Identification of proteolytic processing sites within the Gag and Pol polyproteins of feline immunodeficiency virus.
N-terminal amino acid sequencing, ion spray mass spectrometry, and cleavage of synthetic peptide substrates were used to identify the N and C termini of the mature Gag and Pol proteins of feline immunodeficiency virus (FIV). The Gag polyprotein encodes matrix (MA), capsid (CA), and nucleocapsid (NC) proteins. The Gag-Pol polyprotein encodes, in addition to the above proteins, protease (PR), reverse transcriptase (RT), dUTPase (DU), and integrase (IN). Secondary cleavage of RT at Trp-595-Tyr-596 of Pol yields a truncated form lacking the C-terminal RNase H domain. The observed and expected molecular masses of the viral proteins were in agreement, with three exceptions. (i) The molecular mass of MA was 14,735 Da, compared with a predicted mass of 14,649 Da, based on a single cleavage at Tyr-135-Pro-136 of Gag. The observed molecular mass is consistent with myristoylation of MA, which was confirmed by metabolic labeling of FIV MA with [3H]myristic acid. (ii) The N terminus of the NC protein is generated via cleavage at Gln-366-Val-367 of Gag, which predicts a mass of 25,523 for CA and 9,101 for the major form of NC. The observed mass of CA was 24,569, consistent with loss of nine C-terminal amino acids by a second cleavage of CA at Leu-357-Leu-358. Synthetic FIV protease accurately cleaved synthetic peptide substrates containing this site. (iii) The actual mass of NC (7,120 Da) was approximately 2 kDa smaller than the mass predicted by synthesis to the stop codon at the end of Gag (9,101 Da). Experiments are in progress to characterize additional cleavage(s) in NC.
Department of Molecular Biology, Scripps Research Institute, La Jolla 92037.
J H
JH
Elder
M
M
Schnölzer
C S
CS
Hasselkus-Light
M
M
Henson
D A
DA
Lerner
T R
TR
Phillips
P C
PC
Wagaman
S B
SB
Kent
D000595:Amino Acid Sequence; D015153:Blotting, Western; D010450:Endopeptidases / Q000378:metabolism; D015683:Gene Products, gag / Q000378:metabolism*; D015684:Gene Products, pol / Q000378:metabolism*; D016089:Immunodeficiency Virus, Feline / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry
D015683:Gene Products, gag; D015684:Gene Products, pol; D009227:Myristic Acids; D011506:Proteins; D019814:Myristic Acid; D010450:Endopeptidases