Expression profiling of baculovirus genes in permissive and nonpermissive cell lines

doi:10.1016/j.bbrc.2004.08.114

Biochemical and Biophysical Research Communications

Volume 323, Issue 2, 15 October 2004, Pages 599-614

https://doi.org/10.1016/j.bbrc.2004.08.114 Get rights and content

Abstract

The baculoviruses Bombyx mori nucleopolyhedrovirus (BmNPV) and Autographa californica multicapsid NPV (AcMNPV) are highly homologous at the genomic level, but they have essentially nonoverlapping host ranges. In order to characterize baculovirus replication in permissive and nonpermissive cell lines, the expression profiles of baculovirus-specific genes (at 2, 6, 12, 24, 36, 48 or 72 h post-infection) were examined in BmN (BmNPV-permissive) or Sf-9 (AcMNPV-permissive) cells that were inoculated with BmNPV or AcMNPV. Surprisingly, nearly all of the 154 genes of AcMNPV appeared to be expressed in both Sf-9 and BmN cells although the peak expression levels of these genes were delayed by roughly 12 h in the nonpermissive BmN cells. In addition, the expression levels of the very late AcMNPV polyhedrin and p10 genes were dramatically reduced in BmN cells, which presumably led to the inability of AcMNPV to form polyhedral inclusion bodies in BmN cells. Nearly all of the 136 genes of BmNPV appeared to be expressed in BmN cells, however, BmNPV gene expression was dramatically reduced in Sf-9 cells inoculated with BmNPV. Experiments in which BmNPV DNAs were transfected to Sf-9 cells suggested that this dramatic reduction in gene expression was not the result of poor attachment, penetration or uncoating of the BmNPV virion into Sf-9 cells. In conclusion, we established a system to monitor global gene expression patterns during baculovirus infection in permissive and nonpermissive cell lines. This system was used to identify global trends in the transcription of baculovirus genes during productive and nonproductive infection.

Section snippets

Materials and methods

Cell lines, viruses, plaque assay, and virus growth curves. The Sf-9 and BmN cell lines were maintained as described previously on TC-100 medium supplemented with 10% fetal bovine serum [21]. The AcMNPV E2 [24] isolate was propagated in Sf-9 cells. The BmNPV T3 [25] and eh2-AcMNPV [12] isolates were propagated in BmN cells. Virus growth curves were determined by plaque assay as described previously [26], [27]. Titration of eh2-AcMNPV was performed on BmN cells.

Microarray analysis. Microarray

Gene expression patterns in Sf-9 and BmN cells infected with AcMNPV or BmNPV

As reported previously, BmNPV and AcMNPV are highly homologous at DNA sequence and genome organization levels, but their host ranges are nonoverlapping [10], [11]. In our hands, BmNPV strongly replicated (titer of >10⁸ pfu/ml at 48 h p.i.) and produced PIBs in the B. mori-derived BmN cell line, but it replicated very weakly in the Spodoptera frugiperda-derived Sf-9 cell line (Figs. 1A and B). In contrast, AcMNPV strongly replicated (titer of about 10⁸ pfu/ml at 48 h p.i.) and produced PIBs in Sf-9

Acknowledgments

This work was supported in part by grants from BRAIN (to M.K.) and JSPS (Nos. 14360032 and 14656023) (to T.S.), Japan.

References (36)

M.D. Ayres et al.
The complete DNA sequence of Autographa californica nuclear polyhedrosis virus
Virology
(1994)
T. Hayakawa et al.
Patterns of genome organization and content in lepidopteran baculoviruses
Virology
(2000)
T.D. Morris et al.
Characterization of productive and non-productive AcMNPV infection in selected insect cell lines
Virology
(1993)
T. Noji et al.
Isolation and comparison of different ecdysone-responsive cuticle protein genes in wing discs of Bombyx mori
Insect Biochem. Mol. Biol.
(2003)
S. Katsuma et al.
Functional genomic search of G-protein-coupled receptors using microarrays with normalized cDNA library
Methods Enzymol.
(2002)
K. Takagaki et al.
cDNA microarray analysis of altered gene expression in Ara-C-treated leukemia cells
Biochem. Biophys. Res. Commun.
(2003)
S. Katsuma et al.
Transcriptional profiling of gene expression patterns during sphingosine 1-phosphate-induced mesangial cell proliferation
Biochem. Biophys. Res. Commun.
(2003)
D.R. O’Reilly et al.
Baculovirus Expression Vectors: A Laboratory Manual
(1992)
P.V. Choudary et al.
Expression of foreign genes in Bombyx mori larvae using baculovirus vectors
J.E. Maruniak et al.
Physical maps and virulence of Anticarsia gemmatalis nucleopolyhedrovirus genomic variants
Arch. Virol.
(1999)

A.B. Inceoglu et al.

Recombinant baculoviruses for insect control

Pest. Manag. Sci.

(2001)

F.M. Boyce et al.

Baculovirus-mediated gene transfer into mammalian cells

Proc. Natl. Acad. Sci. USA

(1996)

I. Shoji et al.

Efficient gene transfer into various mammalian cells, including non-hepatic cells, by baculovirus vectors

J. Gen. Virol.

(1997)

S. Gomi et al.

Sequence analysis of the genome of Bombyx mori nucleopolyhedrovirus

J. Gen. Virol.

(1999)

S. Maeda et al.

Characteristically distinct isolates of the nuclear polyhedrosis virus from Spodoptera litura

J. Gen. Virol.

(1990)

M. Ikeda et al.

Characterization of Autographa californica nucleopolyhedrovirus infection in cell lines from Bombyx mori

J. Insect Biotechnol. Sericol.

(2001)

S. Maeda et al.

Host range expansion of Autographa californica nuclear polyhedrosis virus (NPV) following recombination of a 0.6-kbp DNA fragment originating from Bombyx mori NPV

J. Virol.

(1993)

G. Croizier et al.

Extension of Autographa californica nuclear polyhedrosis virus host range by interspecific replacement of a short DNA sequence in the p143 helicase gene

Proc. Natl. Acad. Sci. USA

(1994)

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Bombyx mori nucleopolyhedrovirus (BmNPV) is known to replicate in many tissues of Bombyx mori larvae. However, the cell lines used for BmNPV research are predominantly derived from B. mori ovaries or early embryos. In the present study, we examined the properties of NIAS-Bm-aff3 (aff3), a cell line that was established from B. mori larval fat body, which is one of the major tissues for BmNPV propagation. aff3 is a floating cell line, and cell adhesion was enhanced following the coating of the culture dish with poly-d-lysine. RT-qPCR assays demonstrated that the expression of germ cell markers, Vasa, Siwi, and BmAgo3, was much lower in aff3 cells as compared to the B. mori ovary-derived cell line BmN-4. Conversely, aff3 cells express an adipocyte marker, Fabp1, at higher levels, indicating that this cell line retains the characteristics of fat body cells. BmNPV infection induces unique cell fusion in aff3 cells, which was also observed following infection with Autographa californica multiple nucleopolyhedrovirus, a virus that does not cause productive infection in B. mori cells. Occlusion bodies (OBs) produced in BmNPV-infected aff3 cells exhibit large cuboidal shapes as compared to those produced in BmN-4 cells. Furthermore, extremely large OBs (~25 μm in side length) were produced in aff3 cells when infected with a cuboidal polyhedrin mutant. Taking into account these unusual properties, we conclude that aff3 could prove to be a useful resource for conducting baculovirus research.
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Baculoviruses are insect-specific DNA viruses with a restricted host range. Two examples of such include Autographa californica nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). AcMNPV is a commercially available and widely used baculovirus prototype which can infect 39 species in 13 families. On the other hand, BmNPV is a major pathogen of silkworms which has developed high host specificity to Bombyx mori. Coincidentally, AcMNPV and BmNPV are highly homologous, but share no overlapping host range on a genomic level. In fact, these two similar viruses have extremely different infection outcomes in Bombyx mori. We hypothesized that the determination of host specificity may depend on virus-host interactions and several genes may be involved in determining host specificity. Therefore, we used next generation sequencing (NGS) to analyze transcriptome responses of hosts to these viruses. A transcriptome library was constructed, annotated, and grouped after sequence assembly. The comparison of gene expressions showed several significant differences in the gene expression profiles of BmNPV and AcMNPV, especially in cases where genes involved with immune responses were verified by RNA interference. In addition, studies have shown that small RNA plays a role in virus-host interaction, and further determines host specificity. Therefore, we screened microRNA induced by AcMNPV infection, and then combined NGS data from cellular gens to predict possible regulation networks. The manipulation of virus-host specificity can provide a breakthrough for the application of baculovirus in protein expression systems and the development of bio-control agents.
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Citation Excerpt :
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We previously established the first Bombyx mori macula-like virus (BmMLV)-free cell line (BmVF cells) from a B. mori embryo. In this study, we evaluated the expression of recombinant proteins in BmVF cells using a B. mori nucleopolyhedrovirus (BmNPV)-derived expression vector. Our results showed that BmVF cells are susceptible to BmNPV, and both the promoter activity of the polyhedrin gene and the post-translated modifications of a recombinant protein are equivalent between BmMLV-negative BmVF and -positive BmN4 cells. These findings indicate that persistent infection with BmMLV has no discernible effect on BmNPV-mediated protein production in B. mori cells.
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Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious viral pathogen of silkworm, and no drug or specific protection against BmNPV infection is available at present time. Although functions of most BmNPV genes were depicted in recent years, knowledge on the mechanism of BmNPV entry into insect cells is still limited. Here BmNPV cell entry mechanism is investigated by different endocytic inhibitor application and subcellular analysis. Results indicated that BmNPV enters BmN cells by clathrin-independent macropinocytic endocytosis, which is mediated by cholesterol in a dose-dependent manner, and cholesterol replenishment rescued the BmNPV infection partially.

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¹: These three authors contributed equally to this work.

²: Present address: New Frontiers Research Laboratory, Toray Industries, Inc., Tebiro 1111, Kamakura, Kanagawa 248-8555, Japan.

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Expression profiling of baculovirus genes in permissive and nonpermissive cell lines

Abstract

Section snippets

Materials and methods

Gene expression patterns in Sf-9 and BmN cells infected with AcMNPV or BmNPV

Acknowledgments

Virology

Virology

Virology

Insect Biochem. Mol. Biol.

Methods Enzymol.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Baculovirus Expression Vectors: A Laboratory Manual

Expression of foreign genes in Bombyx mori larvae using baculovirus vectors

Physical maps and virulence of Anticarsia gemmatalis nucleopolyhedrovirus genomic variants

Arch. Virol.

Recombinant baculoviruses for insect control

Pest. Manag. Sci.

Baculovirus-mediated gene transfer into mammalian cells

Proc. Natl. Acad. Sci. USA

Efficient gene transfer into various mammalian cells, including non-hepatic cells, by baculovirus vectors

J. Gen. Virol.

Sequence analysis of the genome of Bombyx mori nucleopolyhedrovirus

J. Gen. Virol.

Characteristically distinct isolates of the nuclear polyhedrosis virus from Spodoptera litura

J. Gen. Virol.

Characterization of Autographa californica nucleopolyhedrovirus infection in cell lines from Bombyx mori

J. Insect Biotechnol. Sericol.

Host range expansion of Autographa californica nuclear polyhedrosis virus (NPV) following recombination of a 0.6-kbp DNA fragment originating from Bombyx mori NPV

J. Virol.

Extension of Autographa californica nuclear polyhedrosis virus host range by interspecific replacement of a short DNA sequence in the p143 helicase gene

Proc. Natl. Acad. Sci. USA