Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55Gag -- Data Supplement - Supplemental Data -- Proceedings of the National Academy of Sciences

Supplementary material for VerPlank et al. (June 26, 2001) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.131059198

Supplemental Materials and Methods
Plasmid construction.
Oligonucleotides and procedures used for PCR and mutagenesis to construct Pr55^Gag, p1-p6, and Tsg101 GAL4-hybrids for expression in yeast; human Tsg101 for in vitro expression; and Pr55^GagDp6 for expression in mammalian cells are described in Table 1 and this supplementary text. Pr55^Gag and p1-p6 hybrids for expression in yeast.
HIV-1 sequences were amplified by PCR by using plasmid gpVI as template to synthesize inserts for ligation into yeast expression vectors pGBT9 or pMA424 and pGAD424. pgpVI encodes Pr55^Gag and part of Pol and an inactivating mutation in the catalytic site of PR (D₂₅A) within Pol. The 5¢ end primer, oligo no. 1 in Table 1 and the 3¢ end primer (no. 2) were used to synthesize a PCR fragment of full-length gag. The primers annealed to nt 333 of gag and the end of the PR coding region, respectively. BamHI and BglII sites were engineered in the insert for ligation into pGBT9 and pGAD424. For the MA-CA insert, we used the same 5¢ end primer (no. 1) and a 3¢ end primer (no. 3) that annealed at position 1421 in the gag gene at the C terminus of CA. These oligonucleotides contained the restriction sites BglII and BamHI, respectively. For the CA-p2-NC insert, we used a 5¢ primer (no. 4) that annealed to the gag gene at position 730 at the N terminus of CA and a 3¢ end primer (no. 5) complementary to position 1673 of the gag gene at the C terminus of p1. The PCR product was cut with BglII and ligated into the vector at that site. For the p1-p6 insert, we used a 5¢ end primer (no. 6) that annealed to the gag gene at position 1636 and a 3¢ end primer (no. 7) that annealed at position 2116 in the pol gene. The insert obtained spanned the p1-p6 region of Gag in the gag frame and the first 99 amino acids of the pol frame. It lacked the frameshift site located in the p1 region upstream of nucleotide 1636 so that only expression of the gag frame was obtained. Both oligonucleotides contained BglII sites. The Pr55^GagDp1-p6 insert was created by restriction digestion at the unique BglII site. gag sequences were ultimately placed in the pGBT9 vector background by transferring inserts from pMA424 to pGBT9 using the BamHI and SalI sites. Deletions and site-directed mutations in pGBT9-p1-p6 were created by mutagenesis by using the Gene Editor (Promega). Mutagenic oligonucleotides (nos. 8-15) were used according to the manufacturer's protocol. Mismatch regions, introduced for point mutations leading to amino acid changes or deletion mutations are underlined or indicated by /, respectively. Double mutations were created by using a second selection oligonucleotide (no. 13) that obliterates a SnaBI restriction site within the vector. Resistance to SnaBI digestion selected for the mutant plasmids. The double mutant K_487,493R was synthesized by using the second-selection oligonucleotide and the K₄₉₃R mutagenic oligo (no. 11) with the mutant K₄₈₇R as the template. The double mutant DPTAPP_1,2was synthesized by using the second-selection oligonucleotide and the DPTAPP₂mutagenic oligo (no. 9), with DPTAPP₁as the template. Point mutations in the second PTAPP motif were synthesized from the mutant DPTAPP₁ by using the second selection oligo and oligos nos. 14 and 15, as indicated in Table 1. Tsg101 hybrids for expression in yeast.
Tsg101, as isolated from a B cell library in a pACT vector, was used as template for PCR of inserts for the yeast two-hybrid system. The inserts were engineered with EcoRI and SalI sites for ligation into pGAD424. For the wild-type tsg101 insert, the forward (5¢ end) primer (no. 16) was complementary to the beginning of the coding region of Tsg101. The reverse (3¢ end) primer was no. 17. Truncations at the 3¢ end were introduced using the tsg101 forward primer (no. 16) and reverse primers nos. 18-20, all with translational stop codons and SalI sites introduced at the downstream terminus of the gene. Truncations at the 5¢ end were introduced by using the tsg101 reverse primer (no. 17) and forward primers nos. 21-23, each with an EcoRI site introduced before the 5¢ terminus of the sequence. Site-directed mutations in tsg101 were created using the Gene Editor and oligos nos. 24-28. In vitro
expression. Human tsg101 was subcloned into the BamHI site of the pET3a vector under the control of the T7 promoter (Novagen). The insert was produced by PCR using pACT-tsg101 as template with 5¢ primer no. 29, which anneals at the position corresponding to amino acid 10 in the protein, and 3¢ primer no. 30, which anneals to the C-terminus of the coding region. Pr55^GagDp6
for expression in mammalian cells. pgp-RRE-r was used as a template to synthesize three point mutations in the first codon of p6 by PCR, thereby converting it into a stop (ochre) codon. The 5¢ primer (no. 31) anneals to nucleotides 1637–1687 of BH10 gag and includes a unique BglII site. The 3¢ primer (no. 32) anneals to nucleotides 2655–2682 in the pol gene downstream of a unique EcoRV site. The pgp-RRE-r plasmid and the fragment produced from PCR were cut with BglII and EcoRV, and the desired fragments were ligated.

All constructs were confirmed by sequencing using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit (Perkin–Elmer, Foster City, CA) according to the manufacturer’s instructions.

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