Several potential mechanisms by which restriction factors inhibit infection at a very early postentry phase could be envisaged, including ubiquitination and proteasome-mediated degradation of capsid components. Notably, a RING domain at the amino terminus of TRIM5 proteins has been shown to exhibit self-ubiquitination activity (30
). Previous studies demonstrated a requirement for the RING domain for full TRIM5α activity (10
) but could not distinguish whether it is required for capsid recognition or for secondary activities associated with restriction. Because TRIM-CypA carries an autonomous CA-binding domain (CypA), that should not, in principle, require the RING domain for capsid recognition, we tested the HIV-1-restricting activity of full-length and δRING versions of C-terminally HA-tagged TRIM-CypA. Each protein was stably expressed at equivalent levels in MDTF cells (Fig. ) and was similarly localized in a diffuse or fine punctate pattern in the cytoplasm (Fig. ). As expected, TRIM-CypA inhibited vesicular stomatitis virus G (VSV-G)-pseudotyped HIV-1 vector infection by about 40-fold but did not inhibit infection by HIV-1(SIV CA) (Fig. ). The δRING TRIM-CypA protein also specifically inhibited HIV-1 infection, albeit modestly (approximately fourfold). Thus, TRIM5 proteins lacking the RING domain can be weak restriction factors, but as is the case in TRIM5α restriction of HIV-1 or MLV, the RING domain is required for full activity.
FIG. 3. Requirement for the RING domain but not ubiquitin-activating (E1) enzyme or proteasome activity in TRIM-CypA restriction. (A) Expression of TRIM-CypA and δRING TRIM-CypA (lacking residues 2 to 60). Lysates from G418-resistant pools of Mus dunni (more ...)
Therefore, to address whether ubiquitination and proteasome activities are required for TRIM-CypA restriction, we took two approaches. The first employed a mutant of Chinese hamster E36 cells, termed Ts20, which carries a temperature-sensitive allele of the sole ubiquitin-activating (E1) enzyme in mammals, which is fully active at 30°C but completely inactivated within 1 h at 40°C (12
). While incubation of Ts20 cells above 40°C has been shown to inactivate E1, block de novo ubiquitination activity, and inhibit ubiquitin-dependent processes (12
), we adopted a more stringent Western blot assay to monitor E1 protein depletion. Incubation of Ts20 cells but not control wild-type E36 cells at 42°C resulted in nearly complete removal of E1 protein within 3 h (Fig. ). Conversely, engineered expression of TRIM-CypA or TRIM5α was only marginally affected, if at all (Fig. ). Incubation of Ts20 or E36 cells at 42°C for 3 h followed by challenge with a VSV-G-pseudotyped HIV-1 vector for 2 h resulted in a severalfold increase in infection, but this was independent of the presence or absence of E1 or TRIM-CypA (Fig. ) and also occurred to some extent with unrestricted HIV-1(SIV CA) (Fig. ). Indeed, TRIM-CypA conferred strong (>40-fold) and specific resistance to HIV-1 at either 30°C or 42°C (Fig. ). A similar experiment carried out using E36 and Ts20 cells expressing human TRIM5α showed that N-MLV-specific restriction by human TRIM5α is also efficient in the absence of E1 (Fig. ).
A second approach was to treat target cells with a proteasome inhibitor, lactacystin. As was previously reported (5
), proteasome inhibition caused modest enhancement of HIV-1 infection under nonrestricting conditions (Fig. ). However, treatment of OMK target cells with clastolactacystin-β-lactone for 1 h prior to, 2 h during, and 1 h after challenge with VSV-G-pseudotyped HIV-1 (4 h total treatment) failed to influence restriction in OMK cells endogenously expressing TRIM-CypA (Fig. ) or MDTF cells engineered to express TRIM-CypA-HA (data not shown). To verify that proteasome activity was inhibited by clastolactacystin-β-lactone treatment of OMK cells, they were transfected with plasmids expressing HA-tagged ubiquitin (29
) or HIV-1 integrase, an unstable proteasome substrate (14
). Subsequent clastolactacystin-β-lactone treatment, for 1 h or 4 h, resulted in the marked accumulation of high-molecular-weight HA-ubiquitin conjugates that would normally be removed as a result of proteasome activity (Fig. ). Similarly, expression of HIV-1 integrase (which bears an N-terminal Phe residue and, because of the N-end rule, is rapidly degraded by proteasomes [14
]) was markedly stabilized by 4 h of clastolactacystin-β-lactone treatment of OMK cells (Fig. ). Conversely, expression of a relatively stable protein (GFP) was unaffected (Fig. ). Thus, we conclude that while full TRIM-CypA restriction activity requires the RING domain, neither an E1 enzyme nor active proteasomes appear essential. However, we cannot definitively exclude the possibility that long-lived ubiquitin-E2 conjugates and ubiquitin-dependent but proteasome-independent mechanisms might have a role in restriction.