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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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Precursor autoprocessing of various 77–93 covariance pairs.Autoprocessing (A) and quantification (B) of L-MBP fusion precursors carrying the indicated variances in transfected HEK293T cells. Autoprocessing efficiency was determined by quantifying the amounts of L-MBP-Flag-p6* and L-MBP-Flag (the proximal and distal cleavage products) as a percentage of the sum of those products plus the full-length unprocessed precursor detected by Flag antibody in each lane. Constructs are arranged in the order of decreasing autoprocessing efficiency from left to right. Brackets indicate an increase in autoprocessing efficiency observed when a V77I mutation is combined with I93L and I93V mutations; the asterisk denotes statistical significance (p = 0.0162). Quantification values are based on at least 4 independent biological samples, and error bars represent standard deviations.
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pone.0123561.g005: Precursor autoprocessing of various 77–93 covariance pairs.Autoprocessing (A) and quantification (B) of L-MBP fusion precursors carrying the indicated variances in transfected HEK293T cells. Autoprocessing efficiency was determined by quantifying the amounts of L-MBP-Flag-p6* and L-MBP-Flag (the proximal and distal cleavage products) as a percentage of the sum of those products plus the full-length unprocessed precursor detected by Flag antibody in each lane. Constructs are arranged in the order of decreasing autoprocessing efficiency from left to right. Brackets indicate an increase in autoprocessing efficiency observed when a V77I mutation is combined with I93L and I93V mutations; the asterisk denotes statistical significance (p = 0.0162). Quantification values are based on at least 4 independent biological samples, and error bars represent standard deviations.

Mentions: Because individual substitutions of charged or polar amino acids at positions 77 and 93 abolish precursor autoprocessing, we didn’t examine mutations with oppositely charged residues placed at these two positions for complementation. Instead, we postulated that the distribution of naturally occurring variants at sites 77 and 93 would be positively correlated with autoprocessing activity, i.e., higher autoprocessing efficiency would be observed with more commonly occurring variances. Subsequently, we engineered fusion precursors carrying variations (V, I, or L), individually at each position or in combination, in the context of the L-MBP fusion backbone to test this thought. A monoclonal Flag antibody was used to detect the full-length precursor, proximal cleavage product (L-MBP-Flag-p6*), and distal cleavage product (L-MBP-Flag) in each lane. Band intensity was quantified to represent the amount of product each band represents. This allowed us to directly examine the fusion precursor and its autoprocessing products at the steady state in the cell lysate. We defined autoprocessing efficiency as the percentage of released products relative to the sum of the full-length precursor plus the released products. Accordingly, high autoprocessing efficiencies correspond to high percentages of released products, while low autoprocessing efficiencies correspond to low percentages of release products and accumulation of full length precursors (Fig 5). We did not quantify PR-containing products for autoprocessing efficiency analysis as these fragments are known to undergo self-degradation [18,20,48] and thus would compromise data interpretation.


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)

Precursor autoprocessing of various 77–93 covariance pairs.Autoprocessing (A) and quantification (B) of L-MBP fusion precursors carrying the indicated variances in transfected HEK293T cells. Autoprocessing efficiency was determined by quantifying the amounts of L-MBP-Flag-p6* and L-MBP-Flag (the proximal and distal cleavage products) as a percentage of the sum of those products plus the full-length unprocessed precursor detected by Flag antibody in each lane. Constructs are arranged in the order of decreasing autoprocessing efficiency from left to right. Brackets indicate an increase in autoprocessing efficiency observed when a V77I mutation is combined with I93L and I93V mutations; the asterisk denotes statistical significance (p = 0.0162). Quantification values are based on at least 4 independent biological samples, and error bars represent standard deviations.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123561.g005: Precursor autoprocessing of various 77–93 covariance pairs.Autoprocessing (A) and quantification (B) of L-MBP fusion precursors carrying the indicated variances in transfected HEK293T cells. Autoprocessing efficiency was determined by quantifying the amounts of L-MBP-Flag-p6* and L-MBP-Flag (the proximal and distal cleavage products) as a percentage of the sum of those products plus the full-length unprocessed precursor detected by Flag antibody in each lane. Constructs are arranged in the order of decreasing autoprocessing efficiency from left to right. Brackets indicate an increase in autoprocessing efficiency observed when a V77I mutation is combined with I93L and I93V mutations; the asterisk denotes statistical significance (p = 0.0162). Quantification values are based on at least 4 independent biological samples, and error bars represent standard deviations.
Mentions: Because individual substitutions of charged or polar amino acids at positions 77 and 93 abolish precursor autoprocessing, we didn’t examine mutations with oppositely charged residues placed at these two positions for complementation. Instead, we postulated that the distribution of naturally occurring variants at sites 77 and 93 would be positively correlated with autoprocessing activity, i.e., higher autoprocessing efficiency would be observed with more commonly occurring variances. Subsequently, we engineered fusion precursors carrying variations (V, I, or L), individually at each position or in combination, in the context of the L-MBP fusion backbone to test this thought. A monoclonal Flag antibody was used to detect the full-length precursor, proximal cleavage product (L-MBP-Flag-p6*), and distal cleavage product (L-MBP-Flag) in each lane. Band intensity was quantified to represent the amount of product each band represents. This allowed us to directly examine the fusion precursor and its autoprocessing products at the steady state in the cell lysate. We defined autoprocessing efficiency as the percentage of released products relative to the sum of the full-length precursor plus the released products. Accordingly, high autoprocessing efficiencies correspond to high percentages of released products, while low autoprocessing efficiencies correspond to low percentages of release products and accumulation of full length precursors (Fig 5). We did not quantify PR-containing products for autoprocessing efficiency analysis as these fragments are known to undergo self-degradation [18,20,48] and thus would compromise data interpretation.

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