SUPPORTING INFORMATION
Figure S1. Expression and HIV-1-restricting activity of TRIM5AGM variants. (A)
The steady-state expression levels of TRIM5AGM(Pyg), TRIM5AGM(Tan) and
the TRIM5AGM(Tan) chimeras with Loop 1 of the RING domain replaced by
those of human (hu), rhesus monkey (rh) or squirrel monkey (sq) TRIM5
in Cf2Th cells are shown. The same cell lysates were Western blotted
with an antibody directed against -actin as a control. (B) HeLa cells
expressing the indicated TRIM5AGM variants or transduced with the empty
LPCX vectors were challenged with HIV-1-GFP. The results of these
infections are shown in Figures 1C and 1D, and were re-plotted here on a
semi-log scale to allow comparison of the relative potency of HIV-1
restriction mediated by the TRIM5AGM variants. (C) Cf2Th cells
expressing TRIM5AGM(Pyg), TRIM5AGM(Tan) or the indicated TRIM5AGM(Tan)
variants were incubated with HIV-1-GFP viruses. Forty-eight hours later,
GFP expression in the cultures was analyzed by FACS. The results
shown are typical of those obtained in two independent experiments.
Figure S2. In vitro auto-ubiquitylation of TRIM5AGM(Tan). (A) The time course at
37°C of the in vitro auto-ubiquitylation of the TRIM5AGM(Tan) protein
immunoprecipitated by the anti-HA epitope antibody is shown. The
reactions were stopped at the indicated times and Western blotted with
either an anti-HA antibody to detect TRIM5AGM(Tan) (left panel) or an anti- myc antibody to detect myc-ubiquitin (right panel). (B) The effect of pre-
washing with a NaCl solution on the in vitro auto-ubiquitylation of
TRIM5AGM(Tan) is shown. The TRIM5AGM(Tan) protein precipitated by the
anti-HA antibody and magnetic protein G beads was washed with lysis
buffer containing the indicated concentration of NaCl and then used for the
in vitro auto-ubiquitylation assay. The reactions were Western blotted with
the anti-HA antibody. (C) The auto-ubiquitylation of TRIM5rh was
examined in the presence of different E2 ubiquitin-conjugating enzymes
(UbcH 2-13, Boston Biochem), in the absence or presence of ATP. The
UbcH13 used in this experiment was purchased as a complex with its
cofactor Uev1a. The reactions were Western blotted with an anti-HA
antibody to detect TRIM5rh (upper panel) or an anti-myc antibody to
detect myc-ubiquitin (lower panel). (D) The ubiquitin chain specificity of
TRIM5rh auto-ubiquitylation was investigated by using a panel of ubiquitin
mutants containing only one lysine residue. The position of the single
lysine residue is indicated by the number above the lane. (E) Methylated
ubiquitin was used as a single source of ubiquitin in the TRIM5rh auto-
ubiquitylation reaction, thus preventing ubiquitin-ubiquitin chain formation.
The indicated multi-monoubiquitylated forms of TRIM5 were detected by
Western blotting with an anti-HA antibody.
Figure S3. Formation of capsid-like structures and cylindrical tubes by the
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
the assembly reaction to proceed. The resulting complexes were
negatively stained and examined by electron microscopy.
Figure S4. Measurement of the turnover rates of TRIM5rh-TRIM5AGM chimeras.
(A) Cf2Th cells expressing the indicated TRIM5rh-TRIM5AGM chimeric
proteins were treated with cycloheximide for the indicated times and
harvested. The same amounts of lysate were Western blotted with an
anti-HA antibody. (B) The band intensities were quantitated with a
densitometer and plotted, relative to the starting level, which was set at
100%.
Figure S5. Effect of cyclosporine A treatment on reverse transcription in
SIVmac(HIV 4/5)-challenged cells expressing TRIM5rh-TRIM5AGM
chimeras. Cf2Th cells expressing the indicated TRIM5rh-TRIM5AGM
chimeras or transduced with the empty LPCX vector were preincubated
for 1 hour with either DMSO or 20 M cyclosporine A (CsA) prior to
infection with SIVmac-GFP or SIVmac(HIV 4/5)-GFP. The cyclosporine A
was also present in the medium during the incubation with the viruses.
After 3 hours at 37°C, the cells were lysed and the single-strand strong-
stop DNA was PCR-amplified from 100 ng of the total DNA.