Abstract
We identified a novel, 6,513-bp-long RNA, termed Bombyx mori macula-like latent virus (BmMLV) RNA, which abundantly expressed in B. mori cultured BmN cells. BmMLV RNA potentially encodes two proteins, putative RNA replicase and coat protein, which share structural features and sequence similarities with those of a plant RNA virus, the genus Maculavirus. Northern blot analysis showed that two transcripts were expressed in BmN cells: a 6.5-kb-long RNA, which contains both putative RNA replicase and coat protein genes, and a 1.2-kb-long RNA, which contains only a coat protein gene. Southern blot analysis showed that BmMLV RNA is not carried by the B. mori genome. RT-PCR analysis also revealed the presence of BmMLV RNA in several B. mori cell lines other than BmN cells, suggesting that BmMLV RNA latently exists in B. mori cultured cells. Infection studies showed that BmMLV virions were able to infect BmMLV-negative Spodoptera frugiperda Sf-9 cells and B. mori larvae. Electron microscopy and Northern blot analysis of a purified BmMLV revealed that isometric virions appear to be 28 to 30 nm in diameter and contain a 6.5-kb genomic RNA. These results showed that BmMLV is a novel macula-like virus infectious to and replicable in B. mori-derived cells.
The family Tymoviridae comprises the genus Tymovirus (11), the genus Marafivirus (5), and the genus Maculavirus (12). Maculavirus is a new genus of plant viruses typified by Grapevine fleck virus (GFkV). A possible second member is Grapevine redglobe virus (GRGV). Maculaviruses are nonmechanically transmissible phloem-limited RNA-containing viruses with 30-nm isometric particles that have a rounded contour and a prominent surface structure. The genome of GFkV is a 7,564-bp-long single-stranded RNA, excluding the poly(A) tail, and contains four putative open reading frames (ORFs) that encode a 215-kDa polypeptide with the conserved motifs of replication-associated proteins of positive-strand RNA viruses (ORF1), the coat protein (CP) (ORF2), and one (GRGV) or two (GFkV) proline-rich proteins of 31 kDa (ORF3) and 16 kDa (ORF4), respectively, with unknown functions. Replication-associated proteins and CP are phylogenetically related to those members of the genera Tymovirus and Marafivirus.
The natural and artificial host ranges of GFkV and GRGV are restricted to European and American Vitis species. Neither virus is transmitted by mechanical inoculation of sap, but both are readily transmitted by grafting. It is known that GFkV spreads naturally in the field (3), but no vector has been identified. GFkV is not seed transmissible (6), but its transmission through dodder has been reported (21). GFkV-infected grapevine cells contain vesiculated mitochondria, the possible site of RNA replication. In the field, GFkV particles accumulate in great quantity, sometimes in crystalline arrays, in differentiating sieve tubes, and in companion cells of naturally infected grapevines (1).
It is well known that baculovirus infection alters both the host protein and the host mRNA levels. A baculovirus infection causes a global shutoff of host protein synthesis and gene expression in insect cells beginning around 12 to 18 h postinfection (hpi) (15, 17). The mechanism for this down-regulation of host mRNA levels mediated by a baculovirus infection has not been elucidated. To investigate host gene expression during baculovirus infection in more detail, we monitored the global gene expression using a cDNA microarray (16) in Bombyx mori-cultured BmN cells infected with B. mori nucleopolyhedrovirus (BmNPV). We identified a clone, N0071, that exhibited high-level expression in BmNPV-infected BmN cells at a late stage of BmNPV infection. Surprisingly, N0071 has significant homology to RNA replicase and CP genes of the genus Maculavirus. We termed this B. mori macula-like latent virus (BmMLV) RNA and cloned a full-length 6,513-bp-long RNA. Infection experiments showed that BmMLV virions were able to infect BmMLV-negative Spodoptera frugiperda Sf-9 cells and B. mori larvae. Electron microscopy and Northern blot analysis of putative virus particles purified from BmN cells revealed that they are isometric, are 28 to 30 nm in diameter, and contain a 6.5-kb genomic RNA. This is the first report of a macula-like virus that can replicate in Lepidoptera.
MATERIALS AND METHODS
Insects, cells, and viruses.
B. mori larvae (F1 hybrid Kinshu × Showa and inbred strain Cambodge) were reared as described previously (2). The B. mori cultured cells used in this study were as follows: BmN-4 (BmN, ovary) (20), NIAS-Bm-ao1 (ovary), NIAS-Bm-aff3 (fat body), NIAS-Bm-oyanagi1 (embryo), SES-BoMo-15A (embryo) (8), SES-BoMo-J125K5 (embryo) (7), and NIAS-BoMo-Cam1 (ovary). The wild-type BmNPV T3 isolate was used in the infection experiments.
cDNA cloning and DNA sequencing.
To determine the sequence of the full-length cDNA of BmMLV RNA, we screened cDNA libraries prepared from BmN cells and found four different but overlapping clones showing homology to the genome sequence of GFkV (Fig. 2). NV021287, N-1030, and N-0773 were isolated from cDNA libraries prepared from BmN cells as described previously (13). BmNP08_K23 was isolated from a BmN cDNA library constructed by an oligo-capping method (T. Shimada et al., unpublished data; http://www.ab.a.u-tokyo.ac.jp/Bombyx_EST/). By sequencing and assembling these cDNA clones, we obtained a 6,532-bp-long cDNA sequence [with 19-bp poly(A)] encoding a putative RNA replicase and a CP (Fig. 2). The nucleotide sequence was determined with the ABI Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) and the ABI Prism 3100 DNA sequencer (Applied Biosystems).
FIG. 2.
Structure of the full-length cDNA of BmMLV RNA encoding putative RNA replicase and CP. Four overlapping cDNA clones (BmNP08_K23, NV021287, N-1030, and N-0773) showing homology to the genome sequence of GFkV were isolated from BmN cDNA libraries. NV021287, N-1030, and N-0773 were isolated from cDNA libraries prepared from BmN cells (13). BmNP08_K23 was isolated from a BmN cDNA library constructed by an oligo-capping method (Shimada et al., unpublished; http://www.ab.a.u-tokyo.ac.jp/Bombyx_EST/). By sequencing and assembling these cDNA clones, we obtained a 6,532-bp-long cDNA sequence [with 19-bp poly(A)]. The locations of putative ORFs are shown at the top. The length of each cDNA clone is also shown in parentheses.
Northern blot analysis.
Total RNA from BmN cells and fat bodies of silkworms was prepared using the Trizol reagent (Invitrogen). Poly(A)+ RNA was purified by using the FastTrack 2.0 mRNA isolation kit (Invitrogen). Northern blotting was performed as described previously (2). Probes for BmMLV RNA were amplified by PCR with primers N0071-1 (5′-CAAGACTCAAGCTGTCATCC-3′) and N0071-4 (5′-TGGTAAATCACTTGATCTGC-3′) for RNA replicase, N0071-7 (5′-GTCTCCTCCATCATCAAAGG-3′) and N0071-10 (5′-GGATCGAAGACGTAGACTCG-3′) for CP, and N0071-1 and N0071-10 for both RNA replicase and CP, using cDNA prepared from BmN cells as a template. A probe for B. mori Actin3 was amplified by PCR with primers BA3F1 (5′-AGATGACCCAGATCATGTTCG-3′) and BA3R1 (5′-GAGATCCACATCTGTTGGAAG-3′) using cDNA prepared from BmN cells as a template.
Southern blot analysis.
Genomic DNA of BmN cells was extracted as described previously (2). Genomic DNA of silkworms was extracted from the posterior silk glands of mid-fifth-instar larvae according to the protocol described by Suzuki et al. (18). Genomic DNA was fully digested with restriction enzymes at 37°C overnight and subjected to Southern blot analysis as described previously (18). A probe for BmChi-h was amplified by PCR with primers BmChi-hF1 (5′-GTAGTAGCCGATACTGATGG-3′) and BmChi-hR1 (5′-AGCTGAACGTATACATCTCC-3′) using a plasmid containing BmChi-h as a template.
Reverse transcription-PCR (RT-PCR).
Total RNA was reverse transcribed, diluted, and used for PCR as described previously (9). Primers for PCR were described above. Real-time PCR experiments were performed as described previously (10).
Infection of B. mori larvae with BmMLV.
The silkworm strain Kinshu × Showa was used. The larvae at day 1 in the fifth instar were injected with 30 μl of virus solution in phosphate-buffered saline (PBS). This amount of virus solution is equivalent to 0.2 × 102 BmN cells. The virus solution was prepared as follows: 3.2 × 107 BmN cells were homogenized in 15 ml of PBS and centrifuged at 6,000 × g for 15 min at 4°C. After centrifugation, the supernatants were filtered (0.22-μm-pore-size filter) and used as the virus solution. RT-PCR analysis was performed as described above.
Electron microscopy analysis of purified BmMLV.
BmN cells were harvested, washed with PBS, and sonicated in 20 volumes of PBS. After low-speed centrifugation, the supernatants were filtered (0.22-μm-pore-size filter) and concentrated with an Amicon Ultra filter (Millipore). The resultant was subjected to cesium chloride (CsCl) gradient ultracentrifugation (ρ = 1.3 to 1.7). Putative virus bands were collected, diluted in 10 mM Tris buffer (pH 7.5), and subjected again to ultracentrifugation. Virus particles were suspended in 10 mM Tris buffer and used for electron microscopy analysis. The purified preparation was negatively stained with 2% phosphotungstate solution (pH 6.0) and observed under an electron microscope (JEOL JEM-1010).
Phylogenetic analysis.
We performed a phylogenetic analysis of the amino acid sequences encoded in ORFs of the BmMLV and 12 related viruses. The sequences were aligned using CLUSTAL W (19). With the aid of PHYLIP, version 3.572 (4); the distances were calculated using the PAM matrix; and neighbor-joining trees were constructed. The branching patterns were evaluated by a bootstrap of 1,000 replicates.
Nucleotide sequence accession number.
The nucleotide sequence reported in this paper has been submitted to the DDBJ, EMBL, and GenBank data banks under accession number AB186123.
RESULTS
Identification of BmMLV RNA, which was abundantly expressed in BmN cells.
It is known that a baculovirus infection results in a shutdown of host gene expression (15, 17). While searching the host genes, which were still expressed in BmN cells at the late stage of BmNPV infection, we identified a clone, N0071, that exhibited a high level of expression in BmNPV-infected BmN cells. Sequence analysis revealed that N0071 contains a 2,379-bp-long cDNA, which has significant homology to the RNA replicase and CP genes of the genus Maculavirus. We termed this BmMLV RNA. By Northern blot analysis with a probe containing both RNA replicase and CP genes, two bands at 6.5 and 1.25 kb were detected. These transcripts were highly expressed throughout BmNPV infection, although a housekeeping gene, Actin3, was completely diminished by 24 hpi (Fig. 1A). To investigate the structure of these two transcripts, we carried out Northern blot analysis using two probes, each one containing an RNA replicase gene and a CP gene. As shown in Fig. 1B, the larger transcript (6.5 kb) was detected by both probes, whereas the smaller one was detected only when a probe containing a CP gene was used. This clearly showed that a 6.5-kb-long transcript contained both genes, whereas a 1.25-kb-long transcript contained only a CP gene. In addition, we examined the copy number of this transcript by real-time PCR. As shown in Fig. 1C, we found that the copy number of the putative CP transcript was almost same as that of a housekeeping gene, Actin3, in BmN cells.
FIG. 1.
Northern blot and real-time PCR analysis of BmMLV RNA. (A) BmN cells were infected with BmNPV T3 at a multiplicity of infection of 5, and total RNA was prepared at 0, 24, 48, and 72 hpi. Northern blot analysis was performed by using a probe containing both RNA replicase and CP genes. A rRNA band stained with ethidium bromide is shown as a loading control. Actin3 was used as a control. (B) Poly(A)+ RNA was prepared from BmNPV-infected BmN cells at 0, 24, 48, and 72 hpi at a multiplicity of infection of 5. Northern blot analysis was performed by using two probes containing RNA replicase and CP genes. Actin3 was used as a control. (C) Total RNA was prepared from BmN cells, reverse transcribed, and used for real-time PCR analysis of Actin3 and the CP gene (CP). Data show means ± standard error of three independent experiments.
Structure of the full-length cDNA of BmMLV RNA encoding putative RNA replicase and CP.
To determine the sequence of the full-length cDNA of BmMLV RNA, we screened cDNA libraries prepared from BmN cells and found four different, but overlapped clones showing homology to the genome sequence of GFkV (Fig. 2). By sequencing and assembling these cDNA clones, we identified a full-length cDNA of 6,513 bp without the poly(A) tail that contained two ORFs encoding 1,747 (ORF1) and 237 (ORF2) amino acid residues, respectively (Fig. 2). A putative protein, p15, was also found (Fig. 2). ORF1 (nucleotides [nt] 58 to 5,299) potentially encodes a 196-kDa polypeptide, which shares structural features and sequence similarities with RNA replicases of the genus Maculavirus. ORF2 (nt 5,312 to 6,022) encodes a putative viral CP (25 kDa) that is also expressed via a 1.25-kb-long subgenomic RNA (Fig. 1 and 2). Screening of several cDNA libraries prepared from BmN cells revealed that a large number of clones contained cDNAs beginning at nt 5,300, suggesting that a transcription start site of a 1.25-kb-long subgenomic RNA was near this point.
Phylogenetic analysis of BmMLV.
The amino acid sequence of the RNA replicase of BmMLV was aligned with the replicases of one known maculavirus, two known marafiviruses, and five known tymoviruses. For the analysis of RNA replicases, white clover mosaic virus was used as the outgroup. For CPs, no outgroup was included because its homology was limited to these three genera. The sources of the amino acid sequences are listed in Table 1. As a result, the topology of both trees for RNA replicases and CPs was shown to coincide well. Both trees showed two distinct clades of Tymovirus and Marafivirus plus Maculavirus (Fig. 3A). They indicate that BmMLV belongs to the latter clade and is relatively closer to Maculavirus than Marafivirus. The amino acid identities of RNA replicases and CPs were 44 and 41%, respectively, between BmMLV and GFkV, which belongs to the genus Maculavirus (Fig. 3B).
TABLE 1.
Sources of amino acid sequences for the phylogenetic analysis
| Genus | Virus | Accession no. (database)
|
|
|---|---|---|---|
| RNA replicase | CP | ||
| ? | Bombyx mori macula-like latent virus (BmMLV) | AB186123 (GenBank) | AB186123 (GenBank) |
| Marafivirus | Oat blue dwarf marafivirus | 2315524A (PRF) | P89921 (TrEMBL) |
| Maize rayado fino virus | 2714327A (PRF) | 2714327A (PRF) | |
| Poinsettia mosaic virus | 2618414A (PRF) | Q9IW07 (TrEMBL) | |
| Maculavirus | Grapevine fleck virus | Q8UZB6 (TrEMBL) | Q8UZB5 (TrEMBL) |
| Grapevine red globe virus | not reported | Q71EB5 (TrEMBL) | |
| Tymovirus | Chayote mosaic tymovirus | Q9QCX3 (TrEMBL) | Q9QCX2 (TrEMBL) |
| Ononis yellow mosaic virus | P20127 (SwissProt) | P20124 (SwissProt) | |
| Turnip yellow mosaic virus | P10358 (SwissProt) | Q8V150 (TrEMBL) | |
| Erysimum latent virus | P35928 (SwissProt) | P35927 (SwissProt) | |
| Kennedya yellow mosaic virus | P36304 (SwissProt) | P36303 (SwissProt) | |
| Physalis mottle virus | Q91260 (TrEMBL) | P36351 (SwissProt) | |
| Potexvirus (outgroup) | White clover mosaic virus | P09498 (SwissProt) | No homology |
FIG. 3.
(A) Phylogenetic analysis of RNA replicases and CPs. See Table 1 for the sources of amino acid sequences. The alignments can be downloaded at http://www.ab.a.u-tokyo.ac.jp/data/bmmlv/. The trees were constructed by the neighbor-joining method as described in Materials and Methods. The numbers above the branches indicate the supported times in 1,000 bootstrap replicates. (B) Comparison of proteins encoded in BmMLV and GFkV genomes. Alignment of amino acid sequences of RNA replicases (pane1 1) and CPs (panel 2) is shown. Black backgrounds indicate identical amino acid residues, and gray backgrounds indicate similar residues. Underlining indicates conserved motifs of methyltransferase (MTR I to -III), helicase (HEL I to -VI), and polymerase (REP I to -VIII), which were inferred from comparison with the sequence of maize rayado fino virus (5). (C) Diagram showing the putative organization and expression strategy of BmMLV and GFkV. The ORFs are drawn as rectangles. The location of MTR, PRO (papain-like protease), HEL, REP, and CP are indicated. A putative protein of BmMLV, p15, and two GFkV proteins, p31 and p16, are also shown.
Members of the family Tymoviridae possess a polyprotein containing the conserved signature motifs of methyltransferase (MTR), helicase (HEL), and RNA polymerase (replicase, or REP) (5). Alignment of amino acid sequences of RNA replicases of BmMLV and GFkV revealed that these motifs (MTR I to -III, HEL I to -VI, and REP I to -VIII) are highly conserved between two viruses (Fig. 3B). The putative organization and expression strategy of BmMLV and GFkV are shown in Fig. 3C. Although ORF1 (RNA replicase) and ORF2 (CP) showed high sequence homology between two viruses (Fig. 3B), the homologues of ORF3 (p31) and ORF4 (p16), which encode proline-rich proteins with unknown functions (12), were not found in the BmMLV sequence. A putative BmMLV protein, p15, has no homology to all known proteins. Combined with these data, the replication strategies of BmMLV and GFkV may involve autoproteolytic cleavage of the 200-kDa polypeptide by the papain-like protease encoded by ORF1 and the production of subgenomic RNA.
BmMLV RNA is not encoded by the B. mori genome.
To further examine whether BmMLV RNA is encoded by the B. mori genome, we performed PCR and Southern blot analysis with the genomic DNAs of BmN cells and B. mori (F1 hybrid Kinshu × Showa). Genomic PCR analysis showed that the BmMLV sequence could not be amplified with the genomic DNAs of BmN cells and B. mori (Fig. 4A). Southern blot analysis also revealed that BmMLV RNA was not detected in the genome of BmN cells and B. mori with a PCR product containing both RNA replicase and CP genes, whereas a positive band was detected for BmChi-h with a PCR fragment containing BmChi-h (Fig. 4), which is located on chromosome 7 of B. mori (2). This indicates that BmMLV RNA is not encoded by the B. mori genome. Furthermore, the BmMLV RNA sequence is not found in the draft sequence of the B. mori genome (14), strongly supporting the idea that BmMLV RNA is a foreign RNA derived from a putative RNA virus by which BmN cells are infected.
FIG. 4.
BmMLV RNA is not encoded by the B. mori genome. (A) PCR analysis of RNA replicase and CP genes by using BmN cDNA (lane 1), the BmN genome (lane 2), and the B. mori genome (lane 3). Actin3 is used as a control. (B) Southern blot analysis. Genomic DNAs prepared from BmN cells (lanes 1) and B. mori (lanes 2) were digested with EcoRI and SphI, electrophoresed, and hybridized with a probe containing RNA replicase and CP genes. As a positive control, we used a probe containing the B. mori BmChi-h gene that is located on chromosome 7 of B. mori (2).
Presence of BmMLV RNA in several B. mori cell lines other than BmN cells.
To investigate whether BmMLV RNA is present in B. mori cell lines other than BmN cells, we performed RT-PCR analysis using cDNAs prepared from other B. mori cell lines. As shown in Fig. 5, among six cell lines examined, BmMLV RNA was present in two embryo-derived cell lines, NIAS-Bm-oyanagi1 and SES-BoMo-J125K5, and one ovary-derived cell line, NIAS-BoMo-Cam1. We cloned the RT-PCR products and confirmed by DNA sequencing that the PCR-amplified products were almost identical to the BmMLV cDNAs derived from BmN cells (data not shown). These results indicate that BmMLV RNA is latently present in various B. mori cultured cells.
FIG. 5.
Presence of BmMLV RNA in several cultured B. mori cell lines other than BmN cells. RT-PCR analysis of RNA replicase and CP genes was performed using RNA prepared from several B. mori cell lines. Actin3 was used as a control. Lanes: 1, NIAS-Bm-ao1 (ovary); 2, NIAS-Bm-aff3 (fat body); 3, NIAS-Bm-oyanagi1 (embryo); 4, SES-BoMo-15A (embryo); 5, SES-BoMo-J125K5 (embryo); 6, NIAS-BoMo-Cam1 (ovary); M, DNA size marker.
BmMLV RNA as the genomic RNA of an infectious virus.
We next examined whether infectious virus particles containing BmMLV RNA were released into the culture medium of BmN cells. RNA was prepared from the culture medium or BmN cells, and RT-PCR analyses of RNA replicase, CP, and Actin3 genes were performed using gene-specific primers. As shown in Fig. 6A, BmMLV RNA was present in the culture medium as well as in BmN cells, suggesting that BmMLV can bud from the cells. To investigate the infectivity of BmMLV, we used S. frugiperda Sf-9 cells, because we revealed by Northern blot analysis that this cell line as well as the Trichoplusia ni High Five cell line did not express BmMLV RNA (Fig. 6B). BmMLV-negative Sf-9 cells were cultured with a BmN- or Sf-9-cultured conditioned medium. Three days later, poly(A)+ RNA was prepared, and Northern blot analysis was performed. Two transcripts corresponding to 6.5- and 1.25-kb RNAs of BmMLV were clearly detected in Sf-9 cells cultured with a BmN-conditioned medium but not with a Sf-9-conditioned medium (Fig. 6C), suggesting that BmMLV is an infectious virus. In addition, we examined whether BmMLV can infect B. mori larvae. We first investigated the existence of BmNLV in the tissues of B. mori strain Kinshu × Showa, which we planned to use in the infection experiments. As shown in Fig. 7A, we did not detect the transcripts of either the BmMLV replicase gene or the CP gene by RT-PCR using RNA prepared from fat body, testis, and ovary of this strain. Fifth-instar B. mori larvae were injected with 30 μl of the BmN extract containing BmMLV, and we then examined the existence of BmMLV at 2, 4, 6, and 14 days after RT-PCR injection. As shown in Fig. 7B, we detected the transcripts of both replicase and CP genes in fat body, midgut, and gonads at 2, 4, 6, and 14 days after the injection, although the larvae and pupae did not show any symptoms. This suggests that the latent and continuous infection of BmMLV occurred. The larval-pupal transformation did not affect the persistent infection of this virus in these tissues. However, the virus RNA was not detected in the unfertilized eggs laid by BmMLV-infected females or in the 3-day-old fertilized eggs generated by infected females and males (data not shown). This result suggests that BmMLV is hardly transmitted vertically to the next generation through eggs or sperms.
FIG. 6.
BmMLV infection on BmMLV-negative Sf-9 cells. (A) Presence of BmMLV in both the culture medium and BmN cells. Total RNA was prepared from the culture medium and BmN cells. RT-PCR analysis of RNA replicase and CP genes was carried out using gene-specific primers. Actin3 was used as a control. (B) Expression of BmMLV RNA in non-Bombyx insect cell lines. Northern blot analysis was performed by using total RNA prepared from BmN (lane 1), Sf-9 (lane 2), and High Five cells (lane 3). Actin3 was used as a control. (C) BmMLV infection on Sf-9 cells. Sf-9 cells were cultured with Sf-9- or BmN-cultured conditioned medium. Three days after, poly(A)+ RNA was prepared, and Northern blot analysis was performed by using a CP gene as a probe. Lanes: 1, poly(A)+ RNA prepared from Sf-9 cells cultured with Sf-9-cultured conditioned medium; 2, poly(A)+ RNA prepared from Sf-9 cells cultured with BmN-cultured conditioned medium. Actin3 was used as a control.
FIG. 7.
BmMLV infection on B. mori larvae. (A) BmMLV does not latently exist in the Kinshu × Showa strain of B. mori larvae. The existence of BmMLV in fat body (FB), testis (TES), and ovary (OV) was examined by RT-PCR. cDNA prepared from BmN total RNA was used as a positive control. EF1α was used as a control. (B) Infection of B. mori larvae with BmMLV. Fifth-instar B. mori larvae (Kinshu × Showa) were starved for several hours and then injected with 30 μl of the BmN extract containing BmMLV. Total RNA was prepared from fat body, midgut, and gonads of BmMLV-injected larvae at 2, 4, 6, and 14 days after the injection. The existence of BmMLV was examined by RT-PCR. Actin3 was used as a control.
Finally, we tried to purify BmMLV virions from BmN cells by CsCl gradient ultracentrifugation. RT-PCR and Northern blot analysis of RNA prepared from a putative BmMLV fraction showed that this fraction contained an approximately 6.5-kb RNA, presumably derived from BmMLV genomic RNA (Fig. 8A and B). To explore the presence of BmMLV particles in this fraction, electron microscopy analysis was carried out. As shown in Fig. 8C, isometric virions 28 to 30 nm in diameter were observed, confirming that BmMLV RNA is the genomic RNA of an infectious virus that latently exists in BmN cells.
FIG. 8.
Purification of BmMLV. RT-PCR (A) and Northern blot analysis (B) of purified BmMLV. BmMLV was purified by CsCl-gradient ultracentrifugation as described in the Materials and Methods section. RNA was isolated from a virus fraction. RT-PCR was performed using specific primers for BmMLV replicase, CP, and B. mori Actin3 genes. Northern blot analysis was carried out using BmMLV replicase, CP, and B. mori Actin3 genes as probes. (C) Electron micrograph of purified BmMLV. Bar, 100 nm.
DISCUSSION
In the present study, we identified a novel, 6,513-bp-long RNA, termed BmMLV RNA, which was abundantly expressed in B. mori-derived BmN cells. The identification of BmMLV RNA as a viral genome-sense RNA was based on the following data. (i) BmMLV RNA encodes two proteins highly homologous to RNA replicase and CP of members of the family Tymoviridae (Fig. 2 and 3). (ii) BmMLV RNA is not encoded by the B. mori genome (Fig. 4). (iii) BmMLV RNA exists in several B. mori cell lines (Fig. 5). (iv) Putative BmMLV virions are infectious to Sf-9 cells and the tissues of B. mori larvae, which do not latently express BmMLV RNA (Fig. 6 and 7). (v) BmMLV has a 6.5-kb-long genomic RNA and a 1.25-kb-long subgenomic RNA (Fig. 1 and 8). (vi) Electron microscopic observation revealed putative BmMLV virions 28 to 30 nm in diameter (Fig. 8). To our knowledge, this is the first report to characterize a macula-like virus that exists latently and can replicate in lepidopteran cell lines.
The question of how and when BmN cells were infected with this virus deserves attention. RT-PCR and DNA sequencing showed that several other B. mori cell lines that were derived from different B. mori strains and were established by different researchers also express BmMLV RNA (Fig. 5). This led us to speculate that some B. mori strains were latently infected with BmMLV. To resolve this, we tried to detect BmMLV RNA in the ovary of a B. mori strain, Cambodge, from which the BmMLV-positive NIAS-BoMo-Cam1 cell line originated. However, we could not detect the transcripts of the BmMLV CP and replicase genes (data not shown). This may be due to the low levels of BmMLV in the ovary of this strain. Other B. mori strains expressing high levels of BmMLV RNA in their tissues need to be searched.
Northern blot analysis showed that BmMLV RNA is represented by quite abundant levels of mRNA in BmN cells. In addition, using BLAST search analysis against BmN expressed sequence tags in Silkbase (13), a number of clones were shown to have significant homology to this RNA. Thus, we carried out real-time PCR analysis to estimate the copy number of this RNA. Surprisingly, we found that the copy number of the BmMLV CP gene was almost same as that of a housekeeping gene, Actin3, in BmN cells (Fig. 1C). Furthermore, we showed that the BmMLV RNA was still highly expressed in BmN cells at the late stage of BmNPV infection (72 hpi), at which host gene expression is completely suppressed (Fig. 1). This strongly suggests that the replication of this virus is mainly independent on host gene expression and that the expression level of this RNA is comparable to that of host housekeeping genes.
The phylogenetic analysis (Fig. 3A) elucidated that BmMLV clearly belonged to the family Tymoviridae and was the most closely related to GFkV in the genus Maculavirus. Both the replication protein and CP of BmMLV showed the highest or near-highest homology to GFkV (Fig. 3B). Therefore, it is probable that the close ancestor of BmMLV was a plant virus in Tymoviridae. The viruses in the genus Marafivirus are transmitted by hemipteran insects (plant hoppers; Cicadellidae) in a persistent manner. The viruses in the genus Tymovirus are persistently or semipersistently transmitted by coleopteran insects (beetles) belonging to the families Chrysomelidae and Curculionidae, which are nonsucking insects (11). The vectors of Maculavirus (GFkV and GRGV) are unknown. Although so far no lepidopteran insects have been reported as vectors of plant viruses, we can easily imagine that a plant virus in Tymovirudae was transferred to a lepidopteran insect (moth) through its host plant, and started to replicate in the insect away from the plant because tymoviruses can potentially replicate both in plants and in insects.
In conclusion, we identified a novel macula-like virus, BmMLV, and purified putative virus particles from BmN cells. Further analyses are required to clarify the origin of this virus and its replication strategy.
Acknowledgments
We thank Hiroaki Abe, Tokyo University of Agriculture and Technology, for his help in the analysis of Cambodge's RNA.
This work was supported by grants from MEXT (no. 16658023 and 16011209) (to T.S.), BRAIN (to M.K. and T.S.), and MAFF-NIAS (Insect Technology) (to T.S.), Japan.
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