Reovirus protein sigmaNS binds in multiple copies to single-stranded RNA and shares properties with single-stranded DNA binding proteins

Abstract

Reovirus nonstructural protein sigmaNS interacts with reovirus plus-strand RNAs in infected cells, but little is known about the nature of those interactions or their roles in viral replication. In this study, a recombinant form of sigmaNS was analyzed for in vitro binding to nucleic acids using gel mobility shift assays. Multiple units of sigmaNS bound to single-stranded RNA molecules with positive cooperativity and with each unit covering about 25 nucleotides at saturation. The sigmaNS protein did not bind preferentially to reovirus RNA over nonreovirus RNA in competition experiments but did bind preferentially to single-stranded over double-stranded nucleic acids and with a slight preference for RNA over DNA. In addition, sigmaNS bound to single-stranded RNA to which a 19-base DNA oligonucleotide was hybridized at either end or near the middle. When present in saturative amounts, sigmaNS displaced this oligonucleotide from the partial duplex. The strand displacement activity did not require ATP hydrolysis and was inhibited by MgCl(2), distinguishing it from a classical ATP-dependent helicase. These properties of sigmaNS are similar to those of single-stranded DNA binding proteins that are known to participate in genomic DNA replication, suggesting a related role for sigmaNS in replication of the reovirus RNA genome.

FIG. 1

Gel mobility shift assay with a 151-nucleotide fragment of reovirus S4 plus strand and immunoblot analysis of a similar assay with anti-ςNS serum. (A) Increasing amounts of purified ςNS were incubated with 0.21 pmol of radiolabeled ssRNA comprising the 5′ 151 nucleotides of the reovirus S4 plus strand (5′S4-151). The samples were then subjected to electrophoresis in a 5% Tris-glycine polyacrylamide native gel until the free RNA was near the bottom of the gel. The radiolabeled RNA in the dried gel was visualized by phosphorimaging. ςNS-RNA complexes with six distinct mobilities are indicated at left. (B) Increasing amounts of ςNS were incubated with and without unlabeled 5′S4-151 RNA (0.8 pmol) and subjected to electrophoresis as described above. An immunoblot assay was performed to detect the protein. The amounts of RNA and protein in these samples were increased so that the ratios were similar to those in panel A but the protein was more easily detected. Arrows, comparable bands in panels A and B.

FIG. 2

Gel mobility shift assays with S4 plus-strand RNA fragments ranging in size from 121 to 211 nucleotides in 30-base increments. Radiolabeled ssRNA fragments comprising the following 5′ portions of the S4 plus strand were synthesized: 121 nucleotides, 5′S4-121; 151 nucleotides, 5′S4-151; 181 nucleotides, 5′S4-181; and 211 nucleotides, 5′S4-211. (A) Increasing amounts of purified ςNS were separately incubated with 0.21 to 0.22 pmol of each RNA, and the samples were analyzed as described for Fig. 1A. ςNS-RNA complexes with five to eight distinct mobilities are indicated to the left of each gel. (B) Purified ςNS (18 pmol) was incubated with each of the four RNAs as described for panel A (RNA sizes are indicated above the lanes) and subjected to electrophoresis as described for Fig. 1A. To provide better separation of the shifted complexes, the gel was run longer in this experiment such that the free RNA was run off the bottom.

FIG. 3

Hill plot of ςNS binding to ssRNA to determine cooperativity. A gel shift assay with 0.24 pmol of radiolabeled 5′S4-121 RNA was performed as described for Fig. 2A except that the concentrations of purified ςNS ranged from 0.24 to 6 pmol. The amount of ςNS bound at each concentration was calculated as described in Materials and Methods. Log₁₀ (bound ςNS/1 − bound ςNS) was plotted relative to log₁₀ (ςNS concentration), and a best-fit line was calculated for the linear portion of the graph (the equation and coefficient of determination for the line are shown in the box).

FIG. 4

Competition assays for ςNS binding to ssRNA using ssRNA, dsRNA, ssDNA, or dsDNA as the competitor. Each data point represents the mean from three experiments, and the error bars represent the standard deviation of the mean. (A) Increasing concentrations of unlabeled competitor ssRNA, dsRNA, dsDNA, or ssDNA (see Materials and Methods) were combined with 0.24 pmol of radiolabeled 5′S4-121 ssRNA. Purified ςNS (4.8 pmol) was added to each sample, and the sample was incubated. The samples were subjected to electrophoresis and visualized as described for Fig. 1A. The upper four shifted RNA bands were included in the quantitation of bound RNA. The ratios of competitor to probe RNA were calculated from weights rather than molar amounts of nucleic acid to reflect numbers of potential ςNS binding sites rather than numbers of nucleic acid molecules. (B) The assay was performed as described for panel A except that the unlabeled competitor ssRNAs were 121 nucleotides from the 5′ end of the S4 plus strand (5′S4-121), the 3′ end of the S4 plus strand (3′S4-121), or the vector sequence (pGEM-121). For one set of samples, ςNS was added to the labeled RNA prior to addition of the 5′S4-121 competitor.

FIG. 5

Gel shift assay with ssRNA having an RNA-DNA duplex region at its 3′ end. A 19-nucleotide DNA oligonucleotide that was exactly complementary to the 3′ end of the RNA was ³²P-labeled at its 5′ end. (A) The oligonucleotide was hybridized to unlabeled ssRNA (5′S4-121) and purified. Differing amounts of purified ςNS were then mixed with 0.26 pmol of hybrid and assayed as described for Fig. 1A. (B) An unhybridized ³²P-labeled oligonucleotide was incubated in the presence of increasing amounts of ςNS and also assayed as described for Fig. 1A. For both panels, the amounts of ςNS added to samples are indicated beneath the lanes, and the positions of the RNA-DNA hybrid and the DNA oligonucleotide are also indicated.

FIG. 6

Analysis of ATP hydrolysis during strand displacement by ςNS. Either 12.5 mM EDTA (+) or H₂O (−) was added to 75 pmol of purified ςNS (ςNS) or 6 × 10⁹ reovirus core particles (cores) in each sample. Cores were included as a positive control for ATP hydrolysis. All samples were analyzed in duplicate. (A) The RNA-DNA hybrid described for Fig. 5 (0.11 pmol) was added to each sample in one set. These samples were then analyzed as described for Fig. 1A. The positions of the RNA-DNA hybrid and the DNA oligonucleotide are indicated. (B) Three microcuries of [α-³²P]ATP and 0.11 pmol of the RNA-DNA hybrid were added to each sample in the other set. Products of the reaction with ATP were resolved by thin-layer chromatography and analyzed by phosphorimaging. The positions of unlabeled ATP, ADP, and AMP markers, as determined by UV absorption, are indicated.