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Originally published In Press as doi:10.1074/jbc.M306928200 on September 8, 2003

J. Biol. Chem., Vol. 278, Issue 48, 48084-48091, November 28, 2003
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RNA Packaging Device of Double-stranded RNA Bacteriophages, Possibly as Simple as Hexamer of P4 Protein*

Denis E. Kainov{ddagger}§, Markus Pirttimaa{ddagger}, Roman Tuma{ddagger}, Sarah J. Butcher{ddagger}, George J. Thomas, Jr.¶, Dennis H. Bamford{ddagger}||, and Eugene V. Makeyev{ddagger}**

From the {ddagger}Department of Biosciences and Institute of Biotechnology, FIN-00014, University of Helsinki, Finland and the Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110

Received for publication, June 30, 2003 , and in revised form, August 28, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Genomes of complex viruses have been demonstrated, in many cases, to be packaged into preformed empty capsids (procapsids). This reaction is performed by molecular motors translocating nucleic acid against the concentration gradient at the expense of NTP hydrolysis. At present, the molecular mechanisms of packaging remain elusive due to the complex nature of packaging motors. In the case of the double-stranded RNA bacteriophage {varphi}6 from the Cystoviridae family, packaging of single-stranded genomic precursors requires a hexameric NTPase, P4. In the present study, the purified P4 proteins from two other cystoviruses, {varphi}8 and {varphi}13, were characterized and compared with {varphi}6 P4. All three proteins are hexameric, single-stranded RNA-stimulated NTPases with {alpha}/{beta} folds. Using a direct motor assay, we found that {varphi}8 and {varphi}13 P4 hexamers translocate 5' to 3' along ssRNA, whereas the analogous activity of {varphi}6 P4 requires association with the procapsid. This difference is explained by the intrinsically high affinity of {varphi}8 and {varphi}13 P4s for nucleic acids. The unidirectional translocation results in RNA helicase activity. Thus, P4 proteins of Cystoviridae exhibit extensive similarity to hexameric helicases and are simple models for studying viral packaging motor mechanisms.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A number of biological systems rely on active transport of nucleic acids (NA)1 powered by the hydrolysis of NTPs. This is observed in prokaryotic organisms, for example, during cell division, conjugation, and sporulation (1). NTP-dependent translocation is also utilized by many DNA viruses for genome encapsidation, or "packaging," into preformed capsids, examples including herpesviruses and adenoviruses, as well as tailed bacteriophages, such as {varphi}29, {lambda}, P22, and T4 (2–7).

Genome packaging is best understood for the dsDNA bacteriophage {varphi}29, whose portal complex serves as a packaging engine (8). The complex contains three components: 1) the head-tail connector, a dodecamer of protein p10, attached to a unique 5-fold prohead vertex; 2) a multimeric ring of packaging RNAs; and 3) a homomultimeric ATPase composed of protein p16 (6, 8). The portal of {varphi}29 can translocate DNA against loads up to 57 piconewtons and therefore is one of the most powerful molecular motors in nature (9).

Portal complexes of many other DNA bacteriophages are organized similarly to {varphi}29, although the packaging RNA requirement is limited to {varphi}29-related phages (10). In addition, DNA translocation in these systems is often accompanied by the processing of genomic DNA concatemers (terminase activity) (3, 11). Because the {varphi}29 p16 and terminases of others bacteriophages have been shown to catalyze NTP hydrolysis in vitro, these proteins are thought to generate the energy for the packaging reaction (12). It has been suggested that the energy is somehow transmitted from the transiently associated terminase to the packaging RNA ring and connector, which in turn effects unidirectional DNA translocation (6, 13). However, the complexity of the portal structures in DNA viruses has made it difficult to elucidate the packaging mechanism experimentally.

In vitro packaging of dsRNA viruses has been accomplished only for bacteriophages {varphi}6 and {varphi}8 of the Cystoviridae family, which infect the plant pathogenic bacterium Pseudomonas syringae (14, 15). All cystoviruses contain a lipid envelope and a tripartite dsRNA genome composed of S, M, and L segments.

Similarly to {varphi}29, {varphi}6 packaging can be assayed in vitro using recombinant empty particles, called procapsids (PCs) and (+) sense ssRNA genome precursors s+, m+, and l+, respectively (14). Segment s+ is packaged first, followed by m+ and l+ (16). The specificity of packaging is due to the pac sites located within the 5'-terminal ~300-nt regions of all three RNA precursors (14, 17).

The {varphi}6 PC consists of four virus-encoded proteins, P1, P2, P4, and P7. P1 forms the structural framework of the PC (18). P2 is an RNA-dependent RNA polymerase that replicates and transcribes the packaged RNA (19, 20). P7 is important for PC stability and proper functioning (21, 22). Comparison between the wild-type and protein-deficient {varphi}6 PC has shown that P4 is essential for RNA packaging (23, 24). P4 sequence contains characteristic NTPase motifs, which are conserved among the Cystoviridae (Fig. 1), although there is no other detectable sequence identity. Indeed, the purified {varphi}6 P4 is an unspecific NTPase, hydrolyzing ribo-, deoxyribo-, and dideoxyribonucleoside triphosphates (25, 26).



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  		FIG. 1.
Cystoviral P4 proteins contain two highly conserved regions. Amino acid sequences of P4 NTPases from {varphi}6, {varphi}8, and {varphi}13 (NCBI accession numbers P11125 [GenBank] , AAF63301 [GenBank] , and AAG00445 [GenBank] , respectively) were aligned using the ClustalW algorithm (50). The similarity plot, built up on the alignment data using a 20-amino acid sliding window, reveals two peaks (black) corresponding to the Walker A and B motifs (51). Interestingly, the two spans show detectable similarity to the corresponding regions of hexameric DNA helicase gp4 of bacteriophage T7 (NCBI accession number CAA24407 [GenBank] ). In the alignment, residues identical in all four proteins are highlighted black; similar residues are shown in gray.

 
The {varphi}6 P4 forms doughnut-shaped hexamers in the presence of divalent cations and ATP or ADP, and the NTPase activity is associated only with the multimeric form. The NTPase activity is weakly stimulated by ssRNA (27). Cryoelectron microscopy reconstruction of {varphi}6 PC has localized P4 hexamers to the particle 5-fold vertices, thus constituting a symmetry mismatch (28). Our recent micrographs of {varphi}8 subviral particles show that the localization of P4 is likely to be similar for other cystoviruses (29). The symmetry mismatch is also typical for packaging motors of tailed bacteriophages, and it has been proposed to facilitate rotation of the portal during nucleic acid translocation (11).

Although P4 NTPase activity is essential for RNA packaging (24, 27), it is still unknown whether P4 alone constitutes the packaging motor or if other PC proteins are also involved. In addition, the details of the mechanochemical coupling during RNA packaging in dsRNA viruses are poorly understood, a situation similar to the genome packaging in DNA viruses.

The existing RNA packaging assays are based on precursor RNase protection, cosedimentation with the PC, and replication inside the PC (14, 22, 24). Obviously, these assays do not permit direct demonstration of ssRNA translocation, and new approaches are needed to monitor the motor action. To address these issues, we developed a novel packaging assay based on complementary oligonucleotide displacement (COD), which is conceptually similar to helicase assays (30). Using this approach, we showed that P4 proteins, isolated from bacteriophages {varphi}8 and {varphi}13 (31, 32), specifically translocate ssRNA in the 5' to 3' direction at the expense of NTP hydrolysis. Other types of nucleic acids are bound but not translocated. We also demonstrated that the packaging motors of the Cystoviridae share structural and biochemical characteristics with hexameric helicases. Because of their simplicity, these P4 proteins constitute an attractive model system for delineating the translocation mechanism and mechano-chemical coupling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
P4 Expression and Purification—{varphi}6 and {varphi}8 P4s were purified as described (27, 33). To construct a {varphi}13 P4 expression plasmid, pDK3, gene 4 was PCR-amplified from the {varphi}13 L segment cDNA (plasmid pLM2200) (32) using recombinant Pfu DNA polymerase (Stratagene) and the oligonucleotides 5'-AGGGATGTCATATGACTGACGAAAAGA-3' and 5'-GCGGAAGCTTCAACATAAACAGCTCCT-3'. The PCR fragment was inserted into the vector pT7–7 (34) at the NdeI-HindIII sites. Soluble {varphi}13 P4 was produced in Escherichia coli strain BL21(DE3) transformed with pDK3 as described for {varphi}8 P4 (33).

NTPase Assays—The NTPase activity of the purified P4 proteins was assayed in 20 mM Tris (pH 8.0), 75 mM NaCl, 7.5 mM MgCl2, 4 mM unlabeled NTP, and 0.5 µCi of the relevant [{alpha}-32P]NTP (3000 Ci/mmol; Amersham Biosciences). The final P4 concentration was 0.1 mg/ml. Reactions were incubated for 1 h at 37 °C, and 1 µl of the reactions were analyzed on polyethyleneimine-cellulose F TLC plates (Merck). Chromatograms were developed as described (26). The amount of radioactivity in the nucleotide spots was measured using a phosphor imager (Fuji BAS 1500).

The steady-state rate of NTP hydrolysis was studied as the rate of phosphate release using the EnzChek phosphate assay kit (Molecular Probes, Inc., Eugene, OR). The concentration of Pi was proportional to the absorbance at 360 nm. The calibration was done using standard KH2PO4 solutions. Steady-state kinetics of phosphate release was measured using a Victor2 plate reader (Wallac-PerkinElmer Life Sciences). ATP was purchased from Amersham Biosciences, ribohomopolymers (poly(G), poly(A), poly(U), and poly(G)) were from Sigma (concentrations reported in moles of nucleotide), and E. coli rRNA (16 and 23 S) was from Roche Applied Science.

The rate of NTP hydrolysis (measured by phosphate release), v, was described by Michaelis-Menten kinetics as follows,

(Eq. 1)
where Km represents the Michaelis constant and kcat is the enzyme turnover number.

RNA Substrates—RNA oligonucleotides RNA2 (5'-GUUUUCACCCUAUCCUCCCC-3') and RNA3 (5'-CGACUCAUGGACCUUGGGAG-3') were labeled by T4 polynucleotide kinase in the presence of [{gamma}-32P]ATP (35) and purified through P-6 spin columns (Bio-Rad) equilibrated with 10 mM Tris-HCl, pH 7.4. Synthetic ssRNAs were produced by run-off transcription in vitro with T7 RNA polymerase (19). RNAs sR5, s({Delta}11–23)R5, s({Delta}11–32)R5, and s({Delta}11–43)R5 were transcribed from the EcoRV-cut plasmids pLM659, pLM1771, pLM1772, and pLM1773, respectively (16, 36). RNAs (residues 72–136) was transcribed from a pLM659 PCR fragment amplified using oligonucleotides 5'-GCGTAATACGACTCACTATAGGGGAGGATAGGGT-3' (T7 promoter sequence italicized) and 5'-CGACTCATGGACCTTGGGAG-3' as up- and downstream primers, respectively.

To produce the RNA1 probe, plasmid pEM21 was constructed by inserting the duplex of two complementary oligonucleotides, 5'-CTAGATCGGTAACCTCGGTCAGGTAC-3' and 5'-CTGACCGAGGTTACCGAT-3', at the XbaI-KpnI sites of the pGEM3Zf(+) vector (Promega). The resultant plasmid was linearized with HindIII and transcribed with T7 RNA polymerase in the presence of [{alpha}-32P]UTP. All transcripts were precipitated with 3 M LiCl, reprecipitated with ethanol, and dissolved in sterile water.

COD Assays—The COD activity of PCs was assayed under the conditions optimized for RNA packaging (37). Complete and protein-deficient PCs were produced as described (24). To prepare RNA duplex substrates, unlabeled RNAs were mixed with 32P-labeled RNA probes in hybridization buffer containing 15 mM HEPES-KOH, pH 7.5, 150 mM NaCl, and 0.75 mM EDTA. The solution was boiled for 2 min and slowly cooled to room temperature. If necessary, residual nonannealed probe was removed by passing the mixture through a Sephacryl S-300 spin column (Amersham Biosciences) equilibrated with 10 mM Tris-HCl (pH 7.5), 2 mM EDTA. COD assay mixtures (25 µl) contained 50 mM Tris-HCl (pH 8.9), 2 mM dithiothreitol, 0.1 mM EDTA, 5 mM MgCl2, 80 mM NH4Ac, 6% polyethyleneglycol 4000, 1 mM ATP or its nonhydrolyzable analog AMP-PCP, 1 unit/µl rRNasin (Promega), ~1.5 µg of PCs, and 0.2 µg of an RNA duplex. The reactions were usually incubated for 1–2 h at 30 °C and transferred to ice. The volume was then brought to 100 µl with 0.13 mg/ml yeast tRNA (Sigma) in water, followed by phenolchloroform extraction and ethanol precipitation. The samples were dissolved in Tris-EDTA and analyzed by native 7% PAGE (in standard Tris borate-EDTA buffer) at room temperature. The gels were dried and analyzed by autoradiography and/or phosphorimaging (Fuji BAS 1500).

For P4 proteins, the COD assay was performed in 10-µl reaction mixtures containing 20 mM Tris-HCl (pH 7.5), 7.5 mM MgCl2, 75 mM NaCl, 5 mM nucleotide (NTP, NDP, or AMP-PNP), 0.1 µg of RNA duplex, and 0.05 µg of P4. The reactions were incubated at 23 °C for 1 h; stopped by adding 2 µl of 15% glycerol, 20 mM EDTA, 3% SDS; and analyzed by native PAGE as above.

Gel Shift Assays—{varphi}6, {varphi}8, and {varphi}13 P4s (at 0.1, 0.05, and 0.01 mg/ml, respectively) were incubated for 15 min on ice with RNA or DNA substrates (0.1 mg/ml) in 7.5-µl mixtures containing 20 mM Tris-HCl (pH 8.0), 7.5 mM MgCl2, 75 mM NaCl. Thereafter, 2.5 µl of 15% glycerol, 20 mM Tris-HCl (pH 8.0), 7.5 mM MgCl2, 75 mM NaCl, 0.25% bromphenol blue was added, and the samples were electrophoresed in a 1.2% agarose gel (x Tris borate-EDTA buffer, 5 V/cm, 90 min).

Dynamic Light Scattering—The hydrodynamic properties of individual proteins were studied using a batch dynamic light scattering instrument (Precision Detectors) equipped with deconvolution software for correlation function analysis (33).

Raman Spectroscopy—Protein solutions were concentrated (to ~20 mg/ml each), sealed in sterile capillaries (Kimax 34504), and thermostated at 5 °C. Raman spectra were recorded on a Spex 500M spectrograph equipped with a notch filter and charge-coupled device detector (SpectrumOne; Instruments S.A., Edison, NJ) using a spectral slit width of 4 cm–1. A solid state, diode-pumped, frequency-doubled Nd: YVO4 laser (model Verdi {lambda}em = 532 nm; Coherent, Santa Clara, CA) was used to obtain 150 milliwatts at the sample. Each spectrum represents the accumulation of 15 exposures of 2-min duration on each of two independently prepared protein solutions. Spectra were corrected for buffer and NTP contributions and normalized for difference computations as described previously (38). The positions of strong bands are reported with ±1-cm–1 accuracy, whereas those of weak and broad bands are reported with ±2-cm–1 accuracy.

Small Angle Neutron Scattering—{varphi}8 P4 was concentrated to 4 mg/ml and dialyzed against D2O buffer solution (20 mM Tris-DCl, 50 mM NaCl, 7.5 mM MgCl2). The sample was filtered and centrifuged (Beckman Airfuge; rotor A-95, 90,000 rpm, 10 min), and supernatant was transferred into a thermostated quartz sample cell (2-mm path, 20 °C). The SANS data were collected on beam line D22 (Institute Laue Langevin; Grenoble, France) using a collimation distance of 4.4 m, neutron wave-length of 0.6 nm, and a 4-m sample-detector distance. The data were processed as described (39) to obtain an averaged and corrected scattering curve. The curve was modeled by scattering from a set of dummy atoms using the DAMMIN program (40) and imposing 6-fold symmetry.

Cryoelectron Microscopy—For cryoelectron microscopy, samples of 0.1 mg/ml {varphi}8 P4 and 0.25 mg/ml {varphi}13 P4 in buffer (20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 7.5 mM MgCl2) were vitrified as described elsewhere (29). The data were collected at –180 °C (Oxford CT3500 cryospecimen holder) in an FEI Tecnai F20 field emission gun transmission electron microscope (Electron Microscopy Unit, University of Helsinki) operating at 200 kV. The images were recorded under low dose conditions on Eastman Kodak Co. SO-163 film at a magnification of x50,000 ({varphi}8) or x62,000 ({varphi}13).

The micrographs were scanned on a Z/I imaging Photoscan TD flat bed scanner in 12-bit gray scale mode at a raster step of 7 µm, corresponding to 1.4 Å/pixel ({varphi}8 P4) and 1.13 Å/pixel ({varphi}13 P4) in the image. Individual particles were picked manually from the scans using EMAN (41) and analyzed using the Spider program package (42). Reference-free alignment was used for the averaging (43). No symmetry constraints were applied.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
P4 Proteins Are Ring-shaped Hexamers with an {alpha}/{beta} Fold—{varphi}6 P4 has been previously shown to assemble into donut-like hexamers in the presence of nucleotides and divalent metal ions (27, 28). The quaternary structures of the purified P4s were characterized by several methods (Table I, Fig. 2). The apparent molecular masses of {varphi}6, {varphi}8, and {varphi}13 P4s deduced by gel filtration and light scattering were ~212, ~202, and ~220 kDa, respectively (Fig. 2C), as expected for the hexameric forms (predicted masses of 210, 204.6, and 225.6 kDa, respectively).


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  		TABLE I
Properties of purified P4 proteins

 



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  		FIG. 2.
P4s from different cystoviruses are {alpha}/{beta} proteins with ring-like hexameric architecture. A, SDS-PAGE analysis of purified P4s from {varphi}6, {varphi}8, and {varphi}13. Homogeneous {varphi}6 P4 is known to migrate as a double band in the electrophoretic system used (27). B, amide I region in Raman spectra of P4 proteins: {varphi}6 P4, 50 mg/ml (1 mM ADP was added to stabilize the hexamer); {varphi}8 P4, 22 mg/ml; and {varphi}13 P4, 20 mg/ml. All spectra were normalized with respect to concentration. C, purified P4 proteins were analyzed on a Superdex 200 gel filtration column. The traces represent absorbance at 280 nm. The arrows indicate the injection time (inject) and positions of the molecular mass standards: blue dextran (BD) (2000 kDa); {beta}-amylase ({beta} Am) (200 kDa); mouse immunoglobulin G (IgG) (150 kDa); bovine serum albumin (BSA) (67 kDa); ovalbumin (OA) (45 kDa); soybean trypsin inhibitor (STI) (20.1 kDa). D, electron microscopy of vitrified P4. The image on the left represents the average of 206 {varphi}8 P4 projections. The image on the right resulted from averaging 953 {varphi}13 P4 projections. Scale bar, 5 nm. E, side and top views of a dummy atom model of {varphi}8 P4 hexamer based on solution neutron scattering. Scale bar, 5 nm.

 
The oligomeric status and shape of each P4 was confirmed by electron microscopy. Examination of several thousand vitrified P4 molecules revealed primarily ring-shaped molecules, indicating a preferential orientation for the particles in a vitrified water layer. Reference-free alignment and averaging images revealed hexameric rings for both {varphi}8 and {varphi}13 P4 (Fig. 2D). The inner diameter of the {varphi}8 P4 ring was ~3.0 nm, and the outer diameter was ~12.1 nm. Similarly, the {varphi}13 P4 ring exhibits an inner diameter of ~2.8 nm and outer diameter of ~12.1 nm. These data agree well with those published earlier for the {varphi}6 P4 (27, 28). The average image of {varphi}8 P4 is in good agreement with a small angle solution scattering-based model, which revealed a funnel shaped hexamer with a two-dimensional projection similar to the electron microscopy average (Fig. 2E).

We also examined the secondary structure of the three P4 proteins using Raman spectroscopy. Raman spectra have been widely employed to compare the secondary structures of viral proteins (44, 45). In particular, Raman spectral intensities of the backbone amide I vibrations (range 1630–1700 cm–1) corresponding to pure classes of secondary structures (i.e. {alpha}-helices, {beta}-sheet, and undefined structures) are well documented both experimentally and theoretically (46). The amide I signatures of P4 proteins from the three cystoviruses are compared in Fig. 2B. Each exhibits a broad amide I band with an apparent maximum between 1660 and 1670 cm–1 ({beta}-strand) and shoulders at both higher (turns and extended structures) and lower ({alpha}-helix) wave number values. This spectral signature is typical of {alpha}/{beta} proteins that bind and hydrolyze nucleotides (27).

Despite the overall similarity of the subunit folds, significant differences are apparent. The P4 amide I band of {varphi}8 is much sharper and displays a distinct peak position (1663 cm–1) reflecting lesser {alpha}-helical structure and greater {beta}-strand structure than the P4s of {varphi}6 and {varphi}13. On the other hand, the broader amide I band of the {varphi}13 P4 spectrum indicates a greater {alpha}-helical content.

P4 Proteins Are RNA-stimulated NTPases—{varphi}6 P4 is known to be an unspecific NTPase (26). We assayed NTPase activities of P4s from the other two phages. The proteins were incubated for 1 h at 37 °C in the presence of either purine (ATP) or pyrimidine (UTP) NTPs, and the reaction products were analyzed by TLC (Fig. 3A). It is obvious that isolated {varphi}13 P4 also possesses a readily detectable NTPase activity, hydrolyzing both types of NTPs into NDP and inorganic phosphate (Fig. 3A, lanes 2 and 5). In comparison, hexameric {varphi}8 P4 appears to be a very poor NTPase (Fig. 3A).



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  		FIG. 3.
NTPase activity of P4 proteins. A, P4s from {varphi}6, {varphi}8, or {varphi}13 were incubated at 37 °C for 1 h with [{alpha}-32P]ATP or UTP, as indicated, and the reaction mixtures were analyzed by TLC. Migration of ATP, ADP, UTP, and UDP is indicated. B, concentration dependence of phosphate release kinetics in the presence of 0.1 µM P4 proteins at 25 °C for ATP and UTP substrates. C, the effect of different ssRNA (nucleotide unit concentration was 1 mM) on the ATPase activity of the three P4 proteins was measured using steady state kinetics (left panel) and kcat determination (right panel, ATP concentration 1 mM). The analysis was repeated three times, and error bars show the S.E. The color scheme employed in the left panel corresponds to that in the right panel and designates the type of RNA used.

 
In order to establish nucleotide substrate specificity, we have determined the kcat and Km for the P4 proteins and nucleotides ATP and UTP (Fig. 3B and Table II). Notably, no hydrolysis was detected for {varphi}8 P4; therefore, Km constants have not been determined. The Km value for ATP and UTP hydrolysis is similar for {varphi}6 P4, whereas {varphi}13 P4 exhibits higher affinity for UTP (Table II and Fig. 3B). However, judged by kcat/Km, both enzymes prefer the UTP substrate (Table II).


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  		TABLE II
Steady state NTPase parameters of P4 proteins

 
It has been reported that the NTPase activity of {varphi}6 P4 was enhanced 1.6-fold by the addition of ssRNA (27). We therefore examined the effect of ssRNA on the NTPase activities (kcat) of the P4 proteins (Fig. 3C). A weak stimulation of the {varphi}13 P4 NTPase activity, comparable with that of the {varphi}6 P4, was observed in the presence of ribosomal RNA. Interestingly, an NTPase rate comparable with that of the {varphi}6 and {varphi}13 proteins was detected for {varphi}8 P4 in the presence of rRNA. This suggests that the {varphi}8 NTPase is strongly coupled to the presence of ssRNA. NTPase activity also became cooperative in the presence of RNA (data not shown).

To find the best ssRNA cofactor for each P4 NTPase, we analyzed the effect of synthetic polyribonucleotides on kcat (Fig. 3C). Poly(C) and poly(A) acted as the best cofactors for the {varphi}8 P4. {varphi}6 P4 was also stimulated by poly(C) and poly(A) with a preference for poly(A). On the other hand, all polynucleotides except poly(G) stimulated {varphi}13 P4. Poly(G) had an inhibitory effect.

Nucleic Acid Binding Properties of P4 Proteins—NA binding to P4 proteins was assessed by an electrophoretic mobility shift assay (gel shift), which detects stable nucleoprotein complexes (nanomolar affinity). P4 proteins from {varphi}6, {varphi}8, and {varphi}13 were incubated with long ssRNA, dsRNA, ssDNA, or dsDNA and analyzed by nondenaturing agarose gel electrophoresis (Fig. 4A). No stable complexes were detected for {varphi}6 P4. In the case of {varphi}8 and {varphi}13 proteins, slowly migrating complexes were formed with all four types of NA. The extent of the retardation increased with the P4 amount added. Notably, the ssDNA probe used in this assay was the circular genome of bacteriophage M13, which indicated that free NA ends were dispensable for P4 binding.



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  		FIG. 4.
{varphi}8 and {varphi}13 P4s bind all types of NA, but only ssRNA stimulates the NTPase activity. A, ethidium bromide-stained agarose gel shift analyses of {varphi}8 P4 binding to different NA. ssRNA, T7 transcript from plasmid pLM659 cut with SmaI; dsRNA, S, M, and L segments purified from bacteriophage {varphi}6; ssDNA, circular genome of bacteriophage M13; dsDNA, PCR fragment comprising {varphi}8 gene 1 (amplified using oligonucleotides 5'-GGAGTTGACATATGAGTAAGCTTGATCT-3' and 5'-TAGGATCCGTCATGTCACATACCTT-3'). Lane 1, free NA; lanes 2–4, {varphi}8 P4 added to 0.1, 0.05, and 0.01 mg/ml; lanes 5–7, {varphi}13 P4 added to 0.1, 0.05, and 0.01 mg/ml; lanes 8 and 9, {varphi}6 P4 added to 0.1 and 0.01 mg/ml, respectively. Positions of free and P4-bound (shift) NA are marked on the left. B, stimulation of NTPase activity of {varphi}6, {varphi}8, and {varphi}13 P4s by different nucleic acids; poly(C) was used as ssRNA, and dsRNA, ssDNA, and dsDNA were as described for A.

 
Since {varphi}8 and {varphi}13 P4s bind all types of NA, we studied the possible effect of dsRNA, ssDNA, and dsDNA on the NTPase activity of these proteins. Of these, only dsRNA slightly stimulated the {varphi}8 P4 NTPase, whereas no activation was detected in other combinations (Fig. 4B).

COD Assay Strategy—The current {varphi}6 packaging assays require the presence of intact PC particles. We sought to develop a more direct method for detecting RNA translocation activity of isolated as well as procapsid-associated P4 proteins. Electron microscopy reconstructions indicated that the genomic RNA might be packaged via a narrow central opening in the P4 protein (28). Given the extensive secondary structure of ssRNA precursors (17), it is likely that packaging would lead to separation of short complementary regions.

Accordingly, an RNA substrate was prepared by annealing an unlabeled 700-nt-long 5'-terminal fragment of the s+ segment (sR5 RNA) with the 32P-labeled 66-nt-long RNA oligonucleotide (RNA1). RNA1 was designed to target the 3'-proximal region of sR5, ~300 nt downstream of the pac site. Only the middle 18 nt of RNA1 can form a duplex with the corresponding sequence (590–607 nt) of sR5, the 5' and 3' termini forming single-stranded overhangs (Fig. 5A). If the packaging apparatus recognizes sR5/RNA1, RNA1 may be displaced in the course of sR5 translocation. The liberated RNA1 probe can be separated from the input duplex in PAGE analysis under native conditions (30).



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  		FIG. 5.
RNA translocase activity of P4 proteins. A, schematics of the COD assay. Portal corresponds to either procapsid-associated P4 or isolated P4 hexamer. B, P4 COD activity was assayed with sR5/RNA1 in the presence of ATP or ADP or without nucleotides, as indicated. Lanes 1–3, {varphi}6 P4; lanes 4–6, {varphi}13 P4; lanes 7–9, {varphi}8 P4. Migration of the sR5/RNA1 duplex (D) and liberated RNA1 probe (P) is shown on the right. C, directionality of translocation was assayed by COD activity of {varphi}8 P4 using specially designed substrates s (positions 72–136)/RNA2 and s (positions 72–136)/RNA3 depicted at the top of the panel. Reactions contained ATP or ADP, as indicated below the panel. D, COD activity of wild-type PC particles was assayed with RNA substrates where RNA1 probe (P) was duplexed (D) with either sR5 RNA, containing the intact packaging region of the {varphi}6 s+ segment (wt) or one of the three sR5 variants containing deletions in the packaging region ({Delta}11–23, {Delta}11–32, and {Delta}11–43). The reactions were carried out at 30 °C for 1 h in the presence of either ATP or its nonhydrolyzable analog AMP-PCP, as indicated. Lane P, RNA1 probe only. E, time course of the COD reaction catalyzed by WT PC particles in the presence of ATP or AMP-PCP.

 
P4 Proteins of {varphi}8 and {varphi}13 but Not {varphi}6 Can Translocate RNA Substrates—We examined the COD activity of isolated P4 proteins. For this purpose, P4 enzymes were incubated with the R5/RNA1 duplex under various conditions. In contrast to {varphi}6P4 (Fig. 5B, lane 1), both {varphi}8 and {varphi}13 proteins displaced the RNA1 probe from the duplex in the presence of ATP (Fig. 5B, lanes 4 and 7), {varphi}8 P4 showing noticeably higher activity. No COD activity was detected when ATP was omitted or substituted with either ADP or AMP-PNP (Fig. 5B, lanes 5, 6, 8, and 9, and data not shown). Thus, isolated {varphi}8 and {varphi}13 P4 can translocate RNA.

{varphi}8 P4 Translocates in the 5' to 3' Direction—To determine the direction of RNA translocation, two additional RNA duplex substrates were designed containing unlabeled RNAs (positions 72–136) (a 65-nt fragment of {varphi}6 s+) and either of the two 32P-labeled RNA oligonucleotides, RNA2 and RNA3, complementary to the 5' and 3' regions of s (positions 72–136), respectively (Fig. 5C). Both duplexes were incubated with {varphi}8 P4, and the reaction products were separated by nondenaturing PAGE. Only the RNA3 and not the RNA2 probe was displaced from the duplex, thus indicating that P4 translocates s (positions 72–136) RNA in the 5' to 3' direction. Notably, genome precursors have been shown to enter {varphi}6 PC in the 5' to 3' direction (14).

RNA Packaging into {varphi}6 PC—No displaced radioactive probe was detected for isolated {varphi}6 P4 even after prolonged incubations (not shown), thus suggesting that {varphi}6 P4 either requires additional PC component(s) or needs to be physically associated with the particle to support RNA translocation.

When the COD activity of the WT {varphi}6 PC was assayed in the presence of ATP, ~20% of the RNA1 was indeed released from the sR5/RNA1 duplex (Fig. 5D, lane 3). No RNA1 bands appeared when PC particles were omitted from the reaction or when ATP was replaced by a nonhydrolyzable analog AMP-PCP (Fig. 5D, lanes 1 and 2). Prolonged incubations in the presence of ATP but not AMP-PCP led to the displacement of >40% of RNA1 (Fig. 5E).

To confirm that the RNA1 displacement was due to sR5 packaging, three control substrates were assayed with the WT PCs. These contained small deletions in the 5' region, s({Delta}11–23)R5, s({Delta}11–32)R5, or s({Delta}11–43)R5 annealed with RNA1. Both {Delta}11–32 nt and {Delta}11–43 nt deletions are known to disrupt the s+ pac activity, whereas the {Delta}11–23 nt deletion is neutral (16). In line with these results, RNA1 was efficiently displaced from the s({Delta}11–23)R5/RNA1 duplex, but not from s({Delta}11–32)R5/RNA1 or s({Delta}11–43)R5/RNA1 (Fig. 5D, lanes 4–9).

RNA Translocation by PC Requires the Presence of P4 — Previous experiments with mutated {varphi}6 PC have shown that P4 is indispensable for RNA packaging (24). We therefore tested the COD activities of several recombinant PC variants. PC containing ~10% of the normal P4 amount due to the point mutation S250Q in P4 (P1P2P4*P7) displaced the RNA1 probe with an efficiency close to that of the WT PC (Fig. 6A). Furthermore, PC containing the normal amount of P4 but missing protein P2 (P1P4P7) also supported oligonucleotide displacement, albeit with 10-fold reduced efficiency compared with the WT particles. As expected, PCs missing P4 (P1P2P7) did not displace RNA1.



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  		FIG. 6.
{varphi}6 P4 requires additional PC components for COD activity. A, COD activity of different recombinant {varphi}6 PC particles produced in E. coli. wt PC, wild type particles containing normal amount of P1, P2, P4, and P7 proteins. P1P2P4*P7, particles containing ~10% of P4 protein due to the S250Q mutation in P4. P1P2P7 and P1P4P7, particles missing proteins P4 or P2 altogether. B, COD activity of {varphi}6 PC particles assembled in vitro from purified individual proteins (22), as indicated below the bars. Wild-type recombinant PC, which were expressed and assembled in E. coli (wt PC), were used as a control. Amounts of proteins used in the assembly mixtures (P1, P2, P4, and P7) were 0.4, 0.05, 0.2, and 0.15 mg/ml, respectively.

 
We also assayed the COD activity of PCs assembled in vitro (22). For this purpose, different combinations of individual PC proteins were incubated with sR5/RNA1 under conditions promoting PC assembly (22). The RNA1 probe was efficiently displaced in the mixture containing all four PC proteins, whereas other mixtures showed very little COD activity (Fig. 6D).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Many viruses encapsidate their genomes through NTP-dependent packaging into preformed capsids. This process requires a portal complex that operates as a molecular motor converting chemical energy into mechanical work. Although DNA packaging in tailed bacteriophages has been studied to a great extent, the molecular basis of mechanochemical coupling is far from being understood. This may reflect the complexity of the packaging motor in which the terminase only transiently associates with the portal protein and the consequent need to study translocation in the context of assembled capsids.

RNA translocation of dsRNA bacteriophages (Cystoviridae) seems to be a much simpler system. As demonstrated here, it can be assayed without the need to monitor RNA inclusion to the PC particles using the COD technique (Figs. 5 and 6), which can also be applied to purified packaging proteins.

Only two short amino acid spans, apparently important for NTP binding and catalysis, are noticeably conserved between {varphi}6, {varphi}8, and {varphi}13 P4 sequences (Fig. 1). Nevertheless, all three P4 proteins share a common hexameric ringlike architecture and {alpha}/{beta} fold (Fig. 2). They differ only in the detailed shape and the amount of helical secondary structure (Fig. 2 and Table I), indicating the presence of accessory {alpha}-helical domains for {varphi}13 and {varphi}6. Although the NTPase activity of all three proteins is stimulated by RNA, only the {varphi}8 and {varphi}13 proteins have nanomolar affinity for nucleic acids (Figs. 3 and 4). The high affinity for nucleic acids correlates with the ability of these proteins to translocate along RNA (Fig. 5 and Table I).

The results of this study indicate that RNA packaging in Cystoviridae is catalyzed by a single protein species, P4, which therefore represents one of the simplest nucleic acid pumps described so far. The isolated {varphi}8 P4 exhibits the properties of a tightly coupled motor hydrolyzing NTP only in the presence of RNA, whereas {varphi}6 P4 may need to be attached to the PC particle to become an RNA-dependent translocase. In line with this idea, previous Raman spectroscopy studies have shown that {varphi}6 P4 undergoes extensive conformational changes upon its binding to the PC particle (38).

Although all or most of the PC 5-fold vertices seem to be occupied by P4 hexamers (18, 28), a model has been suggested, where only one of the 12 vertices operates as a packaging portal (24). Consistently, particles containing only ~10% of P4 displace complementary oligonucleotides almost as efficiently as WT PCs (Fig. 2D). It is also notable that no specific sequence requirements were observed in the COD assays for purified {varphi}8 and {varphi}13 P4s. Because packaging in Cystoviridae requires the presence of pac sites, located at the 5' termini of genome precursors (14), other PC proteins (e.g. P1) are likely to be responsible for the RNA selection.

Our data suggest that cystoviral P4s are structurally and functionally related to other hexameric enzymes that can couple NTP hydrolysis with unidirectional translocation along nucleic acids and other types of mechanical work. These include replicative helicases (e.g. bacterial DnaB and phage T4 gp41), transcription termination factor Rho, cell-encoded DNA pumps, and the AAA family ATPases (47). For some of these proteins, such as the DNA helicase gp4 of bacteriophage T7, a high resolution structure is available, offering a model for translocation (48). Interestingly, gp4 translocates DNA in the 5' to 3' direction, similarly to the polarity of RNA translocation by {varphi}8 P4 (Fig. 5C). Furthermore, cystoviral P4 proteins show substantial sequence homology with T7 gp4 in both of the two conserved regions (Fig. 1). This encourages us to propose that the molecular basis of P4 catalyzed RNA translocation could be similar to that suggested for T7 gp4 (48).

In conclusion, isolated P4 proteins of cystoviruses {varphi}8 and {varphi}13 function as NTP dependent RNA translocases in vitro, which argues that the packaging device of at least some viruses can be exceptionally simple and perhaps mechanistically similar to the hexameric helicases. Notably, a helicase-like protein has been implicated in genome encapsidation of the adeno-associated virus 2 (49), indicating a wider spread of this paradigm. P4 hexamers of {varphi}8 and {varphi}13 can bind all types of nucleic acids, but only ssRNA stimulates the NTPase activity and supports translocation. Thus, the nucleic acid is likely to play an active role in the coupling between NTPase activity and translocation.


   FOOTNOTES
 
* This work was supported by the Academy of Finland ("Finnish Centre of Excellence Program 2000–2005" Grants 172623 (to R. T.), 178778 (to S. J. B.), and 1202855 and 1202108 (to D. H. B.)) and National Institutes of Health Grant GM50776 (to G. J. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Fellow of the National Graduate School in Informational and Structural Biology. Back

** Present address: Dept. of Molecular and Cellular Biology, Harvard University, 7 Divinity Ave., Cambridge, MA 02138. Back

|| To whom correspondence should be addressed: P.O. Box 56, Viikinkaari 5, FIN-00014, University of Helsinki, Finland. Tel.: 358-9-191-59100; Fax: 358-9-191-59098; E-mail: dbamford{at}mappi.helsinki.fi.

1 The abbreviations used are: NA, nucleic acid(s); dsRNA, double-stranded RNA; dsDNA, double-stranded DNA; PC, procapsid; ssRNA, single-stranded RNA; ssDNA, single-stranded DNA; nt, nucleotide; COD, complementary oligonucleotide displacement; AMP-PCP, adenosine 5'-({beta},{gamma}-methylenetriphosphate); AMP-PNP, adenosine 5'-({beta},{gamma}-imino)triphosphate; WT, wild type. Back


   ACKNOWLEDGMENTS
 
We are grateful to Riitta Tarkiainen and Pasi Laurinmäki for technical assistance. Teemu Ikonen and Peter Timmins are thanked for help with SANS data collection and Jiri Lisal for the help with NTPase kinetic experiments.



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