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.

Picornaviruses, including human pathogens such as poliovirus, rhinoviruses, and hepatitis A virus (HAV), are small nonenveloped, icosahedral viruses. Their genomes are single-stranded, positive-sense RNAs of 7,200 to 8,500 nucleotides, and each encodes a single long polyprotein (22). After virus cell entry, the genomic RNA released into the cytoplasm serves as a mRNA for the synthesis of polyproteins that are processed to functional proteins by virus-encoded proteases. The genomic RNA also acts as a template for negative-strand RNAs which, in turn, are transcribed into positive strands. The synthesized positive-strand RNAs are encapsidated to form virions.

Picornavirus positive-strand RNAs are selectively encapsidated despite the coexistence of viral negative-strand RNAs and cellular RNAs in infected cells. The mechanism of the RNA encapsidation process is not well understood. The RNA sequence essential for encapsidation or for interaction with capsid proteins during encapsidation has not been identified in picornaviruses. However, studies on poliovirus have provided some clues for the identification of determinants of specific encapsidation. Poliovirus RNA in which the capsid-coding region is replaced with a foreign gene can be encapsidated by capsid proteins that are provided in trans by a helper virus (4, 5, 20). In addition, chimeric polioviruses harboring a different picornaviral 5′ untranslated region (5′-UTR) or 2A protein-coding region are viable (16, 26). These observations suggest that neither the poliovirus 5′-UTR, the capsid-coding region nor the 2A-coding region contains a signal essential for specific encapsidation. Based on the finding that only newly synthesized positive-strand RNAs are encapsidated, it is proposed that viral RNA replication and encapsidation are functionally coupled to each other and that the specific interaction between the RNA replication complex and the assembling capsids determines the specificity of poliovirus RNA encapsidation (17).

Aichi virus, which is associated with acute gastroenteritis in humans (27), is a new member of the family Picornaviridae (28). Computer-based secondary-structure prediction suggested the presence of three stem-loop structures (SL-A, SL-B, and SL-C) within the 5′-end 120 nucleotides of the Aichi virus genome (Fig. 1A). In the previous study, we investigated the function of the most 5′-end stem-loop structure (SL-A) by using various site-directed mutants derived from an infectious cDNA clone, and we showed that SL-A is an element required for viral RNA replication (24). One (mut6) of the mutants showed an interesting property. In mut6, the 7-nucleotide stretches of the middle part of the stem were exchanged with each other to maintain the base pairings of the stem (Fig. 1B). mut6 exhibited efficient RNA replication ability in transfected cells but formed no plaques (24). This finding suggested two possibilities; one is that mut6 RNA is not encapsidated, and the other is that mut6 RNA can be encapsidated, but the mutant has a defect in a certain early step of the infection cycle.

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.

In the present study, we showed that mut6 has a severe defect in encapsidation, and we precisely mapped the region in SL-A relevant to encapsidation by characterizing several more mutants bearing changes in the middle part of SL-A. As a result, it was indicated that the 7 bp of the middle part of the stem of SL-A at the 5′ end of the Aichi virus genome are critical for viral RNA encapsidation. This is the first report of the identification of an RNA element critical for encapsidation in picornaviruses.

MATERIALS AND METHODS

Cell culture and viruses.

Vero cells were grown in Eagle minimal essential medium (MEM) containing 5% fetal calf serum (growth medium) at 37°C. As wild-type Aichi virus, we used viruses derived from Vero cells transfected with in vitro transcripts synthesized from a full-length cDNA clone, pAV-FL (24). Poliovirus Sabin 1, as a reference virus, was propagated in Vero cells.

Mutants of pAV-FL.

mut6 was constructed in the previous study (24). Other mutants were created by PCR-based site-directed mutagenesis as described previously (24). The sequences of the primers used were as follows: for 11-15/34-38, 5′-CGGCCCCCTGTGGGTCTTTTCCGGTGGTCT and 5′-AGGCCCCCCGTGGGCCTTTTCAAACCTATAGTGA; for 12-14/35-37, 5′-CGGCCCCCTCTGGCTCTTTTCCGGTGGTCT and 5′-AGGCCCCCCCTGGCCCTTTTCAAACCTATAGTGA; for 11-16/33-38, 5′-CGGCCCCCGGTGGGTCTTTTCCGGTGGTCT and 5′-AGGCCCCCAGTGGGCCTTTTCAAACCTATAGTGA; and for 10-15/34-39, 5′-CGGCCCCCTGTGGGGCTTTTCCGGTGGTCT and 5′-AGGCCCCCCGTGGGACTTTTCAAACCTATAGTGA.

In vitro transcription.

pAV-FL and its mutants were linearized by digestion with HindIII, and RNA transcripts were synthesized with T7 RNA polymerase as described previously (24).

Electroporation.

RNA transcripts (20 μg) were electroporated into 10⁷ Vero cells by using a Gene Pulser (Bio-Rad) as described previously (24).

Preparation of radiolabeled viruses and sucrose gradient analysis.

A Vero cell monolayer in a 100-mm plate was incubated with viruses at a multiplicity of infection of 5 to 10 for 1 h at 37°C. The inoculum was removed, and the cells were incubated in growth medium for an additional 2 h. For preparation of ³⁵S-labeled viruses from RNA transcripts, Vero cells were transfected with RNAs by electroporation, and then the cells were incubated with growth medium in a 100-mm plate for 2 h. The cells were then washed with phosphate-buffered saline (PBS) two times, and 3.7 MBq of l-[³⁵S]methionine (Amersham Pharmacia) in 2 ml of MEM without methionine was added. After incubation for 4 h, the cells were lysed with Nonidet P-40 and sodium deoxycholate at final concentrations of 1% (vol/vol) and 0.5% (wt/vol), respectively. Cell debris was pelleted, and the supernatant was centrifuged through a 30% sucrose cushion at 30,000 rpm for 3 h in a P40ST rotor (Hitachi). The pelleted viral particles were resuspended in PBS and sedimented through a 10 to 30% sucrose gradient at 39,000 rpm for 80 min in a P40ST rotor. The gradients were fractionated, and the radioactivity in each fraction was counted with a liquid scintillation counter.

For preparation of ³H-labeled viruses, a Vero cell monolayer in a 100-mm plate was treated with 1.25 μg of actinomycin D/ml for 30 min before infection. Virus infection was performed as described above, followed by incubation in growth medium containing 1.25 μg of actinomycin D/ml for 2 h. After incubation, the medium was replaced with 0.5 ml of growth medium containing 1.25 μg of actinomycin D/ml and 3.7 MBq of [5,6-³H]uridine (Amersham Pharmacia). After additional incubation for 4 h, the cells were lysed, and ³H-labeled viruses were prepared by sucrose gradient centrifugation as described above.

Immunofluorescence staining.

Three sets of serial dilutions of a lysate of cells harvested at 6 h after electroporation with AV-FL or mut6 RNAs were prepared. For one set of serial dilutions, RNase A was added at the concentration of 20 μg/ml. For the other two sets of serial dilutions, either guinea pig antiserum raised against purified Aichi virus particles or preimmune serum at a dilution of 1/30 and RNase A were added. These lysates were incubated at 37°C for 1 h. The treated lysates were added into Vero cells and, after incubation at 37°C for 1 h, the cells were washed with MEM two times and then cultured in growth medium. At 6 and 24 h after infection, the medium was discarded, and the cells were fixed with cold (−80°C) methanol for 10 min. After removal of the methanol, the cells were incubated with guinea pig antiserum raised against purified Aichi virus particles for 1 h at 37°C. The cells were washed with PBS and then incubated with fluorescein isothiocyanate-conjugated goat anti-guinea pig immunoglobulin G for 1 h at 37°C. The cells were washed with PBS and then viewed under a fluorescence microscope.

Dot blot hybridization.

RNA transcripts were transfected into Vero cells by electroporation. Total RNA was extracted from the transfected cells by using Trizol reagent (Life Technologies, Inc.) at 3, 6, 9, 12, and 24 h after electroporation. Dot blot hybridization was carried out to detect plus-strand viral RNA as described in a previous study (24).

Western blotting.

Vero cells electroporated with RNAs were lysed at 3 and 9 h after electroporation, and then the lysates were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The proteins were transferred onto a polyvinylidene difluoride membrane, and then immunoblot analysis with antiserum against virus particles was performed as described previously (24).

Titration of viable viruses in cells transfected with RNA transcripts.

Vero cells electroporated with RNAs were frozen at 3, 6, 9, 12, and 24 h postelectroporation. The cells were disrupted by three consecutive freeze-thaw cycles, and the lysates were used for the plaque assay. The number of plaques was determined at 72 h after infection.

RESULTS

mut6 has a defect in encapsidation.

We previously examined the function of SL-A by using various site-directed mutants derived from an infectious cDNA clone of Aichi virus, pAV-FL. mut6 was constructed by exchanging the seven-nucleotide stretches (nucleotide numbers 10 to 16 and 33 to 39) of the middle part of the stem of SL-A with each other to maintain the base pairings of the stem (Fig. 1B). It was found that mut6 exhibited efficient RNA replication ability in transfected cells but had lost the plaque-forming ability (24). We assumed two possible causes of the inability of mut6 to form plaques; one is that mut6 RNA is not encapsidated, and the other is that mut6 RNA can be encapsidated but the resultant viral particles have a defect in a certain early step of the infection cycle, e.g., the adsorption or uncoating step.

To determine whether mut6 RNA is encapsidated, we carried out sedimentation analysis of virus particles. We performed a preliminary experiment to compare the sedimentation profile of Aichi virus with that of poliovirus Sabin 1, whose sedimentation profile has been well analyzed. Analysis of [³⁵S]methionine- and [³H]uridine-labeled viral and subviral particles showed that the sedimentation profiles of virions and empty capsids of Aichi virus were similar to those of poliovirus (data not shown).

We next analyzed the sedimentation profile of mut6. Vero cells were electroporated with mut6 and AV-FL RNAs, and then [³⁵S]methionine-labeled viral and subviral particles were prepared and sedimented through sucrose gradients. For AV-FL, well-separated peaks of virions and empty capsids were found, as expected (Fig. 2). In contrast, in mut6, empty capsids were accumulated, whereas few virions were formed, demonstrating that mut6 has a severe defect in encapsidation. Thus, a defect at the stage of encapsidation would be a primary cause of the inability of mut6 to form plaques.

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.

mut6 virions are infectious.

Although mut6 has a severe defect in encapsidation, a small portion of mut6 RNA appeared to be encapsidated (Fig. 2, fractions 18 and 19). This suggests that infectious mut6 virus particles are generated at very low efficiency.

To confirm this possibility, Vero cells were infected with serial dilutions of the lysates of the cells electroporated with AV-FL and mut6 RNAs, and then capsid proteins synthesized in the infected cells were detected at 6 and 24 h after infection by means of an immunofluorescence assay with antiserum raised against purified virions (Fig. 3). The cell lysates used for infection were pretreated with RNase A to rule out the possibility of infection by naked viral RNAs. At 6 h after infection, synthesis of capsid proteins was observed in cells infected with mut6, although the number of cells synthesizing capsid proteins was much lower than that of cells infected with AV-FL (Fig. 3). Infection of mut6, as well as AV-FL, was inhibited by incubation of the cell lysates with guinea pig antiserum against purified virions prior to infection; the number of the AV-FL-infected cells decreased to <1%, and infection by mut6 was almost perfectly blocked, whereas preimmune guinea pig serum exhibited no inhibitory effect (Fig. 3). These results indicate that infectious mut6 virions are produced at very low efficiency in mut6 RNA-transfected cells. At 24 h after infection, AV-FL viruses spread cell to cell and formed fluorescent focuses, whereas mut6 viruses hardly did so (Fig. 3). This is consistent with the observation that mut6 formed no plaques in the plaque assay (24). The encapsidation efficiency of mut6 RNA would be too low for the formation of plaques.

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.

Further mapping of the region important for encapsidation.

To further define the region important for encapsidation, we constructed several mutants in which the mutated region is reduced compared with mut6 (Fig. 4A). Vero cells were electroporated with the mutant RNAs, and then the accumulation of plus-strand viral RNA, synthesis of capsid proteins, and yield of viable viruses in the transfected cells were examined.

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.

11-15/34-38 and 12-14/35-37 were constructed by exchanging the five- and three-nucleotide streches of the middle part of the stem, respectively, with each other to maintain the base pairings of the stem (Fig. 4A). Both mutant RNAs replicated and expressed capsid proteins as efficiently as AV-FL (Fig. 4B and C). In the plaque assay, 11-15/34-38 formed approximately 10-fold fewer plaques than AV-FL, and 12-14/35-37 produced a similar amount of viruses compared to the wild type (Fig. 4D). This indicates that as the mutated region is narrowed, the encapsidation efficiency is improved. Interestingly, only reducing the mutated regions from 7 to 5 bp recovered the yield of viruses up to ca. 10% of that of wild type (Fig. 4D, compare mut6 and 11-15/34-38). To investigate which of the changes of the G₁₀-U₃₉ and the G₁₆-U₃₃ pairs affects the encapsidation efficiency more severely, we constructed two more mutants, 11-16/33-38 and 10-15/34-39 (Fig. 4A). These mutants also exhibited efficient RNA replication and translation abilities (Fig. 4B and C). 11-16/33-38 produced infectious viruses at almost the same efficiency as 11-15/34-38 (Fig. 4D), whereas 10-15/34-39 generated approximately 10-fold fewer viruses than 11-15/34-38 (Fig. 4D) and formed pinpoint plaques (data not shown). Thus, mutation of the G₁₀-U₃₉ pair affected the encapsidation efficiency more severely than did mutation of the G₁₆-U₃₃ pair. However, mutation of the 7 bp was responsible for abolishing the plaque-forming ability.

DISCUSSION

In the present study, we showed that the RNA sequence at the 5′ end of the Aichi virus genome, which folds into a stem-loop structure (SL-A), contains an element important for viral RNA encapsidation. The mutation introduced into both the seven-nucleotide stretches (nucleotides 10 to 16 and 33 to 39) in the middle part of the stem remarkably reduced the efficiency of RNA encapsidation (Fig. 2). As the mutated region is narrowed, the encapsidation efficiency was improved (Fig. 4). As a result, the mutation of the 7 bp was found to be required to abolish the plaque-forming ability.

This is the first detailed identification of a cis-acting element critical for encapsidation in picornaviruses. Chimeric poliovirus in which the internal ribosome entry site (IRES) within the 5′-UTR is replaced by the encephalomyocarditis virus (EMCV) IRES is viable, but the yield of the chimeric virus is slightly decreased compared to that of the wild-type virus (1, 23, 26). Johansen and Morrow (14) performed a trans-encapsidation assay using poliovirus replicons in which the capsid-coding region is replaced with a luciferase gene and a recombinant vaccinia virus expressing poliovirus capsid proteins. They showed that when the IRES of the replicon is replaced by the EMCV IRES, the efficiency of encapsidation is reduced. However, further mapping of the region within the poliovirus IRES responsible for encapsidation efficiency has not been carried out.

The 5′-end 90 nucleotides of the poliovirus genome fold into a cloverleaf-like structure, and this structure is known to be a cis-acting element important for viral RNA replication (3, 6, 12). Many site-directed mutants of the 5′ cloverleaf structure in poliovirus have been constructed and characterized (2, 3, 18, 26), but there has been no report showing that the structure acts as a packaging signal. In some picornaviruses, including cardioviruses, parechoviruses, and HAV, a stem-loop structure is formed at the 5′ end of the genome (7, 8, 11, 15). The function of the stem-loop structure of these viruses has not been sufficiently analyzed. The Aichi virus genome also has a stem-loop structure at the 5′ end. We have previously shown that SL-A is a structural element required for viral RNA replication (24). In addition, we elucidated in the present study that SL-A is critical for RNA encapsidation. Thus, SL-A has been shown to be bifunctional. It would be significant to investigate whether the 5′ end of other picornaviral genomes is involved in the encapsidation process because, in other groups of viruses, the location of the packaging signal on the genome is not necessarily conserved among related viruses. The region containing the packaging signal is different between two alphaviruses, Sindbis virus and Ross River virus (10).

The 5′ end of the HAV genome folds into three stem-loop structures. The most 5′-end stem-loop (stem-loop I) consists of 41 nucleotides including a 4-nucleotide loop and a single unpaired U residue in the middle part of the stem (7) (Fig. 1B). On the whole, SL-A is similar to HAV stem-loop I in size and shape, although its primary sequence is different. In a previous study (24), we characterized a mutant in which SL-A was replaced with the stem-loop I of HAV. The mutant had a severe defect in RNA replication but yielded viable viruses, albeit with low efficiency. This suggests that only SL-A is not sufficient for determining the specificity of encapsidation. Nugent et al. (17) showed the coupling of poliovirus RNA encapsidation to RNA replication and proposed a model for encapsidation. According to this model, specific interactions occur between capsid proteins and proteins in the viral RNA replication complex, and a newly synthesized positive-strand RNA emerging from the replication complex is encapsidated through interaction between capsid proteins and the viral RNA. The specificity of encapsidation has thus been proposed to be determined by specific interactions between capsid proteins and proteins constituting the RNA replication complex. This model may be applied to Aichi virus. Aichi virus SL-A may serve as a binding site for capsid proteins. Alternatively, it is possible that another region, in addition to SL-A, is required for determining the specificity of encapsidation. Further studies are needed to clarify the role of SL-A in encapsidation and the requirement for the specificity of encapsidation.

In various viruses, including Sindbis virus (25), a nodavirus (flock house virus) (29), a coronavirus (mouse hepatitis virus) (9), a tombusvirus (turnip crinkle virus) (21), a hepadnavirus (HBV) (19), and retroviruses (13), it has been reported that the region containing the RNA encapsidation signal folds into one or more stem-loop structures. In Aichi virus, the nucleotide sequence of the middle part of the stem of SL-A was shown to be critical for encapsidation. It remains to be elucidated whether the secondary structure of SL-A in addition to the primary sequence is important for encapsidation, although it is currently difficult to resolve this question. Since disruption of the secondary structure of SL-A abolished RNA replication (24), the site-directed mutations to be introduced into SL-A should be restricted to those that maintain the secondary structure. An assay system by which encapsidation can be evaluated independently of RNA replication is needed. For example, if encapsidation of mut6 RNA is restored by insertion of wild-type SL-A into another region of the RNA, the resultant mutant would be useful. We are trying to develop such a system.

This and the previous studies (24) showed that the 5′ end of the Aichi virus genome is involved in both viral RNA replication and encapsidation. The phenotypic property of mut6 indicates that the requirements in SL-A for viral RNA encapsidation and RNA replication are distinct. We presume that the 5′ end of the genome would interact with different factors during RNA replication and encapsidation. The 5′ end of the Aichi virus genome may play a role in determining which process the newly synthesized positive-strand RNAs are used for: RNA replication or encapsidation.