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. 2002 Aug;76(15):7485-94.
doi: 10.1128/jvi.76.15.7485-7494.2002.

Sequence requirements for viral RNA replication and VPg uridylylation directed by the internal cis-acting replication element (cre) of human rhinovirus type 14

Yan Yang  1 , Rene Rijnbrand, Kevin L McKnight, Eckard Wimmer, Aniko Paul, Annette Martin, Stanley M Lemon
Affiliations

Sequence requirements for viral RNA replication and VPg uridylylation directed by the internal cis-acting replication element (cre) of human rhinovirus type 14

Yan Yang et al. J Virol. 2002 Aug.

Abstract

Until recently, the cis-acting signals required for replication of picornaviral RNAs were believed to be restricted to the 5' and 3' noncoding regions of the genome. However, an RNA stem-loop in the VP1-coding sequence of human rhinovirus type 14 (HRV-14) is essential for viral minus-strand RNA synthesis (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998). The nucleotide sequence of the apical loop of this internal cis-acting replication element (cre) was critical for RNA synthesis, while secondary RNA structure, but not primary sequence, was shown to be important within the duplex stem. Similar cres have since been identified in other picornaviral genomes. These RNA segments appear to serve as template for the uridylylation of the genome-linked protein, VPg, providing the VPg-pUpU primer required for viral RNA transcription (A. V. Paul et al., J. Virol. 74:10359-10370, 2000). Here, we show that the minimal functional HRV-14 cre resides within a 33-nucleotide (nt) RNA segment that is predicted to form a simple stem-loop with a 14-nt loop sequence. An extensive mutational analysis involving every possible base substitution at each position within the loop segment defined the sequence that is required within this loop for efficient replication of subgenomic HRV-14 replicon RNAs. These results indicate that three consecutive adenosine residues (nt 2367 to 2369) within the 5' half of this loop are critically important for cre function and suggest that a common RNNNAARNNNNNNR loop motif exists among the cre sequences of enteroviruses and rhinoviruses. We found a direct, positive correlation between the capacity of mutated cres to support RNA replication and their ability to function as template in an in vitro VPg uridylylation reaction, suggesting that these functions are intimately linked. These data thus define more precisely the sequence and structural requirements of the HRV-14 cre and provide additional support for a model in which the role of the cre in RNA replication is to act as template for VPg uridylylation.

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Figures

FIG. 1.
FIG. 1.
Organization of the subgenomic HRV-14 replicon, ΔP1LucCRE. In ΔP1LucCRE, all of the P1 region was replaced with luciferase coding sequence, except for the 21 nt coding for the carboxy-terminal 7 aa of VP1 (12). The HRV-14 cre was inserted in frame between the luciferase and residual VP1 coding sequences at two engineered restriction sites, XhoI and NheI. D1, D2, and D3 deletion mutants contain progressively lengthier deletion mutations flanking the apical stem-loop of the HRV-14 cre and were constructed as described in Materials and Methods. The MFOLD-predicted secondary structures of the cre in the parental HRV-14 cre and the three deletion mutants are shown to the right of each construct. ΔP1LucXN is identical to ΔP1LucCRE, but lacks any cre sequence and is replication incompetent when transfected into HeLa cells (12).
FIG. 2.
FIG. 2.
(A) Luciferase activity derived from triplicate transfections of RNAs transcribed from plasmids pΔP1LucCRE and pΔP1LucXN. Transfected cells were processed and analyzed for luciferase activity at 3, 6, 12, and 24 h following transfection: ⧫, ΔP1LucCRE in the absence of guanidine; ∗, ΔP1LucCRE in the presence of 2 mM guanidine, added immediately after transfection; and ΔP1LucXN in the absence (▪) or presence (▴) of guanidine. (B) Luciferase activities expressed by ΔP1LucCRE, ΔP1LucXN, and the deletion mutants shown in Fig. 1, following RNA transfection of HeLa cells. At 3 h (open columns) and 24 h (solid columns) following transfection, cell lysates were harvested and assayed for luciferase activity. The results are the average of three independent transfections with error bars indicating the standard deviation. ∗, less than 100 light units.
FIG. 3.
FIG. 3.
Mutagenesis analysis of the loop of the HRV-14 cre. (A) Multiple single-nucleotide substitutions (shown in italic type outside of the circle) were created at each nucleotide between G63 and 76A in the loop of the cre. The underlined substitutions are those that were predicted by MFOLD to introduce changes in the secondary structure of the loop region. (B) MFOLD-predicted structures of the single nt 69U, 76C, or 76U mutants. The predicted structures of the 76C and 76U mutants were identical. (C) MS1 and MS2 are cre mutants with multiple nucleotide substitutions: MS1 contains 63U, 64U, 75U, and 76U substitutions; and MS2 contains 72U and 73C. In this and latter figures, the nucleotide position is indicated by the last two unique digits of the nucleotide map position (i.e., “63U” is nt 2363).
FIG. 4.
FIG. 4.
Luciferase expression as a measure of replication of HRV-14 cre mutants in transfected HeLa cells. At 3 and 24 h following transfection, cell lysates were harvested and assayed for luciferase activity as described in Materials and Methods. Each column represents the fold increase in luciferase activity observed with a single RNA construct. The efficiency of RNA amplification was calculated as the ratio of luciferase expression at 24 h relative to luciferase expression at 3 h and compared with that observed with the parent ΔPLucCRE (set as 100%). The dashed line indicates a fold increase in luciferase activity equivalent to 15% of that observed with the parent RNA. Greater fold increases in luciferase activity were considered indicative of significant RNA amplification, while lesser increases were considered to be due primarily to translation from input RNA (see text). wt, wild type.
FIG. 5.
FIG. 5.
Summary of the effect of nucleotide substitutions within the HRV-14 cre loop on RNA replication. Only the loop and the proximal 4 bp within the stem are shown in this figure. (A) Replication-competent HRV-14 cre mutants. (B) Mutations with a lethal effect on HRV-14 replication that represent those contributing critically to cre function (see legend to Fig. 4). Underlined substitutions are those predicted by MFOLD to introduce changes in the secondary structure of the RNA. The nucleotides in triangles are those for which no substitution was permissible, while the nucleotides in the squares are those for which some substitutions were permissible. At the other loop positions, any nucleotide substitution was permissible, provided that it did not introduce a stop codon.
FIG. 6.
FIG. 6.
Capacity of mutated HRV-14 cre RNA transcripts to support in vitro VPg uridylylation. (A) The products of in vitro VPg uridylylation reactions were separated on Tricine-SDS-PAGE gels (13.5% polyacrylamide) and visualized by autoradiography. WT, wild type (ΔPLucCRE). (B) Quantitation of VPg uridylylation reaction products by PhosphorImager. Each column represents the relative abundance of uridylylated VPg relative to the amount produced in control reactions containing the parental cre RNA (set at 100%). The dashed line indicates 15% of the amount of product produced with the parent RNA.
FIG. 7.
FIG. 7.
Least-squares-fit plot showing the correlation between the efficiencies of each mutant cre to support in vitro VPg uridylylation and to function in RNA replication, based on the data presented in Fig. 4 and 6. The dotted lines represent values for replication capacity and uridylylation activity equal to 15% of the wild-type cre. The R2 value obtained in a transformed regression model was 0.7685.
FIG. 8.
FIG. 8.
Comparison of the sequences and predicted structures of the cres of human rhinoviruses and enteroviruses. (A) Alignments of the sequence of the HRV-14 cre with the PV-1-type 1 cre sequence (left) and the HRV-2 cre sequence (right). The brackets depict the nucleotide segments predicted by MFOLD to form the terminal loops of the cre hairpins. (B) MFOLD predictions of the structures of the cres of HRV-14, PV-1 (5), HRV-2 (4), and the predicted cres of HRV-1b and -16 (4). The conserved critical bases identified by mutational analysis of the HRV-14 cre are shown in boldface. The proposed equivalent structures in the PV-1 and HRV-2 cres are enclosed in dashed boxes. At the far right is shown the proposed common structure for HRV and enterovirus cres. R = A/G; W = A/U; M = A/C; N = any nucleotide.
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References

    1. Andino, R., G. E. Rieckhof, P. L. Achacoso, and D. Baltimore. 1993. PV-1 RNA synthesis utilizes an RNP complex formed around the 5′-end of viral RNA. EMBO J. 12:3587-3598. - PMC - PubMed
    1. Duconge, F., C. Di Primo, and J. J. Toulme. 2000. Is a closing “GA pair” a rule for stable loop-loop RNA complexes? J. Biol. Chem. 275:21287-21294. - PubMed
    1. Flanegan, J. B., R. F. Petterson, V. Ambros, N. J. Hewlett, and D. Baltimore. 1977. Covalent linkage of a protein to a defined nucleotide sequence at the 5′-terminus of virion and replicative intermediate RNAs of PV-1. Proc. Natl. Acad. Sci. USA 74:961-965. - PMC - PubMed
    1. Gerber, K., E. Wimmer, and A. V. Paul. 2001. Biochemical and genetic studies of the initiation of human rhinovirus 2 RNA replication: identification of a cis-replicating element in the coding sequence of 2Apro. J. Virol. 75:10979-10990. - PMC - PubMed
    1. Goodfellow, I., Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans. 2000. Identification of a cis-acting replication element within the poliovirus coding region. J. Virol. 74:4590-4600. - PMC - PubMed

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