HIV-1 reverse transcriptase: crystallization and analysis of domain structure by limited proteolysis

Abstract

Bacterially expressed recombinant HIV-1 reverse transcriptase is active as both a homodimer of Mr 66,000 subunits and a heterodimer of Mr 66,000 and 51,000 subunits. The heterodimer is formed by cleavage of a C-terminal fragment from one Mr 66,000 polypeptide, which occurs during purification and crystallization of reverse transcriptase. Thus, crystals obtained from purified Mr 66,000 polypeptide preparations consisted of an apparently equimolar mixture of Mr 66,000 and 51,000 polypeptides, which were apparently analogous to the Mr 66,000 and 51,000 polypeptides detected in HIV-infected cells and in virions. Limited proteolysis of the homodimer with alpha-chymotrypsin also resulted in cleavage to a stable Mr 66,000/51,000 mixture, and proteolysis with trypsin resulted in the transient formation of some Mr 51,000 polypeptide. These results are consistent with the reverse transcriptase molecule having a protease-sensitive linker region following a structured domain of Mr 51,000. Further digestion with trypsin resulted in cleavage of the Mr 51,000 polypeptide after residue 223, yielding peptides of apparent Mr 29,000 and 30,000. A minor peptide of Mr 40,000 was also produced by cleavage of the Mr 66,000 polypeptide after residue 223. About half the original Mr 66,000 polypeptides remained resistant to proteolysis and existed in complex with the above peptides in solution. During both chymotrypsin and trypsin digestion there was an increase in the reverse transcriptase activity caused by a doubling of Vmax with little change in Km for dTTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunologic and proteolytic analysis of HIV-1 reverse transcriptase structure

“…There is a region of the 66-kDa protein that is susceptible to the viral protease and to unrelated proteases. We have not seen as much specificity in the cleavage of the 66-kDa HIV-1 RT when we incubated the protein with nonviral proteases at 23" as Lowe et al (1988) observed when they performed digestions at 0". They reported additional cleavages when the incubation temperature was raised from 0 to 21" and the incubation time was increased.…”

“…These results suggest that cleavage occurs either at or very near the same site in the in vitro digestion and in virions. The 66-kDa form can be cleaved to a 5 1 -kDa form by a protease present in E. co/i (Lowe et al, 1988). Although the HIV-l protease made in E. co/i was purified by HPLC, we considered the possibility that a contaminating bacterial protease could have been responsible for cleaving the HIV-1 RT in vitro.…”

“…The sequences in the 66-kDa protein that are thought to be cleaved to yield the 51-kDa protein are not cleaved by the HIV-1 protease if presented as a synthetic peptide (Billich et a/., 1988). Nonviral proteases have been reported to cleave the 66-kDa form of HIV-l RT to yield a protein similar to the 51 -kDa protein (Lowe et al, 1988). Of the proteases we have tested, the viral protease displays the most selectivity in cleaving the 66-kDa form of HIV-l.…”

“…The 51 -kDa recombinant proteins have levels of ,RNA-dependent DNA polymerase activity that are at most only a few percent of the activity possessed by the 66-kDa form . It has been suggested that the 66-and 51-kDa proteins are the two subunits of a dimer, and that this heterodimer is more active in RNA-dependent DNA polymerase assays than is the pure 66-kDa form (Lowe et a/., 1988).…”

Review

“…The HIV-1 PR generates F440 as the carboxy end of the p51 subunit (Mizrahi et al, 1989;Bathurst etal., 1990;Graves etal., 1990). Bacterially expressed HIV-1 RT, on the other hand, has a heterogeneous C-terminus centered around P433 (Lowe et al, 1988). The crystal structure of the HIV-1 PR with and without substrate analogs (Miller et al, 1989) shows that it is highly unlikely that the protease could reach the cleavage site unless the RNase H domain of HIV-1 RT p66 is partially or totally unfolded upon dimerization (Jacobo-Molina and Arnold, 1991).…”