Moloney murine sarcoma virions synthesize full-genome-length double-stranded DNA in vitro.

Abstract

Moloney murine sarcoma virus (MSV) virions incubated under optimal conditions were shown to support extensive synthesis of double-stranded DNA. The major product, a 5950-base-pair (6-kilobase-pair DNA) double-stranded DNA, was characterized by cleavage with restriction endonucleases and shown to contain a 600-nucleotide-ong direct repeat at both ends of the MSV genome. Linear DNA molecules made in vivo shortly after infection were compared to the linear doublestranded DNA synthesized in vitro. The restriction maps of both viral DNA products were indistinguishable. The 600-base-pair repeat results in a progeny DNA molecule that is longer than the parental MSV genomic RNA. The generation of this repeat must involve a mechanism that allows the viral reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) to copy 5'-and 3'-terminal genomic (+) Synthesis of DNA in Detergent-Disrupted Virions. Virions at a concentration of 5 mg/ml were disrupted with Triton X-100 (final concentration 0.028-0.030%) in a reaction mixture containing 50 mM Tris-HCI (pH 7.5), 20 mM dithiothreitol, 2mM MgCl2, and the four dNTPs at 1 mM each. DNA was labeled with [a-32P]dNTPs (Amersham or New England Nuclear) at a specific activity of 5-50 cpm/pmol or with 125I-labeled dCTP, made by a modification of the method of Commerford (5, 10). Incubations were at 37"C for 16-18 hr. These conditions optimized the synthesis of full-length double-stranded products.The DNA products were extracted with phenol saturated wth 50 mM Tris-HCI, pH 7.5/0.1 M NaCl/1 mM EDTA/0.1% sodium dodecyl sulfate (NaDodSO4) and precipitated twice with 2 vol of ethanol. This material was digested with an excess of nuclease S1 (Sigma, 5 units per ,ug of DNA) in 100 mM sodium acetate buffer, pH 4.5/0.2 M NaCI/1 mM ZnCl2 at 37°C for 30 min.Isolation and Purification of Viral DNA. Full-length viral dsDNA was isolated by electrophoresis on 1.5% neutral agarose (Sea Kem, HGT) horizontal slab gels in 50 mM borate/i mM EDTA at pH 8 at 150 V for about 16 hr. Restriction fragments of T7 DNA (11) or phage a3 replicative form II [32PJDNA served as molecular weight markers. The DNA was stained with ethidium bromide (0.1 ,ug/ml in 0.25 M ammonium acetate) for 30 min and visualized by UV illumination, and the bands were cut out. The DNA was eluted by crushing the agarose in 0.5 M ammonium acetate/10 mM EDTA/0.1% NaDodSO4, incubating for 16 hr at 370C, and separating the agarose from the DNA by centrifuging the mixture over silanized glass wool in a micropipet tip. The DNA was precipitated twice with 2 vol of ethanol at -70°C and collected by centrifugation. When necessary, DNA preparations were further purified by Bio-Gel A-5M chromatography in 4 ml of Quik-Sep (Isolab, Akron, OH) columns with 50 mM Tris-HCI/0. 1 M NaCI/2.5 mM EDTA/ 0.1% NaDodSO4/10% glycerol/0.01% diethylpyrocarbonate as solvent, precipitated twice with ethanol, and stored in 10 mM The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby...

A novel approach to cloning transcriptionally active retrovirus-like genetic elements from mouse cells

“…The reaction takes place within the virion which is partially lysed to allow the entry of deoxyribonucleoside triphosphates. Using optimum conditions Nucleic Acids Research large quantities of full-length proviral DNA can be obtained [12,13,15]. For Ki-MuLV maximum synthesis of full-length DNA was obtained at a virus concentration of 4 mg mV'1 in the presence of 0.02% (v/v) Triton X-100.…”

“…To overcome these problems we have developed an alternative strategy for cloning retrovirus-transmitted VL30 genes. This exploits the ability of detergentdisrupted retrovirus particles to synthesize abundant quantities of retroviral DNA [12,13]. In this report we show that VL30 DNA can also be synthesized in such endogenous reactions.…”

“…Detection of Ki-MuLV and VL30 Nucleic Acid by Filter Hybridization Filter hybridization, post hybridization washing and autoradiography were performed as described previously [15]. Ki-MuLV/VL30 cDNA probe (specific activity 109 cpm pg-1) was synthesized from purified virion RNA templates as described by Avery et al [16] [12,13]. Such in vitro synthesized DNA is identical to that found in virus-infected cells [12,,13,15] and has been shown to possess biological integrity by infectivity assays [13,21].…”

“…Otherwise, reaction conditions were as described by Benz and Dina [12]. Endogenous reaction mixtures were incubated at 370C for 12 h (longer incubations resulted in degradation of reaction products).…”

An X-linked gene affecting mouse cell DNA synthesis also affects production of unintegrated linear and supercoiled DNA of murine leukemia virus.

“…Several of these steps seem to be carried out by reverse transcriptase alone or possibly in combination with other virion proteins. Indeed, infectious double-stranded linear viral DNA, as well as circular viral DNA with one LTR copy can be synthesized in vitro by purified virions (1,2,5,6,10). This in vitro reaction, however, does not yield closed circular viral DNA with two LTR copies.…”