Bombyx mori Nucleopolyhedrovirus p26 Is Associated with Viral Late Stage Replication

Bombyx mori nucleopolyhedrovirus (BmNPV) p26 is conserved among all Lepidoptera baculoviruses that have been completely sequenced thus far, and some baculoviruses even have two copies of p26, which suggested that p26 may play an important role in the virus infection cycle. This study aimed to characterize BmNPV p26. We found that BmNPV p26 transcripts were detectable as early as 3 h post-infection (hpi), and the transcript levels rapidly increased starting from 12 hpi. Western blot analysis using an anti-p26 polyclonal antibody demonstrated that the corresponding protein was also detectable from 6 hpi in BmNPV-infected cell lysates. Immunofluorescence analysis demonstrated that p26 was mainly dispersed in the infected cell cytoplasm, whereas the over-expressed fusion protein EGFP-p26 also accumulated in the nucleus. These results indicated that p26 is an early BmNPV gene and has functions both in the cytoplasm and the nucleus. RNAi-based knockdown of p26 could produce infectious virus and normal-appearing virions but decreased budded virus (BV) production in BmNPV-infected cells at 72 hpi. Moreover, the results of further quantitative PCR (Q-PCR) analysis indicated that the gp64 and p74 transcripts levels decreased significantly. These results indicated that BmNPV p26 may be associated with BmNPV replication during the late infection stage.

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Characterization of a Late Expression Gene of Bombyx mori Nucleopolyhedrovirus

Zeitschrift für Naturforschung C ◽

10.1515/znc-2010-7-815 ◽

2010 ◽

Vol 65 (7-8) ◽

pp. 508-518 ◽

Cited By ~ 3

Author(s):

Yang Zhou ◽

Keping Chen ◽

Qin Yao ◽

Hongxing Shen ◽

Guiting Liang ◽

...

Keyword(s):

Confocal Microscopy ◽

Infected Cell ◽

Bombyx Mori ◽

Polyclonal Antiserum ◽

Late Gene ◽

Structural Protein ◽

Immunofluorescence Analysis ◽

Bombyx Mori Nucleopolyhedrovirus ◽

Late Expression

Bombyx mori nucleopolyhedrovirus (BmNPV) ORF5 (Bm5) is a gene present in many lepidopteran nucleopolyhedroviruses (NPVs), but its function is unknown. In this study, Bm5 was characterized. The transcript of Bm5 was detected 12 - 72 h post infection (p.i.). Polyclonal antiserum raised to a His-BM5 fusion protein recognized BM5 in infected cell lysates from 24 to 72 h p.i., suggesting that Bm5 is a late gene. Immunofluorescence analysis by confocal microscopy showed that the BM5 protein is localized primarily in the cytoplasm. Localization of BM5 in budded virion (BV) and occlusion-derived virion (ODV) by Western analyses demonstrated that BM5 is not a structural protein associated with BV or ODV.

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Identification of differentially expressed host genes in Bombyx mori nucleopolyhedrovirus infected cells by using subtractive hybridization

Applied Entomology and Zoology ◽

10.1303/aez.2007.151 ◽

2007 ◽

Vol 42 (1) ◽

pp. 151-159 ◽

Cited By ~ 10

Author(s):

Masashi Iwanaga ◽

Toru Shimada ◽

Masahiko Kobayashi ◽

WonKyung Kang

Keyword(s):

Bombyx Mori ◽

Subtractive Hybridization ◽

Differentially Expressed ◽

Bombyx Mori Nucleopolyhedrovirus ◽

Infected Cells ◽

Host Genes

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Functional analysis of the putative antiapoptotic genes, p49 and iap4, of Spodoptera litura nucleopolyhedrovirus with RNAi

Journal of General Virology ◽

10.1099/vir.0.2008/001008-0 ◽

2008 ◽

Vol 89 (8) ◽

pp. 1873-1880 ◽

Cited By ~ 20

Author(s):

Qian Yu ◽

Tiehao Lin ◽

Guozhong Feng ◽

Kai Yang ◽

Yi Pang

Keyword(s):

Spodoptera Litura ◽

Virus Production ◽

Host Cells ◽

Homology Search ◽

Pcr Analysis ◽

Viability Analysis ◽

Infected Cells ◽

Budded Virus ◽

Almost All

A homology search of a public database revealed that Spodoptera litura nucleopolyhedrovirus (SpltNPV) possesses two putative, antiapoptotic genes, p49 and inhibitor of apoptosis 4 (iap4), but their function has not been investigated in its native host cells. In the present study, we used RNA interference (RNAi) to silence the expression of Splt-iap4 and Splt-p49, independently or together, to determine their roles during the SpltNPV life cycle. RT-PCR analysis and Western blot analysis showed the target gene expression had been knocked out in the SpltNPV-infected SpLi-221 cells after treatment with Splt-p49 or Splt-iap4 double-stranded RNA (dsRNA), respectively, confirming that the two genes were effectively silenced. In SpltNPV-infected cells treated with Splt-p49 dsRNA, apoptosis was observed beginning at 14 h, and almost all cells had undergone apoptosis by 48 h. In contrast, budded virus production and polyhedra formation progressed normally in infected cells treated with Splt-iap4 dsRNA. Cell viability analysis showed that Splt-IAP4 had no synergistic effect on the inhibition of apoptosis of SpLi-221 cells induced by SpltNPV infection. Interestingly, after Splt-iap4 dsRNA treatment, cells did not congregate like those infected with SpltNPV in the early infection phase, implying an unknown role of baculovirus iap4. Our results determine that Splt-p49 is necessary to prevent apoptosis; however, Splt-iap4 has no antiapoptotic function during SpltNPV infection.

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Open reading frame 60 of the Bombyx mori nucleopolyhedrovirus plays a role in budded virus production

Virus Research ◽

10.1016/j.virusres.2010.05.003 ◽

2010 ◽

Vol 151 (2) ◽

pp. 185-191 ◽

Cited By ~ 8

Author(s):

Zhong-Jian Guo ◽

Li-Hua Qiu ◽

Shi-Heng An ◽

Qin Yao ◽

Enoch Y. Park ◽

...

Keyword(s):

Bombyx Mori ◽

Virus Production ◽

Open Reading Frame ◽

Bombyx Mori Nucleopolyhedrovirus ◽

Reading Frame ◽

Budded Virus

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Bombyx mori nucleopolyhedrovirus ORF56 encodes an occlusion-derived virus protein and is not essential for budded virus production

Journal of General Virology ◽

10.1099/vir.0.83633-0 ◽

2008 ◽

Vol 89 (5) ◽

pp. 1212-1219 ◽

Cited By ~ 30

Author(s):

Hai-Jun Xu ◽

Zhang-Nv Yang ◽

Jin-Fang Zhao ◽

Cai-Hong Tian ◽

Jun-Qing Ge ◽

...

Keyword(s):

Bombyx Mori ◽

Cultured Cells ◽

Virus Production ◽

Core Gene ◽

Virus Protein ◽

Infected Cells ◽

Budded Virus ◽

Larval Bioassay

Bombyx mori nucleopolyhedrovirus ORF56 (Bm56) is a baculovirus core gene that is highly conserved in all baculoviruses that have had their genomes sequenced to date. Its transcripts in BmNPV-infected cells could be detected from 12 h post-infection (p.i.) and the encoded protein could be detected at 16 h p.i. by using a polyclonal antibody against glutathione S-transferase–Bm56 fusion protein. Western blot analysis showed that Bm56 is a structural component of the occlusion-derived virus nucleocapsid. Subsequent confocal microscopy revealed that Bm56 was distributed in the outer nuclear membrane and the intranuclear region of infected cells. To investigate the role of Bm56 in virus replication, a Bm56-knockout bacmid of BmNPV was constructed via homologous recombination in Escherichia coli. The Bm56 deletion had no effect on budded virus (BV) production in cultured cells; however, the deletion affected occlusion-body morphogenesis. A larval bioassay demonstrated that the Bm56 deletion did not reduce infectivity, whereas it resulted in a 50 % lethal time that was 16–18 h longer than that of the wild-type bacmid at every dose used in this study. These results indicate that Bm56 facilitates efficient virus production in vivo; however, it is not essential for BV production in vitro.

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Characterization of Bombyx mori Nucleopolyhedrovirus orf68 Gene That Encodes a Novel Structural Protein of Budded Virus

Virology ◽

10.1006/viro.2002.1443 ◽

2002 ◽

Vol 297 (1) ◽

pp. 39-47 ◽

Cited By ~ 19

Author(s):

Masashi Iwanaga ◽

Masaaki Kurihara ◽

Masahiko Kobayashi ◽

WonKyung Kang

Keyword(s):

Bombyx Mori ◽

Structural Protein ◽

Bombyx Mori Nucleopolyhedrovirus ◽

Budded Virus

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Ubiquitins of Bombyx mori nucleopolyhedrovirus and Helicoverpa armigera nucleopolyhedrovirus show distinct subcellular localization in infected cells

Acta Virologica ◽

10.4149/av_2011_02_101 ◽

2011 ◽

Vol 55 (2) ◽

pp. 101-106 ◽

Cited By ~ 1

Author(s):

J. GUO ◽

Y. ZHU ◽

G. LI ◽

K. CHEN ◽

C. ZHANG

Keyword(s):

Subcellular Localization ◽

Bombyx Mori ◽

Helicoverpa Armigera ◽

Bombyx Mori Nucleopolyhedrovirus ◽

Helicoverpa Armígera ◽

Infected Cells

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Bombyx mori nucleopolyhedrovirus BmP95 plays an essential role in budded virus production and nucleocapsid assembly

Journal of General Virology ◽

10.1099/vir.0.050583-0 ◽

2013 ◽

Vol 94 (7) ◽

pp. 1669-1679 ◽

Cited By ~ 16

Author(s):

Xingwei Xiang ◽

Yunwang Shen ◽

Rui Yang ◽

Lin Chen ◽

Xiaolong Hu ◽

...

Keyword(s):

Bombyx Mori ◽

Virus Production ◽

Full Length ◽

Tubular Structures ◽

Bombyx Mori Nucleopolyhedrovirus ◽

Budded Virus ◽

Conserved Gene ◽

Fluorescence And Light Microscopy

Bombyx mori nucleopolyhedrovirus (BmNPV) BmP95 is a highly conserved gene that is found in all of the baculovirus genomes sequenced to date and is also found in nudiviruses. To investigate the role of BmP95 in virus infection in vitro, a BmP95 deletion virus (vBmP95-De) was generated by homologous recombination in Escherichia coli. Fluorescence and light microscopy and titration analysis indicated that the BmP95 deletion bacmid led to a defect in production of infectious budded virus (BV). However, deletion of BmP95 did not affect viral DNA replication. Electron microscopy showed that masses of aberrant tubular structures were present in cells transfected with the BmP95 deletion bacmid, indicating that deletion of BmP95 affected assembly of the nucleocapsid. This defect could be rescued by insertion of full-length BmP95 into the polyhedrin locus of the BmP95-knockout bacmid but not the N-terminal domain of BmP95. Together, these results showed that full-length BmP95 is essential for BV production and is required for nucleocapsid assembly.

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Human Cytomegalovirus UL99-Encoded pp28 Is Required for the Cytoplasmic Envelopment of Tegument-Associated Capsids

Journal of Virology ◽

10.1128/jvi.77.19.10594-10605.2003 ◽

2003 ◽

Vol 77 (19) ◽

pp. 10594-10605 ◽

Cited By ~ 137

Author(s):

Maria C. Silva ◽

Qian-Chun Yu ◽

Lynn Enquist ◽

Thomas Shenk

Keyword(s):

Golgi Apparatus ◽

Infected Cell ◽

Human Cytomegalovirus ◽

Infectious Virus ◽

Protein A ◽

Viral Dna ◽

Point Mutant ◽

Large Numbers ◽

Infected Cells ◽

Substitution Mutant

ABSTRACT The human cytomegalovirus UL99-encoded pp28 is a myristylated phosphoprotein that is a constituent of the virion. The pp28 protein is positioned within the tegument of the virus particle, a protein structure that resides between the capsid and envelope. In the infected cell, pp28 is found in a cytoplasmic compartment derived from the Golgi apparatus, where the virus buds into vesicles to acquire its final membrane. We have constructed two mutants of human cytomegalovirus that fail to produce the pp28 protein, a substitution mutant (BADsubUL99) and a point mutant (BADpmUL99), and we have propagated them by complementation in pp28-expressing fibroblasts. Both mutant viruses are profoundly defective for growth in normal fibroblasts; no infectious virus could be detected after infection. Whereas normal levels of viral DNA and late proteins were observed in mutant virus-infected cells, large numbers of tegument-associated capsids accumulated in the cytoplasm that failed to acquire an envelope. We conclude that pp28 is required for the final envelopment of the human cytomegalovirus virion in the cytoplasm.

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