(A) TK-ts13 cell clones expressing MT4-TRIM5α (□), CV1-TRIM5α-type1 (•), CV1-TRIM5α-type2 (▪), HSC-F-TRIM5α (▴), or empty vector (○) were exposed to the indicated GFP-expressing HIV-1 vector. GFP-positive cells were counted with a flow cytometer. Data typical of at least three independent clones for each TRIM5α are shown. (B) Lysates of MT4 cells infected with recombinant SeV expressing CV1-TRIM5α-tag, HSC-F-TRIM5α-tag, or the parental Z strain were immunoprecipitated by anti-HA antibody. Resultant immunoprecipitates were visualized by Western blotting with an antibody to HA. A representative result of four independent experiments is shown. (C) MT4 cells were mock infected (○), or infected with SeV expressing CV1-TRIM5α-tag (▪), HSC-F-TRIM5α-tag (▵), or the parental Z strain (•). At 9 h after infection, cells were inoculated with an HIV-1 strain, NL43, and culture supernatants were periodically assayed for levels of p24. The datum points are means for triplicate samples with the SD.

(A) Schematic representation of chimeric TRIM5α and summary of the results. Filled and open bars denote CV1 and HSC-F sequences, respectively. D, the CV-1-TRIM5α-specific 20-amino-acid duplication. The +, ±, and − symbols denote full, partial, and nosuppression, respectively. (B) Lysates of C143 cells expressing CV1-TRIM5α-tag (lane 1), HSC-F-TRIM5α-tag (lane 2), HSC-F+60tag (lane 3), CV1-60tag (lane 4), or empty vector (lane 5) were immunoprecipitated by using anti-HA antibody. The resultant immunoprecipitates were visualized by Western blotting with an antibody to HA. A representative result of three independent experiments is shown. (C) C143 cells expressing CV1-TRIM5α-tag (▪), HSC-F-TRIM5α-tag (□), CV1-60tag (▵), HSC-F+60tag (▴), or empty vector (○) were infected with VSV-pseudotyped NL43 or SIVmac239, and culture supernatants were periodically assayed for levels of p24 or p27. The datum points are means for triplicate samples with the SD. (D) MT4 cells infected with SeV expressing CV1-TRIM5α-tag (▪), HSC-F-TRIM5α-tag (□), CV1-60tag (▵), HSC-F+60tag (▴), or empty vector (○) were infected with NL43 or SIVmac239, and culture supernatants were assayed for levels of p24 and p27. The datum points are means for triplicate samples with the SD. (E) Lysates of MT4 cells infected with recombinant SeVs expressing CV1-60tag, HSC-F+60tag, HSC-delete-tag, CV1-delete-tag, HSC-insert-tag, or parental Z strain were immunoprecipitated by using anti-HA antibody. Resultant immunoprecipitates were visualized by Western blotting with an antibody to HA. A representative result of three independent experiments is shown. The relative amounts of TRIM5α protein to that of HSC-F+60tag were calculated, and means and SD values of three independent experiments are shown. (F) MT4 cells infected with SeV expressing CV1-60tag (▵), HSC-F+60tag (▴), HSC-delete-tag (asterisks with blue lines), HSC-insert-tag (•), CV1-delete-tag (diamonds with red lines), or empty vector (○) were infected with NL43 or SIVmac239, and culture supernatants were assayed for levels of p24 and p27. The datum points are means for triplicate samples with the SD.

(A) Alignment of amino acid sequences of African green monkey (AGM, CV1-TRIM5α-type1), cynomolgus monkey (CM, HSC-F-TRIM5α), and human TRIM5α (Hu, MT4-TRIM5α) predicted from the sequences of the cDNAs used in the present study with key domains indicated. (B) Alignment of amino acid sequences of the highly variable region within the SPRY (B30.2) domain of TRIM5α. The rhesus monkey TRIM5α (Rh) sequence published by Stremlau et al. (33) was added. A box indicates the 20-amino-acid duplication within African green monkey TRIM5α. An open arrowhead denotes the recombination point in chimeric TRIM5αs, HSC-delete-tag, HSC-insert-tag, and CV1-delete-tag (see Fig. 4A). (C) Phylogenetic tree of various TRIM5α sequences produced by the UPGMA (unweighted pair-group method with arithmetic averages) method.

(A) MT4 (○), HSC-F (▵), or CV1 (▪) cells were infected with VSV-pseudotyped NL43 or VSV-pseudotyped SIVmac239, and culture supernatants were periodically assayed for levels of p24 or p27. The datum points are means for triplicate samples with the SD. (B) MT4 cells were mock infected (○) or infected with SeV expressing CV1-TRIM5α-tag (▪) or HSC-F-TRIM5α-tag (▵). At 9 h after infection, cells were inoculated with SIVmac239, and culture supernatants were periodically assayed for levels of p27. The datum points are means for triplicate samples with the SD.

C143 cells expressing CV1-TRIM5α-tag (▪), HSC-F-TRIM5α-tag (□), CV1-60tag (▵), HSC-F+60tag (▴), or empty vector (○) were infected with VSV-pseudotyped GH123, and culture supernatants were periodically assayed for levels of p25. The datum points are means for triplicate samples with the SD.