Secondary structural elements within the 3' untranslated region of mouse hepatitis virus strain JHM genomic RNA

Abstract

Previously, we characterized two host protein binding elements located within the 3'-terminal 166 nucleotides of the mouse hepatitis virus (MHV) genome and assessed their functions in defective-interfering (DI) RNA replication. To determine the role of RNA secondary structures within these two host protein binding elements in viral replication, we explored the secondary structure of the 3'-terminal 166 nucleotides of the MHV strain JHM genome using limited RNase digestion assays. Our data indicate that multiple stem-loop and hairpin-loop structures exist within this region. Mutant and wild-type DIssEs were employed to test the function of secondary structure elements in DI RNA replication. Three stem structures were chosen as targets for the introduction of transversion mutations designed to destroy base pairing structures. Mutations predicted to destroy the base pairing of nucleotides 142 to 136 with nucleotides 68 to 74 exhibited a deleterious effect on DIssE replication. Destruction of base pairing between positions 96 to 99 and 116 to 113 also decreased DI RNA replication. Mutations interfering with the pairing of nucleotides 67 to 63 with nucleotides 52 to 56 had only minor effects on DIssE replication. The introduction of second complementary mutations which restored the predicted base pairing of positions 142 to 136 with 68 to 74 and nucleotides 96 to 99 with 116 to 113 largely ameliorated defects in replication ability, restoring DI RNA replication to levels comparable to that of wild-type DIssE RNA, suggesting that these secondary structures are important for efficient MHV replication. We also identified a conserved 23-nucleotide stem-loop structure involving nucleotides 142 to 132 and nucleotides 68 to 79. The upstream side of this conserved stem-loop is contained within a host protein binding element (nucleotides 166 to 129).

FIG. 1

Computer-predicted secondary structure model of the 3′-terminal 166 nt of MHV-JHM genomic RNA. Mfold, version 3.0, was used to generate thermodynamically stable secondary structures, and the model which best fits the experimental data is shown. The nucleotide position was numbered such that nt 1 is immediately 5′ to the poly(A) tail. The summary of enzymatic probing data is noted at each nucleotide: ++, strongly cut by single-strand-specific enzyme; +, weakly cut by single-strand-specific enzyme; ∗∗, strongly cut by double-strand-specific enzyme; ∗, weakly cut by double-strand-specific enzyme, ∗∗+, strongly cut by double-strand-specific enzyme and weakly cut by single-strand-specific enzyme, ++∗, strongly cut by single-strand-specific enzyme and weakly cut by double-strand-specific enzyme.

FIG. 2

Enzymatic probing of the 3′-terminal 166 + 11A RNA. Dephosphorylated, 5′-end-labeled, and gel-purified 166 + 11A RNA was subjected to limited digestion with RNase T₁ (0.0002 U), A (0.0002 U), U2 (2 U), and CV1 (0.07 U) on ice for 30 min. The digested products were resolved by electrophoresis in 7 M urea–20% polyacrylamide gels (A) or 7 M urea–10% polyacrylamide gels (B) for 4 h. Six hours of 7 M urea–10% PAGE was also employed (C) to enlarge the readable region. Lanes RNA (panels A and B), undigested 166 + 11A RNA; lanes OH, RNA ladders generated by limited alkaline hydrolysis of 166 + 11A RNA.

FIG. 3

Primer extension mapping of the secondary structure of the 3′-most 44 nt. Primer extension reactions with 5′-end-labeled oligonucleotide 5638B (Table 1) were performed with 166 + 11A RNA which had been subjected to limited digestion with single- and double-strand-specific RNases as described in Materials and Methods. Primer extension reaction products and DNA sequencing reaction products were loaded on the same gel to locate the primer extension products.

FIG. 4

A 23-nt stem-loop in 3′-terminal 166-nt genomic RNA is conserved in both MHV and BCoV. The primary sequences of nt 142 to 132 and 68 to 79 of MHV and nt 130 to 120 and 65 to 76 of BCoV are conserved with the exception of the boxed 6 nt, which covary to maintain identical secondary structures.

FIG. 5

Replication of wild-type DE25, mutants A1 and A2, and complementary mutant A12. 17Cl-1 cells were infected with MHV-JHM and transfected with DI RNAs 1 h later. Cells were labeled with [³²P]orthophosphate in the presence of actinomycin D when 20% of the cells had formed syncytia. Total RNA was isolated when syncytia involved 80% of the culture. RNAs (5 μg) were resolved in a formaldehyde–1% agarose gel at 110 V for 6 h. Arrows, positions of viral RNA1 to RNA7 and DI RNA.

FIG. 6

Replication of wild-type DE25, mutants B1 and B2, and complementary mutant B12. Replication assays were performed as described for Fig. 5. Arrows, positions of viral RNAs and DI RNA.