Rotavirus NSP2 interferes with the core lattice protein VP2 in initiation of minus-strand synthesis

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Virology 313 (2003) 261–273

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Rotavirus NSP2 interferes with the core lattice protein VP2 in initiation of minus-strand synthesis Patrice Vende,1 M. Alejandra Tortorici, Zenobia F. Taraporewala, and John T. Patton* Laboratory of Infectious Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, 50 South Drive MSC 8026, Bethesda, MD 20892, USA Received 26 November 2002; returned to author for revision 31 January 2003; accepted 28 March 2003

Abstract The rotavirus nonstructural protein NSP2 self-assembles into stable octameric structures that possess nonspecific affinity for singlestranded (ss)RNA and RNA–RNA helix-destabilizing and NTPase activities. Furthermore, NSP2 is a component of replication intermediates with replicase activity and plays a critical role in the packaging and replication of the segmented dsRNA genome of rotavirus. To better understand the function of the protein in genome replication, we examined the effect that purified recombinant NSP2 had on the synthesis of dsRNA by the open core replication system. The results showed that NSP2 inhibited the synthesis of dsRNA from viral mRNA in vitro, in a concentration-dependent manner. The inhibition was overcome by adding increasing amounts of viral mRNA or nonviral ssRNA to the system, indicating that the inhibition was mediated by the nonspecific RNA-binding activity of NSP2. Further analysis revealed that NSP2 interfered with the ability of the open core proteins, GTP, and viral mRNA to form the initiation complex for (⫺) strand synthesis. Additional experiments indicated that NSP2 did not perturb recognition of viral mRNA by the viral RNA polymerase VP1, but rather interfered with the function of VP2, a protein that is essential for (⫺) strand initiation and dsRNA synthesis and that forms the T ⫽ 1 lattice of the virion core. In contrast to initiation, NSP2 did not inhibit (⫺) strand elongation. Collectively, the findings provide evidence that the temporal order of interaction of RNA-binding proteins with viral mRNA is a crucial factor impacting the formation of replication intermediates. © 2003 Elsevier Science (USA). All rights reserved.

Introduction Rotaviruses, members of the family Reoviridae, are a major cause of acute dehydrating diarrhea in infants and young children (Kapikian et al., 2001). The rotavirus genome consists of 11 segments of double-stranded RNA (dsRNA) which encode six-structural (VP) and six nonstructural (NS) proteins. The capsid of the virion is an icosahedron formed by three concentric layers of protein (Prasad et al., 1988). The innermost layer is composed of 60 asymmetric dimers of VP2, arranged as a T ⫽ 1 lattice. Associated with each of the vertices of the VP2 lattice is a copy of the RNA-dependent RNA polymerase VP1 and the * Corresponding author. Fax: ⫹1-301-496-8312. E-mail address: [email protected] (J.T. Patton). 1 Present address: Laboratoire de Virologie et Immunologie Mole´culaires, INRA, C.R.J.J., Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France.

multifunctional mRNA-capping enzyme VP3 (Lawton et al., 1997b; Pizarro et al., 1991; Chen et al., 1999). Together, the proteins VP1, VP2, and VP3 and the dsRNA genome make up the core of the virion. Double-layered particles (DLPs) consist of cores surrounded by the intermediate layer protein, VP6. Transcriptase activity associated with DLPs catalyzes the synthesis of 11 capped mRNAs (Lawton et al., 1997a). The mRNAs direct the synthesis of viral proteins and serve as templates for the synthesis of (⫺) strand RNA to produce the dsRNA genome segments. Analysis of replication intermediates (RIs) recovered from infected cells indicates that the mRNA templates undergo replication concurrently with their packaging into precapsid structures associated with core and double-layered RIs (Patton and Gallegos, 1990). Core RIs consist of the structural proteins VP1, VP2, and VP3 and the nonstructural proteins NSP2 and NSP5 (Gallegos and Patton, 1989). Double-layered RIs, in addition,

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contain VP6. Core RIs are thought to be formed by the interaction of VP2, NSP2, and NSP5 with complexes, termed precore RIs, which contain viral mRNA and VP1 and VP3. Replicase activity (mRNA 3 dsRNA) is associated with core RIs, and not with precore RIs, a reflection of the need for VP2 in the synthesis of (⫺) strand RNA (Mansell and Patton, 1990; Patton et al., 1997). Genome packaging and replication and the formation of cores and DLPs occur in perinuclear cytoplasmic inclusions, termed viroplasms (Petrie et al., 1984). Of the viral proteins in viroplasms, only VP1 has specific affinity for viral mRNAs while several others, i.e., VP2, VP3, NSP2, and NSP5, have nonspecific affinity for single-stranded (ss)RNA and thus can also interact with the mRNAs (Kattoura et al., 1992; Labbe et al., 1994; Patton and Chen, 1999; Vende et al., 2002). How the interaction of the RNAbinding proteins with the template mRNAs is temporally regulated in the assembly of RIs is not known. NSP2 and NSP5 not only accumulate in viroplasms but also are necessary for the formation of viroplasms (Fabbretti et al., 1999). Biochemical analyses have shown that NSP2 has NTPase activity and that the protein may be involved in the hyperphosphorylation of NSP5 (Afrikanova et al., 1998; Taraporewala et al., 1999; Vende et al., 2002). NSP2 also possesses RNA–RNA helix destabilizing activity which may remove higher order structures from mRNA templates that impede RNA packaging (Taraporewala and Patton, 2001). Structural studies have shown that NSP2 self assembles into octamers by the head-to-head interaction of doughnut-shaped tetramers (Jayaram et al., 2002; Schuck et al., 2001). An NSP2 octamer contains four potential RNAbinding grooves and undergoes a conformational shift in the presence of NTPs. These properties are consistent with the octamer functioning as a molecular motor to facilitate the packaging of mRNA templates into the precapsid structures of RIs during genome replication. In this study, we have found that incubation of recombinant (r)NSP2 with virion-derived open cores or with purified rVP1 and rVP2 in cell-free replication assays inhibits dsRNA synthesis by interfering with the formation of the initiation complex for (⫺) strand synthesis. Further analysis indicated that although VP1 could function in the formation of the initiation complex in the presence of NSP2, the ability of VP2 to do so was abrogated. Once assembled, the initiation complex was able to promote (⫺) strand synthesis even in the presence of NSP2. These data establish that the order of interaction of viral RNA-binding proteins with template mRNAs is a critical factor influencing the formation of functional RIs.

Results rNSP2 inhibits dsRNA synthesis by open cores rNSP2 linked to a C-terminal His tag was expressed in bacteria and purified by Ni-nitrilotriacetic acid (NTA)-af-

finity column chromatography. Sucrose gradient sedimentation and polyacrylamide gel electrophoresis indicated that rNSP2 in the preparation was homogeneous in size and structure, consisting of rNSP2 assembled into 10S octameric complexes (data not shown). The open core replication system supports the initiation and elongation of (⫺) strand RNA from input template mRNA, forming dsRNA in vitro that is indistinguishable in size from the dsRNA genome segments. To analyze the effect of NSP2 on the synthesis of rotavirus dsRNA, concentrations of rNSP2 octamer up to 10 pmol were included in the open core system. Reaction mixtures also included 1 pmol of gene 11 mRNA as the exogenous (⫹) strand template and [32P] UTP to radiolabel newly made RNAs. The 32 P-labeled gene 11 dsRNA products were detected by PAGE and quantified with a phosphorimager (Fig. 1). The results showed that rNSP2 inhibited gene 11 dsRNA synthesis in a biphasic manner with amounts of octamer up to ⬃1 pmol having a greater interfering effect than amounts beyond 1 pmol. Thus, rNSP2 inhibited more effectively when the molar ratio of octamer to template RNA was ⱕ1 than when the molar ratio was ⬎1. Notably, gene 11 dsRNA synthesis was inhibited by ⬃50% when equal molar concentrations of rNSP2 octamer and template RNA were present in reaction mixtures. As the concentration of rNSP2 increased in the replication assays, an increase was also noted in the the synthesis of dsRNAs from the endogenous genome segments contained in the open cores preparation (Fig. 1). The fact that open cores have polymerase activity which can produce background levels of dsRNA products from the endogenous segments has been reported before (Chen et al., 1994). Replication of exogenous template RNAs in the open core system competitively interferes with the synthesis of dsRNAs from the endogenous segments (Patton and Chen, 1999). The presence of NSP2 in the open core system not only inhibited replication of the exogenous mRNA but also overcame the inhibitory effect that the addition of exogenous mRNA normally has on the synthesis of dsRNA from the endogenous segments (Fig. 1). To test whether the inhibition of RNA replication was a nonspecific event resulting simply from the addition of large amounts of protein to the open core system, the impact of including bovine serum albumin (BSA) in reaction mixtures in place of rNSP2 was examined. The results showed that although the presence of 1 and 5 pmol of rNSP2 octamer, i.e., 8 and 40 pmol of rNSP2 monomer, inhibited gene 11 replication by ⬃50 and 90%, respectively, the presence of 8 and 40 pmol of BSA had little or no effect on replication (Fig. 2). Hence, rNSP2 interfered with dsRNA synthesis in the open core system by a protein-specific mechanism. To assess the possibility that the rNSP2 preparation contained a contaminating RNase that degraded the (⫹) strand template for dsRNA synthesis in open core assays, the purified protein was incubated with 32P-labeled gene 11 mRNA. Electrophoretic analysis showed that the RNA was not degraded,

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containing rNSP2 was affected by the concentration of (⫹) strand template RNA. For this analysis, reaction mixtures were prepared which contained 2.5 pmol of rNSP2 octamers and either 1, 2, or 4 pmol (0.22, 0.44, or 0.88 ␮g, respectively) of gene 11 mRNA. Examination of the products of the assays showed that as the concentration of template RNA in reaction mixtures was increased fourfold from 1 to 4 pmol, an ⬃threefold increase was observed in the level of dsRNA synthesis (Fig. 3A). As a consequence of adding 4 pmol of template RNA, the level of dsRNA synthesis in reaction mixtures containing rNSP2 reached ⬃75% of the level of dsRNA synthesis occurring in reaction mixtures lacking rNSP2. These results provided evidence that rNSP2 inhibited replication by binding to the (⫹) strand template for (⫺) strand synthesis, which could be overcome by increasing the ratio of template to rNSP2 in reaction mixtures. Since the RNA-binding activity of NSP2 is nonspecific, we tested whether the addition of nonviral ssRNA to open core assays would also reduce the inhibitory effect that rNSP2 had on dsRNA synthesis. In this analysis, the reaction mixtures contained 2.5 pmol of rNSP2; 1 pmol (0.22 ␮g) of gene 11 mRNA; and 0, 0.22, or 0.66 ␮g of luciferase RNA. Therefore, the total amount of RNA in the mixtures was 0.22, 0.44, or 0.88 ␮g, respectively, the same as in the experiment described above (Fig. 3A). Analysis of the

Fig. 1. Inhibitory effect of NSP2 on dsRNA synthesis by open cores. Replication assays containing 60 fmol of open cores, 1 pmol of gene 11 mRNA, and 0 to 10 pmol of rNSP2 octamer were incubated for 2 h. 32 P-labeled dsRNA products were resolved by PAGE and detected by autoradiography. The positions of genome segments 1 to 11 are labeled (A). To calculate percentage replication, the intensities of the bands for gene 11 dsRNA were determined and adjusted relative to those obtained for gene 11 dsRNA made in the assay containing no rNSP2, which was set at 100%. The results of two independent experiments were averaged; the error bar represents the standard deviation (B).

ruling out the possibility that rNSP2 preparation contained RNase activity which interfered with the synthesis of dsRNA by open cores (data not shown). RNA-binding activity of NSP2 mediates inhibition of replication To gain further insight into the mechanism by which rNSP2 inhibited dsRNA synthesis in vitro, we tested whether the extent of RNA replication in open core assays

Fig. 2. Effect of protein levels on dsRNA synthesis. Replication assays were executed which contained 60 fmol of open cores, 1 pmol of gene 11 mRNA, and either no added protein or 8 or 40 pmol of NSP2 monomer or BSA. 32P-labeled dsRNA products were resolved by PAGE and detected by autoradiography (A). Percentage replication was calculated as for Fig. 1 (B).

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that activity sequence specific (Patton and Chen, 1999). From the experimental results described above (Fig. 3), it is possible to conclude that rNSP2 inhibited gene 11 dsRNA synthesis by competing with the binding of one or more of the core proteins for the (⫹) strand template, thereby preventing the formation of RNA–protein complexes that have replicase activity. As a test of this hypothesis, assays were performed in which reaction mixtures contained constant amounts of rNSP2 (2.5 pmol) and gene 11 mRNA (1 pmol) but quantities of open cores that were one, two, or four times greater than that used in the standard reaction mixture. Analysis of the reaction products (Fig. 4) showed that, as the amount of open cores added to the reaction mixtures increased, there was a proportional increase in the level of dsRNA synthesis. Remarkably, the reaction mixtures which contained rNSP2, gene 11 mRNA, and a fourfold increase in open cores produced dsRNA at levels only slightly less than those produced by corresponding reaction mixtures lacking rNSP2. Thus, the increased levels of open cores in the reaction mixtures nearly fully overcame the inhibitory effect of rNSP2 on dsRNA synthesis. These data indicated

Fig. 3. Impact of RNA concentration on inhibition of dsRNA synthesis by NSP2. Replication assays were carried out which contained open cores, 2.5 pmol of rNSP2 octamer, and the indicated amount of gene 11 mRNA (A) or gene 11 mRNA and luciferase RNA (B) (0.22 ␮g ⬇ 1 pmol gene 11 mRNA). The products were resolved by PAGE and the levels of 32Plabeled gene 11 dsRNA quantified with a phosphorimager. The levels were adjusted relative to the amount of gene 11 dsRNA made in an assay containing open cores, 0.22 ␮g of gene 11 mRNA, and no rNSP2, which was defined as 100% replication.

dsRNA products showed that the addition of nonviral ssRNA in the reaction mixtures caused a decrease in the inhibitory effect of rNSP2 (Fig. 3B). The extent of inhibition was quantitatively similar to that observed when a total of 0.22, 0.44, or 0.88 ␮g of gene 11 mRNA was included in reaction mixtures (Fig. 3A). Thus, the addition of either viral mRNA or nonviral RNA to reaction mixtures reduced the extent to which rNSP2 inhibited the replication of exogenous viral mRNA by open cores. This finding indicated that the sequence-independent ssRNA-binding activity of rNSP2 was responsible for the ability of the protein to interfere with RNA replication in vitro. Core protein concentration affects inhibition of replication by NSP2 The three core proteins, VP1, VP2, and VP3, possess RNA-binding activity, although only in the case of VP1 is

Fig. 4. Impact of open core concentration on inhibition of dsRNA synthesis by NSP2. Replication assays were performed which included no or 2.5 pmol of rNSP2 octamer, 1 pmol of gene 11 mRNA, and the indicated amount of open cores (1.5 ␮l ⫽ 60 fmol). The products were resolved by PAGE, detected by autoradiography (A), and quantified with a phosphorimager. Levels of gene 11 dsRNA made in assays containing rNSP2 were divided by levels of gene 11 dsRNA made in assays lacking rNSP2, but containing the equivalent amount of open cores. To obtain percentage replication, the values were multiplied by 100 (B).

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that the ratio of open core proteins to rNSP2 influenced RNA replication with relatively higher ratios favoring increased dsRNA synthesis and lower ratios resulting in decreased dsRNA synthesis. Considered with the results of experiments presented above (Fig. 3), the data also indicated that rNSP2 impeded dsRNA synthesis by competitively interfering with the binding of one or more of the core proteins to the template RNA. Order of rNSP2 and core protein interaction with mRNA affects replication In evaluating the effect of rNSP2 on the synthesis of dsRNA by open cores, we found that the order in which rNSP2, open cores, and template RNA were added to reaction mixtures had a significant impact on the extent of replication. In standard reactions, where open cores and rNSP2 (2.5 pmol octamers) were combined in reaction mixtures prior to the addition of template RNA, dsRNA was produced at levels ⬃15% of that of reaction mixtures which lacked rNSP2 (Fig. 5). In contrast, combining rNSP2 with template RNA prior to the addition of open cores resulted in a near complete blockage of dsRNA synthesis. And when open cores were mixed with template RNA prior to the addition of rNSP2, dsRNA was produced at levels 30 – 40% of that made in reaction mixtures which lacked rNSP2. These results showed that the sequence of interaction of core proteins and rNSP2 with template RNA is a significant factor influencing the formation of RNA–protein complexes with replicase activity. Specifically, the data indicated that the template RNA must interact with one or more core proteins prior to interaction with NSP2, a major component of viroplasms and core RIs, if complexes are to be assembled that can catalyze (⫺) strand synthesis. rNSP2 interferes with the formation of the initiation complex for (⫺) strand synthesis Formation of the initiation complex for (⫺) strand synthesis requires open core proteins, the mRNA template, and GTP and is a process blocked by concentrations of monovalent salts of ⱖ200 mM (Chen and Patton, 2000). Once formed, the initiation complex is resistant to salt destabilization. The RNA polymerase can elongate the (⫺) strand even when salt concentrations exceed 1 M. To assess whether rNSP2 inhibited dsRNA synthesis in vitro by interfering with (⫺) strand initiation or elongation, or both, reaction mixtures were prepared that contained the components (open cores, gene 11 mRNA, and GTP) necessary for formation of initiation complexes. Ten picomoles of rNSP2 octamers, an amount that nearly blocks all replication of exogenous viral mRNA by open cores (see Fig. 1), was included in one of the reaction mixtures (Fig. 6A, lane 3). After incubation for 1 h, all reaction mixtures were supplemented with the four NTPs and [32P] UTP. Then 10 pmol of rNSP2 octamers was added to one of the reaction mixtures

Fig. 5. Importance of order of addition of reaction components on dsRNA synthesis. Open cores (60 fmol), 1 pmol of gene 11 mRNA, and 2.5 pmol of rNSP2 octamers were added to reaction mixtures in the order indicated and the mixtures were kept on ice until all components had been added, at which point incubation was carried out at 32°C. Reaction mixtures were thoroughly mixed in between addition of reaction components and the time that lapsed between addition of each component was ⬍1 min. The 32Plabeled gene 11 dsRNA products were resolved by PAGE and detected by autoradiography (A). Percentage replication was determined as for Fig. 1. Gene 11 dsRNA made by reaction mixture containing only cores and mRNA was defined as 100% replication (B).

not already containing the protein (lane 4). Following an additional incubation of 2 h, dsRNA produced in mixtures was detected by PAGE and quantified with a phosphorimager (Fig. 6B). The analysis showed that dsRNA was not made by reaction mixtures in which rNSP2 was added at the same time as components necessary for forming initiation complexes (lane 3). In contrast, dsRNA was produced in reaction mixtures in which initiation complexes had been allowed to form prior to the addition of rNSP2 (lane 4). Indeed, the level of dsRNA synthesis in the latter reaction mixtures was as high as those made by reaction mixtures in which 200 mM NaCl was added subsequent to the formation of initiation complexes (lane 1). Collectively, these data indicated that in the open cores system rNSP2 interfered with the formation of (⫺) strand initiation complexes but not with (⫺) strand elongation. As an alternative test of the idea that (⫺) strand elongation was not inhibited by rNSP2, intracellular subviral particles (SVPs) were recovered from rotavirus-infected cells (Gallegos and Patton, 1989). Such SVPs have replicase

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light. RNA in the pools was recovered by pelleting through CsCl gradients and assayed for the presence of associated rNSP2 by Western blot assay using anti-His antibody. As shown in Fig. 7B, rNSP2 was present in the RNA pellet of pool 1 but was absent in the RNA pellets of pools 2 and 3 (Fig. 7B). These results indicate that rNSP2 was associated with RNA contained in the SVP preparation and that the association required UV crosslinking and was lost if the RNA of the SVP preparation was removed by RNase digestion. This analysis provides evidence that rNSP2 can interact with RNA contained in SVP preparations. Binding of the RNA polymerase to template RNA is not affected by rNSP2 Electrophoretic mobility shift assays (EMSAs) have shown that VP1 specifically recognizes the 3⬘-end of rota-

Fig. 6. Effect of NSP2 on formation of (⫺) strand initiation complex by open cores. Assays supporting initiation complex formation, but not elongation, contained open cores, 2.5 mM GTP (no other NTP), 1 pmol of gene 11 mRNA, and, in some cases, 0.2 M NaCl or 10 pmol of rNSP2 octamer. After incubation for 1 h, the reaction mixtures were modified to allow for (⫺) strand elongation by the addition of the four NTPs and [32P]UTP. NaCl or rNSP2 was also added at this time to reaction mixtures not already containing either one of these components. The 32P-labeled gene 11 dsRNA products were resolved by PAGE and detected by autoradiography (A). The intensities of the bands for gene 11 dsRNA were determined and adjusted relative to those obtained for gene 11 dsRNA made in the assay in which NaCl was present during the elongation phase but not the initiation phase (lane 1) (B).

activity that synthesize dsRNA by the elongation of (⫺) strand RNAs on endogenous (⫹) strand templates associated with the particles (Fig. 7A, lane 4). The SVPs are not able to catalyze dsRNA by initiating and elongating (⫺) strand RNAs from exogenous template mRNAs (lane 5). The impact of rNSP2 on the synthesis of dsRNA by SVPs and open cores was compared by incubating each of these with gene 11 mRNA and 0, 1, or 5 pmol of rNSP2 octamer. As expected, analysis of the reaction products showed that rNSP2 inhibited the synthesis of dsRNA by open cores in a concentration-dependent manner (Fig. 7A, lanes 1–3). In contrast, rNSP2 had no measurable effect on the synthesis of dsRNA by SVPs, supporting the hypothesis that rNSP2 does not interfere with (⫺) strand elongation (lanes 4, 6, and 7). To test the possibility that rNSP2 did not inhibit dsRNA synthesis by SVPs because the protein lacked access to the RNA associated with the particles, we prepared SVPs from infected cell lysates which had been incubated with rNSP2. The SVPs were then divided into three pools: the first was exposed to UV light (to promote RNA–protein crosslinking), the second was mock exposed to UV light, and the third was incubated with RNase before exposure to UV

Fig. 7. Impact of NSP2 on synthesis of dsRNA by intracellular subviral particles. (A) Replication assays were performed which contained either virion-derived open cores or intracellular subviral particles (SVPs) and 1 pmol of added gene 11 mRNA and 0, 1, or 5 pmol of rNSP2 octamer. The 32 P-labeled dsRNA products were resolved by PAGE and detected by autoradiography. (B) SVPs were recovered from infected cell lysates which had been incubated with His-tagged rNSP2. Portions of the SVPs were either exposed to UV light, mock exposed to UV light, or exposed to UV light after RNAse treatment. After detergent treatment, RNA was recovered from the samples by pelleting in CsCl gradients and analyzed for associated rNSP2 by SDS–PAGE and Western blot assay using anti-His antibody and chemiluminescence.

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Fig. 8. Effect of NSP2 on the interaction of VP1 with viral RNA. (A) Reaction mixtures containing the 32P-labeled g8-3⬘ 60 RNA probe (lane 1), the probe and open cores (2), the probe and 1 pmol of rNSP2 octamer (3), or the probe and 10 pmol of rNSP2 octamer (4) were incubated at room temperature for 1 h. In parallel, reaction mixtures containing the RNA probe and open cores were incubated for 30 min. Then following the addition of 1 or 10 pmol of rNSP2 octamer (lane 5 and 6, respectively), the mixtures were incubated for another 30 min. Also in parallel, reaction mixtures containing the RNA probe and 1 or 10 pmol of rNSP2 octamer were incubated for 30 min (lanes 7 and 8, respectively). Then following the addition of open cores, the mixtures were incubated for 30 min more. (B) Reaction mixtures containing probe alone, probe and 2 pmol of rVP1, or probe and the indicated amount of rNSP2 in the presence or absence of rVP1 were incubated for 30 min. RNA–protein complexes in the reaction mixtures were detected by nondenaturing gel electrophoresis and autoradiography. Positions of VP1–, VP2–, and NSP2–probe complexes are indicated (Patton and Chen, 1999). (C) Portions of the gel shown in (B) labeled as “A”, “B”, and “C” were recovered and analyzed for the presence of rVP1 by SDS–PAGE and Western blot assay using guinea pig anti-open core antisera and chemiluminescence.

virus mRNAs (Patton and Chen, 1999) and reconstitution assays have determined that both VP1 and VP2 are required for the formation of the initiation complex for (⫺) strand synthesis (Tortorici et al., submitted for publication). The possibility that rNSP2 interfered with dsRNA in the open core system by preventing the binding of VP1 to the template mRNA was explored by EMSA using as probe, a 32 P-labeled RNA with a 3⬘-end that was identical in sequence to the last 60 nucleotides of the SA11 gene 8 mRNA (g8-3⬘60 probe). In the first step of the assay, reaction mixtures containing the g8-3⬘60 probe, GTP, and MgCl2 and either open cores or rNSP2 were incubated for 30 min. Afterward, rNSP2 and open cores were added to some of the mixtures and all the mixtures were incubated for an additional 30 min (Fig. 8A). As expected from earlier studies (Patton and Chen, 1999), EMSA of mixtures containing only open cores revealed two species of RNA–protein complexes in the gel, resulting from the interaction of probe with VP1 and VP3 (lane 2). Radiolabel detected in the well of lane 2 predominantly results from the binding of the probe to VP2 multimers that are too large to enter into the

polyacrylamide gel. Analysis of reaction mixtures containing rNSP2 yielded multiple species of slowly migrating RNA–protein complexes, produced by binding of one or more octameric units of the protein to individual molecules of probe (lanes 3 and 4). Cooperativity in the binding of octamers to RNA resulted in a shift in the formation of lower order rNSP2–probe complexes to higher order complexes as the concentration of rNSP2 in the reaction mixtures was increased (lanes 3 and 4) (Taraporewala et al., 1999). Comparison of the levels of VP1–probe complexes formed in reaction mixtures in which open cores were incubated with the probe prior to the addition of rNSP2 (lanes 5 and 6) versus reaction mixtures in which rNSP2 was incubated with the probe prior to the addition of open cores (lanes 7 and 8) showed that they were equivalent (Fig. 8A). This result indicated that rNSP2 did not affect the extent of interaction of VP1 with the 3⬘-end of viral RNA regardless of whether the RNA was incubated with rNSP2 before or after incubation with VP1. In contrast, the results obtained from open core replication assays (Fig. 6) revealed

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that preincubation of rNSP2 with template mRNA and the presence of rNSP2 in reaction mixtures inhibited dsRNA synthesis by interfering with the formation of the initiation complex for (⫺) strand synthesis (above). Taken together, the results suggested that rNSP2 inhibited dsRNA synthesis not by preventing interaction of the RNA polymerase with template mRNA, but rather by interfering with another event crucial for formation of initiation complexes. Although incubation of the g8-3⬘60 probe with rNSP2 before or after VP1 did not influence the levels of VP1– probe complexes that were formed (Fig. 8A, lanes 5 and 6 versus 7 and 8), fewer VP1–probe complexes were detected in reaction mixtures containing a higher concentration of rNSP2 (lanes 6 and 8) than were detected in mixtures containing a lower concentration of rNSP2 (lanes 5 and 7). One explanation for this is that the higher concentration of rNSP2 in reaction mixtures may have favored the interaction of both VP1 and rNSP2 with molecules of probe to form super VP1–rNSP2–probe complexes that would have migrated more slowly in the nondenaturing gel than VP1– probe complexes. To test this possibility, rVP1 was produced in insect cells infected with recombinant baculovirus containing a gene 1 cDNA of SA11 rotavirus. Purified rVP1 was incubated with the g8-3⬘60 probe in the absence of rNSP2 (Fig. 8B, lane 2) or in the presence of increasing amounts of rNSP2 (lanes 3– 6). The results showed that in some reaction mixtures containing both rVP1 and rNSP2 (lanes 3– 4), a novel slow-migrating complex was formed that was absent in reaction mixtures that lacked either rVP1 (lanes 7–10) or rNSP2 (lane 2). Analysis of the novel complex by SDS–PAGE and Western blot assay showed that it contained rVP1 (Fig. 8C). Together, these results suggest that the reduction in the levels of VP1–probe complexes observed as a consequence of adding higher levels of rNSP2 to open cores (Fig. 8A) was due to the conversion of these complexes to supercomplexes containing rVP1, rNSP2, and the probe. rNSP2 interferes with the function of VP2 in replication Both VP1 and VP2 are necessary for the formation of the (⫺) strand initiation complexes. To gain a better understanding of the mechanism by which rNSP2 interfered with dsRNA synthesis, rVP1 and rVP2 were produced in insect

Fig. 9. Synthesis of dsRNA by recombinant VP1 and VP2. Replication assays contained 6 pmol of gene 8 mRNA and either rVP1 (2 pmol) or rVP2 (40 pmol) or both rVP1 and rVP2. The reaction mixtures were incubated for 4 h at 37°C and 32P-labeled gene 8 dsRNA products were detected by PAGE and autoradiography.

cells infected with recombinant baculoviruses containing gene 1 or 2 cDNAs of SA11 rotavirus. Incubation of purified rVP1 (2 pmol) or rVP2 (40 pmol) with NTPs and gene 8 mRNA (6 pmol) showed that neither protein alone possessed the necessary replicase activity required to synthesize dsRNA (Fig. 9, lanes 1 and 2). In contrast, reaction mixtures containing both rVP1 and rVP2 had replicase activity that catalyzed the synthesis of gene 8 dsRNA (lane 3). These results are in agreement with those of a previous study which showed that VP1 requires VP2 for polymerase activity (Patton et al., 1997). Coincubation of rVP1, rVP2, mRNA, and GTP for 1 h under conditions favoring the formation of initiation complexes (initiation phase), followed by addition of NTPs and [32P]UTP and further incubation for 4 h (elongation phase), also supported the synthesis of gene 8 dsRNA (Fig. 10, lane 1). If rNSP2 octamers (10 pmol) were added to such an assay after the initiation phase, but prior to the elongation phase, the amount of dsRNA produced was reduced by ⬃50% (lane 2). Most likely, this reduction stemmed from rNSP2 preventing de novo formation of initiation complexes during the elongation phase of the assay (see Fig. 6). Coincubation of rVP1, mRNA, and GTP in reaction mixtures prior to the addition of rVP2, NTPs, and [32P]UTP (lane 3) supported dsRNA synthesis, although at a level slightly less (⬃30%) than that made by reaction mixtures in which both rVP1 and rVP2 were present during the initiation phase (lane 1). If the order of addition of rVP1 and rVP2 to the reaction mixtures was reversed, such that rVP2 was included in the initiation phase, and rVP1 was added for the elongation phase, the synthesis of dsRNA was reduced to ⬃50% (lane 5) of that made in assays in which rVP1 was added before rVP2 (lane 3). These results indicate that the order of interaction of rVP1 and rVP2 with mRNA templates in the reaction mixtures was an important factor affecting formation of RNA–protein complexes that support dsRNA synthesis. In particular, the data suggest that RNA– protein complexes with replicase activity are formed more efficiently if the RNA polymerase interacts with the template prior to the core matrix protein. Little or no dsRNA synthesis occurred in replication assays in which rNSP2 was added to reaction mixtures after the coincubation of rVP1, mRNA, and GTP but before the addition of rVP2, NTPs, and [32P]UTP (lane 4). In contrast, an intermediate level of dsRNA was produced in assays in which rNSP2 was added after the coincubation of rVP2, mRNA, and GTP, but before the addition of rVP1, NTPs, and [32P]UTP (lane 6). Indeed, the levels of dsRNA made in the latter assays were similar to those made in assays which differed only in that they lacked the added rNSP2 (lane 5). Thus, rNSP2 strongly inhibited dsRNA synthesis when allowed to interact with template mRNA prior to rVP2 but had little effect on dsRNA synthesis if incubated with template mRNA subsequent to rVP2 (lane 6). The presence of rNSP2 did not impede the ability of rVP1 to associate with the template mRNA to form initiation complexes (lane 6

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et al., 1996, 1998; Vende et al., 2002). To examine the impact of NSP5 on RNA replication, His-tagged rNSP5 was expressed in bacteria, purified by NTA-affinity column chromatography, and included in replication assays containing open cores. Analysis of the reaction products showed that the presence of 2 or 5 pmol of rNSP5 did not significantly effect the synthesis of dsRNA (Fig. 11). Likewise, the presence of 2 or 5 pmol of rNSP5 did not alter the inhibition of dsRNA synthesis caused by including 2.5 pmol of NSP2 octamers in replication assays (Fig. 11). These results indicate that, under these experimental conditions, rNSP5 neither interferes with RNA replication nor modulates the ability of rNSP2 to inhibit RNA replication.

Discussion Several rotavirus proteins with ssRNA-binding activity accumulate in viroplasms and are components of RIs with replicase activity. These proteins include the RNA polymerase VP1, the capping enzyme VP3, the core lattice protein VP2, the NTPase NSP2, and the phosphoprotein NSP5. The temporal order of interaction of these proteins with viral

Fig. 10. Impact of NSP2 on the assembly of the (⫺) strand initiation complex from recombinant proteins. Duplicated replication assays containing 5 mM GTP (but no ATP, CTP, or UTP), 6 pmol of gene 8 mRNA, and 2 pmol of rVP1, and/or 40 pmol of rVP2 were incubated for 1 h at 37°C. Subsequently, 10 pmol of rNSP2 octamer was added to one of each pair of reaction mixtures. After incubation for 1 h, ATP, CTP, UTP, and [32P]UTP were added to all mixtures, and rVP1 and rVP2 were added to those mixtures in which these proteins were absent. The mixtures were then incubated for another 4 h. The 32P-labeled dsRNA products of the assays were resolved by PAGE and detected by autoradiography. The intensities of the gene 8 dsRNA bands were normalized to that of the gene 8 dsRNA made in the reaction mixture in which rVP1 and rVP2 were present in the absence of rNSP2 through all stages of incubation (lane 1).

and Fig. 8). Taken together, the data indicate that rNSP2 inhibited dsRNA synthesis by preventing VP2 from participating in the formation of initiation complexes for (⫺) strand synthesis. rNSP5 does not modulate the inhibitory effect of NSP2 NSP5 is a dimeric nonspecific RNA-binding protein whose phosphorylation is upregulated by NSP2 (Afrikanova

Fig. 11. Effect of NSP5 on RNA replication in vitro. Replication assays contained 60 fmol of open cores, 1 pmol of gene 11 mRNA, and the indicated amount of rNSP2 and rNSP5 and were incubated for 2 h. 32 P-labeled gene 11 dsRNAs were detected by PAGE and autoradiography (A). The intensities of the gene 11 bands were determined and adjusted relative to the intensity of the gene 11 band of the reaction mixture lacking recombinant protein, which was set at 100%. The results of two independent experiments were averaged; the error bar represents the standard deviation (B).

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mRNA leading to the assembly of RIs has not been defined. However, characterization of RIs isolated from infected cells suggests that VP1 and VP3 precedes VP2, NSP2, and NSP5 in interacting with mRNA (Gallegos and Patton, 1989). Analysis of rotavirus RNA replication in vitro has indicated that two of these proteins, VP1 and VP2, are required for formation of the initiation complex for (⫺) strand synthesis (Tortorici et al., submitted for publication). In the study described herein, we have found that NSP2 inhibits dsRNA synthesis in vitro in a concentration-dependent manner, by interfering with the synthesis of (⫺) strand RNA from the mRNA template. Further analysis of this phenomenon revealed that NSP2 inhibited (⫺) strand initiation, but not elongation, and that the inhibition was a consequence of the nonspecific ssRNA-binding activity of NSP2. The use of rVP1 and rVP2 allowed us to determine that NSP2 impeded replication by interfering with the role of VP2 in forming the (⫺) strand initiation complex. In contrast, NSP2 did not prevent rVP1 from interacting with the 3⬘-end of viral mRNA, thereby suggesting that the RNA polymerase can participate in the formation of the initiation complex even in the presence of NSP2. In toto, these results suggest that the most efficient pathway for the assembly of RIs with replicase activity is to allow the core structural proteins to interact with the mRNA template prior to the nonstructural proteins. On the other hand, given that core RIs are assembled in viroplasms, the intracellular site at which both VP2 and NSP2 accumulate, it is difficult to understand how events in RI assembly could be orchestrated to allow VP2 to interact with the mRNA template before NSP2. Instead, we propose that VP2 and NSP2 compete for the mRNA and that under conditions in which the molar ratio of VP2 to NSP2 is high, the formation of core RIs is favored, thus leading to the packaging/assortment and replication of the genome. In situations where the ratio of VP2 to NSP2 is low, the interaction of NSP2 with the mRNA would impede dsRNA synthesis until sufficient VP2 became available to relieve the NSP2-dependent block of assembly of initiation complexes and core RIs. Since inhibition of dsRNA synthesis by NSP2 was suppressed by adding increasing amounts of ssRNA to open core assays, it may be concluded that a high molar ratio of mRNA to NSP2 in the viroplasm also provides an environment more favorable to the formation of initiation complexes and core RIs. Assays performed with rVP1 and rVP2 indicated that the order of interaction of these proteins with the mRNA template influences the efficiency of formation of complexes with replicase activity (Fig. 10). In particular, the results showed that the sequential interaction of VP1 and VP2 with the template produced approximately twice the number of replicase complexes than formed if the template was allowed to interact with VP2 prior to VP1. It may be extrapolated from this finding that the most efficient pathway for the assembly of RIs in vivo would be to allow VP1 to initially interact with template mRNA in an environment free of VP2, followed by movement

of the VP1–mRNA complex to an environment rich in VP2. In the context of the infected cell, this could be accomplished by allowing the mRNA to interact with VP1 outside viroplasms, producing a complex which then moves into viroplasms where subsequent interaction with VP2 could occur. This suggestion mirrors an earlier one which proposed that precore RIs were assembled outside viroplasms and subsequently moved into viroplasms where they underwent maturation into core RIs (Patton, 1994). The nature of the contribution that rVP2 makes to the formation of the initiation complex for (⫺) strand synthesis and to the induction of viral replicase activity is unknown. Based on earlier studies with purified recombinant proteins (Patton et al., 1997), the molar ratio of rVP1 to rVP2 required for maximal levels of dsRNA synthesis in vitro is ⬃1:10, a ratio which is similar to that for VP1 and VP2 (1:5 asymmetric dimers) present at the vertices of the T ⫽ 1 virion cores. The five dimers of VP2 have been proposed to form an RNA-binding platform on which the RNA polymerase can catalyze RNA synthesis (Prasad et al., 1996). Prior work with tsF, a rotavirus mutant with a temperaturesensitive (ts) lesion in its VP2 gene, has also provided in vivo evidence that VP2 must assemble into higher order structures for the RNA polymerase to carry out dsRNA synthesis (Mansell and Patton, 1990). Considering these findings, it is possible to speculate that rNSP2 inhibits dsRNA synthesis in vitro by impeding VP1–mRNA complexes from acting as nucleation points for the assembly of VP2 pentameric platforms. NSP2 may be recruited to VP1– mRNA complexes via its affinity not only for the ssRNA template but also for the viral RNA polymerase (Aponte et al., 1996; Kattoura et al., 1994). Although NSP2 is an inhibitor of initiation, our studies have shown that the protein does not interfere with elongation of (⫺) strand RNA in vitro by either open cores or SVPs. Indeed, studies of tsE, a mutant rotavirus with a ts lesion in the NSP2 gene, have provided evidence that NSP2 is necessary for packaging of rotavirus RNAs (Ramig and Petrie, 1984). Analysis of intracellular SVPs has indicated that dsRNA synthesis and mRNA packaging are concurrent events and that the mRNA template moves from the exterior to the interior of RIs during replication (Patton and Gallegos, 1990). Hence, mRNA packaging is a process that continues even after the synthesis of (⫺) strand RNA has commenced, and therefore, any role that NSP2 plays in packaging may likewise continue after the onset of (⫺) strand synthesis. The fact that NSP2 is a component of RIs recovered from infected cells that elongate associated nascent (⫺) strand RNAs is consistent with the protein having a role in replication postinitiation (Aponte et al., 1996; Patton and Gallegos, 1990). From the results of the earlier studies mentioned above and those reported herein, we suggest that NSP2 has a dual role in rotavirus genome replication, regulating the initiation of (⫺) strand synthesis and facilitating the packaging of the (⫹) strand template into core-like intermediates.

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Like NSP2, NSP5 has affinity for RNA (Vende et al., 2002), is a component of core RIs (Gallegos and Patton, 1989), and accumulates in viroplasms (Fabretti et al., 1999). NSP5 also interacts with two proteins, VP2 and NSP2, that are necessary for genome replication (Afrikanova et al., 1998; Berios et al., 2003). Its collective properties are highly suggestive of a role for NSP5 in dsRNA synthesis. However, our analysis of rNSP5 in replication assays failed to provide evidence of such a role, indicating instead that NSP5 does not affect (⫺) strand synthesis. Our analysis also suggests that NSP5 does not modulate the inhibitory activity of NSP2 on RNA replication. In the infected cell, NSP5 undergoes extensive posttranslational modification including phosphorylation and O-linked glycosylation (Afrikanova et al., 1996; Gonza´ lez and Burrone, 1991). The absence of such modifications for the bacterial-expressed rNSP5 used in our experiments may yield a form of the protein that is functionally deficient, lacking the capacity to affect RNA replication or to modulate NSP2 activity. As a consequence, experiments need to be performed with the appropriately modified form of NSP5 before a role for the protein in these processes can be fully excluded.

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was precipitated with ammonium sulfate from the soluble fraction of a homogenate prepared from rBVg1-infected Sf9 cells. rVP1 was isolated from the solubilized precipitate by sequential chromatography on heparin–Sepharose, cation exchange, and Superdex-200 exclusion columns. The purified protein was contained in 50 mM Hepes–NaOH, pH 7.8, 150 mM NaCl, 1 mM EDTA, 10% glycerol, and 2 mM ␤-mercaptoethanol. To prepare rVP2, rBVg2-infected cells were pelleted by low-speed centrifugation, washed with phosphate-buffered saline, and resuspended in hypotonic buffer (3 mM Tris– HCl, pH 8.1, 10 mM NaCl, 1.5 mM MgCl2) containing 0.5% Triton X-100 (Patton et al., 1997). Following incubation on ice for 3 min, the sample was subjected to low-speed centrifugation. The pellet was resuspended in hypotonic buffer containing 1.31 g/cm3 of CsCl. After centrifugation for 18 h at 120,000g in a Beckman SW55 rotor, rVP2 banding at a density of 1.30 g/cm3 was collected and dialyzed extensively against LSB. Concentrations of purified proteins were determined by Bradford assay using bovine serum albumin (BSA) as the standard and by comparison with known amounts of BSA coelectrophoresed on sodium dodecyl sulfate–polyacrylamide gels and stained with Coomassie blue.

Materials and methods RNAs Expression and purification of recombinant proteins rNSP2 and rNSP5 were expressed in Escherichia coli M15[pREP4] from the vectors pQE60g8 and pQE30g11 and contained N- and C-terminal tags, respectively, of six His residues (Taraporewala et al., 1999; Vende et al., 2002). The proteins were purified from bacterial lysates by NTAaffinity column chromatography (Qiagen). The eluted rNSP2 was dialyzed against low-salt buffer (LSB) (2 mM Tris–HCl, pH 7.5, 0.5 mM EDTA, 0.5 mM dithiothreitol [DTT]), and the eluted rNSP5 was dialyzed against LSB containing 200 mM NaCl. The proteins were stored at 4°C. The rNSP2 preparation was verified to consist of octamers by sedimentation on a 5 to 20% sucrose gradient for 16 h at 200,000g in a Beckman SW40Ti rotor (Taraporewala et al., 1999). Gradient fractions were analyzed by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) and staining with Coomassie blue. The recombinant baculoviruses rBVg1 and rBVg2 contain cDNAs of the VP1 and VP2 genes, respectively, of simian SA11 rotavirus (Patton et al., 1997). To produce rVP1 and rVP2, spinner cultures of Sf21 and Sf9 cells, respectively, were infected at a multiplicity of infection (m.o.i.) of 2 with the appropriate recombinant baculovirus and maintained in TNM-FH medium containing 2% fetal bovine serum. One microgram per milliliter each of the protease inhibitors leupeptin and aprotinin was also included in the medium of rBVg2-infected cells. A detailed protocol for the purification of rVP1 is given elsewhere (Tortorici et al., submitted for publication). Briefly, rVP1

The T7 transcription vectors, SP65g8 and SP65g11cn86, contain full-length cDNAs of rotavirus SA11 gene 8 and CN86 gene 11 RNAs, respectively. Prior to transcription, the vectors were linearized by digestion with SacII and treated with T4 DNA polymerase. Transcripts were made from the linearized vectors using an Ambion T7 transcription kit. The quality of the RNA products was analyzed by electrophoresis on 5% polyacrylamide gels containing 7 M urea (Patton et al., 1996). RNA concentrations were determined from optical densities at 260 nm. To generate the vector T7B/T3-60, polymerase chain reaction (PCR) was used to prepare amplified DNA containing a T3 promoter linked to a sequence corresponding to the last 60 nucleotides of plus-sense gene 8 RNA. The reaction mixture included Elongase (Invitrogen), the plussense primer 5⬘-gggATTCGCTATCAATTTGAGGAT-3⬘ (T3 promoter is underlined, viral-specific sequences in uppercase), the minus-sense primer 5⬘-actcctgcattaggaagcagc3⬘, and the template SP65g8 (Patton et al., 1996). The amplification product was purified by elution from an agarose gel and ligated into the Novagen PCR vector, pT7Blue. Dideoxynucleotide sequencing was used to verify the accuracy of the sequence of T7B/T3-60. To produce the template for synthesis of the RNA probe g8-3⬘60, PCR was use to amplify a portion of T7B/T3-60 extending from upstream of the T3 promoter to the 3⬘-end of the gene 8-specific sequence. The plus-sense and minus-sense primers used in this amplification were 5⬘-ttctagtgtagccgtagttaggcc-3⬘ and 5⬘-GGTCACATAAGCGCTTTCTATTC-3⬘. The product

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was treated with T4 DNA polymerase and transcribed with an Ambion T3 Megascript kit, yielding 32P-labeled g8-3⬘60 RNA. Transcription was performed under the conditions suggested by the supplier of the kit except that the concentration of cold UTP was reduced by one-fourth and 2.5 ␮Ci of [␣-32P]UTP (800 Ci/mmol) was included. The probe was purified by elution from an 8% polyacrylamide gel containing 7 M urea. The probe g8-3⬘ 60 is 63 nucleotides in length with the last 60 nucleotides identical to those at the 3⬘-end of the SA11 gene 8 mRNA. Replication assays Open cores were prepared from CsCl-purified preparations of the mono-reassortant virus D X RRV (Midthun et al., 1985), as described previously (Chen and Patton, 1998). SVPs were recovered at 9 h postinfection from 10 ⫻ 10-cm plates of MA104 cells infected at an m.o.i. of 10 with SA11-4F (Gallegos and Patton, 1989). The SVPs were resuspended in a total of 50 ␮l of HGD buffer (10 mM Hepes–HCl, pH 7.6, 10% glycerol, 2 mM dithioerythritol). Unless otherwise indicated, open core replication assays included 50 mM Tris–HCl, pH 7.1, 10 mM magnesium acetate, 1.5% polyethylene glycol, 2 mM DTT, 20 U RNasin (Promega), 1.25 of each of the four NTPs, 0.5 ␮Ci of [␣-32P]UTP (800 Ci/mmol), approximately 60 fmol of D X RRV open cores (⬃0.6 pmol VP1, 7.5 pmol VP2), and 1 pmol of gene 8 or gene 11 mRNA. In some cases, replication assays were performed in which open cores were replaced with 2.5 ␮l of SVPs. Assays performed with rVP1 and rVP2 contained 50 mM Tris–HCl, pH 7.1; 1.5% polyethylene glycol; 20 U RNasin; 2 mM DTT; ATP, CTP, and UTP (1.25 mM each); 5 mM GTP; 20 mM magnesium acetate; and 2 mM manganese acetate. The final volume of the reaction mixtures was 20 ␮l and the mixtures were typically incubated for 2 h at 32°C. Replication assays performed with rVP1 and rVP2 were incubated for 4 h at 37°C. 32P-labeled dsRNA synthesized by the reaction mixtures was resolved by electrophoresis on 12% polyacrylamide gels containing SDS and detected by autoradiography. Band intensities were quantified with Molecular Dynamics Phosphorimager 445SI. The components of the open core initiation assay were the same as those of the standard open core replication assay except that the initiation assay contained 2.5 mM GTP and lacked cold ATP, CTP, and UTP, and [32P]UTP. In some cases, 0.2 M NaCl or 10 pmol of rNSP2 octamer was also included in the initiation assay. After incubation for 1 h at 32°C, ATP, CTP, GTP, and UTP (2.5 mM each) and [32P]UTP were added to the initiation assay, thereby modifying the reaction components such that elongation was supported. In some assays, NaCl or rNSP2 was also added to the reaction mixtures along with the missing NTPs. The presence of NaCl was shown earlier to prevent initiation but to allow elongation in replication assays (Chen and Patton, 2000).

Electrophoretic mobility shift assays The procedures used to analyze the interaction of viral proteins and RNA probes have been described previously (Patton, 1996). Assay mixtures contained 1 pmol of 32Plabeled g8-3⬘ 60 probe, 1.25 mM GTP, 5 mM magnesium acetate, 2 mM DTT, and the indicated amounts of open cores, rVP1, and/or rNSP2 and were incubated at room temperature. Mixtures were analyzed for the presence of RNA–protein complexes by electrophoresis on nondenaturing 8% polyacrylamide gels and autoradiography (Patton, 1996). Interaction of rNSP2 with RNA of SVPs Cell lysates were prepared at 6 h postinfection from three 10-cm dishes of MA104 cells infected with rotavirus SA11-4F using Dounce homogenization as described before (Gallegos and Patton, 1989). The lysate was incubated with 50 ␮g of His-tagged rNSP2 for 30 min at room temperature. SVPs were recovered from the lysate by pelleting through a 15 to 30% sucrose gradient (Gallegos and Patton, 1989) and resuspended in 100 ␮l of TMN buffer (3 mM Tris–HCl, pH 8.1, 66 mM NH4Cl, 3 mM magnesium acetate, 14 mM potassium acetate, 1 mM dithioerythritol). Ten microliters of the SVP suspension was added to each of three tubes (A, B, and C) along with 10 ␮l of TMN buffer. The Tube A sample was exposed to 254-nm UV-light at a distance of 4 cm for 10 min on ice. Tube B was mocked exposed to UV light. Tube C was incubated with 0.25 mg RNase A for 15 min at 30°C and then exposed to UV light. After addition of 250 ␮l of 1% deoxycholate, all three samples were incubated for 15 min at 37°C. The samples were overlaid onto 4.5-ml gradients of 1.38 g/cm3 of CsCl which were centrifuged at 10°C in a Beckman SW55 rotor for 24 h at 110,000g. The RNA pellets were resuspended in 100 ␮l of TE buffer (10 mM Tris–HCl, pH 7.4, 1 mM EDTA). Portions of the RNA samples (10 ␮l) were digested with 0.2 mg of RNase A for 15 min at 37°C and analyzed for the presence of rNSP2 by Western blot assay using anti-His antibody (1:1000) (Taraporewala et al., 1999).

Acknowledgment We appreciate the help of Dayue Chen on this project.

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