Deletion of highly conserved arginine-rich RNA binding motif in cowpea chlorotic mottle virus capsid protein results in virion structural alterations and RNA packaging constraints

Abstract

The N-proximal region of cowpea chlorotic mottle virus (CCMV) capsid protein (CP) contains an arginine-rich RNA binding motif (ARM) that is also found in the CPs of other members of Bromoviridae and in other RNA binding proteins such as the Tat and Rev proteins of human immunodeficiency virus. To assess the critical role played by this motif during encapsidation, a variant of CCMV RNA3 (C3) precisely lacking the ARM region (C3/Delta919) of its CP gene was constructed. The biology and the competence of the matured CP derived in vivo from C3/Delta919 to assemble and package progeny RNA was examined in whole plants. Image analysis and computer-assisted three-dimensional reconstruction of wild-type and mutant virions revealed that the CP subunits bearing the engineered deletion assembled into polymorphic virions with altered surface topology. Northern blot analysis of virion RNA from mutant progeny demonstrated that the engineered mutation down-regulated packaging of all four viral RNAs; however, the packaging effect was more pronounced on genomic RNA1 and RNA2 than genomic RNA3 and its CP mRNA. In vitro assembly assays with mutant CP subunits and RNA transcripts demonstrated that the mutant CP is inherently not defective in packaging genomic RNA1 (53%) and RNA2 (54%), but their incorporation into virions was competitively inhibited by the presence of other viral RNAs. Northern blot analysis of RNA encapsidation in vivo of two distinct bromovirus RNA3 chimeras, constructed by exchanging CPs having the Delta919 deletion, demonstrated that the role of the conserved N-terminal ARM in recognizing and packaging specific RNA is distinct for each virus.

FIG. 1.

Characteristics of CCMV CP variants. (A) The structure of C3 is shown, with noncoding sequences represented as single lines and MP and CP genes shown as open and stippled boxes, respectively. A filled circle at the 5′ end and a cloverleaf at the 3′ end represent cap and tRNA-like structures, respectively. The first 25 N-proximal amino acids are shown, and the boxed region represents the N-terminal ARM conserved among plant and nonplant viruses. In CCMV, the initiating methionine (enclosed in parenthesis) is removed, and the resultant N-terminal serine is acetylated in the mature CP (21). In variant C3/Δ919, the deletion of the ARM region, located between amino acids 8 and 20, is indicated by a broken line. In variants C3/P10, C3/P13, CP/P18, and C3/CP19, arginine residues located at positions 10, 13, 18, and 19, respectively, are replaced by a proline residue. (B) Replication competence of C3/Δ919 in protoplasts. Protoplasts were transfected with WT C1 and C2 and either WT C3 or C3/Δ919. After a 24-h incubation, total RNA was isolated and subjected to Northern blot analysis. The positions of four WT CCMV RNAs are shown to the left. (C) Electron microscopy of purified virions. Virions were purified from symptomatic leaves, applied to glow-discharged carbon-coated copper grids and negatively stained with 1% uranyl acetate prior to viewing under an electron microscope. Prior to application of the sample onto the grids, WT samples were diluted 1:10 while the Δ919 samples remain undiluted. (D) Analysis of viral capsid protein. Purified virions resulting from infections of either WT C3 or C3/Δ919 were suspended in sample buffer, boiled for 5 min, and subjected to SDS-10% PAGE. The gel was stained with Coomassie brilliant blue prior to photography. An arrow indicates the position of WT CP. Lane M, molecular weight marker proteins.

FIG. 2.

Electron microscopy and image analysis of WT CCMV and C3/Δ919 virions. Electron micrographic images of purified virions of WT CCMV (A to D) and Δ919 (E to F). Images shown in panels A and E represent typical regions of electron micrographs of WT and Δ919 mutant virus particles, respectively, negatively stained with 1% uranyl acetate. Images shown in panels B and F represent class sums after the alignment by classification step. Uniform size was observed for the WT (B), whereas for the Δ919 mutant, two particle sizes were observed In panel F, particles shown in the first three images from the left are WT size, while particles shown in the remaining two images are approximately 1.5 nm smaller in diameter than those of the WT. Images C and G show averages after the final MRA for WT and the larger of the two Δ919 populations, respectively. Images D and H represent the final three-dimensional reconstructions for the WT and the larger Δ919 particles, respectively. The positions of the five-, three-, and twofold symmetry axes are indicated. Scale bars are 60 nm (A and E) and 10 nm (B through H). The resolution of the final models was estimated to 25 Å by the Fourier shell criterion with a cutoff of 0.5 for both WT and mutant virions.

FIG. 3.

Analysis of virion RNA content of C3/Δ919 virions. (A) Native agarose gel analysis of viral RNA. RNA isolated from purified virion preparations of WT C3 or C3/Δ919 was subjected to electrophoresis in 1% agarose and stained with ethidium bromide. (B) Northern blot analysis of total nucleic acid (T) and virion RNA (V) preparations recovered from symptomatic cowpea leaves inoculated with WT C1 and C2 and either C3 or C3/Δ919. Approximately 5 μg of total nucleic acid and 200 ng of virion RNA were denatured with formamide-formaldehyde and subjected to 1.5% agarose gel electrophoresis prior to vacuum blotting to a nylon membrane. The blot was hybridized with a ³²P-labeled riboprobe complementary to the commonly shared 3′ noncoding region of CCMV RNAs. The autoradiograph shown in panel C represents a longer exposure image of panel B. (D) Concentrations of C3/Δ919 virion RNA ranging between 0.4 to 3.2 μg per ml were subjected to Northern hybridization as described above. The autoradiograph shown in panel E represents a longer exposure image of panel D. The positions of four CCMV RNAs are shown to the left.

FIG. 4.

Gel retardation analysis of RNAs by WT and Δ919 CP subunits. The desired CCMV RNA transcript (approximately 30 nM) was titrated with the indicated amounts of CP dimers of either WT or Δ919 for 20 min or 24 h at 20°C. After incubation the samples were loaded onto prepared 1% agarose gels and electrophoresed in Tris-acetate-EDTA buffer. A sample of native CCMV virions, assembled with 90 CP dimers, of purified symptomatic cowpea leaves is shown on the right. The arrowhead shown to the left and the asterisk shown to the right of each panel, respectively, indicate the relative mobility of free viral RNA and purified WT virions (v). The position of the C1 complex formed in a 24-h incubation with RNA1 samples is indicated by a bracket.

FIG. 5.

Encapsidation competence of CCMV RNAs with N-terminal ARM mutants. (A) In vitro assembly assays. Northern blot analysis of RNA isolated from virions assembled in vitro with Δ919 CP subunits and each of the three genomic RNA transcripts (panels I to III), their mixture (panel IV), virion RNA (panel V), and a mixture containing all three BMV RNA transcripts (panel VI). Purified CP subunits and the indicated RNAs were allowed to assemble in vitro as described in Materials and Methods. Conditions for denaturizing RNA, electrophoresis, and hybridization with riboprobes are as described in the legend of Fig. 3. RNA samples shown in panels I to V were hybridized with a probe complementary to the 3′ end of CCMV RNA, whereas the RNA sample shown in panel VI was hybridized with a probe complementary to the 3′ end of BMV RNA. The numbers shown in parentheses below panels I to III represent the percentages of assembly efficiency of Δ919 CP subunits for each WT RNA transcript with respect to WT CP subunits. (B and C) Packaging profiles of CCMV CP bearing N-terminal proline mutations (Fig. 1A). Shown are Northern blots of total nucleic acids or virion RNA recovered from symptomatic leaves of cowpea infected with the indicated C3 mutant. The blots were hybridized with ³²P-labeled RNA probes complementary to the homologous 3′ region present on each of the four CCMV RNAs. The positions of CCMV RNAs are shown to the left of each blot.

FIG. 6.

Characteristic features and biological activity of RNA3 chimeras of CCMV and BMV. (A) Schematic representation of B3 and C3 chimeras bearing heterologous CP with Δ919 mutation. The structures of C3/Δ919 and B3/Δ919 are shown, with noncoding sequences represented as single lines and the MP and CP as rectangle boxes. A filled circle at the 5′ end and a cloverleaf at the 3′ end represent cap and tRNA-like structures, respectively. In each case, the sequence of the N-proximal amino acid region lacking amino acids 9 to 19 (indicated by a dashed line) is shown. (B) Symptom phenotypes induced by WT and chimeras in C. quinoa. (C) Northern hybridization of total nucleic acid (T) and virion RNA (V) preparations recovered from C. quinoa plants inoculated with a mixture containing WTB1+WTB2+B3/CCPΔ919 (B1, BMV RNA1; B2, BMV RNA2) and WTC1+WTC2+C3/BCPΔ919. The conditions for Northern hybridization are as described in the legend of Fig. 3.