Supporting Information s21

SUPPORTING INFORMATION

Figure S1. Expression and HIV-1-restricting activity of TRIM5AGM variants. (A)

The steady-state expression levels of TRIM5AGM(Pyg), TRIM5AGM(Tan) and

the TRIM5AGM(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 TRIM5AGM 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 TRIM5AGM variants. (C) Cf2Th cells

expressing TRIM5AGM(Pyg), TRIM5AGM(Tan) or the indicated TRIM5AGM(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 TRIM5AGM(Tan). (A) The time course at

37°C of the in vitro auto-ubiquitylation of the TRIM5AGM(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 TRIM5AGM(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

TRIM5AGM(Tan) is shown. The TRIM5AGM(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 TRIM5rh 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 TRIM5rh (upper panel) or an anti-myc antibody to

detect myc-ubiquitin (lower panel). (D) The ubiquitin chain specificity of

TRIM5rh 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 TRIM5rh 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 TRIM5rh-TRIM5AGM chimeras.

(A) Cf2Th cells expressing the indicated TRIM5rh-TRIM5AGM 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

Figure S5. Effect of cyclosporine A treatment on reverse transcription in

SIVmac(HIV 4/5)-challenged cells expressing TRIM5rh-TRIM5AGM

chimeras. Cf2Th cells expressing the indicated TRIM5rh-TRIM5AGM

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.