Proteasome inhibition interferes with Gag polyprotein processing, release, and maturation of HIV-1 and HIV-2 -- Data Supplement - Supplemental Text -- Proceedings of the National Academy of Sciences

Supplementary material for Schubert et al. (2000) Proc. Natl. Acad. Sci. USA 97 (24), 13057–13062.

Materials and Methods
Infectivity Assay and Virus Step Gradient.
A3.01 cells acutely infected with HIV-1_NL4-3 were treated with 40 m M N-carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (zLLL) for various times, as indicated. Cell-free supernatants were harvested and filtered, and released virions were quantitated by ELISA. For titration, cultures of C8166 cells were infected by using serial 10-fold dilutions of virus. Infectivity was scored as tissue culture 50% infectious dose (TCID₅₀) by counting syncytia formation after cultivation for 10 d. The infectivity of each sample was normalized for the CA antigen content of the virus inoculum. For sucrose density gradient centrifugation, A3.01 cells acutely infected with HIV-1_NL4-3 were incubated in fresh RPMI medium in the absence or presence of 10 m M zLLL and 10 m M lactacystin (LC). Cell-free supernatants were collected after 24 h of treatment and filtered, and virions were concentrated by ultracentrifugation (1.5 h, 35,000 rpm, 4°C, in a Beckman SW55 rotor) and subjected to 10–60% discontinuous sucrose density gradient centrifugation (2 h, 35,000 rpm, 4°C, in a Beckman SW55 rotor). Fractions were collected beginning at the top of the gradient. Electron Microscopy.
MT-4 cells acutely infected with HIV-1_NL4-3 were cultivated for 2.5 h in fresh medium with or without 50 m M zLLL. Subsequently, cells were sedimented and drawn into cellulose tubes by capillary action. Tubes were incubated for 2.5 h with or without 50 m M zLLL, washed, fixed for 1 h with 2.5% glutaraldehyde in PBS, and washed, and cells were postfixed for 30 min with 1% OsO₄ in PBS followed by washing in water and 30-min staining in 1% uranyl acetate. Ultrathin sections were counterstained with 2% uranyl acetate and lead citrate. Micrographs were taken on a Philips CM120 transmission electron microscope at 80 kV.

Discussion

The most direct explanation for the second model would be the inhibition of PR activity by proteasome inhibitors. Although PR and proteasomes use unrelated active-site mechanisms, recent reports indicate that the HIV-1 PR inhibitor Ritonavir affects proteasomes in vivo (1) by inhibiting the chymotrypsin-like activity (2). However, we excluded the potential crossinhibition of PR by proteasome inhibitors by demonstrating that zLLL and LC have no measurable inhibitory effect on HIV-1 PR activity in vitro. We have not completely eliminated the possibility that proteasome inhibitors interfere with release of active PR from the Gag-Pol polyprotein or dimerization of the released PR necessary for full activation of PR. The mechanism of PR liberation from the Gag-Pol precursor is currently not known, and it is unclear whether cleavage occurs autocatalytically or, alternatively, whether cellular factors are involved that might be regulated by the Ub/proteasome pathway. Direct effects of proteasome inhibitors on PR activation appear highly unlikely, however, given that inhibitors affect Gag processing shortly after addition to acutely infected T cells cultures that possess relatively large amounts of active PR.

Monoubiquitination is insufficient to serve as a proteasomal targeting signal (3) and therefore must have other functional consequences if it has a major influence on virus budding and maturation. Of potential relevance to HIV are findings regarding epidermal growth factor (EGF) receptor-associated protein 15 (Eps15), which is monoubiquitinated in response to EGF engagement of its receptor (4). Eps15 contains in its COOH terminus a tryptophan-rich "WW" domain that is believed to interact with membrane-associated E3 Ub ligases. Potential WW domain interaction sites have been located within late-budding domains of HIV-1 and other retroviruses. In the case of RSV, interaction of the late-budding domain with the WW motif of the Yap protein has been reported (5). Interestingly, Strack et al. (see accompanying paper, ref. 6) found that different L-domains are able to attract Ub ligase activity to assembling HIV Gag molecules and that a defined cellular peptide ligand for the Ub-ligase Nedd4 can substitute for the viral L-domain function in virus budding.

Alternatively, it is also conceivable that the detrimental effect of proteasome inhibitors on Gag processing and virus release may result from changes in chaperone expression and Gag folding. Proteasome inhibitors dramatically enhance the expression of molecular chaperones (7–9), possibly stemming from a shortage of functional chaperones because of their prolonged interaction with substrates that would normally be degraded by proteasomes. It is uncertain whether the net effect of proteasome inhibition is to decrease or increase the concentration of chaperones able to interact with newly synthesized proteins. Decreases in the availability of functional chaperones could prevent the proper folding of Gag, the liberation of PR, or the proper packaging of Gag into virions. Increases in chaperone expression could interfere with virion maturation by the inappropriate incorporation of certain chaperones into virions or by retaining Gag proteins in nonfunctional complexes. However, using time-course studies, we found that proteasome inhibitors severely decreased HIV formation even when added briefly before pulse labeling at an incubation time where induction of chaperones could not be detected (U.S., unpublished observation).

Another possibility is that inhibition of Gag processing and virus release results from the accumulation of defective forms of newly synthesized Gag that are normally disposed of by proteasomes and that we termed Gag-DRiPs (10). Defective ribosomal products (DRiPs), in general, are short-lived polypeptides that never attain their native structure because of errors in translation or immediate posttranslational processes necessary for proper protein folding (11). Gag-DRiPs may be sufficiently folded to assemble with normal Gag and interfere with its processing by PR, or they may act as competitive noncleavable substrate inhibitors of PR. This process would be particularly driven by the inherent propensity of Gag molecules for self-assembly during the process of virus budding. The defects in virus maturation could, therefore, be explained by Gag-DRiPs being coassembled with native Gag into virus particles, resulting in retarded Gag processing and virion release.

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