tag-free pNL-PR construct (Fig 7B).

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