Effect of internal cleavage site mutations in human immunodeficiency virus type 1 capsid protein on its structure and function

Abstract

The capsid protein of the human immunodeficiency virus type 1 has been found to be a substrate of the retroviral protease in vitro, and its processing was predicted to be strongly dependent on a pH-induced conformational change. Several protease cleavage sites have been identified within the capsid protein, but the importance of its cleavage by the viral protease at the early phase of infection is controversial. To confirm the relevance of this process, we aimed to design, produce, and characterize mutant capsid proteins, in which the protein susceptibility toward HIV-1 protease is altered without affecting other steps of the viral life cycle. Our results indicate that while the introduced mutations changed the cleavage rate at the mutated sites of the capsid protein by HIV-1 protease, some of them caused only negligible or moderate structural changes (A78V, L189F, and L189I). However, the effects of other mutations (W23A, A77P, and L189P) were dramatic, as assessed by secondary structure determination or cyclophilin A-binding assay. Based on our observations, the L189F mutant capsid remains structurally and functionally unchanged and may therefore be the best candidate for use in studies aimed at better understanding the role of the protease in the early postentry events of viral infection or retrovirus-mediated gene transduction.

Keywords: HIV‐1; capsid protein; circular dichroism spectroscopy; cyclophilin A; human immunodeficiency virus type 1; protease, mutagenesis.

Figure 1

Structure of wild‐type HIV‐1 capsid protein and its complex with cyclophilin. (A) Figure shows the ribbon/tube representation of crystal structures of wild‐type HIV‐1 capsid protein (3NTE.pdb) (A) and its N‐terminal domain bound to cyclophilin A (1AK4.pdb) (B). Residues harboring the proteolytic cleavage sites are indicated by spacefill representation (residues are labeled by black color), while residues of cyclophilin A‐binding loop (⁸⁷ HAGPIA ⁹²) by ball‐and‐stick representation (residues are labeled by blue color). Color code: red, α‐helix; yellow, β‐sheet. (C) Sequence of the wild‐type CA. Identified cleavage sites are marked with arrows, and introduced amino acid changes are indicated with red letters under the wild‐type sequence.

Figure 2

Cleavage of wild‐type and mutant HIV‐1 CA proteins by HIV‐1 PR. (A) Representative SDS/PAGE of the digested CA proteins. Recombinant CA proteins were incubated with (+) or without (−) HIV‐1 PR at 37 °C for 4 h at pH 5.5; then the reaction mixture was analyzed by SDS/PAGE. M denotes the molecular weight marker (Precision Plus Protein Dual Xtra Standard). (B) Schematic representation of proteolytic fragments produced by the proteolytic cleavage. Modified cleavage sites are marked by green arrows and missed cleavages contributing to the appearance of each fragments are represented by red crosses. Only those fragments are indicated, which have already been separated and identified previously 12. (C) Comparison of unprocessed CA amount after proteolytic digestion. Values were calculated using three independent proteolytic digestion experiments, as described in Materials and methods. Amounts of uncleaved CA after proteolytic cleavage were quantified by scanning the band densities in the gels and were calculated as percentage of total CA proteins (incubated without HIV‐1 PR). The percentage of proteolytic cleavage for the wild‐type was arbitrarily set at 100%. Error bars represent ±SD (n = 3). ** P < 0.01, *** P < 0.001.

Figure 3

Circular dichroic spectra of recombinant HIV‐1 CA protein and its mutants in the far‐UV region at pH 7.5. The pathlength of cuvette was 0.02 cm.

Figure 4

Limited tryptic digestion of the recombinant CA protein and its mutants. (A) Representative SDS/PAGE of the digested CA proteins. Recombinant CA proteins were incubated with (+) or without (−) trypsin at 37 °C for 2 h at pH 7.5; then the reaction mixture was analyzed by SDS/PAGE. M denotes the molecular weight marker (Precision Plus Protein Dual Xtra Standard). (B) Comparison of unprocessed CA amount after tryptic digestion. Values were calculated using three independent tryptic digestion experiments as described in Materials and methods. Amounts of uncleaved CA after proteolytic cleavage were quantified by scanning the band densities in the gels and were calculated as percentage of total CA proteins (incubated without HIV‐1 PR). The percentage of proteolytic cleavage for the wild‐type was arbitrarily set at 100%. The data were plotted in a bar graph. Error bars represent ±SD (n = 3). * P < 0.05, ** P < 0.01.

Figure 5

Analysis of the His₆‐HIVCA pull‐down assay of the wild‐type and mutant HIV‐1 capsid proteins. (A) Representative SDS/PAGE of the pull‐down assays. M denotes the molecular weight marker. (B) Comparison of CypA binding ability of the HIV‐1 capsid proteins. Values were calculated using three independent His₆‐HIVCA pull‐down assay experiments. Amount of CA and CypA proteins were calculated by scanning the band densities in the gels, and ratios of the two proteins were plotted in a bar graph. Error bars represent ±SD (n = 3). *P < 0.05.