<p>SUPPORTING INFORMATION</p><p>Figure S1. Expression and HIV-1-restricting activity of TRIM5AGM variants. (A)</p><p>The steady-state expression levels of TRIM5AGM(Pyg), TRIM5AGM(Tan) and</p><p> the TRIM5AGM(Tan) chimeras with Loop 1 of the RING domain replaced by</p><p> those of human (hu), rhesus monkey (rh) or squirrel monkey (sq) TRIM5</p><p> in Cf2Th cells are shown. The same cell lysates were Western blotted</p><p> with an antibody directed against -actin as a control. (B) HeLa cells</p><p> expressing the indicated TRIM5AGM variants or transduced with the empty</p><p>LPCX vectors were challenged with HIV-1-GFP. The results of these</p><p> infections are shown in Figures 1C and 1D, and were re-plotted here on a</p><p> semi-log scale to allow comparison of the relative potency of HIV-1</p><p> restriction mediated by the TRIM5AGM variants. (C) Cf2Th cells</p><p> expressing TRIM5AGM(Pyg), TRIM5AGM(Tan) or the indicated TRIM5AGM(Tan)</p><p> variants were incubated with HIV-1-GFP viruses. Forty-eight hours later,</p><p>GFP expression in the cultures was analyzed by FACS. The results</p><p> shown are typical of those obtained in two independent experiments.</p><p>Figure S2. In vitro auto-ubiquitylation of TRIM5AGM(Tan). (A) The time course at</p><p>37°C of the in vitro auto-ubiquitylation of the TRIM5AGM(Tan) protein</p><p> immunoprecipitated by the anti-HA epitope antibody is shown. The</p><p> reactions were stopped at the indicated times and Western blotted with</p><p> either an anti-HA antibody to detect TRIM5AGM(Tan) (left panel) or an anti- myc antibody to detect myc-ubiquitin (right panel). (B) The effect of pre-</p><p> washing with a NaCl solution on the in vitro auto-ubiquitylation of</p><p>TRIM5AGM(Tan) is shown. The TRIM5AGM(Tan) protein precipitated by the</p><p> anti-HA antibody and magnetic protein G beads was washed with lysis</p><p> buffer containing the indicated concentration of NaCl and then used for the</p><p> in vitro auto-ubiquitylation assay. The reactions were Western blotted with</p><p> the anti-HA antibody. (C) The auto-ubiquitylation of TRIM5rh was</p><p> examined in the presence of different E2 ubiquitin-conjugating enzymes</p><p>(UbcH 2-13, Boston Biochem), in the absence or presence of ATP. The</p><p>UbcH13 used in this experiment was purchased as a complex with its</p><p> cofactor Uev1a. The reactions were Western blotted with an anti-HA</p><p> antibody to detect TRIM5rh (upper panel) or an anti-myc antibody to</p><p> detect myc-ubiquitin (lower panel). (D) The ubiquitin chain specificity of</p><p>TRIM5rh auto-ubiquitylation was investigated by using a panel of ubiquitin</p><p> mutants containing only one lysine residue. The position of the single</p><p> lysine residue is indicated by the number above the lane. (E) Methylated</p><p> ubiquitin was used as a single source of ubiquitin in the TRIM5rh auto-</p><p> ubiquitylation reaction, thus preventing ubiquitin-ubiquitin chain formation.</p><p>The indicated multi-monoubiquitylated forms of TRIM5 were detected by</p><p>Western blotting with an anti-HA antibody.</p><p>Figure S3. Formation of capsid-like structures and cylindrical tubes by the</p><p>SIVmac CA-NC protein. The SIVmac CA-NC protein was mixed with (TG)50 DNA oligonucleotide in a high-salt buffer at 37°C for over 24 hours to allow</p><p> the assembly reaction to proceed. The resulting complexes were</p><p> negatively stained and examined by electron microscopy. </p><p>Figure S4. Measurement of the turnover rates of TRIM5rh-TRIM5AGM chimeras.</p><p>(A) Cf2Th cells expressing the indicated TRIM5rh-TRIM5AGM chimeric</p><p> proteins were treated with cycloheximide for the indicated times and</p><p> harvested. The same amounts of lysate were Western blotted with an</p><p> anti-HA antibody. (B) The band intensities were quantitated with a</p><p> densitometer and plotted, relative to the starting level, which was set at</p><p>100%.</p><p>Figure S5. Effect of cyclosporine A treatment on reverse transcription in </p><p>SIVmac(HIV 4/5)-challenged cells expressing TRIM5rh-TRIM5AGM</p><p> chimeras. Cf2Th cells expressing the indicated TRIM5rh-TRIM5AGM</p><p> chimeras or transduced with the empty LPCX vector were preincubated</p><p> for 1 hour with either DMSO or 20 M cyclosporine A (CsA) prior to</p><p> infection with SIVmac-GFP or SIVmac(HIV 4/5)-GFP. The cyclosporine A</p><p> was also present in the medium during the incubation with the viruses.</p><p>After 3 hours at 37°C, the cells were lysed and the single-strand strong-</p><p> stop DNA was PCR-amplified from 100 ng of the total DNA.</p>