Packaging motor from double-stranded RNA bacteriophage phi12 acts as an obligatory passive conduit during transcription

Abstract

Double-stranded RNA viruses sequester their genomes within a protein shell, called the polymerase complex. Translocation of ssRNA into (packaging) and out (transcription) of the polymerase complex are essential steps in the life cycle of the dsRNA bacteriophages of the Cystoviridae family (phi6-phi14). Both processes require a viral molecular motor P4, an NTPase, which bears structural and functional similarities to hexameric helicases. In effect, switching between the packaging and the transcription mode requires the translocation direction of the P4 motor to reverse. However, the mechanism of the reversal remains elusive. Here we characterize the P4 protein from bacteriophage phi12 and exploit its purine nucleotide specificity to delineate P4 role in transcription. The results indicate that while P4 actively translocates RNA during packaging it acts as a passive conduit for RNA export. The directionality switching is accomplished via the regulation of P4 NTPase activity within the polymerase core.

Figure 1

Simplified scheme of the cystoviral life cycle. The three dsRNA genomic segments of a cystovirus are brought into the host cell inside the viral core (a). Upon cell entry, the core catalyzes semi-conservative dsRNA transcription and (+) sense ssRNA transcripts (l⁺, m⁺ and s⁺) are extruded into the cytoplasm (b). The cellular protein synthesis machinery translates l⁺ RNA (c) giving rise to proteins P1, P2, P4 and P7. The newly produced proteins assemble into empty polymerase complexes (PC) (d), which are capable of packaging specifically one copy of each l⁺, m⁺ and s⁺ segments (e). Upon packaging PC expands and replication is initiated (f). The dsRNA-filled PC (core) can enter additional rounds of transcription or mature into infectious virions. The latter pathway uses proteins produced by the translation of m⁺ and s⁺ ssRNA segments, which is followed by the acquisition of the rest of the viral structural proteins together with the lipid membrane (not shown). The mature virus particles are released by lysis of the host cell (32).

Figure 2

Effects of the reaction conditions on the activity of the φ12 P4 NTPase. (A–C) Quantitative analysis by TLC showing the effects of pH [(A) 20 mM Tris–HCL, 50 mM NaCl, 5 mM MgCl₂], divalent metals—Me²⁺ [(B) 20 mM Tris–HCl, pH 8.0, 50 mM NaCl, 0.2 mM MgCl₂, 20 mM Me²⁺] and Mg²⁺ concentration [(C) 20 mM Tris–HCl, pH 8.0, 50 mM NaCl], on the GTPase activity of φ12 P4 (GTP concentration was 2 mM, protein concentration 0.25 mg/ml) at 37°C. (D–F) P4 NTP turnover (k_cat) was measured using steady state kinetics of P_i release (NTP concentration 1 mM) under different conditions: NaCl effect [(D) 20 mM Tris–HCl, pH 9.0, 1 mM MgCl₂, 24°C)], temperature dependence [(E) 20 mM Tris–HCl, pH 9.0, 1 mM MgCl₂, 100 mM NaCl)], hydrolysis of different nucleotides [(F) NTPs, AMPPNP and GMPPNP concentration 1 mM, 20 mM Tris–HCl, pH 9.0, 1 mM MgCl₂, 100 mM NaCl, 24°C], and TLC run showing the hydrolysis of different nucleotides under optimal conditions (insert). The analyses were repeated three times and error bars show standard deviations.

Figure 3

Assessment of transcriptional activity of φ12 cores in vitro under favorable conditions for P4 NTPase activity and in the presence of non-hydrolyzable analogs. (A) Two possible models of semi-conservative transcription in Cystoviruses (left—P4 actively translocates ssRNA from the core; right—P4 acts as a passive conduit). Packaging NTPase and polymerase in the context of the capsid. Polymerase is colored red and packaging NTPase is green. (B) SDS–PAGE analyses of purified φ12 virus and core components. (C) Time course of transcription reaction. Reaction products in the presence of P4-hydrolyzable and non-hydrolyzable nucleotides were analyzed on agarose gel and auotoradiographed. (D and E) Sedimentation analysis of the cores after in vitro transcription in the presence of P4-non-hydrolyzable nucleotides: protein composition analyzed on SDS–PAGE (D) and autoradiograph of RNA products separated on agarose gel (E). Lane 120′ corresponds to labeled products of core transcription which where loaded onto gradient.

Figure 4

A model for switching between RNA packaging and semi-conservative transcription.