Properties of avian sarcoma-leukosis virus pp32-related pol-endonucleases produced in Escherichia coli

Abstract

The gag-pol precursor protein of the avian sarcoma-leukosis virus is processed into three known pol-encoded mature polypeptides; the 95-and 63-kilodalton (kDa) and a subunits, respectively, of reverse transcriptase and the 32-kDa pp32 protein. The pp32 protein possesses DNA endonuclease activity and is produced from the precursor by two proteolytic cleavage events, one of which removes 4.1 kDa of protein from the C terminus. A 36-kDa protein (p36P°') which retains this C-terminal segment is detectable in small quantities in virions. We have constructed Escherichia coli plasmid clones that express the C-terminal domains of pol corresponding to pp32 and p36. These proteins have been purified by column chromatographic methods to near homogeneity. No significant differences could be detected in the enzymatic properties of the bacterially produced p32P"' and p36&"" proteins. Both possess DNA endonuclease activity and, like the pp32 protein isolated from virions, can cleave near the junction of two tandem avian sarcoma-leukosis virus long terminal repeats in double-stranded supercoiled DNA substrates. In the presence of Mg2+, both p32P"' and viral pp32 cleave either strand of DNA 2 nucleotides 5' to the junction.

Moloney murine leukemia virus integration protein produced in yeast binds specifically to viral att sites

“…Unintegrated linear MoMuLV DNA (top) terminates with 18 bp of interrupted inverted repeat sequences contained in the LTRs (31). The 36-bp double-stranded oligonucleotide corresponding to the wild-type sequence [wild type (36)] contains both copies of this sequence. Bars above the sequence indicate the 16 of 18 bp complementarity.…”

“…The protein was preincubated with unlabeled competitors, present at 50-fold the concentration of the subsequently added 32P-labeled probe, and electrophoresis was started after further incubation. The wt-U3(18) probe for lanes 7 to 10 was created by 5' end labeling only the top strand of the 36-bp att site oligonucleotide [wild type (36)] and cleaving with PstI at the site indicated in Fig. 2, thus retaining the label at only the U3 half (Fig.…”

“…MLU-IlId t ype (36 ) n1LU t -U 3 (18 ) Oligonucleotides bearing specific alterations from the wild-type substrate (as shown in Fig. 2) were used as the probes and are indicated below each panel.…”

Monoclonal antibodies against Rous sarcoma virus integrase protein exert differential effects on integrase function in vitro

“…Proteins. RSV IN was expressed in Escherichia coli, purified as previously described (36), and used to immunize mice. Wild-type IN and mutated versions of the protein used in further experiments were purified by a modified procedure (27).…”

“…A number of integrases from different retroviral sources have been purified and partially characterized. Some of these proteins have been expressed in large amounts in bacterial cells, making the enzyme accessible for detailed biochemical and biophysical analysis (19,(35)(36)(37). Various approaches have been applied to obtain information about structure-function relationships of retroviral INs.…”