Ha83, a Chitin Binding Domain Encoding Gene, Is Important to Helicoverpa armigera Nucleopolyhedrovirus Budded Virus Production and Occlusion Body Assembling.

Huan Yu,Qiang Liu,Jian Xu,Dun Wang,Tong-Xian Liu

doi:10.1038/srep11088

Huan Yu, Qiang Liu + Show 3 more

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Helicoerpa armigera nucleopolyhedrovirus (HearNPV) ha83 is a late expressed gene that encodes a chitin binding protein. Chitin domain truncation studies revealed that the cysteine at the 128 amino acid position probably played an important role in both chitin binding ability and protein transmission of Ha83. In order to study the function of ha83 in the HearNPV infection cycle, an ha83 knockout HearNPV (Ha83KO) was constructed via homologous recombination. Viral growth and viral DNA replication curves showed that fewer budded virions were produced in Ha83KO transfected cells, while viral DNA replication was increased. Electron microscopy revealed that fewer nucleocapsids were transmitted from virogenic stroma in the Ha83KO transfected cell nucleus, and the morphology of occlusion bodies was prominently larger and cube-shaped. Furthermore, DNA quantity in occlusion bodies of Ha83KO was significantly lower than the occlusion bodies of HaWT. The transcription analysis indicated that these changes may be due to the decreased expression level of viral structural associated genes, such as polyhedrin, p10, pif-2, or cg30 in Ha83KO infected cells. Above results demonstrated that the cysteine at the 128 amino acid position in Ha83 might be the key amino acid, and Ha83 plays an important role in BVs production and OBs assembling.

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The Baculoviridae encompasses a diverse group of insect specific DNA viruses that have been reported worldwide, predominantly from insects of the orders Lepidoptera, Hymenoptera, and Diptera[1]
A search of the 374 nt 5′ untranslated regions (UTR) showed that there are two TATA boxes located at positions − 107 and − 293 upstream of the ATG; a late strong promoter ATAAG element was at position − 202; another TAAG late promoter element was found at − 27; five early promoter CATT elements were found at − 67, − 187, − 211, − 255, and − 310 upstream of the ATG (Fig. 1B)
To identify the functional cysteine needed for the chitin binding domain in Ha83, we constructed seven pET-28a(+ ) plasmids containing different parts of Helicoerpa armigera nucleopolyhedrovirus (HearNPV) Ha83 fused with His-tag, as well as an ha[81] expression vector to express Ha81 used as a negative control

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Introduction

The Baculoviridae encompasses a diverse group of insect specific DNA viruses that have been reported worldwide, predominantly from insects of the orders Lepidoptera, Hymenoptera, and Diptera[1]. 3′RACE and 5′RACE analysis of ha[83] in HearNPV infected HzAM1 cells. We found that ha[83] is involved in BV production for the deletion mutant and produces fewer BVs than the wild type in one step viral growth curve analysis. The transient eukaryotic expression vectors contained the truncated ha[83] fused with egfp and were transfected into HzAM1 cells and confocal microscopic analysis showed that, in late infection, the first N-terminal 141 amino acids were necessary for the traffic of Ha83 from the cytoplasm into the nucleus. The truncated prokaryotic purified proteins revealed a similar result that the conserved cysteine at the 128 amino acid position was important to Ha83 chitin binding activity

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