Maturation of Parvovirus LuIII in a Subcellular System. I. Optimal Conditions for in vitro Synthesis and Encapsidation of Viral DNA

Abstract

SUMMARYThe development of an in vitro system prepared by lysis of LulII virus-infected cells with Brij-58 has enabled the study of the assembly pathway of a parvovirus. Under optimal conditions, radioactive precursors are incorporated both into viral replicative form double-stranded DNA and into progeny viral DNA (vDNA) during pulses as short as 30 s. Labelled 110S particles can be isolated at the end of such pulses. Therefore, synthesis and encapsidation of progeny viral DNA into pre-existing empty viral capsids appear to be closely related processes. Up to about 10 min after incorporation of vDNA, the 110S particles band at 1.44 g/ml and are relatively unstable in 3-5 M-CsC1. Moreover, newly synthesized vDNA molecules show an abnormal electrophoretic behaviour probably due to the presence of a covalently linked terminal protein. This so far uncharacterized alkali-stable polypeptide is lost (cleaved off?) concomitant with the maturation of the 110S virus particles. Maturation is reflected by a change in the virus stability in CsCI and a shift in density from 1-44 to 1-41 g/ml around 10 to 15 rain after encapsidation of progeny vDNA.

Maturation of Parvovirus LuIII in a Subcellular System. II. Isolation and Characterization of Nucleoprotein Intermediates

“…The digested fractions were electrophoresed in a 1 ~o agarose gel under non-denaturing conditions (Muller & Siegl, 1983). LulII virus particles, either denatured with 0.1 M-NaOH or digested with Pronase-SDS, served as markers.…”

“…Individual fractions pooled from sucrose gradients were dialysed in 10 mM-Tris HCI pH 7-6, 10 m~f-EDTA and digested with 200gg/ml proteinase K in the presence of 0-6~o SDS at 37 °C for 1 h. DNA isolated from these fractions was extracted twice with an equal volume of phenol~chloroform (1 : 1, v/v) and collected from the aqueous phase by precipitation with ethanol in the presence of 0-5 M-NaCI at -20 °C as described by Muller & Siegl (1983). The resulting pellet was resuspended in 10 mM-Tris-HC1 pH 8.0, 1 mM-EDTA (TE buffer).…”

“…Nucleic acid was also analysed by electrophoresis in 1 ~ agarose gels both in the native state and after denaturation with 0.1 M-NaOH. DNA was located in the gels either by staining with ethidium bromide or by autoradiography (Muller & Siegl, 1983). Single-stranded (ss) and double-stranded (ds) viral DNA, as well as fragments of bacteriophage 2 DNA generated by digestion with restriction endonuclease HindlII served as size markers.…”

“…The system was shown to allow both the in vitro synthesis of progeny viral DNA and its subsequent encapsidation into complete virus particles. Recently (Muller & Siegl, 1983), it was shown to be possible to select experimental parameters which, in contrast to previous results, enabled the demonstration of the encapsidation of LulII virus DNA concomitant with DNA synthesis. The virus particles formed under these conditions sediment at about 110S.…”

“…The cell lysate or LullI-infected cells were then broken by 50 strokes in a tighly fitting Dounce homogenizer at 0 °C, the extract was layered onto linear 5 to 40~ sucrose gradients in 2 mM-Tris-HC1 pH 7-4 and centrifuged in a Beckman SW50.1 rotor at 4 °C and 40000 rev/min for 70 min. Fractions of the gradients were collected from the bottom of the tubes and the trichloroacetic acid (TCA)-insoluble radioactivity in 20 p,1 amounts of each fraction was determined (Muller & Siegl, 1983).…”