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.
Elder
J H
JH
Department of Molecular Biology, Scripps Research Institute, La Jolla 92037.
Schnölzer
M
M
Hasselkus-Light
C S
CS
Henson
M
M
Lerner
D A
DA
Phillips
T R
TR
Wagaman
P C
PC
Kent
S B
SB
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