Characterization of Five Cell Lines Established from Species of Heliothis

Journal of General Virology

et al. 2008

A combined functional investigation on the four per os infectivity factors (PIFs) of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) was conducted in this study. HearNPV bacmids with deletions of p74 (Ha20), pif1 (Ha111), pif2 (Ha132) and pif3 (Ha98) were constructed individually by homologous recombination in Escherichia coli cells. Repaired bacmids with respective pifs were also constructed. Western blot analyses revealed that all four PIFs were structural components of the envelope of HearNPV occlusion-derived virus (ODV). Electron microscopy showed that deletion of the pifs did not have any obvious effects on the morphology of the occlusion bodies (OBs). Bioassay analyses indicated that deletion of any of the above pifs resulted in loss of oral infectivity of OBs. The mixtures of the four pif-deletion mutants also resulted in deficiency of oral infectivity, implying that the four PIFs must be structural components of the same ODV to accomplish their function. Repairing of the respective genes into the pif-deletion bacmids could rescue the oral infectivity of the pif-deletion viruses. Calcofluor, which can damage the peritrophic membrane (PM), could not rescue the defects of the oral infectivity of the pif-deletion viruses, indicating that the PM is not likely to be the functional target of the PIFs.

Section: Methodsmentioning

confidence: 99%

Functional studies of per os infectivity factors of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus

Song

Wang

Journal of General Virology

et al. 2008

“…An in vivo-cloned strain of HearNPV (HearNPV-G4) (13,55) was used as the wild-type virus and propagated in H. armigera. Cells of the Heliothis zea HzAM1 line (39) were used for producing BV of HearNPV.…”

Section: Methodsmentioning

confidence: 99%

Proteomics Analysis of Helicoverpa armigera Single Nucleocapsid Nucleopolyhedrovirus Identified Two New Occlusion-Derived Virus-Associated Proteins, HA44 and HA100

Wang

Mao

et al. 2007

J Virol

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry were used to analyze the structural proteins of the occlusion-derived virus (ODV) of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV), a group II NPV. Twenty-three structural proteins of HearNPV ODV were identified, 21 of which have been reported previously as structural proteins or ODV-associated proteins in other baculoviruses. These include polyhedrin, P78/83, P49, ODV-E18, ODV-EC27, ODV-E56, P74, LEF-3, HA66 (AC66), DNA polymerase, GP41, VP39, P33, ODV-E25, helicase, P6.9, ODV/BV-C42, VP80, ODV-EC43, ODV-E66, and PIF-1. Two proteins encoded by HearNPV ORF44 (ha44) and ORF100 (ha100) were discovered as ODV-associated proteins for the first time. ha44 encodes a protein of 378 aa with a predicted mass of 42.8 kDa. ha100 encodes a protein of 510 aa with a predicted mass of 58.1 kDa and is a homologue of the gene for poly(ADP-ribose) glycohydrolase (parg). Western blot analysis and immunoelectron microscopy confirmed that HA44 is associated with the nucleocapsid and HA100 is associated with both the nucleocapsid and the envelope of HearNPV ODV. HA44 is conserved in group II NPVs and granuloviruses but does not exist in group I NPVs, while HA100 is conserved only in group II NPVs.

“…A Helicoverpa zea cell line, HzAM1 (10), was maintained at 27°C in Grace's insect medium supplemented with 10% fetal bovine serum (FBS). The HearNPV G4 strain (9) was used in this study.…”

Section: Cell Cultures Virus Infection Virion Purification and Framentioning

confidence: 99%

Comparative Proteomics Reveal Fundamental Structural and Functional Differences between the Two Progeny Phenotypes of a Baculovirus

Hou

Zhang

et al. 2013

J Virol

100

The replication of lepidopteran baculoviruses is characterized by the production of two progeny phenotypes: the occlusion-derived virus (ODV), which establishes infection in midgut cells, and the budded virus (BV), which disseminates infection to different tissues within a susceptible host. To understand the structural, and hence functional, differences between BV and ODV, we employed multiple proteomic methods to reveal the protein compositions and posttranslational modifications of the two phenotypes of Helicoverpa armigera nucleopolyhedrovirus. In addition, Western blotting and quantitative mass spectrometry were used to identify the localization of proteins in the envelope or nucleocapsid fractions. Comparative protein portfolios of BV and ODV showing the distribution of 54 proteins, encompassing the 21 proteins shared by BV and ODV, the 12 BV-specific proteins, and the 21 ODV-specific proteins, were obtained. Among the 11 ODV-specific envelope proteins, 8 either are essential for or contribute to oral infection. Twenty-three phosphorylated and 6 N-glycosylated viral proteins were also identified. While the proteins that are shared by the two phenotypes appear to be important for nucleocapsid assembly and trafficking, the structural and functional differences between the two phenotypes are evidently characterized by the envelope proteins and posttranslational modifications. This comparative proteomics study provides new insight into how BV and ODV are formed and why they function differently. Baculoviruses are insect-specific pathogens containing large circular double-stranded DNA genomes. Over millions of years of interdependence between viruses and their natural insect hosts, both have undergone a coevolution, such that lepidopteran baculoviruses have developed a unique biphasic replication cycle that generates two progeny phenotypes, the budded virus (BV) and the occlusion-derived virus (ODV). ODVs are embedded in occlusion bodies (OBs) that offer the virions a certain amount of protection against environmental degradation. Once ingested by a susceptible insect, ODVs are released from OBs within the larval midgut and initiate oral infection. After infecting midgut epithelial cells, BVs are synthesized and released to disseminate systemic infection of different tissues within the larval host. The two phenotypes have been used in a wide range of applications. Due to their expandable genome and the presence of very strong promoters, BVs have been established as successful vectors for the expression of thousands of proteins and have also been studied as potential vectors for gene therapy (1). The OBs of certain baculoviruses have been widely used in agriculture and forestry as viable alternatives to chemical insecticides in insect pest control (2).The broad applications of baculoviruses provide a strong rationale for identifying the proteins associated with both phenotypes and for understanding their roles in baculovirus infection. While previous proteomic studies have elucidated the protein compositions of a...