A defined system for in vitro packaging of DNA-gp3 of the Bacillus subtilis bacteriophage phi 29.

Abstract

The bacteriophage 429 DNA-gene product 3 complex (DNA-gp3) has been efficiently packaged into proheads in a completely defined in vito system. The 429 DNA packaging protein gpl6, the product of gene 16, was overproduced in Escherichia coli and purified to near homogeneity.The purified gpl6 packaged 23% of the DNA-gp3 added to purified proheads in the dermed mixture, while gpl6 in an extract of phage-infected cells packaged 26% of the DNA-gp3.No host proteins were required in the defined system. ATPdependent packaging of DNA-gp3 in the defined system was optimal with %100 copies of gpl6 per DNA-gp3, an amount similar to the production of gpl6 per DNA-gp3 in +29-infected cells.Bacteriophage 429 of Bacillus subtilis is an attractive model for studying the mechanism of viral DNA packaging because of its relative simplicity and the high efficiency of in vitro assembly (1)(2)(3)(4)(5) MATERIALS AND METHODSBacteria, Phage, and Plasmids. The nonpermissive host B. subtilis SpoA12 (6) and the permissive hosts B. subtilis sup+4 (7) and L15 (6) gpl6 accumulated as large lumps in the cells (Fig. lb). The cells (200 ml) were sedimented, resuspended in 100 ml of buffer for forming spheroplasts (0.01 M Tris-HCl, pH 8.0/0.01 M EDTA/0.125 M NaCl/500 ,ug of lysozyme per ml), and incubated for 5 min at ambient temperature. All subsequent manipulations were performed at 4°C except as noted. Spheroplasts were centrifuged at 3000 X g for 5 min, and the pellet was resuspended in 100 ml of 0.01 M Tris HCl, pH 7.5/0.001 M dithiothreitol. The spheroplasts were lysed in the French pressure cell at 69 MPa. Urea (Schwarz/Mann; 8 M, adjusted to pH 5.2 with glacial acetic acid) was added to the lysate to a final concentration of 0.7 M. After a clarifying centrifugation at 750 x g for 10 min, the supernatant was centrifuged at 7000 x g for 5 min to sediment the gpl6 lumps. The pellet was dissolved in 10 ml of solubilizing buffer [0.01 M Tris HCl, pH 7.5/0.005 M dithiothreitol/6 M guanidinium chloride (Serva, Heidelberg; or BDH)], and the solution was centrifuged at 100,000 x g for 1 hr. One milliliter of the supernatant containing gpl6 was applied to a 1 x 55 cm Sephadex G-150 column equilibrated with 7.5 M urea/0.001 M dithiothreitol adjusted to pH 5.2 as above. The gpl6 was eluted in two peaks with the same solution; fractions of the second peak were pooled, and 0.2 ml of saturated ammonium sulfate was added for each 1.0 ml of eluant. After 3 min at ambient temperature, the precipitate was collected by cenAbbreviations: gp3 and gpl6, products of phage 429 genes 3 and 16;pfu, plaque-forming units. *Permanent address: South China Agricultural University, Guangzhou (Canton), The People's Republic of China. 3505The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

A Viral RNA That Binds ATP and Contains a Motif Similar to an ATP-binding Aptamer from SELEX

“…In vitro Production of Infectious Virions of phi29 Virion Particles with aptRNA and ATP-The purification of procapsids (28,29), gp16 (30), and DNA-gp3 (31,32); the preparation of the tail protein (gp9) (29), neck proteins (gp11, gp12) (29), and the morphogenetic factor (gp13) (29); and the procedure for the assembly of infectious phi29 virion in vitro (29) have been described previously. 1 g of RNA in 1 l RNasefree H 2 O was mixed with 10 l of purified procapsids (0.4 mg/ml) and then dialyzed on a 0.025-m-type VS filter membrane against TBE (2 mM EDTA, 89 mM Tris borate, pH 8.0) for 15 min at room temperature.…”

“…ATPase Assay by Thin Layer Chromatography-The purified DNApackaging components, gp16 (0.24 g), DNA-gp3 (0.1 g), procapsid (3.2 g), and RNA (1 g), were mixed individually or in combination with 0.3 mM of unlabeled ATP and 0.75 Ci (6000 Ci/mmol) of [␥-32 P]ATP in the reaction buffer (30). When one or more components was omitted, the component(s) was replaced with the same volume of TMS.…”

Assembly of a Tailed Bacterial Virus and Its Genome Release Studied in Three Dimensions

“…It requires the prohead with pRNA, the supercoiled DNA substrate with the covalently bound terminal protein (DNAgp3), the packaging ATPase (gp16), and ATP (Guo et al, 1986;Grimes and Anderson, 1997). The head becomes more angular (Bjornsti et al, 1983), and the surface charge decreases during packaging of DNA-gp3 and expulsion of gp7 (D. L. A., unpublished).…”

The Unique Vertex of Bacterial Virus PRD1 Is Connected to the Viral Internal Membrane

“…The 29 portal occupying a pentagonal vertex is a dodecamer of portal protein gp10, forming a propellershaped structure with a central channel (35,64). In addition to the portal, which is attached to the prohead, the packaging machinery consists of five or six copies of the viral ATPase (gp16) and six copies of a 29-encoded packaging RNA (pRNA) (36)(37)(38)(39)74). The DNA, connector, and proheadpRNA-ATPase complex form a set of concentric structures with 10-, 12-, and 5-or 6-fold symmetry, respectively, embedded in the 5-fold vertex of the prohead (35,64).…”