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ISSN: 2155-952X
Journal of Biotechnology & Biomaterials

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Advances on BmNPV Functional Genomics

Zhang-Nv Yang1 and Chuan-Xi Zhang2*

1Zhejiang Provincial Center for Disease Control and Prevention, 310051 Hangzhou, China

2Institute of Insect Science, Zhejiang University, 310058, Hangzhou, China

Corresponding Author:
Chuan-Xi Zhang
Institute of Insect Science, Zhejiang University
310058, Hangzho, China
E-mail: [email protected]

Received date: April 10, 2012; Accepted date: May 10, 2012; Published date: May 12, 2012

Citation: Yang ZN, Zhang CX (2012) Advances on BmNPV Functional Genomics. J Biotechnol Biomaterial S9:006. doi:10.4172/2155-952X.S9-006

Copyright: © 2012 Yang ZN, et al. 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.

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Keywords

Bombyx mori nucleopolyhedrovirus; BmNPV; Functional genomics

Introduction

The baculovirus was originally described when silkworm cultures were threatened by a disease indicated as jaundice [1], and the isolation and chemical composition research of the virus resulted in the new era of the baculovirus research [2].

Baculoviruses are arthropod-specific viruses containing large double- stranded circular DNA genomes of 80-180k bp. The Baculoviridae family can be divided into four genera according to common biological and structural characteristics: Alphabaculovirus, which includes lepidopteran specific baculoviruses and is subdivided into Group I or Group II based on the type of fusogenic protein, Betabaculovirus, comprising lepidopteran-specific granuloviruses, Gammabaculovirus, which includes hymenopteran-specific baculoviruses, and Deltabaculovirus, which, to date, comprises only CuniNPV and possibly still undescribed dipteran-specific baculoviruses [3-6].

There are currently 54 complete baculovirus genomes deposited in GenBank before this paper was published (http://www.ncbi.nlm.nih.gov/genomes/GenomesGroup.cgi?taxid=10442). These include 37 Alphabaculoviruses, 13 Betabaculoviruses, 3 Gammabaculoviruses, and 1 Deltabaculovirus. Despite the diversity in gene content present in different baculovirus genomes, there are now 31 core genes conserved in all sequenced baculovirus genomes, the function of these genes can be divided into replication, transcription, packaging and assembly, cell cycle arrest, and oral infectivity (Table 1) [5]. Most of the data comes from the Autographa californica nucleopolyhedrovirus (AcMNPV), as it is the model type for baculovirus and has been extensively studied. Four core genes have been identified as essential players in viral DNA replication: DNApol, Helicase, Lef-1 and Lef-2. Nearly half of the core genes involved in the packaging and assembly.

Category Name ORF No.in BmNPV ORF No.in AcMNPV Localization Function Reference The phenotype after knock out in BmNPV
Replication
  Lef-1 6 14 nucleosome DNA primase [32] C
  DNApol 53 65 nucleosome DNA polemerase [33,34,35,36] C
  Helicase 78 95 nucleosome DNA helicase [35,37,38,39,] D
  Lef-2 135 6 cell nuclei Bind to both DNA and LEF-1; activate late transcription [40] C
Transcription
  P47 31 40 nuclei RNA polymerase subunit [41,42,] D
  Lef-8 39 50 Nuclei RNA polymerase subunit [43,44] D
  Lef-9 50 62 cell nuclei RNA polymerase subunit [44] D
  Lef-4 73 90 cell nuclei RNA polymerase subunit [41,45,46,47,48] D
  Lef-5 83 99 cell nuclei initiation factor [49] D
Packaging, assembly, and release
  VP1054 43 54 nucleosome structrual protein;
nucleocapsid assembly
[50] C
  Vlf-1 63 77 nucleosome lambda integrase; nucleocapsid assembly [33,42,51,52,53] C
  GP41 66 80 tegument ODV protein; BV production [54,55] B
  ODV -NC42 56 68 capsid ODV protein; Assemble nucleocapsid into ODV [17,56] A
  VP91/P95 69 83 envelope Capsid-associated protein [57] C
  VP39 72 89 capsid major capsid protein [58,59,60,61] C
  P33 75 92 nonstructural sulfhydryloxidase; BV/ODV associated protein [62,63,64] C
  38K 82 98 nucleosome Nucleocapsid assembly [65,66] C
  P6.9 84 100 capsid DNA binding [67,68,69] C
  ODV-EC43 92 109 capsid BV/ODV Protein [70,71,72] C
  Alk-exo 110 133 nucleosome 5'-3'exonulcease [73,74,75] C
  P49 118 142 capsid Nucleocapsid assembly [76] C
  ODV-E18 119 143 envelope BV/ODV Protein; BV production [77] B
  desmoplakin 54 66   BV production [78] C
Cell cycle arrest and/or interaction with host proteins
    67 81   interact with Actin3 in cytoplasm [79] C
  ODV-EC27 120 144 tegument ODV associated protein; cyclin [80,81] C
Oral infectivity
  Pif-2 13 22 envelope mediate binding in midgut [82,83] A
  OEV-E28/19K/Pif4 79 96 envelope primary oral infection [72,83,84,] C
  Pif-3 95 115 envelope form a complex with PIF-1 and PIF-2 [83,72] A
  Pif-1 97 119 envelope mediate binding in midgut [83,85,86] A
  Pif-0/P74 115 138 envelope mediate binding in midgut [87,88,89,90] A
  Pif5/ODV-E56 124 148 envelope primary oral infection [81,91] A

Table 1: Functions of 31 core genes in 54 baculoviruses

In the most well characterized baculoviruses, the life cycle is biphasic with two different phenotypes during virus infection: Occluded Virions (ODV) and Budded Virions (BV) [7]. The ODV transmits infection from insect to insect by infecting midgut columnar epithelial cells, whereas the BV is responsible for causing systemic infection within the host [8]. The two viral forms are efficient for the natural propagation of the occluded baculoviruses.

Over the years, Baculoviruses are extensively being studied for potential use in bioinsecticides around the world and as expression vectors for heterologous genes expression in insect-derived cells as well as in the host caterpillars. Bombyx mori nucleopolyherovirus (BmNPV), the type member of baculovirus, has been successfully developed for surface display [9]. Additionally, baculoviruses are regarded as potentially useful gene therapy vectors [10,11], and has been applied for veterinary and medical applications, including the development of vaccines and diagnostics, putative nonhuman viral vectors for gene delivery, and biological control agents against insect pests [5,6,12]. These applications further trigger the study of molecular basis of baculovirus infection.

BmNPV Functional Genomics

The history of BmNPV is intimately related to the development of the silk industry. Silk production has been of major cultural significance in China and some other Asian Countries. Though BmNPV was the first described NPV, the genome of the BmNPV was completely sequenced and analyzed in 1999. The BmNPV (T3 strain) genome has a size of 12, 843 bp with a G+C content of 40% and encoding 142 predicated open reading frames (136 of the ORFs encoding predicted proteins of over 60 amino acids) [13,14]. Recently, a BmNPV strain (S1) isolated from the wild silkworm (Bombyx mandarina) was also sequenced [15]. The complete nucleotide sequence of the S1 strain was compared with the T3 strain. The S1 strain was 126,770 nucleotides long, with a G+C content of 40.23%. The genome contained 133 potential ORFs encoding predicted proteins of over 60 amino acids. Most of the putative proteins were more than 96% identical to homologs in the T3 strain, except for bro-a, lef-12, bro-c, and bro-d. The S1 genome did not encode the bro-b and bro-e genes. The BmNPV genome was closely related to that of AcMNPV, sharing over 90% similarity to about threequarters of the genome of AcMNPV. And the relatedness of predicted amino acid sequences of corresponding ORFs between BmNPV and AcMNPV was about 90% identical.

As Bombyx mori is well-studied physiologically, biochemically, and molecular biologically, contributed to the genome sequenced through international collaboration between China and Japan [16]. The genome data stimulated the analysis on the BmNPV-silkworm infection system. However, at least two third of the BmNPV viral gene functions are still remain unknown. BmNPV has potential usage not only for expression vector, but also for the display system, thus the fundamental investigation on gene function is important.

Many researchers have made effort on characterization of the BmNPV genes. And the development of the BmNPV bacmid system and the modification of this technology using a lambda red recombinant system allowed for the efficient production of knockouts, make the single gene function analysis more convenient. By using the knockout technology, Ono et al. [16] generated KOVs (knockout viruses) for 141 BmNPV genes and subdivided the KOVs into four phenotypic groups, types A to D [16].

Rohrmann [6] has included pertinent information on the function of all the ORFs present in the AcMNPV genome. Though AcMNPV and BmNPV share high similarities in the genome, some of the homologous genes in the two viruses function quite differently in the regulation of viral life cycle due to adaptation to different hosts. Here, we listed all the genes characterized in BmNPV up to date in Table 2 and marked by the phenotypic groups established by Ono et al. [16].

 Name The phenotype after
 knock out in BmNPV*
References
Bm5 A  [18]
Bm8( BV/ODV-E26) A [92,93]
Bm9 A [94]
Bm21 A [95]
Bm22(BRO-A) A [21]
Bm24(Vfgf) A [92,93]
Bm26(Ubiquitin) A [56,94]
Bm 27(39K) A [95]
Bm33(GTA) A [96]
Bm34 A [97]
Bm41 A [98]
Bm49(FP25k) A [99,100,101]
Bm51 A [102]
Bm56 A  [17]
Bm60 A [103,104]
Bm68(TLP) A [105]
Bm71(P39&CG30) A [106,107]
Bm74 A [108]
Bm80(BRO-B) A [21]
Bm81(BRO-C) A [21]
Bm94 A [109]
Bm102(Lef7) A [95, 110]
Bm103(Chitinase) A [111]
Bm112(P35) A [95,101,111]
Bm114(P10) A [112]
Bm124( Odv-e56) A [113]
Bm 127(Ie2) A [93,95]
Bm132(BRO-E) A [21,]
Bm134 A [56]
Bm131(BRO-D) B [21,]
Bm3(Pk1) C [114]
Bm16(DBP) C [73]
Bm25 C [111]
Bm42 C [115]
Bm54 C [116]
Bm61 C [117]
Bm67 C [118]
Bm69(P95) C [119]
Bm75 C [120]
Bm76 C [121]
Bm79 C [17]
Bm88(VP80) C [22]
Bm 105(GP64) C [104]
Bm109 C [122]
Bm118 C [94]
Bm122 C [123]
Bm39(Lef8) D [24]
Bm50 (lef9) D [24]

Table 2: Characterized genes in BmNPV.

Type-A KOVs showed the ability to expand infections almost equivalent to the wild type virus. The type A genes are likely to be nonessential, as the genes interrupted by insertion/deletion mutagenesis in BmNPV, the virus appeared to be normal, though the deletion may have slight affection on BV/ODV production, virulent, or DNA replication. For instance, it was previously reported that the deletion of Bm56 in BmNPV affected the occlusion body’s morphogenesis [17]. Bm5 is not a structural protein associated with BV or ODV, the Bm5 likely to be nonessential as the KO virus appeared to be viable [18]. And knock out of Bm9 led to a slight reduced production of BV without affect the DNA replication [19]. Bioassays showed that the Bm9-deleted bacmid took approximately 14-22 h longer to kill fifth instar B. mori larvae than wt bacmid, and the LD (50) was about 15 times higher than that of the wt bacmid, indicating that Bm9 is an important but not essential factor in virus production and infectivity in vivo and in vitro. For the core genes of all the baculovirus (Table 1), Oral infectivity genes are type A except Bm79 (OEV-E28/19K/Pif4). Bm79 is an ODV envelope associated protein [20] and in BmNPV and lately was also found associated with the envelope of BV in AcMNPV, and was concluded that Ac96 is a per os infecitivity factor in AcMNPV [6].

Type-B KOVs were characterized by a markedly slow spreading of the infection probably because of the low production of infectious viruses and/or low infectivity of the progeny viruses. As genes have been characterized in BmNPV, only Bm131 (BRO-D) belonged to type B. Bm131 (BRO-D) has been observed as a nucleocytoplasmic shuttling protein, and the KOV of Bm131 was due to a reduction in RNA synthesis [21].

Type-C KOVs expressed report egfp in transfected-BmN cells but the production of infectious viruses was not observed while Type-D showed no ability to express egfp even in the transfection experiments. The core genes of baculovirus involved in replication and transcription are all belonged to type C and type D (Table 1). And most of the type C genes may play important roles in the morphogenesis of BmNPV as most of the genes knocked out in type-C code for virion structural proteins (Table 1). The Bm54-deleted virus produced non-infectious budded virus (BV) and normal nucleocapsids but defect in polyhedra. And disruption of Bmvp80 [22] resulted in single cell infection phenotype. Bmvp80 is essential for normal budded virus production and nucleocapsid maturation, and is functionally divergent between baculovirus species. Also, Bm118 is essential for BV production and nucelocapsid maturation [23].

In the cells transfected with type-D bacmid DNAs, neither egfp expression nor the BV production was detected. Two viral genes of BmNPV have been characterized in type-D KOVs were essential not only for viral replication, but also for polhedrin expression [24].

Take the advantage of silkworm genomic data, researchers analyzed the host-derived protein of the BmNPV [25], the BmNPV resistance in the hemolymph and the fat body in the genomic and proteomic level [26-30]. A dynamic protein-protein interaction network between BmNPV and its host cells was predicted by making comparisons to other insect interactomes recently [31]. This is a comprehensive indepth study analyzing host gene response to baculovirus infection and perhaps the most comprehensive and best described transcriptional analysis available using baculoviruses. Gene enhancements are grouped in functional categories (cell cycle, energy metabolism, etc.) and their potential role during baculovirus infection is well discussed [31]. The protein-protein interaction network prediction provides an excellent resource for others in the community to evaluate certain pathways and to provide more in depth analysis.

Conclusion

In conclusion, two strain genomes of BmNPV have been sequenced and compared. Forty eight genes in a total of 136 BmNPV ORFs have been functionally characterized. As development of new genome sequencing techniques, more and more genomes of baculoviruses have been sequenced, and phylogenetic evolution history of baculoviruses becomes increasingly clear. We have tried to comparatively analyze six various strains from a host at genome level that will enrich our knowledge of evolution of the baculoviruses (data not published). However, many gene functions remain unclear yet. To elucidate the interaction between BmNPV and its host, more works on ORF functions are needed.

References

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