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

The fp25k gene of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) was studied. HearNPV fp25k gene transcription was found starting from about 18 h post-infection, and protein could be detected from the same time with antiserum against FP25K. To study the function of HearNPV fp25k, a recombinant HearNPV (HaBacWD11) with an enhanced green fluorescent protein (GFP) gene replacing the fp25k was constructed using HaBacHZ8, a bacmid of HearNPV that lacks the polyhedrin gene. Growth curve analysis showed that HaBacWD11 produced higher titres of budded viruses (BVs) than its wild-type counterpart HaBacHZ8–GFP. Electron microscopic analysis indicated that at the late stage of infection, the number of intranuclear enveloped nucleocapsids in HaBacWD11-infected cells was much less than that of HaBacHZ8–GFP. A rescue recombinant virus HaBacWD14 was constructed by reintroducing fp25k gene into HaBacWD11. The growth curve and electron microscopic analysis of the rescued recombinant confirmed that the increase of BV yield and the decrease of the virion production in infected cells were the result of fp25k deletion. The expression of membrane fusion protein (Ha133) and ODV-E66 were studied using the FP25K mutants HaBacWD11 and HaBacHZ8–GFP. Unlike FP25K mutants in Autographa californica multicapsid NPV (AcMNPV), which caused an increase in the expression of membrane fusion protein GP64 and a decrease of ODV-E66, no obvious changes at the expression level of Ha133 and ODV-E66 were observed in HearNPV FP25K mutant.

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Virus morphogenesis of Helicoverpa armigera nucleopolyhedrovirus in Helicoverpa zea serum-free suspension culture

Journal of General Virology ◽

10.1099/0022-1317-81-10-2531 ◽

2000 ◽

Vol 81 (10) ◽

pp. 2531-2543 ◽

Cited By ~ 20

Author(s):

Linda H. L. Lua ◽

Steven Reid

Keyword(s):

Suspension Culture ◽

Helicoverpa Armigera ◽

Helicoverpa Zea ◽

Close Association ◽

Fibrillar Structure ◽

Serum Free ◽

Virus Morphogenesis ◽

Helicoverpa Armígera ◽

Infected Cells ◽

Serum Free Suspension

Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV) replication in Helicoverpa zea serum-free suspension culture was studied in detail and the sequence of virus morphogenesis was determined by transmission electron microscopy. By 16 h post-infection (p.i.), virus replication was observed in the virogenic stroma by the appearance of nucleocapsids. Polyhedron formation was detected by 24 h p.i. and the polyhedron envelope (PE) was completely formed by 72 h p.i. PE morphogenesis of HaSNPV is significantly different compared to the extensively studied Autograph californica (Ac)MNPV. In AcMNPV-infected cells, fibrillar structures are found in both cytoplasm and nuclei, and the fibrillar structures in nuclei are in close association with maturing polyhedra during PE formation. Fibrillar structures that resemble the AcMNPV fibrillar structures were detected only in the cytoplasm of HaSNPV-infected cells and appeared to interact with calyx precursors there, but their role in PE formation is unclear. However, prominent calyx precursor structures of various shapes and sizes were observed in the nuclei of HaSNPV-infected cells as well, and they appeared to interact with polyhedra during PE formation. Both the calyx precursor structure and the cytoplasmic fibrillar structure were detected only after HaSNPV virion occlusion had started, indicating that they might have a role in formation of PE. Similar calyx precursor structures and cytoplasmic fibrillar structures were observed in both serum-supplemented and serum-free suspension cultures, as well as in HaSNPV-infected larval tissues, indicating that the structures observed are not cell culture artefacts.

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Bombyx mori Nucleopolyhedrovirus p26 Is Associated with Viral Late Stage Replication

Insects ◽

10.3390/insects12080707 ◽

2021 ◽

Vol 12 (8) ◽

pp. 707

Author(s):

Jun-Qing Ge ◽

Zhu-Hong Wang ◽

Xi Chen ◽

Hua Chen ◽

Jian Huang

Keyword(s):

Infected Cell ◽

Bombyx Mori ◽

Infectious Virus ◽

Pcr Analysis ◽

Immunofluorescence Analysis ◽

Bombyx Mori Nucleopolyhedrovirus ◽

Cell Cytoplasm ◽

Infected Cells ◽

Budded Virus ◽

Infection Stage

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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Comparative Characterization of Chitinases from Silkworm (Bombyx mori) and Bollworm (Helicoverpa armigera)

Cell Biochemistry and Biophysics ◽

10.1007/s12013-011-9196-2 ◽

2011 ◽

Vol 61 (2) ◽

pp. 267-275 ◽

Cited By ~ 13

Author(s):

HongBin Zhang ◽

MingYan Liu ◽

YuJing Tian ◽

XueQin Hu

Keyword(s):

Bombyx Mori ◽

Helicoverpa Armigera ◽

Comparative Characterization ◽

Helicoverpa Armígera ◽

Silkworm Bombyx Mori

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Phenotypic and genotypic analysis of Helicoverpa armigera nucleopolyhedrovirus serially passaged in cell culture

Journal of General Virology ◽

10.1099/0022-1317-83-4-945 ◽

2002 ◽

Vol 83 (4) ◽

pp. 945-955 ◽

Cited By ~ 36

Author(s):

Linda H. L. Lua ◽

Marcia R. S. Pedrini ◽

Steven Reid ◽

Ashley Robertson ◽

David E. Tribe

Keyword(s):

Cell Culture ◽

Helicoverpa Armigera ◽

Base Pair ◽

Point Mutations ◽

Biologically Active ◽

Single Base ◽

Genotypic Analysis ◽

Helicoverpa Armígera ◽

Infected Cells ◽

Single Base Pair

Rapid accumulation of few polyhedra (FP) mutants was detected during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in cell culture. 100% FP infected cells were observed by passage 6. The specific yield decreased from 178 polyhedra per cell at passage 2 to two polyhedra per cell at passage 6. The polyhedra at passage 6 were not biologically active, with a 28-fold reduction in potency compared to passage 3. Electron microscopy studies revealed that very few polyhedra were produced in an FP infected cell (<10 polyhedra per section) and in most cases these polyhedra contained no virions. A specific failure in the intranuclear nucleocapsid envelopment process in the FP infected cells, leading to the accumulation of naked nucleocapsids, was observed. Genomic restriction endonuclease digestion profiles of budded virus DNA from all passages did not indicate any large DNA insertions or deletions that are often associated with such FP phenotypes for the extensively studied Autographa californica nucleopolyhedrovirus and Galleria mellonella nucleopolyhedrovirus. Within an HaSNPV 25K FP gene homologue, a single base-pair insertion (an adenine residue) within a region of repetitive sequences (seven adenine residues) was identified in one plaque-purified HaSNPV FP mutant. Furthermore, the sequences obtained from individual clones of the 25K FP gene PCR products of a late passage revealed point mutations or single base-pair insertions occurring throughout the gene. The mechanism of FP mutation in HaSNPV is likely similar to that seen for Lymantria dispar nucleopolyhedrovirus, involving point mutations or small insertions/deletions of the 25K FP gene.

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