Department of Microbiology, University of the Western Cape, Private Bag X17, Bellville 7535, Cape Town, South Africa1
Author for correspondence: Neil Leat. Fax +27 21 959 2266. e-mail nleat{at}uwc.ac.za
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Complete genome sequences are now available for Drosophila C virus (DCV), Plautia stali intestine virus (PSIV), Rhopalosiphum padi virus (RhPV), sacbrood virus (SBV), infectious flacherie virus (IFV), Himetobi P virus (HiPV) and Acyrthosiphon pisum virus (APV) (Ghosh et al., 1999 ; Isawa et al., 1998
; Johnson & Christian, 1998
; Moon et al., 1998
; Sasaki et al., 1998
; van der Wilk et al., 1997
). The genomes of these viruses are organized in one of three ways. The genomes of DCV, PSIV, HiPV and RhPV are monopartite and bicistronic, with replicase proteins encoded by a 5'-proximal ORF and capsid proteins by a 3'-proximal ORF. Translation initiation of the 3'-proximal ORF has been demonstrated to be dependent on an internal ribosome entry site (IRES) in the case of PSIV (Sasaki & Nakashima, 1999
). Nucleotide sequences highly similar to the IRES of PSIV were also found upstream of the 3'-proximal ORFs of RhPV and DCV, suggesting the presence of an IRES for these viruses as well. The organization of the genome of APV shows some resemblance to that of DCV, PSIV, HiPV and RhPV. It is monopartite and bicistronic, with replicase proteins encoded by the 5' region of the genome and capsid proteins encoded by the 3' region. However, the two ORFs overlap slightly, with the 3'-proximal ORF thought to be translated by a -1 ribosomal frameshift (van der Wilk et al., 1997
). The genomes of SBV and IFV are monopartite and monocistronic and resemble mammalian picornaviruses in that capsid proteins are encoded in the 5' region of the genome while replicase proteins are encoded in the 3' region of the genome.
The results of phylogenetic analyses involving putative RdRp domains reflect the differences in genome structure (Ghosh et al., 1999 ; Moon et al., 1998
). DCV, PSIV and RhPV form a distinct group of related viruses. SBV and IFV are distantly related to one another and APV appears to be unique. Furthermore, while the insect-infecting viruses appear to be related to members of the picorna-like virus lineage, they do not show a close relationship to a specific family.
Black queen-cell virus (BQCV) is one of 18 viruses isolated from honey bees (Allen & Ball, 1996 ; Ball & Bailey, 1991
). It was first isolated from queen prepupae and pupae, found dead in their cells (Bailey & Woods, 1977
). The name of the virus was derived from darkened areas on the walls of cells containing infected pupae. Pupae were found to contain large numbers of isometric virus particles, 30 nm in diameter. Particles contained a single genomic RNA and four capsid proteins, with molecular masses of 34, 32, 29 and 6 kDa. BQCV multiplied readily when injected into pupae, but could not be similarly propagated in caged adult bees. However, it did multiply in adult bees if ingested with spores of the microsporidian parasite Nosema apis (Bailey et al., 1983
). A correlation was also observed between the incidence of BQCV and N. apis in dead field bees from colonies in the UK; both showed peak infections during spring and early summer.
During the present study, a small RNA virus was isolated from adult bees in South Africa. On the basis of a strong serological reaction between this isolate and an antiserum raised against the original BQCV strain, the South African virus was assumed to be an isolate of BQCV. Here, we present the complete genome sequence of the South African isolate and show that its genome organization is most similar to DCV, RhPV, HiPV and PSIV. It is demonstrated that BQCV is not closely related to SBV, the only other honey bee virus for which comprehensive genome sequence is available. Furthermore, BQCV and SBV are shown to differ in genome organization. The South African isolate of BQCV will be referred to as BQCV (SA) to distinguish it from the original isolate, which will be referred to as BQCV (Rothamsted).
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For the purposes of virus propagation, 1·5 µl test samples from extracts of adult bees were injected into drone pupae through a ventral intersegmental membrane. Inoculated pupae were placed on filter paper in Petri dishes and incubated at 30 °C for 7 days. An open tray of water was placed in the incubator to prevent desiccation of the pupae.
RNA isolation.
RNA was isolated by treating purified virus preparations with an equal volume of TE-saturated phenol. Extracted RNA was precipitated from the aqueous phase with ethanol and resuspended in distilled water. RNA was used immediately after preparation.
Synthesis of cDNA.
Purified RNA was used as a template for cDNA synthesis. Reagents for cDNA synthesis were purchased from Promega and used according to the manufacturers instructions. Briefly, AMV reverse transcriptase was used for first-strand cDNA synthesis, while second-strand synthesis was achieved by using RNase H and E. coli DNA polymerase I. cDNA representing the 3' region of the virus was synthesized by initiating first-strand synthesis with an oligo(dT)15 primer. Subsequently, first-strand cDNA synthesis was initiated by using oligonucleotides designed from the sequence of the previous clone. Fragments of cDNA were blunt-ended with T4 DNA polymerase and cloned into the EcoRV site of pBluescript SK (+) (Stratagene). Ligation mixtures were transformed into E. coli JM109.
The 5' RACE system of Roche Molecular Biochemicals was used to generate two independent cDNA clones representing the 5' region of the viral genome. The manufacturers instructions were followed with the exception that the first-strand cDNA was tailed with dCTP. Subsequent PCR was conducted with an oligo(dG)14 primer and primers designed from previously determined genome sequence. PCR products were purified by using the High Pure PCR purification kit (Roche Molecular Biochemicals). PCR products were cloned into a T-vector constructed from pBluescript SK (+), digested with EcoRV and prepared according to the method of Marchuk et al. (1991) .
Nucleotide sequencing and analysis.
Double-stranded templates were sequenced by the dideoxy chain-termination method of Sanger et al. (1977) . Sequencing was conducted by using the Sequitherm kit (Epicentre Technologies) with CY-5-labelled primers. Nucleotide sequence was resolved on an ALFexpress automated DNA sequencer (Pharmacia). Where necessary, deletions were generated in DNA to be sequenced by exonuclease III digestion (Henikoff, 1984
). Both the plus and minus strands of each cDNA clone were sequenced and compared in order to confirm the final nucleotide sequence.
Nucleotide and amino acid sequence manipulation was carried out by using the University of Wisconsin Genetics Computer Group (GCG) sequence analysis package. Default algorithmic search parameters were used throughout. The FASTA program within the GCG suite was used to estimate the amount of nucleotide or amino acid sequence identity between two sequences. The BLAST algorithm of Altschul et al. (1990) was used to compare sequences generated in this study with entries in non-redundant nucleotide and protein sequences databases accessed by the National Center for Biotechnology Information. Multiple sequence alignments were conducted by using the CLUSTAL W program of Thompson et al. (1994)
. Phylogenetic trees were constructed by using the neighbour-joining method as implemented in the CLUSTAL W program. For each tree, confidence levels were estimated by using the bootstrap resampling procedure.
The GenBank accession number for the BQCV (SA) genome sequence is AF183905. A short portion of the BQCV (Rothamsted) genome sequence was accessed from GenBank under the accession number AF125252. Other sequences used in this study (with accession numbers) were: avian encephalomyelitis virus (AEV; CAA12416), APV (AF024514), broad bean wilt virus (BBWV; AAD38152), cowpea mosaic virus (CPMV; P03600), cricket paralysis virus (CrPV; M21938), DCV (AF014388), echovirus 23 (EV23; AAC79756), feline calicivirus (FCV; P27409), foot-and-mouth disease virus (FMDV; P03305), hepatitis A virus (HAV; BAA35107), HiPV (AB017037), IFV (AB000906), maize chlorotic dwarf waikavirus (MCDW; AAB58882), minute virus of mice (MVM; J02275), parsnip yellow fleck virus (PYFV; Q05057), poliovirus Sabin 1 strain (PV; CAA24465), rabbit haemorrhagic disease virus (RHDV; AAB02225), rice tungro spherical virus (RTSV; A46112), RhPV (AF022937), PSIV (AB006531), SBV (AF092924), Sindbis virus (SNBV; J02363), Southampton calicivirus (SRSV; AAA92983) and tomato black ring virus (TBRV; P18522).
SDSPAGE N-terminal sequencing.
Structural proteins were resolved on 12% SDSPAGE gels by using standard protocols (Sambrook et al., 1989 ). Proteins were blotted onto PVDF membranes and N-terminal sequencing was conducted by using an Applied Biosystems Procise Sequencer.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Apparently healthy adult bees were obtained from the Northern Province of South Africa. Extracts, potentially containing viruses, were prepared from these bees and injected into drone pupae. After an appropriate incubation period, preparations from the drone pupae were examined by electron microscopy for the presence of virus particles. Drones injected with extracts from Northern Province bees contained large numbers of isometric virus particles, 30 nm in diameter. Virus particles were not observed in preparations from pupae injected with buffer alone.
The serological relationship between the South African virus and previously isolated honey bee viruses was examined by immunodiffusion (Mansi, 1958 ). A strong positive reaction was observed between the South African isolate and an antiserum raised against BQCV (Rothamsted). In addition, a weak but distinct reaction was observed against SBV antiserum. No reactions were observed when the virus preparation was tested against antisera to other honey bee viruses. On the basis of the serological data, it was concluded that the predominant virus in the preparation was closely related to BQCV (Rothamsted) and was designated BQCV (SA). In addition, it was recognized that at least some SBV was present in the preparation as well.
Nucleotide sequence
The genome sequence of SBV has already been reported (Ghosh et al., 1999 ). In this study, the complete genome sequence of BQCV (SA) was determined. This involved sequencing two overlapping cDNA fragments of 5039 and 2719 bp (Fig. 1
). First-strand synthesis of the 2719 bp cDNA fragment was initiated with a primer complementary to nucleotides 35683585 on the BQCV (SA) genome. Two independent cDNA fragments representing the 5' end of the genome were generated by 5'-RACE and sequenced. The first fragment was generated by using a pair of primers complementary to the region between nucleotides 888 and 935 on the BQCV (SA) genome. The second fragment was generated by using a pair of primers complementary to the region between nucleotides 689 and 734. Since the genome sequence was obtained from a mixed preparation of BQCV and SBV, there was some concern that it might represent a mixed sequence. However, comparisons with the SBV nucleotide sequence revealed no significant similarities.
|
Two large ORFs were identified. The 5'-proximal ORF (ORF 1) was found to have an AUG initiation codon between nucleotides 658 and 660 and a UAG termination codon between nucleotides 5623 and 5625. These codons represent the first potential initiation and termination codons, respectively, of ORF 1.
The 3'-proximal ORF (ORF 2) had an AUG initiation codon between nucleotides 5942 and 5944 and a UAA termination codon between nucleotides 8393 and 8395. While translation termination at UAA (nt 83938395) is not unlikely, inferences from studies on PSIV suggest that translation initiation may not occur at an AUG codon. Sasaki & Nakashima (1999) identified a highly conserved region lying between the two ORFs of the genomes of PSIV, RhPV and DCV. This region was demonstrated to act as an IRES that facilitates the cap-independent translation of the 3'-proximal ORF of PSIV. When the PSIV IRES was compared with the BQCV genome, a highly similar sequence was found between nucleotides 5637 and 5836 (Fig. 2
). Given that the genomes of RNA viruses evolve at a high rate, and yet the intergenic region of the BQCV (SA) genome is similar to the PSIV IRES, it would be reasonable to suggest that it also acts as an IRES. The CUU initiation codon of the PSIV IRES aligns with a CCU codon in ORF 2 of BQCV (SA) (nt 58345836) (Fig. 2
). Direct inference would suggest that translation initiation of ORF 2 is facilitated by an IRES at this codon.
|
Non-structural proteins
The deduced amino acid sequence of ORF 1 was compared with entries in protein sequence databases by using BLAST. This revealed similarity between the predicted product of ORF 1 and the amino acid sequences of proteins involved in the replication of picorna-like viruses. Further analysis involving multiple sequence alignments facilitated the identification of domains within the BQCV (SA) sequence characteristic of helicases, 3C-like cysteine proteases and RdRp (Fig. 3).
|
With the exception of a GXCG domain, 3C-like cysteine proteases of picorna-like viruses are not particularly well conserved. However, a triad of amino acids involving a histidine, either an aspartate or a glutamate and the cysteine within the GXCG domain are recognized as being conserved and essential for protease activity (Koonin & Dolja, 1993 ). An equivalent of the GXCG domain was identified in the deduced amino acid sequence of ORF 1, from amino acids 1056 to 1059. Alignment of previously studied 3C-like cysteine proteases with the BQCV sequence suggests that the catalytic amino acids may be H909, D964 and C1058 (Fig. 3
).
Eight conserved domains have been identified in RdRp amino acid sequences (Koonin & Dolja, 1993 ). Of these, only the fourth, fifth and sixth domains are conserved throughout the three RdRp supergroups. The remaining domains are conserved primarily within the supergroup in which they occur. Conserved motifs typical of RdRps of supergroup 1 were found to lie between amino acids 1317 and 1584 on the deduced amino acid sequence of BQCV ORF 1 (Fig. 3
).
Structural protein analysis
Four mature structural proteins were identified for BQCV (SA) (Table 1). They will be referred to as CP1, CP2, CP3 and CP4 based on their proximity to the N terminus of the capsid polyprotein. N-terminal sequencing by Edman degradation was successful for all but the CP1 protein. The N-terminal sequences obtained correlated to positions on the deduced amino acid sequence of ORF 2 (Table 1
). While the molecular masses of CP3 and CP4 were fairly consistent, whether determined by SDSPAGE or calculated from the conceptual translation of ORF 2, this was not true for CP1 and CP2. The masses of these proteins were smaller when calculated from the deduced amino acid sequence of ORF 2 than when determined by SDSPAGE (Table 1
). Since an N-terminal sequence was not obtained for CP1, it could be argued that the discrepancy in molecular masses for this protein reflects an incorrect prediction of the translation initiation codon CCU (nt 58345836) for ORF 2. However, it is unlikely that this accounts for the discrepancy, since a translation termination codon UAG (nt 58075809) lies only nine codons upstream of the CCU codon. Even if translation initiation occurred immediately after the UAG codon, a protein of only 27·2 kDa would be predicted. It is possible that post-translational modifications of the CP1 and CP2 proteins account for their apparently greater molecular masses when determined by SDSPAGE as opposed to values determined from the deduced amino acid sequence of ORF 2.
|
Phylogenic analysis
The putative helicase and RdRp domains of BQCV were compared with equivalent sequences from picorna-like insect viruses and members of picorna-like virus families (Fig. 4). In each case, a similar result was observed. As expected, members of established virus families were most closely related to one another. In the case of picorna-like insect viruses, BQCV, DCV, PSIV, RhPV and HiPV grouped together irrespective of the domain compared. IFV and SBV appear to be more related to one another than to other viruses included in the study. APV does not show a clear relationship with a specific group of picorna-like viruses.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The South African isolate of BQCV was found to have an 8550 nt genome, excluding the poly(A) tail. The genome contained two ORFs, a 5'-proximal ORF encoding a putative replicase protein and a 3'-proximal ORF encoding a capsid polyprotein. Clearly, this genome organization is unlike that observed for SBV, which resembles mammalian picornaviruses. The overall structure of the BQCV genome does correspond, however, to that reported for members of the Caliciviridae, as well as to the insect viruses HiPV, RhPV, DCV and PSIV and, to some extent, APV. Despite the superficial similarity in the genome organization of these viruses, phylogenetic analyses indicate that the insect viruses are not closely related to members of the Caliciviridae. BQCV, HiPV, RhPV, DCV and PSIV form a group distinct from other picorna-like viruses. Within this group, BQCV appeared to be more closely related to PSIV and HiPV than to DCV and RhPV, irrespective of whether RdRp or helicase domains were compared.
Picorna-like viruses with monopartite bicistronic genomes employ one of three mechanisms to facilitate translation initiation of their 3'-proximal ORFs. Members of the Caliciviridae produce a subgenomic RNA. APV appears to initiate translation of its 3'-proximal ORF by a -1 ribosomal frameshift (van der Wilk et al., 1997 ). PSIV has been demonstrated to have an IRES immediately upstream and overlapping its 3'-proximal ORF (Sasaki & Nakashima, 1999
). Analysis of those viruses phylogenetically related to PSIV revealed motifs highly similar to the PSIV IRES immediately preceding their 3'-proximal ORFs. Given the degree to which these domains have been conserved despite the high rate of evolution of RNA viruses, it seems likely that they represent IRESs in the respective viruses. The mechanism by which translation initiation of the 5'-proximal ORFs of these viruses is facilitated may also involve IRES elements, as in picornaviruses. However, experimental evidence for this is not yet available.
The N-terminal sequences of the mature capsid proteins of DCV, RhPV and PSIV have been determined by Edman degradation. In the present study, sequences surrounding the capsid cleavage sites of the structural proteins of DCV, PSIV, RhPV and BQCV were compared (Fig. 5). For convenience these sites will be referred to as cleavage positions (1), (2) or (3), in accordance with Fig. 5
. The sequences at positions (1) or (3) correspond to available data on sites at which 3C-like proteases are likely to cleave, particularly as a glutamate residue is conserved immediately prior to the point of each cleavage. In contrast, the amino acid prior to the point of cleavage at position (2) is either tryptophan or phenylalanine. Furthermore, the sequence surrounding cleavage position (2) appears to be more highly conserved than that at positions (1) and (3). It would follow that the mechanism facilitating cleavage or processing at position (2) is different from that at positions (1) and (3). Two possibilities have been proposed to account for this. It has been suggested that capsid processing may involve cellular proteases with different recognition sites (Moon et al., 1998
). This was based on the observation that mature capsid proteins of CrPV were only observed in vitro when supplemented with Drosophila cell extracts. Alternatively, Sasaki et al. (1998)
suggested that cleavage at position (2) in PSIV may be analogous to the cleavage that occurs in the PV VP0 protein, yielding the VP4 and VP2 proteins. This occurs within the capsid during capsid maturation and appears to be dependent on the packaging of viral RNA.
|
![]() |
Footnotes |
---|
b Present address: IACR, Rothamsted, Harpenden, Herts AL5 2JQ, UK.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool.Journal of Molecular Biology215, 403-410.[Medline]
Bailey, L. (1967). The incidence of virus diseases in the honey bee.Annals of Applied Biology60, 43-48.[Medline]
Bailey, L. & Gibbs, A. J. (1964). Infection of bees with acute paralysis virus.Journal of Insect Pathology6, 395-407.
Bailey, L. & Woods, R. D. (1974). Three previously undescribed viruses from the honey bee.Journal of General Virology25, 175-186.[Medline]
Bailey, L. & Woods, R. D. (1977). Two more small RNA viruses from honey bees and further observations on sacbrood and acute bee-paralysis viruses.Journal of General Virology37, 175-182.
Bailey, L., Ball, B. V. & Perry, J. N. (1983). Association of viruses with two protozoal pathogens of the honey bee.Annals of Applied Biology103, 13-20.
Ball, B. & Bailey, L. (1991). Viruses of honey bees. In Atlas of Invertebrate Viruses, pp. 525-551. Edited by J. R. Adams & J. R. Bonami. Boca Raton, FL: CRC Press.
Ghosh, R. C., Ball, B. V., Willcocks, M. M. & Carter, M. J. (1999). The nucleotide sequence of sacbrood virus of the honey bee: an insect picorna-like virus. Journal of General Virology80, 1541-1549.[Abstract]
Gorbalenya, A. E., Koonin, E. V. & Wolf, Y. I. (1990). A new superfamily of putative NTP-binding domains encoded by genomes of small DNA and RNA viruses.FEBS Letters262, 145-148.[Medline]
Henikoff, S. (1984). Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing.Gene28, 351-359.[Medline]
Isawa, H., Asano, S., Sahara, K., Iizuka, T. & Bando, H. (1998). Analysis of genetic information of an insect picorna-like virus, infectious flacherie virus of silkworm: evidence for evolutionary relationships among insect, mammalian and plant picorna(-like) viruses.Archives of Virology143, 127-143.[Medline]
Johnson, K. N. & Christian, P. D. (1998). The novel genome organization of the insect picorna-like virus Drosophila C virus suggests this virus belongs to a previously undescribed virus family.Journal of General Virology79, 191-203.[Abstract]
Koonin, E. V. & Dolja, V. V. (1993). Evolution and taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences.Critical Reviews in Biochemistry and Molecular Biology28, 375-430.[Abstract]
Mansi, W. (1958). Slide gel diffusion precipitin test. Nature181, 1289.[Medline]
Marchuk, D., Drumm, M., Saulino, A. & Collins, F. S. (1991). Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products.Nucleic Acids Research19, 1154.[Medline]
Moon, J. S., Domier, L. L., McCoppin, N. K., DArcy, C. J. & Jin, H. (1998). Nucleotide sequence analysis shows that Rhopalosiphum padi virus is a member of a novel group of insect-infecting RNA viruses.Virology243, 54-65.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors.Proceedings of the National Academy of Sciences, USA74, 5463-5467.[Abstract]
Sasaki, J. & Nakashima, N. (1999). Translation initiation at the CUU codon is mediated by the internal ribosome entry site of an insect picorna-like virus in vitro.Journal of Virology73, 1219-1226.
Sasaki, J., Nakashima, N., Saito, H. & Noda, H. (1998). An insect picorna-like virus, Plautia stali intestine virus, has genes of capsid proteins in the 3' part of the genome.Virology244, 50-58.[Medline]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Research22, 4673-4680.[Abstract]
van der Wilk, F., Dullemans, A. M., Verbeek, M. & Van den Heuvel, J. F. (1997). Nucleotide sequence and genomic organization of Acyrthosiphon pisum virus.Virology238, 353-362.[Medline]
Received 14 October 1999;
accepted 18 April 2000.