Three isoforms of cyclophilin A associated with human immunodeficiency virus type 1 were found by proteomics by using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry.
Human immunodeficiency virus type 1 (HIV-1) strain LAV-1 (HIV-1(LAV-1)) particles were collected by ultracentrifugation, treated with subtilisin, and then purified by Sepharose CL-4B column chromatography to remove microvesicles. The lysate of the purified HIV-1(LAV-1) particles was subjected to two-dimensional (2D) gel electrophoresis and stained. The 2D gel electrophoresis image suggested that 24 proteins can be identified inside the virion. Furthermore, the stained protein spots were excised and digested with trypsin. The resulting peptide fragments were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Peptide mass fingerprinting data suggested that two isoforms of cyclophilin A (CyPA), one with an isoelectric point (pI) of 6.40 and one with a pI of 6.53, are inside the viral membrane; that another isoform, with a pI of 6.88, is outside the viral membrane; and that the CyPA isoform with a pI of 6.53 is N acetylated. The mechanisms that permit the redistribution of CyPA on the viral surface have not yet been clarified, but it is surmised that the CyPA isoform with a pI of 6.88 may play a critical role in the attachment of virions to the surface of target cells and that both CyPA isoforms with pIs of 6.40 and 6.53 may regulate the conformation of the HIV-1 capsid protein.
Misumi
Shogo
S
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan.
Fuchigami
Takashi
T
Takamune
Nobutoki
N
Takahashi
Ichiro
I
Takama
Michiho
M
Shoji
Shozo
S
D021984:Cyclophilin A / Q000032:analysis* / Q000502:physiology; D015180:Electrophoresis, Gel, Two-Dimensional; D015497:HIV-1 / Q000201:enzymology* / Q000302:isolation & purification; D007527:Isoenzymes / Q000032:analysis*; D011499:Protein Processing, Post-Translational; D020543:Proteome; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020860:Subtilisin / Q000494:pharmacology; D014771:Virion / Q000737:chemistry
D007527:Isoenzymes; D020543:Proteome; D020860:Subtilisin; D021984:Cyclophilin A