Structural characterization of HIV reverse transcriptase: a target for the design of specific virus inhibitors.

The reverse transcriptase (RT) of HIV is an important target for chemotherapy as demonstrated by the effective treatment of AIDS patients with zidovudine, a potent inhibitor of RT. Structural studies of HIV RT were therefore undertaken with a view to designing more effective inhibitors. To obtain sufficient quantities of enzyme for these studies the reverse transcriptase gene of HIV was cloned into a high level expression plasmid yielding reverse transcriptase at a level of 10% of the total Escherichia coli proteins. Monoclonal antibodies to RT were raised in mice and have been used to purify the enzyme by immunoaffinity chromatography. Crystallization of the enzyme has been achieved and studies are underway to determine its three-dimensional structure. In addition, carboxy-terminal truncated mutants were prepared by inserting stop codons into the gene at appropriate sites. The proteins expressed were analysed for RT and RNase H activity and used for mapping RT epitopes. This, together with previous data on site-directed mutagenesis of conserved regions of HIV RT has helped to map some of the structural and functional regions of the enzyme.

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

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)

Structural studies of the acquired immunodeficiency syndrome virus reverse transcriptase.

The clinical success of zidovudine has established the human immunodeficiency virus (HIV) reverse transcriptase (RT) as a valid target for the design of drugs to treat acquired immunodeficiency syndrome. In order to facilitate structural studies of this enzyme, expression systems in Escherichia coli, which allow the production of large amounts of RT, have been established. Using this recombinant material the RT has been purified and crystallized. Crystallographic studies currently underway are aimed at elucidating the three-dimensional structure of HIV RT. The availability of a bacterial expression system has enabled structural/functional studies of the RT by site-directed mutagenesis. These studies have identified amino acid residues that are essential for activity of the enzyme and might be involved in substrate binding. It is hoped that structural information of this nature will allow the rational design of HIV RT inhibitors.

Virus-specific DNA sequences present in cells which carry the herpes simplex virus thymidine kinase gene.

Two independently derived cell lines which carry the herpes simplex type 2 thymidine kinase gene have been examined for the presence of HSV-2-specific DNA sequences. Both cell lines contained 1 to 3 copies per cell of a sequence lying within map co-ordinates 0.2 to 0.4 of the HSV-2 genome. Revertant cells, which contained no detectable thymidine kinase, did not contain this DNA sequence. The failure of EcoR1-restricted HSV-2 DNA to act as a donor of the thymidine kinase gene in transformation experiments suggests that the gene lies close to the EcoR1 restriction site within this sequence at a map position of approx. 0.3. The HSV-2 kinase gene is therefore approximately co-linear with the HSV-1 gene.

Isolation and analysis of the nucleic acids and polysaccharides from Clostridium welchii.

A method previously described for the use of bentonite in the isolation of the nucleic acids from two gram-positive organisms was applied to the isolation of the nucleic acids from two strains of Clostridium welchii. The nucleic acids were separated from polysaccharides by the fractional precipitation of their cetyltrimethyl-ammonium salts from sodium chloride solution, and the base composition of the nucleic acids was determined. One strain of C. welchii investigated (NCTC 10578) was shown to produce considerable quantities of an acidic and also a weakly acidic or neutral polysaccharide; the other strain (ATCC 10543) gave very small quantities of the latter but none of the former polysaccharide. The monosaccharide composition of these polysaccharides was determined and the acidic polysaccharide was shown to resemble dermatan sulfate.