Simian immunodeficiency virus reverse transcriptase

Abstract

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.

Fidelity of reverse transcriptase of the simian immunodeficiency virus from African green monkey

“…Furthermore, this enzyme is the main target for antiviral therapies (13). Therefore, attempts have been undertaken to study the enzymological features of reverse transcriptases from simian monkeys and to compare these with the characteristics of the human-derived enzyme (14,15).…”

Analysis of the Polymerization Kinetics of Homodimeric EIAV p51/51 Reverse Transcriptase Implies the Formation of a Polymerase Active Site Identical to Heterodimeric EIAV p66/51 Reverse Transcriptase

“…66 kDa) subunit and a small (ca. 51-58 kDa) subunit that share a common amino terminus (1)(2)(3)(4)(5). The small subunit lacks the carboxyl-terminal RNase H domain.…”

Soluble Rous Sarcoma Virus Reverse Transcriptases α, αβ, and β Purified from Insect Cells Are Processive DNA Polymerases That Lack an RNase H 3′ → 5′ Directed Processing Activity

“…The Gag-Pol precursor is processed into individual proteins by the viral protease. RTs from closely related lentiviruses like human immunodeficiency virus (HIV-1 and HIV-2), simian immunodeficiency virus and equine infectious anemia virus show similar RT organization (1)(2)(3)(4). The RTs of these viruses are heterodimeric with a large ϳ66-kDa subunit harboring the N-terminal polymerase and the C-terminal RNase H domains, and a small ϳ51-58-kDa subunit lacking the RNase H domain.…”