Simian immunodeficiency virus reverse transcriptase Purification and partial characterization

Native reverse transcriptase from simian immunodeficiency virus was purified from virus with good recovery to near homogeneity. The optimum reaction conditions of the enzyme were determined with respect to divalent cations, p H and ionic strength. The enzyme was shown to possess both RNA-dependent and DNA-dependent DNA synthesis activity. In addition, we could demonstrate an associated RNase H activity. Employing novel assay conditions, activated DNA as a heteropolymeric substrate was used more efficiently than the homopolymeric substrate poly(rA). oligo(dT) which in turn was used twofold more effectively as the template primer than poly(dC). oligo(dG). Other homopolymeric substrates, including poly(rC). oligo(dG), were also tested but were found to be poorly used by the reverse transcriptase. The Miachaelis-Menten constants were determined for each of the four nucleotides needed to elongate a natural template primer. Simultaneously, using dideoxyadenosine triphosphate as nucleotide analogue, we could show that this compound acts as a competitive inhibitor with respect to dATP, whereas it acts as a non-competitive inhibitor with respect to the other nucleotides. Gel electrophoretic analysis showed the enzyme to consist of two polypeptides with apparent molecular masses of 64 and 48 kDa. Using activity gel electrophoresis, we were able to demonstrate that both subunits exhibit DNA synthesis activity. Since the discovery of human immunodeficiency virus (HIV) as the causative agent of acquired immunodeficiency syndrome (AIDS), many efforts have been made to control its replication. These efforts include chemotherapeutic approaches [l], as well as the development of vaccines [2]. For both approaches a suitable animal model is urgently required. However, there is as yet no simple animal model available for rapid testing of experimental vaccines or potential inhibitors of the retroviral life cycle. As one potential animal model [3], we have chosen the immunodeficiency virus of the African green monkey (SIV,,,). This virus is a member of the lentivirus group and has been successfully cultured in human permanent cell lines [4-61. Simian immunodeficiency viruses have also been isolated from rhesus [7], pig tailed macaques [S], man-drills [9], and from African sooty mangabeys [lo]. SIV,,, is serologically distinct from HIV-1 and HIV-2 but its genome hybridizes with DNA probes prepared from those viruses [6]. The genome of SIV,,, has been cloned and fully sequenced [ll]. All these viruses resemble each other morphologically , share CD4 + lymphotropism and cytopathogenicity and possess homologous structural, regulatory, and rep-licative proteins including reverse transcriptase [4]. The latter enzyme is an ideal target for inhibition of retrovirus replication because not only is it vital, but it is also unique for retroviruses as there is no corresponding enzyme in eucaryotic host cells. Before drugs are given to patients it is desirable not only to understand their mode of action, but also to ensure that there are no serious side effects in animal models. The suit-ability of SIV,,, as animal model for the chemotherapy of immunodeficiency viruses, especially of HIV, depends among others on the similarity of the biochemical functions of SIV,,, and HIV reverse transcriptases. In this communication we describe the purification and partial characterization of the reverse transcriptase of SIV,,,. Our results may not only provide a basis for a better understanding of the action of reverse transcriptases of human and simian immunodeficiency viruses, but may also enable us to use SIV,,, as animal model for chemotherapeutic approaches, especially as infected monkeys may successively be treated with several experimental drugs and therapeutic success may be monitored by immunological and virological parameters.