Identification of a Subgenomic mRNA Encoding the Capsid Protein of Mushroom Bacilliform Virus, a Single-Stranded RNA Mycovirus

Virology 260, 273–276 (1999) Article ID viro.1999.9839, available online at http://www.idealibrary.com on

Identification of a Subgenomic mRNA Encoding the Capsid Protein of Mushroom Bacilliform Virus, a Single-Stranded RNA Mycovirus Peter A. Revill, 1 Andrew D. Davidson, and Peter J. Wright 2 Department of Microbiology, Monash University, Clayton, Victoria, 3168, Australia Received February 10, 1999; returned to author for revision May 13, 1999; accepted June 3, 1999 Mushroom bacilliform virus is unique among mycoviruses of the higher fungi in having a genome of positive-sense single-stranded RNA. We have identified a subgenomic mRNA molecule encoding the viral capsid protein in mushroom bacilliform virus-infected mycelium of Agaricus bisporus. Transcription of subgenomic RNA commences at the sequence ACAAAA, 47 nucleotides upstream of the initiating AUG. © 1999 Academic Press

encoded at the 39-end of the genome by ORF4 (Revill et al., 1994), suggesting it may be translated from a subgenomic mRNA (sgRNA). An sgRNA molecule coterminal with the 39-end of the MBV genome and containing ORF4 (nt 3162–4009) would be a minimum of 0.85 kb and lengthened by an additional leader sequence upstream of the initiating AUG (Fig. 1). However, only genomic RNA and a molecule of 1.8 kb have been reported (Romaine and Schlagnhaufer, 1991). This latter molecule is unlikely to be the main sgRNA encoding the capsid protein, as it would have a 59 leader sequence of approximately 0.9 kb. The genomic nucleotide sequence of MBV commences with ACAAAA. This sequence is also present at the 59-terminus of many subgroup II luteoviruses and the cowpea strain of Southern bean mosaic sobemovirus (SBMV-C) (Keese et al., 1990; Mayo and Ziegler-Graff, 1996; Miller et al., 1997; Veidt et al., 1988; Hacker and Sivakumaran, 1997). This ACAAAA sequence is also present upstream of the initiating AUG in the ORF encoding the capsid protein and has been identified as the sgRNA 59-terminus for the Wageningen strain of potato leafroll virus (PLRV; a subgroup II luteovirus) and SBMV-C (Miller and Mayo, 1991; Hacker and Sivakumaran, 1997). We have now identified in MBV-infected mycelium a 0.9-kb sgRNA molecule that contains ORF4 and encodes the MBV capsid protein and show that it also has the sequence ACAAAA at its 59-terminus.

INTRODUCTION Mushroom bacilliform virus (MBV) infects the cultivated mushroom Agaricus bisporus and is often associated with La France disease, a devastating malady that may result in total crop loss (Hollings, 1978). The role of MBV in causing disease is unclear because a number of icosahedral virus-like particles also infect A. bisporus (Hollings, 1978; Hollings and Stone, 1971; van Zaayen, 1979). One particle with a genome of double-stranded RNA (dsRNA), La France infectious virus (LIV), is strongly implicated as the primary cause of the disease (Goodin et al., 1992; van der Lende et al., 1994). However, MBV is not detected in healthy mushrooms, yet it is not detected in all cases of La France disease (Romaine and Schlagnhaufer, 1995; van Zaayen, 1979). The 19 3 50-nm MBV particle encapsidates a singlestranded (ssRNA) positive-sense monopartite genome of 4 kb (Tavantzis et al., 1980, 1983). MBV is unique among mycoviruses of the higher fungi (ascomycetes and basidiomycetes) in having an ssRNA genome, as all other mycoviruses characterised to date have genomes of dsRNA (Buck, 1986; Ghabrial, 1998). The organisation of the four major MBV open reading frames (ORFs), their deduced amino acid sequences, and the translation strategy at the 59end of the genome have some similarity to plant viruses, particularly the sobemoviruses and subgroup II luteoviruses (Ma¨kinen et al., 1995; Revill, 1995; Revill et al., 1994, 1998). The MBV capsid protein of 22,000 molecular weight is

RESULTS AND DISCUSSION Northern hybridization with a digoxigenin (DIG; Boehringer Mannheim, Mannheim, Germany)-labeled RNA molecule complementary to the 39-terminal 130 nt of MBV showed that the probe hybridized to 4-kb genomic RNA and an additional RNA molecule of approximately 0.9 kb in RNA extracts of infected mycelium (Fig. 2, lane

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Present address: Centre for Plant Molecular Biotechnology, School of Life Sciences, Queensland University of Technology, Gardens Point, Brisbane, Queensland 4001, Australia. 2 To whom reprint requests should be addressed. Fax: 61-3-99054811. E-mail: [email protected]. 273

0042-6822/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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FIG. 1. The MBV genome arrangement, showing the location of the 0.9-kb sgRNA molecule (sgRNA), and the position of ACAAAA sequences in the genome (arrow). The dashes represent sequences upstream of the first AUG codon in ORF3. Pro, putative serine protease. RdRp, putative RNA dependent RNA polymerase; Cp, capsid protein. The GenBank accession number for the MBV genomic sequence is U07551.

2). No hybridization was detected using RNA extracted from uninfected mycelium (Fig. 2, lane 1). Primer extension analysis was performed on RNA extracted from MBV-infected mycelium using a complementary 32P-labeled primer located 70 nt downstream of the AUG initiating ORF4 (nt 3236–3213). This identified a single major band (Fig. 3, lane 5) and positioned the 59-terminus of the sgRNA molecule at nt 3117, 892 nt from the 39 end of the genome and 47 nt upstream of the coat protein initiating AUG (Fig. 1). The predicted size of an sgRNA molecule commencing at this position correlates with the size estimated by Northern hybridization (0.9 kb; Fig. 2, lane 2). The sequence ACAAAA at the transcriptional start point of the sgRNA is identical to that for SBMV-C (Hacker and Sivakumaran, 1997) and the Wageningen strain of PLRV (Miller and Mayo, 1991). However, the distance from the MBV sgRNA transcription start point to the initiating AUG in the capsid protein gene (47 nt) is more similar to SBMV (30 nt) than to PLRV (212 nt).

Additional ACAAAA sequences are present at nt 1, 192, and 2360 in the MBV genome (Fig. 1). An sgRNA molecule commencing at nt 2360 and extending to the 39-end of the genome would be 1649 nt long, similar in size to the 1.8-kb molecule observed by others (Romaine and Schlagnhaufer, 1991). An sgRNA molecule commencing at nt 192 would be 3817 nt long; no molecule of this size has been reported to date. However, using Northern hybridisation, we could find no evidence of these additional sgRNA molecules in RNA extracted from infected mycelium (Fig. 2, lane 2). This suggested that these ACAAAA sites are not transcriptional start points for sgRNA and that the 1.8-kb RNA species observed previously (Romaine and Schlagnhaufer, 1991) may have arisen through degradation of genomic RNA. It is also possible that the MBV isolate used by Romaine and Schlagnhaufer (1991) was a different strain, with a different genome arrangement and/or translation strategy to the isolate used in our studies, but this cannot be confirmed until sequence data are published.

FIG. 2. Northern hybridizaton of total RNA extracted from healthy (H) and infected (MBV) mycelium, probed with a negative-sense DIG labeled RNA probe directed against the 39terminal 150 nt of the MBV genome. The sizes of DIG-labeled RNA markers (Boehringer Mannheim) in kb are indicated (Mw).

FIG. 3. Analysis of the primer extension product (Pr) obtained using RNA extracted from MBV-infected mycelium and a primer complementary to the MBV sequence 70 nt downstream of the AUG codon initiating ORF4. Sequencing ladders obtained with cloned cDNA and the same primer are shown in lanes A, C, G, and T.

SUBGENOMIC mRNA OF MUSHROOM BACILLIFORM VIRUS

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ruses and sobemoviruses than to other plant viruses and mycoviruses (Revill et al., 1994, 1998). This similarity extends to both the presence of an sgRNA molecule coterminal with the 39-end of the genome that encodes the capsid protein and an ACAAAA sequence at the sgRNA transcription start point. Further studies on MBV replication are now under way using fungal protoplasts. These studies will enable us to determine the MBV replication and translation strategies in vivo and may also demonstrate whether MBV can replicate in the absence of other viruses linked with La France disease. MATERIALS AND METHODS Culture conditions and detection of subgenomic RNA

FIG. 4. Predicted RNA secondary structure surrounding the transcriptional start sites of sgRNA for (A) MBV (DG, 23.9 kcal/mol) and (B) SBMV (DG, 18.6 kcal/mol). The location of the first A in each ACAAAA motif is indicated by an asterisk. The secondary structure surrounding the ACAAAA site at nt 192 (C) (DG, 8.9 kcal/mol) and 2360 (D) (DG, 30.2 kcal/mol) in MBV are also shown. The analysis was performed using the mFOLD program (Zuker, 1989), in the GCG package of programs (Deveraux et al., 1984).

Secondary structure analysis of the nucleotides surrounding the transcription start site of the 0.9-kb MBV sgRNA identified a hairpin loop (Fig. 4A, DG, 23.9 kcal/ mol), similar to that observed for SBMV and other sobemoviruses (Fig. 4B) (Hacker and Sivakumaran, 1997). Hairpin loops are important structural elements that provide sites for interaction with proteins and nucleic acids (Nowakowski and Tinoco, 1997). This hairpin, or that in the complementary minus strand, may play a role in sgRNA synthesis (Hacker and Sivakumaran, 1997). Hairpin loops are also possible in the vicinity of the ACAAAA sequences at nt 192 and 2360 (Figs. 4C and 4D). However, we could not detect RNA molecules of the predicted sizes beginning at these sites, suggesting that the presence of an ACAAAA sequence and a hairpin loop do not necessarily define a transcriptional start point. The MBV genome arrangement and in vitro translation strategy show greater similarity to subgroup II luteovi-

Commercially grown A. bisporus sporophores infected with MBV were obtained from the same mushroom farm as that used in earlier studies (Revill et al., 1994). Infection with MBV was confirmed by immunoelectron microscopy and RT-PCR (Revill, 1995). Healthy sporophores were obtained from a farm with no history of infection. The sporadic nature of La France disease outbreaks in Australia in recent years meant that initial experiments were conducted on RNA extracted from sporophores that had been stored at 270°C for 2 years. This proved to be unsatisfactory, as Northern hybridization using the 39terminal RNA probe described below consistently identified a ladder of bands ranging in size from 0.3 to 4 kb, indicative of RNA degradation (data not shown). It was not possible to distinguish a 0.9-kb sgRNA molecule among the many bands present, and primer extension reactions using these RNA preparations were also unsuccessful. To obtain actively growing MBV-infected A. bisporus tissue, mycelial cultures that had been generated from the infected sporophores at the time of harvest were used. These cultures were produced by excising pieces of tissue from the sporophore and growing them on malt agar (Oxoid) for 3 weeks in the dark at 25°C, and they were stored at 4°C until required. The cultures were still viable after 2 years at 4°C, and MBV infection was confirmed by RT-PCR (Revill, 1995). For the experiments described in this report, 10-mm agar plugs were transferred to fresh malt agar plates and grown for 2 weeks at 25°C. Ten agar plugs were then transferred from the growing edge of the mycelium to 100 ml of malt extract broth (Oxoid) and incubated at 25°C for 3 weeks in 500-ml conical flasks without shaking. The mycelium was macerated in a blender, grown for an additional 48 h at 25°C, harvested by filtration, and frozen in liquid nitrogen. Total RNA was isolated from 0.1 g of mycelium using the Qiagen RNeasy kit, according to the manufacturer’s instructions. The RNA was electrophoresed alongside DIG (Boehringer Mannheim)-labeled RNA markers through a 1% agarose gel containing formalde-

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hyde and transferred to a nylon membrane by capillary action. The membrane was probed with a DIG-labeled RNA molecule complementary to the 39-terminal 130 nt of MBV RNA (nt 3880–4009). The hybridization, washing, and detection procedures used were as recommended by Boehringer Mannheim. Identification of the sgRNA transcription start point Primer extension analysis was performed on RNA extracted from mycelium, using a primer extension kit (Promega, Madison, WI) and a complementary 32P-labeled primer located 70 nt downstream of the AUG initiating ORF4 (primer located at nt 3236–3213). A dideoxy terminator sequencing reaction was performed on MBV cDNA (nt 19–3825) using the same primer, and the products were coelectrophoresed with the primer extension product through an 8% urea–polyacrylamide denaturing gel (Fig. 3). The gel was dried, and the products were detected by autoradiography. ACKNOWLEDGMENT This research was supported by the Australian Research Council.

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