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Journal of General Virology (2005), 86, 91–105 DOI 10.1099/vir.0.80488-0 Complete comparative genomic analysis of two field isolates of Mamestra configurata nucleopolyhedrovirus-A Lulin Li,13 Qianjun Li,24 Leslie G. Willis,1 Martin Erlandson,2 David A. Theilmann1 and Cam Donly3 Correspondence 1 Pacific Agri-Food Research Centre, AAFC, Summerland, BC, Canada Cam Donly 2 [email protected] 3 Saskatoon Research Centre, AAFC-Saskatoon, SK, Canada Received 30 July 2004 Accepted 20 September 2004 Southern Crop Protection and Food Research Centre, AAFC, London, ON, Canada A second genotype of Mamestra configurata nucleopolyhedrovirus-A (MacoNPV-A), variant 90/4 (v90/4), was identified due to its altered restriction endonuclease profile and reduced virulence for the host insect, M. configurata, relative to the archetypal genotype, MacoNPV-A variant 90/2 (v90/2). To investigate the genetic differences between these two variants, the genome of v90/4 was sequenced completely. The MacoNPV-A v90/4 genome is 153 656 bp in size, 1404 bp smaller than the v90/2 genome. Sequence alignment showed that there was 99?5 % nucleotide sequence identity between the genomes of v90/4 and v90/2. However, the v90/4 genome has 521 point mutations and numerous deletions and insertions when compared to the genome of v90/2. Gene content and organization in the genome of v90/4 is identical to that in v90/2, except for an additional bro gene that is found in the v90/2 genome. The region between hr1 and orf31 shows the greatest divergence between the two genomes. This region contains three bro genes, which are among the most variable baculovirus genes. These results, together with other published data, suggest that bro genes may influence baculovirus genome diversity and may be involved in recombination between baculovirus genomes. Many ambiguous residues found in the v90/4 sequence also reveal the presence of 214 sequence polymorphisms. Sequence analysis of cloned HindIII fragments of the original MacoNPV field isolate that the 90/4 variant was derived from indicates that v90/4 is an authentic variant and may represent approximately 25 % of the genotypes in the field isolate. These results provide evidence of extensive sequence variation among the individual genomes comprising a natural baculovirus outbreak in a continuous host population. INTRODUCTION Baculoviruses are pathogenic for arthropods, mainly insects of the Lepidoptera, Hymenoptera and Diptera. These viruses have been investigated because of their potential as 3Present address: Animal Disease Research Institute, 3851 Fallowfield Rd, Ottawa, ON, Canada, K2H 8P9. 4Present address: Department of Medicine/Division of Geographic Medicine, University of Alabama at Birmingham, BBRB 203, 845 South 19th Street, Birmingham, AL 35294-2170, USA. The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AF539999. Figures showing mutations in the promoter regions of lef-7 (orf16) and orf25, an alignment of the LEF-9 C-terminal amino acid sequences of v90/4 and v90/2 with those of 13 lepidopteran baculoviruses and an alignment of the 59-end sequences of bro-b between v90/4 and v90/2 are available as supplementary material in JGV Online. 0008-0488 Printed in Great Britain biological control agents of agricultural and forest pests. Baculoviruses contain circular, double-stranded DNA genomes of 80–180 kb. To date, the genomes of 26 nucleopolyhedroviruses (NPVs) have been sequenced completely. The bertha armyworm, Mamestra configurata, is an important pest of cruciferous oilseed crops in western Canada, from which a number of NPVs have been isolated from field populations. In exploring the potential of these viruses for control of M. configurata and other pest insects, the viral isolates have been characterized with respect to their virulence in M. configurata, as well as their genomic restriction endonuclease (REN) profiles (Erlandson, 1990). Analysis of M. configurata NPV (MacoNPV) isolates has revealed significant diversity in their biological properties and genetics. Recently, we reported the complete genome analysis of two MacoNPV species (Li et al., 2002a, b). These Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 05:37:59 91 L. Li and others two viruses are closely related, but have evolved divergently into two separate baculovirus species, designated MacoNPV-A and MacoNPV-B. Very little is known about the genetic diversity of baculoviruses in field populations. Restriction fragment length polymorphisms have been reported for many species, suggesting that some level of natural genome variation is common among baculoviruses (Croizier & Ribeiro, 1992; Garcia-Maruniak et al., 1996; Hodgson et al., 2001; McIntosh et al., 1987; Muñoz et al., 1999). In addition, it has recently been reported that field isolates of the archetypal baculovirus Autographa californica multiple NPV (AcMNPV) contain additional genes to those in the previously reported genome sequence (Lu et al., 1996; Schetter et al., 1990; Yanase et al., 2000). These observations suggest that baculovirus genomes are quite dynamic and that this variability may provide selective or evolutionary advantages to the virus population. In this study, we describe the sequence of a second MacoNPV-A genome, variant 90/4 (v90/4), which was initially identified due to REN profile and biological differences in comparison to the archetypal MacoNPV-A variant, 90/2 (v90/2). This is the first study to perform a complete comparative analysis of two genomes of the same species from the same virus outbreak in a wild insect population. The genomes of v90/4 and v90/2 reveal that significant sequence variation exists between genotypes within the same virus species. METHODS Insects and viruses. Larvae from a laboratory culture of M. config- urata were maintained on a semi-synthetic diet (Bucher & Bracken, 1976) at 21 uC, 60 % relative humidity and an 18 : 6 light : dark photoperiod. The v90/4 isolate was derived from a single larval cadaver that was collected near Lamont, Alberta, Canada (53u 509 N 112u 389 W), in 1990. The Lamont virus isolate was amplified by infection in vivo. Initial REN analysis indicated that this isolate contained a heterogeneous mixture of several genotypes. Haemolymph was collected from fourth-instar bertha armyworm larvae 4 days after being infected with the Lamont isolate and processed for infection of insect cell culture. Briefly, haemolymph samples were collected into 1?5 ml centrifuge tubes containing 0?5 ml Grace’s tissue-culture medium (Gibco-BRL) on ice. Haemocytes were pelleted by lowspeed centrifugation (1400 g for 5 min) and the supernatant was transferred to 0?45 mm SPIN-X centrifuge tube filters (COSTAR) and centrifuged for 1 min in a benchtop centrifuge (Eppendorf 5415 C). The filtrate was then used to infect Mamestra brassicae cells (IZD-MB-0503, ATCC CRL 8003) in plaque assays. A series of 10 plaques was selected and replaqued a second time. Because the production of virus progeny was not very efficient in this cell line (approx. 105 TCID50 units ml21), plaque isolates were amplified in M. configurata larvae for further study. A single plaque isolate, v90/4, was chosen for sequencing. The v90/2 isolate was derived from a single larval cadaver that was collected near Wilkie, Saskatchewan, Canada (52u 309 N 108u 419 W), in 1990. It was amplified by infection in vivo and cloned by using an in vivo isolation technique, as described by Smith & Crook (1988). REN analysis of the virus isolate did not reveal the presence of 92 submolar fragments (Li et al., 1997) and, in subsequent analysis of the genome sequence data, very few (<50) nucleotide polymorphisms were detected, indicating that the isolate was genetically homogeneous. Stocks of MacoNPV were produced by infection of fourth-instar bertha armyworm larvae by contamination of the diet with 1?46104 PIB per cm2 of diet surface. Virus production and polyhedral inclusion body (PIB) isolation, virion purification and viral DNA extraction essentially followed previously described methods (Erlandson, 1990; Li et al., 1997). REN analysis of viral DNA. REN analysis of viral DNA was per- formed as described previously (Li et al., 1997). Briefly, purified DNA of MacoNPV-A v90/4 and v90/2 was digested with HindIII at 37 uC for 3 h, then separated on 0?7 % agarose gels in 0?56 TBE (45 mM Tris/borate, 1 mM EDTA) at 20–40 V for 15–22 h. Gels were stained with ethidium bromide and photographed. Bioassays. Bioassays were carried out with neonate bertha armyworm larvae by using a droplet-feeding bioassay with five virus doses and 100 larvae per dose, as described previously (Erlandson, 1990). Those larvae consuming virus inoculum during a 30 min exposure period were included in the assay and were transferred to an artificial diet and incubated at 21 uC, with fresh diet added as needed for the duration of the bioassay. Mortality was tabulated daily and mortality response data were analysed on the basis of mortality on day 14 post-infection. LD50 estimates were determined by using SAS-PROBIT (version 8, SAS Institute). DNA sequencing and sequence analysis. The MacoNPV-A (v90/4) genome was sequenced by using a shotgun approach, as described previously (Li et al., 2002a). In total, 1929 sequencing runs of 500–600 readable bases were assembled into 15 contigs by using Sequencher 4.0 software (Gene Codes Corporation). PCR was performed to synthesize DNA fragments bridging the gaps between contigs by using MacoNPV-A (v90/4) genomic DNA as template. PCR products were sequenced from both ends. The sequences were assembled with the initial contigs into a single, circular contig. Sequences were analysed with Wisconsin Genetics Computer Group programs (Devereux et al., 1984), GeneWorks 2.3 (IntelliGenetics) and MacVector 7.1 (Accelrys). Homology searches were carried out with GenBank/EMBL, SWISSPROT and PIR databases by using the BLAST algorithm (Altschul & Lipman, 1990). Multiple sequence alignments were performed by using CLUSTAL W (Thompson et al., 1994). MacoNPV genome sequence accession numbers are AF539999 for MacoNPV-A (v90/4), AF467808 for MacoNPV-A (v90/2) and AY126275 for MacoNPV-B. Sequence analysis of HindIII fragments cloned from fieldisolated virus. Occluded virus of the non-plaque-purified MacoNPV Lamont field isolate, from which MacoNPV-A v90/4 was derived, was purified from infected M. configurata larvae (as described above). Occluded virus DNA was digested with HindIII, separated on 0?7 % agarose gels and selected HindIII fragments were purified from gel slices (QIAquick Gel Extraction kit; Qiagen) and cloned in vector pUC18. Eight clones for each HindIII fragment, 3051–4547 and 67365–70629, were sequenced. The DNA sequences were aligned with respect to MacoNPV-A v90/2 and v90/4 genome sequences (LaserGene, Seqman) and compared. RESULTS LD50 and REN profile of MacoNPV-A (v90/4) The v90/4 virus, along with a number of other MacoNPV isolates, including v90/2, was originally collected from NPV epizootics in an outbreak population of M. configurata Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 05:37:59 Journal of General Virology 86 Comparative genomic analysis of MacoNPV-A M (bp) v90/4 v90/2 (bp) 12 000 7 000 5 000 4 000 3 000 6 602 5 309 4 442 2 923 2 000 1 650 1 527 1 000 850 650 500 Fig. 1. REN profiles of MacoNPV-A v90/4 and v90/2 isolates. DNA was isolated as described in Methods and 1 mg DNA was digested with HindIII and separated through a 0?7 % agarose gel in 0?56 TBE buffer. Names of isolates are given above each lane. M, Marker lane containing 1 kb Plus DNA ladder (Invitrogen), with the sizes of DNA fragments indicated to the left of the panel. The major differences between the two MacoNPV-A lanes, including two fragments (6602 and 4442 bp) unique to v90/2 and three fragments (5309, 2923 and 1527 bp) unique to v90/4, are marked by arrows. throughout western Canada in 1990. Preliminary screening of these isolates by using REN profile analysis showed that v90/4 was very similar to v90/2, but contained some restriction fragment length polymorphisms, as shown in Fig. 1. Bioassays with v90/4 and v90/2 in neonate M. configurata demonstrated that v90/4 was less virulent, as its LD50 value was 128?4 (95 % confidence interval, 96–171) PIB per larva, tenfold higher than that of v90/2 at 11?9 (95 % confidence interval, 8?6–15?6) PIB per larva. Genome sequence comparison of v90/4 and v90/2 The genome of v90/4 is 153 656 bp, 1404 bp smaller than that of v90/2. A complete sequence alignment showed that 99?5 % of the v90/4 genome sequence is identical to that of v90/2. As shown in Fig. 2, there are 521 nucleotide changes in aligned regions when the genomes of v90/4 and v90/2 are compared. In addition, relative to v90/2, the v90/4 genome has 31 deletions and 14 insertions, comprising 1527 and 123 bp, respectively. http://vir.sgmjournals.org Analysis of all variations showed that 398 point mutations, six insertions totalling 30 bp and 20 deletions totalling 1326 bp occur in predicted ORFs; 65 point mutations, three insertions (13 bp) and five deletions (102 bp) occur in intergenic regions; and 58 point mutations, two insertions (79 bp) and six deletions (98 bp) occur in homologous repeated (hr) regions. Only 27 % of the mutations cause amino acid sequence substitutions. Although point mutations, insertions and deletions are dispersed throughout the genome, specific regions have a significantly higher density of changes (Fig. 2). The most variable region is located between hr1 and orf31 (bro-c) (v90/4, 15?0–27?1 kb; v90/2, 15?0–28?3 kb). In this 12?1 kb region (7?7 % of the genome), there are 261 of the 521 point mutations, accounting for 50 % of the total nucleotide changes. Of the 261 point mutations, 82 cause nonsynonymous changes and only the chitinase and orf27 genes do not have any amino acid changes. This region also contains multiple deletions in the v90/4 genome. The largest deletion is a 1165 bp fragment that contains orf21 (bro-a) and a portion of orf20. An alignment of the v90/4 and v90/2 sequences around the junction regions of the 1165 bp deletion is shown in Fig. 3. The 1165 bp fragment contains a palindromic sequence (TCTAATTAGA) at its 59 end and another palindromic sequence (AAATATTT) at the 39 end. In total, 417 ORFs of 150 bp or longer, starting with an ATG, were detected in the v90/4 genome. Of these, 168 have minimal overlap with adjacent ORFs or hr regions, or showed significant homology to genes in GenBank. Gene content and arrangement are almost identical between v90/4 and v90/2 (Fig. 2). However, there is a single gene difference between the two viruses. As indicated above, bro-a is absent in v90/4. Of the 168 common ORFs, 49 ORFs show 1–12 % amino acid sequence variation and 12 ORFs vary in size (see Table 1). Among the 63 ORFs that are common to all lepidopteran baculoviruses (Chen et al., 2002; Li et al., 2002a), eight ORFs have amino acid substitutions in their encoded products. These include me53, lef-1, tlp-20, lef-8, lef-9, orf80, odv-e66 (orf144) and ie-1. Five of 12 ORFs that are unique to v90/2 and v90/4, orf5, orf10, orf18, orf23 and orf64 (Li et al., 2002b), have amino acid sequence substitutions in their putative gene products. Alterations in the regulation of gene expression can have significant effects on gene function; therefore, the promoter regions of all genes were analysed for variations in known regulatory motifs. Table 2 lists the nucleotide variations between v90/2 and v90/4 that occur in promoter regions located within 150 bp of an ORF. The promoter motifs in these regions are also presented. For example, the T to A substitution at 245 upstream of orf16 (lef-7) forms a TATA box in this region in v90/4 that is not found in v90/2 (see Supplementary Fig. S1, available in JGV Online). orf25 in v90/2 contains an early gene motif (249-TATAAA, 221CAGT); in v90/4, there is a C to T substitution at 222 that mutates the potential transcriptional start site, CAGT, into Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 05:37:59 93 L. Li and others Fig. 2. Comparison of genome structure between MacoNPV-A v90/4 and v90/2. The figure depicts a schematic representation of the MacoNPV-A genome, with map positions of the 169 ORFs of MacoNPV-A v90/2 (Li et al., 2002b) represented by arrows indicating transcriptional direction and relative size. Numbers above arrows represent the number of each ORF (Li et al., 2002b). Red vertical lines represent the location of point mutations in the v90/4 genome; dark blue bars (lowered) represent deletions and light blue bars (raised) represent insertions in v90/4, compared to v90/2. Yellow arrows represent ORFs with changes in amino acid sequences. Green arrows represent ORFs with identical amino acid sequences. hr sequences and their positions on the genome are indicated by empty boxes. Numbers below arrows represent the genome position relative to base 1. TAGT (see Supplementary Fig. S1, available in JGV Online). Pullen & Friesen (1995) showed that mutation of this base in the AcMNPV ie-1 promoter reduced gene expression dramatically. The hr sequences have been shown in numerous studies to be important as origins of DNA replication and as transcriptional enhancers (reviewed by Friesen, 1997; Lu et al., 1997). Comparing v90/2 and v90/4, hr1, hr3 and hr4 94 all show sequence variation but, interestingly, no changes were observed in hr2 (Fig. 2). Relative to v90/2, the v90/4 hr3 has a deletion of 78 bp and hr4 has an insertion of 76 bp, both of which represent a single hr repeat unit. This is similar to what was observed in hr elements of fieldisolated variants of Spodoptera exigua multiple NPV (SeMNPV) (Muñoz et al., 1999). An alignment of hr4 sequences of v90/4 and v90/2 shows the significant changes that can occur in the hr elements (Fig. 4). The v90/4 hr4, Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 05:37:59 Journal of General Virology 86 Comparative genomic analysis of MacoNPV-A Fig. 3. Comparison of the sequences in the 1165 bp deletion/insertion region between MacoNPV-A v90/4 and v90/2. The v90/2 1165 bp fragment, missing in v90/4, contains a single bro-a gene (shown as the grey arrow) and a portion of orf20. At the ends of the 1165 bp fragment, there are two different palindromic sequences, TCTAATTAGA and AAATATTT (underlined). The identical sequences between v90/4 and v90/2 are linked by vertical lines, whilst nucleotides missing in either v90/4 or v90/2 are represented by dashes. in addition to the 76 bp insertion, also contains five deletions (totalling 20 bp) and 33 point mutations. The hr1 and hr3 elements have eight and 17 point mutations, respectively, relative to v90/2. mixture of genotypes in the original MacoNPV field isolate. MacoNPV-A v90/4 appears to represent approximately 25 % of the mixed genotype population, as measured by cloned fragment pools. Sequence polymorphism in the MacoNPV-A genome Variations in structural protein genes In the process of assembling the v90/4 sequence from shotgun clones, clear alternative base readings or polymorphisms were occasionally observed for specific positions. After ruling out sequencing errors by close examination of all related electropherograms, 214 sequence polymorphisms were detected. As shown in Table 3, the majority (186) of the polymorphisms occurred in ORF regions, but they only caused 47 amino acid polymorphisms in 26 ORFs. Among the ORFs that contain polymorphisms, 14 are homologous to known genes. These include the potential structural protein genes gp37, gp41, 91k, vef and odv-e66 and the viral DNA replication- and transcription-associated genes lef-8 and ae, as well as homologues of fgf, cg30, hoar, bjdp, bro-d and bro-e. The MacoNPV field isolate from which v90/4 is derived was determined to be heterogeneous, based on REN analysis showing submolar ratios of some REN fragments. In an attempt to determine whether v90/4 is representative of the genotypes within the heterogeneous field isolate, selected HindIII fragments were cloned and sequenced. The sequences of HindIII fragment 3051–4547 clones that were taken directly from the field isolate fell into two groups. Two clones had sequences identical to that of MacoNPV-A v90/4 and the remaining six clones were of a genotype that differed from both v90/4 and v90/2 (Fig. 5). Similarly, two of eight HindIII fragment 67365–70629 clones were identical to MacoNPV-A v90/4 sequence, with the remaining six clones representing two additional genotypes (5 : 1). The sequence data indicate that MacoNPV-A v90/4 is an authentic genotype that was found in the heterogeneous http://vir.sgmjournals.org Seven genes encoding known structural proteins contain variations in amino acid sequences between v90/4 and v90/2 or have amino acid sequence polymorphisms in v90/4. Among these genes is the viral enhancing factor gene (vef). VEF is a metalloprotease that is known to enhance viral infectivity and is present in the viral occlusion bodies of granuloviruses and a few NPVs. MacoNPV-A VEF is 847 aa in size and has been shown to enhance infection of AcMNPV (Li et al., 2003). There are two amino acid substitutions in the putative VEF protein of v90/4 relative to v90/2, both occurring in the C-terminal region. At aa 758 and 779, v90/2 has an asparagine and a threonine, whereas v90/4 has an aspartic acid and an alanine, respectively (Table 1). In addition, v90/4 VEF has three amino acid polymorphisms, an asparagine to aspartic acid and two leucine to phenylalanine polymorphisms, at aa 536, 800 and 804, respectively (Table 3). As these variations occur outside the known functional domain region of VEF, it is unknown whether these amino acid residues are important for activity. Two additional genes encoding structural proteins, p87/ vp80 (orf82) and odv-e66 (orf144), have amino acid sequence substitutions in v90/4 relative to v90/2 (Table 1). P87/VP80 has a single substitution at aa 327, an alanine in v90/2 and serine in v90/4. In v90/4, ORF144 (ODV-E66) has a single substitution at aa 36, where a lysine is replaced by an asparagine, and a leucine to isoleucine polymorphism at aa 405. ORF78 is a second ODV-E66 homologue. It contains a threonine to isoleucine polymorphism and an alanine to serine polymorphism at aa 407 and 451, respectively. It is notable that all of the substitutions and polymorphisms Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 05:37:59 95 L. Li and others Table 1. Amino acid sequence variations between v90/2 and v90/4 -, Deletion of amino acid residues; (+), conserved mutations as defined by ORF Name 5 7 8 10 15 16 18 me53 xe lef-7 19 20 21 23 bro-a 24 bro-b 26 28 29 30 96 Sequence variation Position v90/2 v90/4 49 83 119 288 6–8 251 11 45 154 183 10–12 111 135 151 L E D A DRR G A D K E IIT E M D S K(+) Y T AVG E P N E(+) D(+) V(+)FI V I(+) Y 49 51 74–82 31 34 39, 40 45–47 51, 52 54–57 59 61–63 64–93 96–106 109–111 113–116 169 174, 175 177, 178 186 207 246 249 140 151, 152 268 10 140 155 220 22 80–83 86 28 50 R H P S CFYFELYL FI(+)QI(+) LF(+)KY S P R K(+) K, A V, L VAT I(+)WS(+) FQ EP QRFW K(+)K(+)NL E K(+) KSY Q(+)PF(+) ----------TSL VT(+)S VQAK L(+)HPQ(+) T A VI AV(+) SV A(+)A P L M I(+) D E(+) E Q(+) Y C SK PE(+) Q K(+) Q E(+) R K(+) T I L P E K(+) KDET ----D A M I N D(+) BLAST ORF size Identity ORF (90/2, 90/4) (%) terms. Name Position v90/2 98 319, 330 99 94 99 99 99 98 97 175, 65 95 85, 82 88 372, 331 90 31 bro-c 32 34 35 40 45 55 56 59 he65 61 98 98 110, 106 95 214, 213 90 63 64 66 70 72 80 82 89 Sequence variation lef-1 pkip 52 54 106 164–166 168–170 172 175, 176 179 181 184 186 193 205 210 142 148 207 211 213 219 230 235–237 239 247, 248 91 87 77 99 165, 166 83 283 88/89 112 142 147 109 114 154 159, 160 192 Q L G VSN FAC N YA P E S T N V E K K K Q L M N ALT E KI S K R F KQ S T ----E I D T D R HY R v90/4 -F D DIQ VVR I LP E D(+) A(+) I S(+) I(+) D(+) E(+) R(+) Q(+) K(+) M(+) T G EM(+)K Q(+) NV N(+) R(+) K(+) L NK(+) F A EEEE -V(+) N(+) I N K(+) ----G 9 T -sod 120 E K(+) 105, 106 SM N(+)I(+) 146 Q K(+) 185 I V(+) 280 Q R(+) 207 E D(+) p87/vp80 327 A S(+) vef 758 N D(+) 779 T A Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 05:37:59 ORF size Identity (90/2, 90/4) (%) 97 338, 341 99 99 99 99 98 99 99 98 225, 223 98 120, 145 100 99 99 99 168, 169 99 99 99 99 Journal of General Virology 86 Comparative genomic analysis of MacoNPV-A Table 1. cont. ORF Name Sequence variation Position 90 bro-e 96 102 108 118 123 124 bro-g lef-9 126 p94 tlp20 350 355 172 138 156 20 146 301 341 467 161 202 v90/2 v90/4 T K V S E L S Y I G Y E LI Q(+) A F G V(+) F H(+) M(+) V H(+) D(+) ORF size (90/2, 90/4) Identity (%) 360, 361 99 ORF 99 99 99 99 99 99 127 130 141 144 147 99 154 162 appear at the C termini of the ODV-E66 proteins and do not occur in any of the predicted transmembrane domains or the nuclear-targeting signal (Hong et al., 1994). In addition to VEF and ODV-E66, amino acid sequence polymorphisms also occur in the putative products of gp37, gp41 and 91k in v90/4 (Table 3), but none of the amino acid changes occur in regions that have known or predicted function. The 91k gene contains a polymorphism that causes an insertion of a single serine residue at aa 660/661 in one of the two putative translation products. The gp41 gene, which encodes an ODV-specific protein, contains a C to T polymorphism, changing a glutamine codon to a stop codon. This will result in two different proteins potentially being produced, one that is 333 aa in size and is identical to that of v90/2 and a second that is 146 aa, with the C-terminal sequence truncated. If translated, the smaller protein may have altered or antagonistic functions relative to the full-length GP41. Olszewski & Miller (1997) showed that a single base mutation in the C terminus of gp41 was responsible for a temperaturesensitive mutation that inactivated GP41 in AcMNPVinfected cells. Loss of functional GP41 resulted in the inhibition of virus production. Name bro-h lef-8 odv-e66 ie-1 Sequence variation Position v90/2 v90/4 425 492 585 20 65 545 36 42 84 62 26/27 K I Q G E S K T S I -- R(+) M(+) K(+) A K(+) N(+) N I N(+) V(+) D ORF size (90/2, 90/4) Identity (%) 99 99 99 99 98 606, 607 99 99 amino acid substitutions relative to v90/2 LEF-9: tyrosine to histidine, isoleucine to methionine and glycine to valine at aa 301, 341 and 467, respectively (Table 1). LEF-8 has a single serine to asparagine substitution between v90/2 and v90/4 at aa 545, which is in a less conserved region of this protein. In addition, v90/4 LEF-8 has an arginine to cysteine polymorphism and an asparagine to threonine polymorphism at aa 364 and 632, respectively. LEF-1 and LEF-7 have single amino acid sequence substitutions, from arginine to lysine and aspartic acid to asparagine, at aa 77 and 45, respectively, between v90/2 and v90/4 (Table 1). In view of the importance of these genes in viral DNA replication and/or transcription, these changes could potentially affect virus replication. Kamita & Maeda (1997) reported that mutation of two adjacent nucleotides in the AcMNPV helicase gene, which causes a single amino acid change, resulted in host-range expansion (Argaud et al., 1998; Kamita & Maeda, 1997). Variations in DNA replication and transcription regulatory genes In v90/4 IE-1, a single aspartic acid residue is inserted at aa 26 compared to v90/2, which is within the acidic activation domain. The insertion increases the acidic nature of this domain, possibly increasing the transactivation potential of this region. In addition, AE, an exo-alkaline nuclease that is hypothesized to be involved in the processing of DNA replication intermediates (Li & Rohrmann, 2000), has a glutamine to histidine polymorphism at aa 273 in v90/4. Among the v90/4 homologues of viral genes that are involved in DNA replication and transcription, ie-1, lef-1, lef-7, lef-8 and lef-9 contain sequence variations relative to v90/2 [Table 1, Supplementary Fig. S2 (available in JGV Online)]. In transient assays, AcMNPV IE-1 and LEF-1 are required for viral DNA replication (Kool et al., 1994), whereas LEF-7, LEF-8 and LEF-9 are required for late gene expression (Lu & Miller, 1995). LEF-1 has been characterized as a primase (Mikhailov & Rohrmann, 2002). AcMNPV LEF-8 and LEF-9 are subunits of the viral RNA polymerase II complex (Guarino et al., 1998). v90/4 LEF-9 has three The me53 and cg30 genes are putative transcription regulatory genes, but their actual function during the baculovirus life cycle has yet to be determined. Homologues of me53 are conserved in all lepidopteran baculoviruses that have been sequenced to date. Both ME53 and CG30 contain RING finger and leucine zipper domains that are found in other polypeptides known to be involved in gene regulation (Knebel-Mörsdorf et al., 1993; Thiem & Miller, 1989). The ME53 homologue in v90/4 has a single alanine to threonine substitution at aa 288 relative to v90/2, which is within the conserved RING finger domain region. However, the amino http://vir.sgmjournals.org Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 05:37:59 97 L. Li and others Table 2. Changes in promoter regions between v90/4 and v90/2 ORF 4 5 6 8 16 18 22 23 24 Name hoar odvp-6e lef-7 chit bro-b 25 27 28 29 30 31 50 52 54 57 59 61 62 63 68 77 90 91 97 103 116 117 127 128 129 130 139 141 142 143 147 149 150 157 158 159 bro-c ae xe bro-e bro-h iap3 lef-8 lef-11 39k p10 Change* PromoterD ATTTTAAAAGT>278 A>271 A277G T237A T245A G2141T GA279,78AC, T274C A249G G2114A, CT2110,109TG, G239T, T237A, TACTT234–230-, G228A, G226A, A223T, T215A, A210T, A28T T274C, 260-, A233G, C222T, A29T G2135C, A2131G, A2100G, G255A, G253T, A251G, CC248,247TT, GTA229–227CAC TAC281–279GTG, T257C, C255A, C253T, T28C T2125C, A269T, T261C AG2128,2127GA, A2125G, G2123A, T2121C, CC222,221TT A2109G, C2107T, T2105C, CT2103,2102TC, G278A, A263G C229T, C227G, G224A C2129T G294A A2110G C2146T, T265C T218C, A210G A276G A2128G, T224A C2122T A211C C231T T2129G, C25T T273C C26T G2145A, T2130A, A2109C G2109A G2105A C285T C285T A294G C2137T, A23C297G AA223,222TT T280C A2113G, C257T, G225A T257C A223G T231C A285G T2132C Promoter motifsd E L 274-TATAA, 245-CAGT 263-GTAAG E L 260-TATAAA, 227-CAGT 218-ATAAG E 249-TATAAA E 277-TATAAA, 251-CAGT E 273-TATAAA, 251-CAGT E 243-TATAA, 237-CATT E 262-TATATA, 226-CAGT L L L 242-ATAAG 236-ATAAG 226-ATAAG L 2128-ATAAG, 280-GTAAG L L L L 2148-ATAAG, 2102-ATAAG 244-TTAAG 219-ATAAG 239-ATAAG *Numbers represent the location of the nucleotide changes in each promoter region; letters in front of the numbers are sequences present in v90/4; letters behind the numbers are sequences present in v90/2; sequences in front of ‘>’ are insertions and ‘-’ behind the numbers indicates deletions in v90/4 relative to v90/2. DE, Early; L, late. dNumbers indicate the locations of motifs in each promoter region. 98 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 05:37:59 Journal of General Virology 86 Comparative genomic analysis of MacoNPV-A Fig. 4. Nucleotide alignment of the hr4 sequences of v90/4 (nt 134258–135356) and v90/2 (nt 135725–136764), showing multiple point mutations and insertion/deletions. The large 76 bp insertion in v90/4 hr4 represents a single repeat unit. Individual repeat units are separated by slashes. Identical sequences between v90/4 and v90/2 are linked by vertical lines, whilst nucleotides missing in either v90/4 or v90/2 are represented by dashes. acid at this site is divergent among baculoviruses, so it is unlikely that this will alter the function of the RING finger. The v90/4 CG30 has three amino acid polymorphisms: aspartic acid to asparagine, glutamic acid to aspartic acid and serine to glycine at aa 147, 154 and 158, respectively (see Table 3). bro gene variations There are seven and eight bro genes in the v90/4 and v90/2 genomes, respectively. As described above, bro-a is the sole ORF that exists in v90/2 but is missing in v90/4. v90/2 BROA shows low sequence identity to other BROs in the v90/2 and v90/4 genomes, with 27 % identity to v90/2 BRO-C being the highest. Other proteins related to v90/2 BRO-A are Spodoptera litura NPV (SpltNPV) BRO-B (27 %), Xestia c-nigrum granulovirus (XecnGV) BRO-A (25 %), XecnGV BRO-F (24 %), Helicoverpa armigera single NPV (HearSNPV) BRO-C (25 %), Bombyx mori NPV (BmNPV) BRO-C (23 %) and BmNPV BRO-B (23 %). As shown in Fig. 2, bro-a, bro-b and bro-c are located in the most highly variable region of the MacoNPV-A genome. In addition to http://vir.sgmjournals.org missing bro-a, v90/4 bro-b and bro-c have 73 and 55 point mutations, respectively (see Supplementary Fig. S3, available in JGV Online). The nucleotide point mutations in these two bro genes account for 25 % of the point mutations in the whole genome. All of the bro genes except for bro-d and bro-f contain mutations that cause amino acid substitutions (Table 1). BRO-B has 41 aa deleted between aa 65 and 105, as well as 32 amino acid substitutions; BRO-C has 13 amino acid substitutions; and BRO-E, -G and -H each have a single amino acid substitution. Polymorphisms also cause amino acid variation in BRO-E and -F (Table 3). These variation levels suggest strongly that bro genes, especially bro-a, -b and -c, are hot spots for MacoNPV-A mutations and genome variation. Additional ORFs with amino acid sequence mutations or polymorphisms The other known genes whose encoded protein sequences vary between v90/4 and v90/2 or have polymorphisms in Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 05:37:59 99 L. Li and others Table 3. Sequence polymorphisms in the MacoNPV-A (v90/4) genome int, Intergenic (not including hr elements); :, nucleotide deletion; -, deletion of an amino acid residue; *, stop codon. Numbers between two letters in the amino acid variation columns indicate the positions where the amino acid polymorphisms occurred in the predicted gene products. ORF (name) Position 4 (hoar) 4471 4549 4605 4705 8522 8974 9026+1 9030 9034 9073 11732 18272 18489 22787 22999 23002 23012 23327 23369 23390 32936 33064 33068 33107 33178 33269 33272 37183 37267 37268 38864 43371 43389 43450 43466 43593 44951 44953 44956 45133 45229 45242 45248+1 45248+2 45339 45479 45481 45557 7 int 8 10 19 26 27 37 (gp37) 42 48 int 49 50 int int int int 51 (fgf) 100 Variation nt A/G T/A T/C C/G T/C A/T :/A T/A T/: T/C T/C A/G C/A G/A C/T G/A G/A G/A G/A T/C C/T C/T C/T C/T C/A C/T G/A G/A G/A G/A T/A C/G C/A T/C T/A C/T C/T C/G G/A T/C T/: T/C :/T :/T G/A A/C C/G T/G aa ORF (name) M217V 51 E251G Y129C C140F P211S E212K R215H 52 53 54 (ae) S143L 56 Q181K int 59 60 T750I E218V Q418E 75 S25T P67L 78 (odv-e66) 79 80 L298V R297P N275T Position 45579 45606 45703 45871 45997 46180 46236 46243 46297 46360 46544 46771 47077 47499 47509 47689 47734 47817 47941 47977 48025 48112 49809 49902 50040 50280 52492 52812 52893 53172 53180 53295 65102 65147 67187 67212 67262 67358 68203 68334 68666 68930 69098 69104 69943 69944 70019 70334 Variation nt aa A/G G/A C/T G/T A/G G/A A/G A/T G/A G/A C/T T/C A/G C/T A/G A/T C/G A/G T/C T/C T/C C/T C/T C/T G/A A/G C/T G/A A/G T/G G/A G/A T/C T/A G/A A/C A/G C/T C/T G/T G/A G/T T/C T/A G/A G/A G/A A/G L268V P259S ORF (name) 82 83 89 (vef) 89 A352S 90 (bro-e) H273Q int 91 int 92 95 int 100 (cg30) T407I A451S R69S H151Y 101 (91k) 102 104 (gp41) 106 111 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 05:37:59 Position Variation nt 71336 72022 72128 72583 72631 72754 77387 77549 77723 77717 78182 78327 78626 79121 79131 79279 79290 79304 79308 79309 79310 79981 79984 80001 80332 80386 80548 80567 80746 80870 81277 86167 90037 90499 90522 90532 90654 90723 91407 91410 91415 91416 91419 92273 93776 94905 96263 100124 C/T G/A C/T C/T T/G G/A C/T G/A T/C C/A A/G A/G C/T C/G T/C C/T T/G A/G G/: T/: A/: C/G A/G T/C A/G A/G A/G C/T A/G A/G C/T C/T G/A G/A C/A A/G A/G G/A T/C T/C A/: T/: G/: G/A T/C C/T A/G A/G aa C136W D536N F800L F804L Q356K I351T L350- S119G R100Q D147N E154D S158G S661- Q147* V194A Journal of General Virology 86 Comparative genomic analysis of MacoNPV-A Table 3. cont. ORF (name) 115 int int int 116 117 118 119 122 (bro-f) hr3 int 140 (bjdp) 141 (lef-8) Position Variation nt 100125 107315 107441 107480 107481 107482 107676 107880 108001 108022 108037 108042 108055 108952 109289 110959 110961 123281 123304 123976 125057 125247 126182 127368 A/G A/G C/T C/: A/: A/: G/A A/G G/T T/A T/C G/A A/T T/C T/C G/A T/C C/T G/A C/G A/T A/T G/C C/T ORF (name) aa I297V L338M T26S R364C int int int int int 142 int int 144 (odv-e66) 148 v90/4 include homologues of fgf, p94, lsxe, pkip, tlp-20, sod, hoar and bjdp (Tables 1 and 2). Homologues exist in all of the sequenced lepidopteran baculovirus genomes for the predicted product of AcMNPV fgf, which is similar to fibroblast growth factors (Ayres et al., 1994). It was proposed that the expression of baculovirus FGF might facilitate the infection of tracheal cells, which serve as conduits for establishment of systemic AcMNPV infection (reviewed by Hayakawa et al., 2000). The v90/4 FGF contains five amino acid polymorphisms (Table 3). AcMNPV p94 was hypothesized to be associated with the triggering of apoptosis induced by viral infection (Clem et al., 1994). The v90/4 P94 homologue has five amino acid substitutions relative to v90/2; of note is a methionine residue substituted for a conserved isoleucine residue at aa 492 (Table 1). In addition to the above ORFs, 28 other ORFs that have not been characterized or have no predicted function have mutations resulting in amino acid substitutions in v90/4 relative to v90/2 (Tables 1 and 2). The most variable protein observed was ORF30, which shows 12 % sequence variation. This ORF, previously described as a unique MacoNPV ORF (Li et al., 2002b), is homologous to SpltNPV ORF106 (31 % amino acid sequence identity). http://vir.sgmjournals.org Position Variation nt 128123 128173 128192 128459 128597 128630 128915 128916 128917 128918 128919 128920 128921 128927 128937 129238 129422 129423 129724 130138 130491 136072 136237 136393 G/T A/C T/C T/C G/A G/A A/: T/: A/: G/: A/: C/: A/: C/T T/C C/T A/T A/T G/A T/C G/T T/C C/T C/A aa N632T ORF (name) 156 int 158 int 160 162 I54M int 164 165 L405I 167 168 Position Variation nt 136625 140772 140790 140826 140883 141135 141176 141942 142281 142747 143045 145423 145509 145635 145726 145738 147561 147752 147999 148011 150381 150455 aa C/T T/A T/C T/C C/T C/T A/G C/T C/T A/G T/C G/A G/A A/G A/G G/A T/: G/A G/A A/G G/A C/T DISCUSSION The MacoNPV-A viruses v90/4 and v90/2 were originally isolated from M. configurata larvae in relatively close geographical proximity during the same outbreak of this pest. The complete genome sequence of v90/4, which was found to be at least ten times less virulent for this host, has been determined and compared to that of the previously sequenced v90/2 genome. The results showed that the genome of v90/4 had 99?5 % sequence identity to that of v90/2 and nearly identical gene content and arrangement. Surprisingly, however, 49 ORFs were identified that contained nucleotide point mutations, insertions or deletions resulting in amino acid substitutions, as well as one ORF that was present in v90/2, but not in v90/4. These genetic variations underlie the biological differences between these isolates. This is the first study to determine the extent of sequence variability between baculovirus genomes isolated from the same naturally occurring field population. Variability was not distributed evenly throughout the viral genomes, as the sequence data show that the region with the most mutations in v90/4 relative to v90/2 is located between hr1 and orf31 (bro-c) (Fig. 2). This region contains three genes, orf18, orf23 and orf30, that are unique to MacoNPV-A and it does not contain any genes that are Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 05:37:59 101 L. Li and others 102 Journal of General Virology 86 Fig. 5. Sequence comparison of clones from the MacoNPV Lamont field isolate. The HindIII fragment representing nt 3051–4547 was isolated and cloned from genomic DNA extracted directly from the Lamont field isolate. Eight separate clones were sequenced and compared to the sequence of the plaque-purified MacoNPV v90/4 and the archetypal sequence of MacoNPV v90/2. The eight Lamont clone sequences were of two types, two clones having sequences identical to that of MacoNPV-A v90/4 (Lamont type 2 in the comparison) and six clones were of a genotype differing from both v90/4 and v90/2 (Lamont type 1 in the comparison). Green shading, no identity to either v90/4 or v90/2; red shading, identity only to v90/4; blue shading, identity only to v90/2. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 05:37:59 Comparative genomic analysis of MacoNPV-A conserved in all baculoviruses sequenced to date. Interestingly, we have shown previously that this region is also the most variable region between MacoNPV-A and the closely related species MacoNPV-B (Li et al., 2002a). Even when compared with more distantly related baculoviruses, this region appears to be more variable. For example, the gene arrangement of SeMNPV is highly collinear to that of v90/2; however, comparison of the two genomes shows that the relocation and inversion of a cluster of ORFs, as well as various insertions and deletions, have occurred in this highly variable region (Li et al., 2002b). In addition, an SeMNPV mutant with a single deletion of 25 kb encompassing orf15– orf41, a region homologous to MacoNPV-A orf16–orf54, can be isolated routinely in cell culture (Heldens et al., 1996). The SeMNPV deletion mutant did not cause host larval mortality or morbidity, suggesting that the 25 kb deletion contains information that is critical for virus virulence in vivo. MacoNPV homologues of SeMNPV genes in this region that vary between v90/4 and v90/2 include lef-7, he65, orf34 and orf40. In addition, lef-7, chitinase, orf25, orf50 and orf54 contain variations in their upstream promoter regions that could potentially affect gene expression. The largest single difference between v90/4 and v90/2 is the deletion of the bro-a gene. The bro genes are a family of ORFs with sequence homology to AcMNPV orf2 that have been identified in a number of baculoviruses. Lymantria dispar multiple NPV (LdMNPV) has 16 bro-related genes (Kuzio et al., 1999). AcMNPV, Epiphyas postvittana NPV, Orgyia pseudotsugata NPV, BmNPV, HearSNPV, Helicoverpa zea single NPV (HzSNPV), SpltNPV, Culex nigripalpus NPV, Cydia pomonella granulovirus and XecnGV have between one and seven bro genes (Afonso et al., 2001; Ahrens et al., 1997; Ayres et al., 1994; Chen et al., 2001, 2002; Gomi et al., 1999; Hayakawa et al., 1999; Hyink et al., 2002; Luque et al., 2001; Pang et al., 2001). SeMNPV was previously reported to lack a bro gene, but the SeMNPV ORF13 was recently reported to be a BRO homologue of MacoNPV-A bro-g (IJkel et al., 1999; Li et al., 2002b). Only Plutella xylostella granulovirus has been found to lack a bro gene homologue (Hashimoto et al., 2000). The BmNPV bro genes are transcribed as delayed-early genes. Functional studies suggested that BmNPV bro-a, -c and -d are essential for viral infection, but bro-a and bro-c could complement each other functionally (Kang et al., 1999). BmNPV BRO-A, -C and -D have nucleic acidbinding activities and are located in nucleoprotein complexes in the nuclei of infected cells. It has been proposed that BRO-A and -C may influence host DNA replication and/or transcription (Zemskov et al., 2000). The BRO N domain, proposed to be a DNA-binding domain, has recently been shown to be homologous to a family of proteins from other viruses, bacterial phages and bacteria (Iyer et al., 2002). Based on the apparently essential nature of some of the BmNPV bro genes, it is possible that the virulence difference that we observed between v90/4 and v90/2 may be due to the presence or absence of bro-a. http://vir.sgmjournals.org AcMNPV (C6) was originally reported as having only a single bro gene (Ayres et al., 1994). However, another bro gene was later identified in four AcMNPV variants isolated from Galleria mellonella, S. exigua, S. litura and X. c-nigrum (Yanase et al., 2000). In the variant isolated from S. litura, the second bro gene is contained within a 1?1 kb insert between AcMNPV orf30 and orf31. We have also previously reported that bro genes were found to be associated with a region of the MacoNPV-B genome (orf 53–orf 58) that was possibly derived by recombination with a distantly related virus, XecnGV (Li et al., 2002a). In this study with MacoNPV-A, three bro genes (-a, -b and -c) were found to be located in the most highly variable region of the genome, with bro-b and bro-c containing 25 % of all point mutations in the v90/4 genome relative to that of v90/2. Alignments of the homologous bro genes of MacoNPV-A v90/2 and MacoNPV-B showed an average of 81 % sequence identity, which is much lower than the overall 87?6 % sequence identity between the two genomes (Li et al., 2002a). A high degree of sequence variability has also been observed among the bro genes of other viruses. The sequences of HearSNPV and HzSNPV have recently been reported and they have been shown to be variants of the same virus species. Interestingly, the most divergent ORFs between these two viruses are two bro genes (Chen et al., 2002). In BmNPV, among the five bro genes, bro-d is related most closely to AcMNPV orf2, with 80 % amino acid sequence identity. This is much lower than the average identity level of predicted proteins from these two viruses, which is 93 % (Gomi et al., 1999). These data indicate that bro genes are highly variable relative to other baculovirus genes. Furthermore, where multiple bro genes exist within a baculovirus, they are usually highly divergent amongst themselves, as has been shown in MacoNPV-A v90/2 and LdMNPV (Kuzio et al., 1999; Li et al., 2002b). It is likely that the highly variable bro genes are functionally different genes. Overall, the results of this study and those described above suggest strongly that bro genes are associated with baculovirus genome variation, but the reasons for this association are yet to be determined. Analysis of the v90/4 genome identified 214 nucleotide polymorphisms. Sequence polymorphisms are common in genomes of humans, viruses and other organisms and contribute to important phenotypic diversities. This is the first paper to report all polymorphisms for an entire baculovirus genome. The v90/4 viral DNA used for sequencing was from a plaque-purified clone that was amplified in larvae of M. configurata. The presence of a pool of polymorphisms may be a more efficient mechanism than direct substitution, insertion or deletion in adapting to a changeable environment and may provide an evolutionary advantage by having a population of variable genomes. The results of this study describe the extensive sequence variability that exists in natural baculovirus populations. The sequence does not provide direct answers as to why v90/4 is less virulent than v90/2, but suggests that the bro Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 02 Aug 2017 05:37:59 103 L. Li and others genes are potentially involved and may play a significant role in generating baculovirus diversity. 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