Equine infectious anemia virus and the ubiquitin-proteasome system

Abstract

Some retroviruses contain monoubiquitinated Gag and do not bud efficiently from cells treated with proteasome inhibitors, suggesting an interaction between the ubiquitin-proteasome system and retrovirus assembly. We examined equine infectious anemia virus (EIAV) particles and found that approximately 2% of the p9(Gag) proteins are monoubiquitinated, demonstrating that this Gag protein interacts with an ubiquitinating activity. Different types of proteasome inhibitors were used to determine if proteasome inactivation affects EIAV release from chronically infected cells. Pulse-chase immunoprecipitation and time course immunoblot analyses showed that proteasome inactivation slightly decreased virus release (at most a twofold effect), while it did not affect Gag processing. These results contrast with those obtained with other viruses which are sensitive to these inhibitors. This suggests that, although its Gag is monoubiquitinated, the requirements for EIAV release are somewhat different from those for retroviruses that are sensitive to proteasome inhibitors.

FIG. 1.

Analysis of EIAV virions. (A) Immunoblots of EIAV virions digested in the absence (−) or presence (+) of subtilisin. The antibody or antiserum used is indicated above each blot. Molecular mass markers are indicated at the left, and bands are identified at the right. (B) High-pressure liquid chromatography chromatogram of the region containing Ub and Ub-p9^Gag proteins. Fractions are identified under the A₂₀₆ tracing, with the identities of the sequenced proteins indicated next to their respective peaks.

FIG. 2.

Effect of proteasome inhibitors on EIAV release and Gag processing. (A) (Left) Fluorograms of SDS-PAGE gels of immunoprecipitates from cell or virus lysates produced from EIAV-infected cells cultured in the absence or presence of zLLL and lactacystin (LC) (10 μM each). The treatments used and sample identities are indicated. Sample time points: 0, 1, 2, 4, and 8 h. (Right) Graph of virus released in the presence or absence of zLLL and lactacystin. (B) (Left) Fluorograms of SDS-PAGE gels of immunoprecipitates from cell and virus lysates from EIAV-infected cells cultured in the absence or presence of PS-341 (5 μM), epoxomicin (10 μM), lactacystin (20 μM), zLL (20 μM), and zLLL (20 μM). Labeling is as in panel A. (Right) Graph of virus released in the presence or absence of PS-341.

FIG. 3.

Time course analysis of virus release and cellular Ub levels. (A) Immunoblots of virion samples produced by EIAV-infected cells that were untreated or treated with 20 μM lactacystin, 20 μM zLLL, 1 μM PS-341, or 10 μM epoxomicin. (B) Ub immunoblots of the cell lysates that correspond to the virion samples. The antiserum and antibody used are indicated above each blot, and the samples are labeled above the respective lanes.