Ubiquitin is covalently attached to the p6Gag proteins of human immunodeficiency virus type 1 and simian immunodeficiency virus and to the p12Gag protein of Moloney murine leukemia virus

Abstract

Host proteins are incorporated into retroviral virions during assembly and budding. We have examined three retroviruses, human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and Moloney murine leukemia virus (Mo-MuLV), for the presence of ubiquitin inside each of these virions. After a protease treatment to remove exterior viral as well as contaminating cellular proteins, the proteins remaining inside the virion were analyzed. The results presented here show that all three virions incorporate ubiquitin molecules at approximately 10% of the level of Gag found in virions. In addition to free ubiquitin, covalent ubiquitin-Gag complexes were detected, isolated, and characterized from all three viruses. Our immunoblot and protein sequencing results on treated virions showed that approximately 2% of either HIV-1 or SIV p6Gag was covalently attached to a single ubiquitin molecule inside the respective virions and that approximately 2 to 5% of the p12Gag in Mo-MuLV virions was monoubiquitinated. These results show that ubiquitination of Gag is conserved among these retroviruses and occurs in the p6Gag portion of the Gag polyprotein, a region that is likely to be involved in assembly and budding.

FIG. 1

Immunoblot analysis of digested HIV-1. (A) Immunoblot reacted with the antiubiquitin monoclonal antibody; (B) gp120^SU (top) and p24^CA (bottom) immunoblots of the material analyzed in panel A; (C) p6^Gag immunoblot analysis of the blot presented in panel A after being stripped of the antiubiquitin reagents. Lane 1, 25 ng of purified ubiquitin; lanes 2 and 3, 20 μg of mock-digested and digested HIV-1_MN produced from Clone 4-13, respectively; lanes 4 and 5, 20 μg of mock-digested and digested HIV-1_NL4-3 produced from H9 cells, respectively. Molecular masses of bands are indicated at the left.

FIG. 2

HPLC analysis of ubiquitin in a digested HIV-1 preparation. The region of the HIV-1 HPLC analysis that contains ubiquitin is presented with the results of immunoblot and C-SDS-PAGE analysis of selected fractions arranged under the corresponding regions of the chromatogram. The complete chromatogram is placed in an inset with a box around the region presented here. The antiserum or stain used for each set of results is labeled on the left of the corresponding blot or gel; sizes of the bands as determined by relative mobility are presented on the right. The antiserum to ubiquitin (Anti-Ub) was used for these ubiquitin immunoblots.

FIG. 3

Protein sequence results for ubiquitin-Gag conjugates. Sequences are presented in the standard single-letter amino acid code, and X indicates an ambiguous or missing residue. (A) Protein sequence results from the 17-kDa putative ubiquitin-p6^Gag band that was HPLC purified from HIV-1; (B) protein sequence results from the 16-kDa putative ubiquitin-p6^Gag band that was HPLC purified from SIV_Mne; (C) protein sequence results from the 20-kDa putative ubiquitin-p12^Gag band that was HPLC purified from Mo-MuLV. The missing lysine residue in the 20-kDa sequence is indicated by X within the p12^Gag sequence, with the missing lysine indicated above.

FIG. 4

Immunoblot that was reacted with the antiubiquitin monoclonal antibody (A) then stripped and reprobed with anti-SIV p6^Gag (B). The samples were 40 μg of total protein of mock-digested (lane 1) and digested (lane 2) preparations of SIV_Mne. Molecular masses of bands are indicated on the left.

FIG. 5

HPLC purification of a putative ubiquitin-SIV p6^Gag conjugate from SIV. The HPLC profile of the ubiquitin (Ub)-containing region is presented along with antiubiquitin (using the antiubiquitin antiserum) and anti-SIV p6^Gag immunoblot analysis of fraction 125. Molecular masses of bands are indicated on the left.

FIG. 6

Ubiquitin immunoblot of digested and mock-digested Mo-MuLV virions. The Mo-MuLV samples were 20 μg of total protein of mock-digested (lane 1) and digested (lane 2) preparations. The immunoblot was reacted with the antiubiquitin monoclonal antibody. Molecular masses are presented on the left.