Placement of Leucine Zipper Motifs at the Carboxyl Terminus of HIV-1 Protease Significantly Reduces Virion Production

Pan, Yen-Yu; Wang, Shiu-Mei; Huang, Kuo-Jung; Chiang, Chien-Cheng; Wang, Chin-Tien

To place a leucine zipper (LZ) in frame into the HIV-1 PR Cterminus, we engineered a pBRCla-Sal plasmid cassette containing an HIV-1 coding sequence (from ClaI-nt.831 to SalI-nt.5786) and a pcDNA3.1-myc/hisA polylinker inserted at the IN Cterminus. DNC(wtZip) and DNC(Kzip) [53] served as templates for amplifying the respective wt and mutant LZ domains of human CREB [54] using the forward primer 59-CGGGATCCTGGAG-GAGGACGAGAGTGTCGTAG-AAAGAAG-39 and reverse primer 59-CCAAGCGGCCGCGATTTGTGGCAG-TAT-39. Plasmids containing the wt and mutant LZ (provided by E. Barklis [55] ) were used to construct the DNC(wtZip) and DNC(Kzip). The human CREB LZ sequence is 284-RE-CRRKKKEYVKCLENRVAVLENQNKTLIEELKALKDLYC-HKSD-327. The underlined amino acid residues E298, R300, E305, Q307, I312, E314, and L321 were mutated to Lys, and N308 was mutated to His, yielding the mutant LZ [54] . Amplified fragments were digested with BamHI and NotI and ligated into the pBRCla-Sal cassette, yielding constructs PRWz and PRKz, respectively. To add the LZ copy, PCR-amplified wt and mutant LZ fragments were purified, digested with a restriction enzyme, and ligated into the PRWz and PRKz, yielding the constructs PRWWz, PRWKz, PRKKz, and PRKWz.