The 5'-end sequence of the genome of Aichi virus, a picornavirus, contains an element critical for viral RNA encapsidation

Abstract

Picornavirus positive-strand RNAs are selectively encapsidated despite the coexistence of viral negative-strand RNAs and cellular RNAs in infected cells. However, the precise mechanism of the RNA encapsidation process in picornaviruses remains unclear. Here we report the first identification of an RNA element critical for encapsidation in picornaviruses. The 5' end of the genome of Aichi virus, a member of the family Picornaviridae, folds into three stem-loop structures (SL-A, SL-B, and SL-C, from the most 5' end). In the previous study, we constructed a mutant, termed mut6, by exchanging the seven-nucleotide stretches of the middle part of the stem in SL-A with each other to maintain the base pairings of the stem. mut6 exhibited efficient RNA replication and translation but formed no plaques. The present study showed that in cells transfected with mut6 RNA, empty capsids were accumulated, but few virions containing RNA were formed. This means that mut6 has a severe defect in RNA encapsidation. Site-directed mutational analysis indicated that as the mutated region was narrowed, the encapsidation was improved. As a result, the mutation of the 7 bp of the middle part of the stem in SL-A was required for abolishing the plaque-forming ability. Thus, the 5'-end sequence of the Aichi virus genome was shown to play an important role in encapsidation.

FIG. 1.

(A) Schematic diagram of the Aichi virus genome and the predicted secondary structure of the 5′-end 120 nucleotides of the genome. The open box and bold lines indicate coding and noncoding regions, respectively. Vertical lines within the box represent putative cleavage sites for viral proteinase. The three stem-loop structures are termed SL-A, SL-B, and SL-C. (B) Diagrams of SL-A of AV-FL, mut6, and the stem-loop structure (stem-loop I) formed at the 5′ end of the HAV genome. Mutated nucleotides in mut6 are boxed.

FIG. 2.

mut6 has a defect in RNA encapsidation. Vero cells were electroporated with the AV-FL and mut6 RNAs, and then labeled with [³⁵S]methionine. At 6 h after electroporation, [³⁵S]methionine-labeled viral and subviral particles were collected and centrifuged through a 10 to 30% sucrose gradient. The gradient was fractionated, and the radioactivity in each fraction was counted with a liquid scintillation counter.

FIG. 3.

mut6 virions are infectious. Serial dilutions of the lysates of cells harvested at 6 h after electroporation with the AV-FL and mut6 RNAs were prepared and then treated with RNase A alone or with RNase A and guinea pig antiserum raised against purified virus particles (RNase A + anti-virion) or preimmune guinea pig serum (RNase A + preimmune). Vero cells were infected with the treated lysates. At 6 and 24 h after infection, an immunofluorescence assay was performed with antiserum against purified virions. The dilution factor of the cell lysate is shown in each panel.

FIG. 4.

Effects of mutations introduced into the middle part of the stem of SL-A on viral RNA replication, protein synthesis, and yields of viable viruses. (A) Diagram of SL-A of AV-FL and mutants. Mutated nucleotides in the mutants are boxed. (B) RNA replication of AV-FL and its mutants. Vero cells were electroporated with the AV-FL and mutant RNAs, and total RNAs were extracted from the cells at the indicated times after electroporation. The total RNA samples were dotted and probed with digoxigenin-labeled negative-sense viral RNA. As controls, 10-, 1-, and 0.1-ng portions of the AV-FL transcripts were dotted. (C) Accumulation of capsid proteins in the transfected cells. At the indicated times after electroporation, cell lysates were prepared and subjected to SDS-10% polyacrylamide gel electrophoresis, and capsid proteins were detected by Western blotting with antiserum raised against purified virus particles. As a control, the proteins of purified virions were analyzed. The position of each capsid protein is indicated on the left. (D) Virus yields in cells electroporated with the AV-FL and mutant RNAs. At the indicated times after electroporation, cells were harvested, and the virus titer was determined by a plaque assay. The number of plaques was determined at 72 h after infection.