Ream2013VR

This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/authorsrights Author's personal copy Virus Research 178 (2013) 217–225 Contents lists available at ScienceDirect Virus Research journal homepage: www.elsevier.com/locate/virusres Comparative analysis of error-prone replication mononucleotide repeats across baculovirus genomes夽 David C. Ream a , Samuel T. Murakami a,1 , Emily E. Schmidt b,c , Guo-Hua Huang d , Chun Liang b,c , Iddo Friedberg a,c , Xiao-Wen Cheng a,∗ a Department of Microbiology, Miami University, Oxford, OH, USA Department of Botany, Miami University, Oxford, OH, USA Department of Computer Science and Software Engineering, Miami University, Oxford, OH, USA d Institute of Virology, College of Plant Protection, Hunan Agricultural University, Changsha, Hunan, China b c a r t i c l e i n f o Article history: Received 6 July 2013 Received in revised form 4 October 2013 Accepted 7 October 2013 Available online 17 October 2013 Keywords: Viral genome replication Replication slippage Viral genome evolution fp25k acquisition a b s t r a c t Genome replication by the baculovirus DNA polymerase often generates errors in mononucleotide repeat (MNR) sequences due to replication slippage. This results in the inactivation of genes that affects different stages of the cell infection cycle. Here we mapped these MNRs in the 59 baculovirus genomes. We found that the MNR frequencies of baculovirus genomes are different and not correlated with the genome sizes. Although the average A/T content of baculoviruses is 58.67%, the A/T MNR frequency is significantly higher than that of the G/C MNRs. Furthermore, the A7/T7 MNRs are the most frequent of those we studied. Finally, MNR frequencies in different classes of baculovirus genes, such as immediate early genes, show differences between baculovirus genomes, suggesting that the distribution and frequency of different MNRs are unique to each baculovirus species or strain. Therefore, the results of this study can help select appropriate baculoviruses for the development of biological insecticides. © 2013 The Authors. Published by Elsevier B.V. All rights reserved. 1. Introduction Baculoviridae is a family of insect viruses with a double-stranded circular DNA genome in the size range of 80–180 kbp (Herniou et al., 2012). They are important to agriculture and forestry as a means to control insect pests to reduce the use of chemical pesticides (Caputo et al., 2011; Moscardi, 1999). Additionally, baculoviruses are widely used for exogenous gene expression in insect cells by the pharmaceutical industry for vaccine production and in research laboratories for the elucidation of protein functions (Deschuyteneer et al., 2010; Luckow, 1991). According to the current classification system, the Baculoviridae family has four genera: alpha-, beta-, gamma- and deltabaculovirus. Alphabaculoviruses or nucleopolyhedroviruses (NPV) infect lepidopteran insects and are typified by the baculovirus type species Autographa californica multiple NPV (AcMNPV). Betabaculoviruses or granuloviruses (GV) also infect lepidopteran insects and are represented by the widely used 夽 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. ∗ Corresponding author at: Department of Microbiology, 32 Pearson Hall, Miami University, Oxford, OH 45056, USA. Tel.: +1 513 529 5429; fax: +1 513 529 2431. E-mail address: Chengx@MiamiOH.edu (X.-W. Cheng). 1 Present address: Department of Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA. Cydia pomonella GV (CpGV), which is important to apple production (Rohrmann, 2011a). The gammabaculovirus infects dipteran insects such as mosquito Culex nigripalpus and is represented by C. nigripalpus NPV (CuniNPV) (Afonso et al., 2001), whereas deltabaculoviruses infect hymenopteran insects such as the European pine sawfly Neodiprion sertifer NPV (NeseNPV) (Herniou et al., 2012; Jehle et al., 2006). Among the four groups of baculoviruses, the most studied is the alphabaculovirus at both the molecular and application levels. This is due to the availability of cell culture systems for genetic studies and protein expression applications that use the baculovirus expression vector systems (Lu and Miller, 1997; Moscardi, 1999; Rohrmann, 2011a). Infection of an insect cell by NPVs begins with the attachment of the infectious virion to the cell surface through a putative receptor binding mechanism before the virion is endocytosed into the cytoplasm of the insect cells (Charlton and Volkman, 1993). The virion is then transported to the nucleus by actin filaments, and subsequently enters the nucleus via the nuclear pore complex. In the nucleus, the virion uncoats to expose the viral DNA for transcription (Charlton and Volkman, 1993). Early genes are transcribed by the cellular RNA polymerase (POL), and one of the early gene products is the viral DNA POL which is involved in replication of the viral genome (Lu and Miller, 1997; Vanarsdall et al., 2005). The baculovirus 114 kDa DNA POL (AcMNPV) generates mutations during DNA replication that relates to sequence deletions and additions, and these mutations accumulate in the progeny genomes (Bischoff 0168-1702/$ – see front matter © 2013 The Authors. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.virusres.2013.10.005 Author's personal copy 218 D.C. Ream et al. / Virus Research 178 (2013) 217–225 and Slavicek, 1997; Tomalski et al., 1988). In this study we focused on one type of error, the mononucleotide repeat (MNR). MNRs are sequences in the viral genome composed of a single repeating nucleotide. Due to the repetitive nature of the sequence, the viral DNA POL experiences slippage. This in turn leads to insertion of one or more additional nucleotides. MNR replication errors in non-coding regions may be neutral, but if the slippage occurs in a coding region, a frameshift will occur, causing gene inactivation or a malfunction of the coded protein. MNR frameshift errors have been reported in the fp25k gene of Lymantra dispar MNPV (LdMNPV), Bombyx mori NPV (BmNPV), Helicoverpa armigera SNPV (HearSNPV) and AcMNPV detected by the viral few-polyhedra (FP) phenotype in cell infection (Bischoff and Slavicek, 1997; Cheng et al., 2013a; Katsuma et al., 1999; Lua et al., 2002). After viral DNA replication, late gene transcription starts by the viral RNA POL that has four subunits, which are encoded by four late expression factor (lef) genes (lef4, lef8, lef9 and p47) (Guarino et al., 1998). The very late gene products include structural proteins such as polyhedrin (encoded by the polh gene) that are synthesized in large amounts to occlude virions; P74, which is involved in receptor binding of virions in the midgut of insects and virion occlusion, and FP25K that is involved in viral protein sorting, polh promoter activity regulation and virion occlusion (Braunagel et al., 1999, 2007, 2009; Cheng et al., 2013a,b; Haas-Stapleton et al., 2004; Wang et al., 2009). If MNR replication mutations occur in essential early genes or in those involved in transcription, no progeny will result. Conversely, if MNR mutations occur in late genes, virion structure may be negatively impacted, hampering virus transmission. If mutations occur in genes involved in pathogenesis and late gene expression, progeny viruses will have poor infectivity. All the reported MNR replication errors of baculoviruses involve the conversion of either a seven adenosine (A) run (A7) MNR to an A8 MNR or a seven thymidine (T) run (T7) MNR to a T8 MNR, which leads to the inactivation of the fp25k gene of BmNPV, HearSNPV, AcMNPV and LdMNPV (Bischoff and Slavicek, 1997; Cheng et al., 2013a; Katsuma et al., 1999; Lua et al., 2002). The conversion of the T7 MNR to T8 of LdMNPV fp25k was reported on the template strand whereas the rest were all reported on the coding strand (Bischoff and Slavicek, 1997; Cheng et al., 2013a; Katsuma et al., 1999; Lua et al., 2002). Since the baculovirus genome is replicated by the viral DNA polymerase with other factors such as helicase (Miller and Lu, 1997; Vanarsdall et al., 2005), other baculovirus genes with MNRs may also mutate, leading to inactivation of these genes during cell infection, which may or may not result in an observable phenotype. MNR frequencies of different baculovirus genomes have never been systematically analyzed, although prior works on MNRs in prokaryotic and eukaryotic organisms have been analyzed and reported (Castagnone-Sereno et al., 2010; Lim et al., 2004; Subramanian et al., 2003). In this study, we provide a comparative analysis of the MNRs in the fifty-nine baculovirus genomes deposited in GenBank. We provide important information for selecting baculoviruses with fewer MNRs in the genomes for potential applications in insect biological control, vaccine production and protein expression. 2. Materials and methods 2.1. Data acquisition Fully sequenced baculovirus genomes were obtained from GenBank on September 19, 2013, using a Python script. A Python program (Repeater) was written to search for MNRs with stretches of adenosine (A), cytosine (C), thymidine (T) and guanosine (G) that are equal to or longer than seven nucleotides. Only the longest MNR in any particular stretch of DNA was recorded. For example, only the A8 MNR is counted even though it contains an A7 MNR. MNRs in the coding regions of genes were converted to the mRNA sequences based on information from annotations in the GenBank, but MNRs in the non-coding regions were not converted. 2.2. Data analysis Baculoviruses were further grouped based on their associated insects according to their co-evolutionary history for comparison (Herniou et al., 2004; Trautwein et al., 2012). A website-based search engine was also developed to facilitate MNR search and is available at: http://bioinfolab.muohio.edu/virus homopolymer/. To determine if MNR frequency is correlated with the genome size, the viral genome size was plotted against the number of MNRs in the viral genomes for regression analysis using Excel (Microsoft). Similarly, the A7/T7 MNR frequency of viral genomes was plotted against the genome A/T content. MNRs of genes from different viral life-cycle categories were compared between baculoviruses. The early genes in the MNR comparison included IE0 and IE1. The DNA replication enzyme genes included DNA polymerase and helicase. Late expression genes included lef8 and lef9. Virion occlusion genes included polh, fp25k and p74. 3. Results 3.1. MNR frequencies differ among baculovirus genomes We analyzed 55 lepidopteran, 3 hymenopteran and 1 dipteran baculoviruses in our dataset. Among the 55 lepidopteran baculovirus genomes, 12 of them are GVs and the rest are NPVs. All the baculovirus genomes contain MNRs, but the types and frequencies differ. The lepidopteran Pieris rapae GV (PiraGV) genome contains 215 MNRs which is the largest number found among the examined baculovirus genomes. In contrast, the dipteran C. nigripalpus NPV (CuniNPV) genome contains only 53 MNRs which is the least (Fig. 1A). The majority (99.75%) of baculovirus MNRs are A/T and only 6 baculovirus genomes contain G/C MNRs (Fig. 1A). In the A/T MNRs of baculovirus genomes, the A7/T7 MNR has the highest average frequency (66.56%) among all the baculovirus genomes that have been examined. The frequency of A7/T7 MNRs also differs among baculovirus genomes. The lowest frequency (5.66%) of A7/T7 MNRs was found in the genome of CuniNPV, whereas the highest frequency (84.88%) of A7/T7 MNRs was found in the genome of Epiphyas postvittana NPV (EppoNPV). Following the A7/T7 MNR is the A8/T8 MNR that has an average of 22.16%. Antheraea pernyi NPV (AnpeNPV-L2) has the lowest frequency of A8/T8 MNR (8.93%) and Leucania separata NPV (LeseNPV-AH1) has the highest (34.25%). The longest MNR is the T27 that was found in the non-coding region of Maruca vitrata MNPV (MaviMNPV) (Fig. 1A). The genome size range of baculoviruses is between 80 and 180 kbp (Herniou et al., 2012). Regression analysis found that the MNR frequencies of baculovirus genomes do not correlate with the baculovirus genome size (R2 = 0.0101, Fig. 1B). Therefore, the MNR frequency is a unique feature of each viral genome. Because the A7/T7 is the most frequent MNR in the baculovirus genome, the A7/T7 frequency should be positively correlated with the A/T content of the baculovirus genome. The average A/T content of baculovirus is 58.67% in the range of 42.53–67.56% (Fig. 1C). However, when the A7/T7 MNR frequency was plotted against the A/T content, no correlation could be established (R2 = 0.0862, Fig. 1C). This further suggests that the A7/T7 MNR frequency is an intrinsic feature of each viral genome. Five baculovirus species have two or three strains sequenced. These are HearSNPV (HearSNPV-C1 and HearSNPV-G4), Mamestra configurata NPV (MacoNPV-A 90-2, MacoNPV-A 90-4 and MacoNPV-B), A. Author's personal copy D.C. Ream et al. / Virus Research 178 (2013) 217–225 A 250 A10 G7 A11 G8 A12 T10 A13 T11 A14 T12 A16 T13 A17 T14 A23 T16 A7 T19 A8 T21 219 A9 T27 C7 T7 C8 T8 G10 T9 G14 Total MNRs 200 150 100 50 NeabNPV NeleNPV NeseNPV CuniNPV AdorGV AgseGV ChocGV ClanGV CpGV CrleGV PhopGV PiraGV PlxyGV HearGV PsunGV SpliGV XnGV AcMNPV AdhoNPV AdorNPV AgMNPV-2D AgipNPV AgseNPV AnpeNPV-L2 AnpeNPV-Z ApciNPV BmNPV BomaNPV CfDEFMNPV CfMNPV ChChNPV ClbiNPV EcobNPV EppoNPV EupsNPV HearMNPV HearNPV-C1 HearNPV-G4 HearSNPV-NNg1 HycuNPV HzSNPV LdMNPV LeseNPV-AH1 LyxyMNPV MacoNPV-A_90-2 MacoNPV-A_90-4 MacoNPV-B MaviMNPV OpMNPV OrleNPV PlxyMNPV RoMNPV SeMNPV SfMNPV-19 SfMNPV-3AP2 SpliNPV-G2 SpliNPV-II ThorMNPV TnSNPV 0 Lepidopteran baculovirus Dipteran baculovirus Hymenopteran baculovirus Total MNRs 200 y = -0.0002x + 135.54 R² = 0.0101 150 100 50 0 80000 100000 120000 140000 160000 180000 Genome size (bp) % of A7/T7 MNRs C 100 B 250 90 80 70 60 50 40 30 20 10 0 y = 0.6363x + 29.898 R² = 0.0862 40 45 50 55 60 65 70 A/T content (%) Fig. 1. Comparison of MNR sequences among baculovirus genomes. (A) Comparison of total numbers of MNRs among 48 genome-sequenced baculoviruses showing different frequencies of MNRs among different baculoviruses isolated from three insect families. (B) Relationship between the total numbers of MNRs and genome sizes. No correlation was found. (C) Relationship between baculovirus genome A/T contents and A7/T7 percentage. No correlation was found. pernyi NPV (AnpeNPV-L2 and AnpeNPV-Z), Spodoptera frugiperda MNPV (SfMNPV-19 and SfMNPV-3AP2) and Spodoptera litura NPV (SpliNPV-G2 and SpliNPV-II). MNR comparative analysis between the different strains of the same species revealed that the MNR frequency differs between the strains in both viral species (Fig. 1A). In the case of HearSNPV, HearSNPV-G4 has 3 less MNRs (127) than HearSNPV-C1 (130). In MacoNPV, MacoNPV-B has more MNRs (113) than MacoNPV-A 90-2 (81) and MacoNPV-A 90-4 (86) in their genomes. In the two strains of AnpeNPV, AnpeNPV-L2 has 2 more MNRs (58) than AnpeNPV-Z (56). Differences in the number and type of MNRs also exist in the strains of SfMNPV and SpliNPV (Fig. 1A). This suggests that the differences in the MNRs exist not only among different baculovirus species but also between the strains or isolates of the same species. The different MNR contents between strains of the same viral species may reflect the process of the speciation of baculoviruses. regions were detected in 3 baculovirus genomes that include lepidopteran Agrotis ipsilon MNPV (AgipMNPV) and LeseNPV-AH1 and dipteran CuniNPV (Fig. 2A). One possible explanation for more MNRs in non-coding regions than coding regions in the three baculoviruses could be that there are more non-coding sequences than coding sequences in these genomes. Comparison of percentages of coding sequences among baculovirus genomes shows that an average of 88.83% of baculovirus genomic DNA is used for coding. However, Apocheima cinerarium NPV (ApciNPV) uses only 74.6% which is the lowest, whereas Rachiplusia ou MNPV (RoMNPV) uses 93.5% which is the highest (Fig. 2B). The three baculoviruses that have more MNRs in their non-coding regions than coding regions have 88.1% (CuniNPV), 90.1% (Agrotis segetum NPV or AgipNPV) and 83.2% (LeseNPV-AH1) sequences for coding (Fig. 2B). Therefore, the reason for more MNRs in the non-coding than the coding sequences is not due to the more non-coding sequences in the three viruses. Instead, it is an intrinsic feature of the three viral genomes. 3.2. Comparison of MNRs between coding and non-coding regions Due to the compactness of viral genomes, it is likely that most of the baculovirus genome is devoted to protein coding regions. Therefore we expect to see more MNRs in the coding region than in the non-coding region. However, comparative analysis of MNRs shows that the ratios of MNRs in the coding regions to the noncoding regions differ among baculoviruses. In the sequenced 59 baculovirus genomes, more MNRs in the coding regions than in the non-coding regions were found in 56 baculovirus genomes, whereas more MNRs in the non-coding regions than the coding 3.3. Comparison of MNRs of baculovirus immediate early (IE) genes Immediate early (IE) genes are the first group of genes that are activated and expressed when the viral DNA reaches the nucleus of the host cell (Guarino and Summers, 1987). Three IE genes have been characterized that include IE-0, IE-1 and IE-2. Comparison of IE-0 and IE-1 MNRs shows variances among different baculoviruses. Thirty two percent of the baculovirus IE-0 gene contains A/T MNRs Author's personal copy 220 D.C. Ream et al. / Virus Research 178 (2013) 217–225 A 250 Number of MNRs MNRs in coding region MNRs in non-coding region 200 150 100 50 NeabNPV NeleNPV NeseNPV CuniNPV AdorGV AgseGV ChocGV ClanGV CpGV CrleGV PhopGV PiraGV PlxyGV PsunGV SpliGV XnGV AcMNPV HearGV AdhoNPV AdorNPV AgMNPV-2D AgipNPV AgseNPV AnpeNPV-L2 AnpeNPV-Z ApciNPV BmNPV BomaNPV CfDEFMNPV CfMNPV ChChNPV ClbiNPV EcobNPV EppoNPV EupsNPV HearMNPV HearNPV-C1 HearNPV-G4 HearSNPV-NNg1 HycuNPV HzSNPV LdMNPV LeseNPV-AH1 LyxyMNPV MacoNPV-A_90-2 MacoNPV-A_90-4 MacoNPV-B MaviMNPV OpMNPV OrleNPV PlxyMNPV RoMNPV SeMNPV SfMNPV-19 SfMNPV-3AP2 SpliNPV-G2 SpliNPV-II ThorMNPV TnSNPV 0 Dipteran baculovirus Hymenopteran baculovirus 100 90 80 70 60 50 40 30 20 10 0 NeabNPV NeleNPV NeseNPV CuniNPV AdorGV AgseGV ChocGV ClanGV CpGV CrleGV HearGV PhopGV PiraGV PlxyGV PsunGV SpliGV XnGV AcMNPV AdhoNPV AdorNPV AgMNPV-2D AgipNPV AgseNPV AnpeNPV-L2 AnpeNPV-Z ApciNPV BmNPV BomaNPV CfDEFMNPV CfMNPV ChChNPV ClbiNPV EcobNPV EppoNPV EupsNPV HearMNPV HearNPV-C1 HearNPV-G4 HearSNPV-NNg1 HycuNPV HzSNPV LdMNPV LeseNPV-AH1 LyxyMNPV MacoNPV-A_90-2 MacoNPV-A_90-4 MacoNPV-B MaviMNPV OpMNPV OrleNPV PlxyMNPV RoMNPV SeMNPV SfMNPV-19 SfMNPV-3AP2 SpliNPV-G2 SpliNPV-II ThorMNPV TnSNPV % of coding sequences B Lepidopteran baculovirus Dipteran baculovirus Hymenopteran baculovirus Lepidopteran baculovirus Fig. 2. Comparison of MNRs in the coding regions and non-coding regions of baculovirus genomes of three insect families and baculovirus genome coding sequence analysis. (A) The MNRs ≥7 nucleotides were retrieved from each baculovirus genome and sorted into two groups, coding region and non-coding region for the comparison. It is to show that most of baculoviruses have more MNRs in the coding region than the non-coding regions. (B) Comparison of gene coding sequences in baculovirus genomes. and the majority of baculoviruses do not contain MNRs including Adoxophyes orana GV (AdorGV), Cryptophlebia leucotreta GV (CrleGV), CpGV and S. exigua MNPV (SeMNPV) that are commercially produced for biological control of agricultural insect pests (Fig. 3A) (Rohrmann, 2011a). Compared to IE-0, more than half of the baculovirus IE-1 gene (52.5%) contains MNRs and all of them are A/T MNRs with Ecotropis obliqua NPV (EcobNPV), Euproctis pseudoconspersa NPV (EupsNPV) and Thysanoplusia orichalcea MNPV (ThorMNPV) each having the highest (6). Anticarsia gemmatalis MNPV-2D (AgMNPV-2D), LdMNPV and SpliNPV that are used in biological control of insects do not contain any MNRs in the IE-1 gene (Fig. 3B). 3.4. Comparison of genome replication gene MNRs Even though the baculovirus genome is replicated by a set of proteins such as DNA POL (dnapol), helicase, late expression factor 1 (LEF1), LEF2 and LEF3 (Rohrmann, 2011b), the primary enzymes encoded by dnapol and helicase that are involved in DNA synthesis are used for the MNR comparison. The majorityof the dnapol gene (89.83%) of baculoviruses contains MNRs that include the well-studied AcMNPV with 2 MNRs (Fig. 4A). The dnapol gene of A. orana NPV (AdorNPV) contains 12 MNRs that is the most of the baculoviruses used in this comparison. The dnapol gene of 5 baculoviruses does not contain any ≥7 MNRs that include the dipteran CuniNPV and the commercially produced lepidopteran CpGV (Fig. 4A). Helicase unwinds DNA for DNA POL to use the single-stranded DNA as a template to synthesize new DNA during DNA replication. Mutations in helicase may affect baculovirus DNA synthesis thereby altering the life cycle of baculoviruses. The majority (91.52%) of baculoviruses examined has MNRs in the helicase gene with E. pseudoconspersa NPV (EupsNPV) helicase containing the most MNRs (11) among the baculoviruses examined. Only 5 baculoviruses (8.47%) do not have any MNRs in the helicase gene and these baculoviruses are Chrysodeixis chalcites NPV (ChchNPV), CuniNPV, LeseNPV-AH1, SeMNPV and SpliNPV-II (Fig. 4B). 3.5. Comparison of late expression factor gene MNRs Baculovirus late genes are transcribed by the virally encoded RNA POL that is a complex of four subunits (LEF4, LEF8, LEF9 and P47) (Guarino et al., 1998). Together with other late gene expression factors, the viral RNA POL transcribes very late genes such as polh at very high levels, leading to accumulation of large amounts of polyhedrin protein to form polyhedra to occlude virions. The lef8 and lef9 genes were chosen for the comparison of MNRs in baculovirus genomes due to their pivotal rules in RNA synthesis during late gene expression in the baculovirus infection cycle in insect cells (Guarino et al., 1998; Lu and Miller, 1994). The majority (93.22%) of the baculovirus lef8 gene has MNRs and only 4 baculovirus lef8 do not have MNRs. Among these baculoviruses that have MNRs in their lef8 gene, the lef8 gene of PiraGV has 9 MNRs and AdhoNPV and ChocGV each has 8. The baculoviruses that do not have MNRs include AgipMNPV, CuniNPV, LdMNPV and SpliNPV-G2 (Fig. 5A). The frequency of MNRs in the lef9 gene is not as high as lef8 in baculoviruses since only 71.20% of lef9 has MNRs. The lef9 gene of commercially produced AdorGV and SeMNPV has no MNR. EppoNPV lef9 has 5 MNRs that is the most among the baculoviruses examined (Fig. 5B). Number of MNRs 6 IE-0 4 5 3 2 IE-1 A7 A8 A8 A9 A9 T7 T7 A9 T8 T7 T8 Lepidopteran baculovirus A7 A8 T9 Lepidopteran baculovirus A7 T8 Lepidopteran baculovirus T7 Author's personal copy A10 A11 A12 A9 D.C. Ream et al. / Virus Research 178 (2013) 217–225 Dipteran baculovirus Hymenopteran baculovirus A10 A11 A8 T9 NeabNPV NeleNPV NeseNPV CuniNPV AdorGV AgseGV ChocGV ClanGV CpGV CrleGV PhopGV PiraGV PlxyGV HearGV PsunGV SpliGV XnGV AcMNPV AdhoNPV AdorNPV AgMNPV-2D AgipNPV AgseNPV AnpeNPV-L2 AnpeNPV-Z ApciNPV BmNPV BomaNPV CfDEFMNPV CfMNPV ChChNPV ClbiNPV EcobNPV EppoNPV EupsNPV HearMNPV HearNPV-C1 HearNPV-G4 HearSNPV-NNg1 HycuNPV HzSNPV LdMNPV LeseNPV-AH1 LyxyMNPV MacoNPV-A_90-2 MacoNPV-A_90-4 MacoNPV-B MaviMNPV OpMNPV OrleNPV PlxyMNPV RoMNPV SeMNPV SfMNPV-19 SfMNPV-3AP2 SpliNPV-G2 SpliNPV-II ThorMNPV TnSNPV 1 0 7 6 5 4 3 2 1 0 A7 NeabNPV NeleNPV NeseNPV CuniNPV AdorGV AgseGV ChocGV ClanGV CpGV CrleGV HearGV PhopGV PiraGV PlxyGV PsunGV SpliGV XnGV AcMNPV AdhoNPV AdorNPV AgMNPV-2D AgipNPV AgseNPV AnpeNPV-L2 AnpeNPV-Z ApciNPV BmNPV BomaNPV CfDEFMNPV CfMNPV ChChNPV ClbiNPV EcobNPV EppoNPV EupsNPV HearMNPV HearNPV-C1 HearNPV-G4 HearSNPV-NNg1 HycuNPV HzSNPV LdMNPV LeseNPV-AH1 LyxyMNPV MacoNPV-A_90-2 MacoNPV-A_90-4 MacoNPV-B MaviMNPV OpMNPV OrleNPV PlxyMNPV RoMNPV SeMNPV SfMNPV-19 SfMNPV-3AP2 SpliNPV-G2 SpliNPV-II ThorMNPV TnSNPV A B Number of MNRs Dipteran baculovirus Hymenopteran baculovirus A10 A10 Dipteran baculovirus Hymenopteran baculovirus DNA polymerase Helicase NeabNPV NeleNPV NeseNPV CuniNPV AdorGV AgseGV ChocGV ClanGV CpGV CrleGV HearGV PhopGV PiraGV PlxyGV PsunGV SpliGV XnGV AcMNPV AdhoNPV AdorNPV AgMNPV-2D AgipNPV AgseNPV AnpeNPV-L2 AnpeNPV-Z ApciNPV BmNPV BomaNPV CfDEFMNPV CfMNPV ChChNPV ClbiNPV EcobNPV EppoNPV EupsNPV HearMNPV HearNPV-C1 HearNPV-G4 HearSNPV-NNg1 HycuNPV HzSNPV LdMNPV LeseNPV-AH1 LyxyMNPV MacoNPV-A_90-2 MacoNPV-A_90-4 MacoNPV-B MaviMNPV OpMNPV OrleNPV PlxyMNPV RoMNPV SeMNPV SfMNPV-19 SfMNPV-3AP2 SpliNPV-G2 SpliNPV-II ThorMNPV TnSNPV 221 Fig. 3. Comparison of MNRs in immediate early genes among different baculovirus genomes of three insect families. (A) Comparison of MNRs in the IE-0 gene of baculovirus genomes showing differences in the frequencies of MNR types. (B) Comparison of MNRs in the IE-1 gene of baculovirus genomes showing differences in the frequencies of MNR types. 14 12 10 8 6 4 2 0 12 Number of MNRs A B 8 10 6 4 Lepidopteran baculovirus NeabNPV NeleNPV NeseNPV CuniNPV AdorGV AgseGV ChocGV ClanGV CpGV CrleGV HearGV PhopGV PiraGV PlxyGV PsunGV SpliGV XnGV AcMNPV AdhoNPV AdorNPV AgMNPV-2D AgipNPV AgseNPV AnpeNPV-L2 AnpeNPV-Z ApciNPV BmNPV BomaNPV CfDEFMNPV CfMNPV ChChNPV ClbiNPV EcobNPV EppoNPV EupsNPV HearMNPV HearNPV-C1 HearNPV-G4 HearSNPV-NNg1 HycuNPV HzSNPV LdMNPV LeseNPV-AH1 LyxyMNPV MacoNPV-A_90-2 MacoNPV-A_90-4 MacoNPV-B MaviMNPV OpMNPV OrleNPV PlxyMNPV RoMNPV SeMNPV SfMNPV-19 SfMNPV-3AP2 SpliNPV-G2 SpliNPV-II ThorMNPV TnSNPV 2 0 Dipteran baculovirus Hymenopteran baculovirus Fig. 4. Comparison of MNRs in genes involved in viral DNA replication among different baculovirus genomes of three insect families. (A) Comparison of MNRs in the DNA polymerase gene (dnapol) of baculovirus genomes showing differences in the frequencies of MNR types. (B) Comparison of MNRs in the helicase gene of baculovirus genomes showing differences in the frequencies of MNR types. Number of MNRs Author's personal copy 222 D.C. Ream et al. / Virus Research 178 (2013) 217–225 10 lef8 9 8 7 6 5 4 3 2 1 0 A10 A12 A7 A8 A9 T7 T8 NeabNPV NeleNPV NeseNPV CuniNPV AdorGV AgseGV ChocGV ClanGV CpGV CrleGV HearGV PhopGV PiraGV PlxyGV PsunGV SpliGV XnGV AcMNPV AdhoNPV AdorNPV AgMNPV-2D AgipNPV AgseNPV AnpeNPV-L2 AnpeNPV-Z ApciNPV BmNPV BomaNPV CfDEFMNPV CfMNPV ChChNPV ClbiNPV EcobNPV EppoNPV EupsNPV HearMNPV HearNPV-C1 HearNPV-G4 HearSNPV-NNg1 HycuNPV HzSNPV LdMNPV LeseNPV-AH1 LyxyMNPV MacoNPV-A_90-2 MacoNPV-A_90-4 MacoNPV-B MaviMNPV OpMNPV OrleNPV PlxyMNPV RoMNPV SeMNPV SfMNPV-19 SfMNPV-3AP2 SpliNPV-G2 SpliNPV-II ThorMNPV TnSNPV Number of MNRs A Lepidopteran baculovirus Dipteran baculovirus Hymenopteran baculovirus 6 lef9 A10 A11 A7 A8 A9 G7 T7 T8 5 4 3 2 1 0 NeabNPV NeleNPV NeseNPV CuniNPV AdorGV AgseGV ChocGV ClanGV CpGV CrleGV HearGV PhopGV PiraGV PlxyGV SpliGV PsunGV XnGV AcMNPV AdhoNPV AdorNPV AgMNPV-2D AgipNPV AgseNPV AnpeNPV-L2 AnpeNPV-Z ApciNPV BmNPV BomaNPV CfDEFMNPV CfMNPV ChChNPV ClbiNPV EcobNPV EppoNPV EupsNPV HearMNPV HearNPV-C1 HearNPV-G4 HearSNPV-NNg1 HycuNPV HzSNPV LdMNPV LeseNPV-AH1 LyxyMNPV MacoNPV-A_90-2 MacoNPV-A_90-4 MacoNPV-B MaviMNPV OpMNPV OrleNPV PlxyMNPV RoMNPV SeMNPV SfMNPV-19 SfMNPV-3AP2 SpliNPV-G2 SpliNPV-II ThorMNPV TnSNPV Number of MNRs B Dipteran baculovirus Hymenopteran baculovirus Lepidopteran baculovirus Fig. 5. Comparison of MNRs in genes involved in late gene expression among different baculovirus genomes. (A) Comparison of MNRs in the lef8 gene of baculovirus genomes showing differences in the frequencies of MNR types. (B) Comparison of MNRs in the lef9 gene of baculovirus genomes showing differences in the frequencies of MNR types. 3.6. Comparison of virion occlusion gene MNRs In the late phase of baculovirus infection in cells, newly assembled virions are occluded in the polyhedra in the nucleus. The genes polh, fp25k and p74 have been suggested to play roles in virion occlusion. Detrimental mutations of any of these genes lead to reduced virion occlusion efficiency, limiting their efficacy as insect pest control agents. The fp25k gene was not reported in the genomes of hymenopteran and dipteran baculoviruses (Duffy et al., 2006; Garcia-Maruniak et al., 2004; Lauzon et al., 2004). Of the 55 sequenced lepidopteran baculoviruses, 39 have MNRs including those commercially produced for application in agriculture (AgMNPV-2D, HearSNPV, HzSNPV, Fig. 6A) (Rohrmann, 2011a). SeMNPV, CpGV, AdorGV and CrleGV that are manufactured commercially for insect pest control do not have MNRs in the fp25k gene (Fig. 6A) (Rohrmann, 2011a). This may suggest that the lack of MNRs in the fp25k gene contributes to the success of these commercially produced viruses. Thirty six of the 39 lepidopteran baculoviruses which have MNRs in the fp25k gene have the A7 MNR most frequently. It is also noted that AgMNPV-2D is the only virus that has a T7 MNR in the coding strand of the fp25k gene (Fig. 6A). Five baculovirus fp25k has the A8 MNR that includes ApciNPV, Choristoneura fumiferana MNPV (CfMNPV), EcobNPV, EupsNPV and LdMNPV. However, the A8 MNR in the fp25k gene of LdMNPV was derived from the sequence of an LdMNPV fp25k mutant that has the conversion of the A7 to A8 MNR of the coding strand resulting in the report of two truncated ORFs of fp25k in the LdMNPV genome (Kuzio et al., 1999). Even though FP25K is a small protein in baculoviruses (Ayres et al., 1994), 4 MNRs were found in the fp25k gene of ApciNPV and three were found in the fp25k gene of nine baculoviruses (Fig. 6A). However, of the nine baculoviruses with MNRs in the fp25k gene, only the A7 MNR in the fp25k gene of BmNPV was reported mutated to the A8 MNR (Katsuma et al., 1999). MNRs are also present in the p74 gene of some baculoviruses, although no MNR mutation has been reported. This might be due to the difficulties in isolating the p74 mutants since no phenotype is readily discernible in infected insect cells. Of the 59 sequenced baculovirus genomes, the p74 gene of 34 baculoviruses has MNRs in the coding region with ThorMNPV having the largest number (4) of MNRs (Fig. 6B). In the commercially produced baculoviruses, the p74 gene of SeMNPV is the only one that does not have MNRs, whereas the p74 gene of HearSNPV (both C1 and G4 strains) contains a T7 MNR and CpGV has a T8 MNR (Fig. 6B). The extensively studied AcMNPV p74 gene also has an A7 MNR (Fig. 6B). The lack of MNRs in both p74 and fp25k in SeMNPV may suggest that this is a suitable NPV for high quality commercial production for S. exigua control in different countries due to the stability in mass production of polyhedra with proper virion occlusion for application in agriculture. The polh gene of baculovirus is paramount during baculovirus evolution since persistent transmission of baculoviruses in insects in the natural environment requires protection of the virions by the polyhedra against desiccation. It is interesting to note that only one A7 MNR is found in the polh gene of NeabNPV and XnGV but no MNR is found in the polh gene of other sequenced baculovirus genomes examined (Fig. S1). 4. Discussion 4.1. Motivation of MNR search in baculoviruses The first discovery of an A7 MNR (T7 on the template strand) mutation to A8 in the fp25k gene of LdMNPV occurred in the late Author's personal copy D.C. Ream et al. / Virus Research 178 (2013) 217–225 A Number of MNRs 5 223 fp25k A7 A8 A9 T7 4 3 2 1 AdorGV AgseGV ChocGV ClanGV CpGV CrleGV PhopGV PiraGV PlxyGV HearGV PsunGV SpliGV XnGV AcMNPV AdhoNPV AdorNPV AgMNPV-2D AgipNPV AgseNPV AnpeNPV-L2 AnpeNPV-Z ApciNPV BmNPV BomaNPV CfDEFMNPV CfMNPV ChChNPV ClbiNPV EcobNPV EppoNPV EupsNPV HearMNPV HearNPV-C1 HearNPV-G4 HearSNPV-NNg1 HycuNPV HzSNPV LdMNPV LeseNPV-AH1 LyxyMNPV MacoNPV-A_90-2 MacoNPV-A_90-4 MacoNPV-B MaviMNPV OpMNPV OrleNPV PlxyMNPV RoMNPV SeMNPV SfMNPV-19 SfMNPV-3AP2 SpliNPV-G2 SpliNPV-II ThorMNPV TnSNPV 0 Lepidopteran baculovirus B Number of MNRs 5 p74 A7 A8 T7 T8 4 3 2 1 NeabNPV NeleNPV NeseNPV CuniNPV AdorGV AgseGV ChocGV ClanGV CpGV CrleGV HearGV PhopGV PiraGV PlxyGV PsunGV SpliGV XnGV AcMNPV AdhoNPV AdorNPV AgMNPV-2D AgipNPV AgseNPV AnpeNPV-L2 AnpeNPV-Z ApciNPV BmNPV BomaNPV CfDEFMNPV CfMNPV ChChNPV ClbiNPV EcobNPV EppoNPV EupsNPV HearMNPV HearNPV-C1 HearNPV-G4 HearSNPV-NNg1 HycuNPV HzSNPV LdMNPV LeseNPV-AH1 LyxyMNPV MacoNPV-A_90-2 MacoNPV-A_90-4 MacoNPV-B MaviMNPV OpMNPV OrleNPV PlxyMNPV RoMNPV SeMNPV SfMNPV-19 SfMNPV-3AP2 SpliNPV-G2 SpliNPV-II ThorMNPV TnSNPV 0 Dipteran baculovirus Hymenopteran baculovirus Lepidopteran baculovirus Fig. 6. Comparison of MNRs in genes involved in virion occlusion among different baculovirus genomes of three insect families. (A) Comparison of MNRs in the fp25k gene of baculovirus genomes showing differences in the frequencies of MNR types. (B) Comparison of MNRs in the p74 gene of baculovirus genomes showing differences in the frequencies of MNR types. 1990s (Bischoff and Slavicek, 1997). Since then, conversions of A7 to A8 MNRs in the fp25k gene of HearSNPV, BmNPV and AcMNPV have been reported (Cheng et al., 2013a; Katsuma et al., 1999; Lua et al., 2002). The detection of the A7 to A8 MNR conversion in the fp25k gene in these four lepidopteran NPVs is largely due to the reduced polyhedrin expression phenotype that results from fp25k mutants of NPVs (Bischoff and Slavicek, 1997; Cheng et al., 2013a; Katsuma et al., 1999). These pioneering discoveries of A7 to A8 MNR mutations prompted us to search MNRs in other genes within baculovirus genomes. To the best of our knowledge, this is the first comprehensive systematic analysis of MNRs in the baculovirus genomes. Data from this report will provide useful information on MNR mutation mechanisms, baculovirus-host co-evolution and selection of baculoviruses for biological control in agriculture and forestry. Only a few selected baculovirus genes with well-studied biological functions were used in the comparison of MNRs without showing their positions within the genes. Detailed positions of these MNRs in the coding region of the selected genes are provided in the supplementary table (Table S2). Furthermore, genes of interest can be readily analyzed for MNRs using the web-site MNR search engine of genome sequence data. The portable MNR prediction program (Repeater) can also be requested through the corresponding author. MNRs than G/C MNRs (Subramanian et al., 2003). The higher frequency of A/T MNRs than G/C MNRs in the baculovirus genome may suggest higher DNA replication slippage errors by the viral DNA POL on the tracts of A/T MNRs in the viral genome. This is in contrast to the Saccharomyces cerevisiae and human MNR replication slippage errors that have a higher prevalence on the G/C MNRs than the A/T MNR tracts, even though the frequency of A/T MNRs is higher than the G/C MNRs (Gragg et al., 2002; Subramanian et al., 2003; Zhang et al., 2001). One exception to higher G/C MNR replication errors than A/T MNRs in humans is the conversion of the A8–A9 MNR by DNA replication slippage that leads to familial colorectal cancer development in the Ashkenazi Jewish population (Laken et al., 1997). It is also suggested that the minimum length for MNR replication slippage is about 8 mononucleotides in humans (Rose and Falush, 1998). This may reflect the minor structural differences between the DNA POL of baculovirus and human. It is possible that the 114 kDa single peptide viral DNA POL functions in a complex with cellular subunits such as the helicase (Tomalski et al., 1988), whereas the human DNA POL delta is a heterotetramer consisting of a 125 kDa protein that performs the catalytic reaction associated with three more subunits of the gene products of p50, p68 and p12 during genome replication (Hughes et al., 1999; Liu et al., 2000; Zhou et al., 2011). The size difference between the two DNA polymerases or other factors may determine the minimum length of MNRs for replication slippage of DNA. 4.2. MNR comparison between baculovirus and cellular genomes The most abundant MNRs in the baculovirus genomes are the A/T MNRs, although the A/T contents of baculovirus genomes are averaged at 58.67% in the range of 42.53–67.56% (Fig. 1A–C). Similarly, the human genome has a significant higher frequency of A/T 4.3. Rational for choosing MNRs equal to or longer than 7 nucleotides We chose the cutoff of A7/T7 MNRs for this investigation because there is no study linking shorter MNRs to polymerase slippage on Author's personal copy 224 D.C. Ream et al. / Virus Research 178 (2013) 217–225 A6/T6 MNRs of baculovirus replication in insect cells (Bischoff and Slavicek, 1997; Cheng et al., 2013a; Katsuma et al., 1999; Lua et al., 2002). It should be noted that A6/T6 MNR are more abundant than A7/T7 MNRs in all the baculoviruses examined in this study. This may suggest that the A7/T7 MNR is the minimum length of MNRs that trigger replication slippage of DNA by the viral DNA POL. It is still unknown if the MNRs longer than the 7 bp MNR in the baculovirus genomes are the results of replication slippage by the viral DNA POL, since most of these mutations are not fixed in the population due to their lethality. For example, these genes affected are involved in immediate early transcription and viral DNA replication (Figs. 3 and 4). 4.4. MNR expansion mechanisms Although the mechanism behind the baculovirus MNR slippage replication has never been studied, DNA replication is a universal biochemical process so the mechanism should be conserved. The slippage of DNA polymerase has been experimentally studied in vitro using bacteriophage T7. Studies found that the viral T7 DNA polymerase pauses at MNRs and disassociates transiently from the MNR template, leading to the newly synthesized DNA strand separating from the template during DNA replication. The T7 DNA polymerase resumes DNA synthesis after a unit of repeats anneals to the newly synthesized DNA. This leads to a one unit extension of the MNR in the newly synthesized strand of DNA. It is likely that the baculovirus DNA replication slippage mechanism is similar to the bacteriophage T7 DNA POL since both of them are of viral origin and smaller than the cellular DNA POL (Reutimann et al., 1985; Viguera et al., 2001). DNA replication slippage on MNRs leading to insertion of repeating units has been well documented in humans and baculoviruses (Bischoff and Slavicek, 1997; Cheng et al., 2013a; Katsuma et al., 1999; Laken et al., 1997; Lua et al., 2002). It has also been suggested that a repeating unit can also be deleted during replication slippage on MNRs (Gragg et al., 2002). If this is true, it is likely that the baculovirus genome is dynamic on these MNRs. For example the A7 and A8 MNRs of non-essential baculovirus genes in DNA replication such as the well-studied fp25k gene can be mutated back and forth. This is supported by the fact that microsatellites with MNRs are highly variable in humans. Malfunctions in DNA error repair systems often result in longer or shorter repeat units in microsatellites of humans (Weber and Wong, 1993). 4.5. Acquisition of baculovirus fp25k during evolution Phylogenetic analysis using baculovirus lef8 and Ac22 predicts that the hymenopteran baculoviruses (NeabNPV, NeleNPV and NeseNPV) and the dipteran baculovirus (CuniNPV) evolved earlier than lepidopteran baculoviruses (Castagnone-Sereno et al., 2010). The evolutionary history of the three insect families that these baculoviruses are associated with is also predicted with Hymenoptera and Diptera evolved earlier than Lepidoptera (Trautwein et al., 2012). It is also interesting to note that the ancient hymenopteran and dipteran baculoviruses have smaller genomes (84 kbp for hymenopteran baculoviruses and 108 kbp for dipteran CuniNPV) than lepidopteran baculovirus with an average genome size of 134.52 kbp. Similarly, hymenopteran and dipteran baculoviruses do not have the fp25k gene along with other non-essential genes like the lepidopteran baculoviruses do (Fig. 6A). In one possible scenario, the lepidopteran baculovirus fp25k gene is of host origin, and lepidopteran baculoviruses acquired it from their hosts. Another possible source of the lepidopteran baculovirus fp25k is through evolution of a gene of hymenopteran or dipteran baculoviruses. 4.6. Conclusions MNRs found within genes involved in the quality of baculovirus for commercial pesticides can be altered by site-directed mutagenesis to eliminate their likelihood of DNA replication slippage mutations. MNRs in genes such as fp25k involved in polh promoter activity in commercial AcMNPV and BmNPV expression vectors can be similarly eliminated to improve protein expression yields. Acknowledgements The authors of this publication would like to thank Mrs. Yesim A. Dizman for initial work on the baculovirus MNR comparison. 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