Autographa californica M nucleopolyhedrovirus open reading frame 109 affects infectious budded virus production and nucleocapsid envelopment in the nucleus of cells

Open reading frame 132 (Ha132) of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) is a homologue of per os infectivity factor 2 (pif-2) of Spodoptera exigua multiple nucleopolyhedrovirus. Sequence analysis indicated that Ha132 encoded a protein of 383 aa with a predicted molecular mass of 44.5 kDa. Alignment of HA132 and its baculovirus homologues revealed that HA132 was highly conserved among baculoviruses, with 14 absolutely conserved cysteine residues. RT-PCR indicated that Ha132 was first transcribed at 24 h post-infection. Western blot analysis showed that a 43 kDa band was detectable in HearNPV-infected HzAM1 cells from 36 h post-infection. Western blots also indicated that HA132 was a component of the occlusion-derived virus, but not of budded virus. Deletion of Ha132 from HearNPV abolished per os infectivity, but had no effect on the infectivity of the budded virus phenotype.

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Identification of a Domain of the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus Single-Strand DNA-Binding Protein LEF-3 Essential for Viral DNA Replication

Journal of Virology ◽

10.1128/jvi.00115-10 ◽

2010 ◽

Vol 84 (12) ◽

pp. 6153-6162 ◽

Cited By ~ 13

Author(s):

Mei Yu ◽

Eric B. Carstens

Keyword(s):

Gene Expression ◽

Dna Replication ◽

Viral Genome ◽

Virus Production ◽

Autographa Californica ◽

Late Gene ◽

Late Gene Expression ◽

Viral Dna ◽

Viral Dna Replication ◽

Budded Virus

ABSTRACT Autographa californica multiple nucleopolyhedrovirus (AcMNPV) lef-3 is one of nine genes required for viral DNA replication in transient assays. LEF-3 is predicted to contain several domains related to its functions, including nuclear localization, single-strand DNA binding, oligomerization, interaction with P143 helicase, and interaction with a viral alkaline nuclease. To investigate the essential nature of LEF-3 and the roles it may play during baculovirus DNA replication, a lef-3 null bacmid (bKO-lef3) was constructed in Escherichia coli and characterized in Sf21 cells. The results showed that AcMNPV lef-3 is essential for DNA replication, budded virus production, and late gene expression in vivo. Cells transfected with the lef-3 knockout bacmid produced low levels of early proteins (P143, DNA polymerase, and early GP64) and no late proteins (P47, VP39, or late GP64). To investigate the functional role of domains within the LEF-3 open reading frame in the presence of the whole viral genome, plasmids expressing various LEF-3 truncations were transfected into Sf21 cells together with bKO-lef3 DNA. The results showed that expression of AcMNPV LEF-3 amino acids 1 to 125 was sufficient to stimulate viral DNA replication and to support late gene expression. Expression of Choristoneura fumiferana MNPV lef-3 did not rescue any LEF-3 functions. The construction of a LEF-3 amino acid 1 to 125 rescue bacmid revealed that this region of LEF-3, when expressed in the presence of the rest of the viral genome, stimulated viral DNA replication and late and very late protein expression, as well as budded virus production.

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Autographa californica Multiple Nucleopolyhedrovirus me53 (ac140) Is a Nonessential Gene Required for Efficient Budded-Virus Production

Journal of Virology ◽

10.1128/jvi.02390-08 ◽

2009 ◽

Vol 83 (15) ◽

pp. 7440-7448 ◽

Cited By ~ 19

Author(s):

Jondavid de Jong ◽

Basil M. Arif ◽

David A. Theilmann ◽

Peter J. Krell

Keyword(s):

Transcriptional Start Site ◽

Virus Production ◽

Autographa Californica ◽

Cell Fractionation ◽

Late Promoter ◽

Log Reduction ◽

Baculovirus Gene ◽

Fold Reduction ◽

Budded Virus ◽

Autographa Californica Multiple Nucleopolyhedrovirus

ABSTRACT me53 is a highly conserved baculovirus gene found in all lepidopteran baculoviruses that have been fully sequenced to date. The putative ME53 protein contains a zinc finger domain and has been previously described as a major early transcript. We generated an me53-null bacmid (AcΔme53GFP), as well as a repair virus (AcRepME53:HA-GFP) carrying me53 with a C-terminal hemagglutinin (HA) tag, under the control of its native early and late promoter elements. Sf9 and BTI-Tn-5b1 cells transfected with AcΔme53GFP resulted in a 3-log reduction in budded-virus (BV) production compared to both the parental Autographa californica multiple nucleopolyhedrosis virus and the repair bacmids, demonstrating that although me53 is not essential for replication, replication is compromised in its absence. Our data also suggest that me53 does not affect DNA replication. Cell fractionation showed that ME53 is found in both the nucleus and the cytoplasm as early as 6 h postinfection. Deletion of the early transcriptional start site resulted in a 10- to 360-fold reduction of BV yield; however, deletion of the late promoter (ATAAG) resulted in a 160- to 1,000-fold reduction, suggesting that, in the context of BV production, ME53 is required both early and late in the infection cycle. Additional Western blot analysis of purified virions from the repair virus revealed that ME53:HA is associated with both BV and occlusion-derived virions. Together, these results indicate that me53, although not essential for viral replication, is required for efficient BV production.

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Functional analysis of a 603 nucleotide open reading frame upstream of the polyhedrin gene of Autographa californica nuclear polyhedrosis virus

Journal of General Virology ◽

10.1099/0022-1317-71-2-251 ◽

1990 ◽

Vol 71 (2) ◽

pp. 251-262 ◽

Cited By ~ 14

Author(s):

K. L. Gearing ◽

R. D. Possee

Keyword(s):

Functional Analysis ◽

Nuclear Polyhedrosis Virus ◽

Autographa Californica ◽

Open Reading Frame ◽

Polyhedrin Gene ◽

Reading Frame ◽

Polyhedrosis Virus ◽

Nuclear Polyhedrosis

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The Herpesvirus Saimiri Open Reading Frame (ORF) 50 (Rta) Protein Contains an AT Hook Required for Binding to the ORF 50 Response Element in Delayed-Early Promoters

Journal of Virology ◽

10.1128/jvi.78.9.4936-4942.2004 ◽

2004 ◽

Vol 78 (9) ◽

pp. 4936-4942 ◽

Cited By ~ 9

Author(s):

Matthew S. Walters ◽

Kersten T. Hall ◽

Adrian Whitehouse

Keyword(s):

Dna Binding ◽

Virus Production ◽

Open Reading Frame ◽

Site Directed Mutagenesis ◽

Herpesvirus Saimiri ◽

Binding Motif ◽

Core Element ◽

Response Element ◽

Reading Frame ◽

Glycine Residue

ABSTRACT The herpesvirus saimiri open reading frame (ORF) 50 encodes two proteins, which activate transcription directly, following interactions with delayed-early (DE) promoters containing a specific motif. In this report, we demonstrate that ORF 50 contains a DNA binding domain that has homology to an AT hook DNA binding motif. Deletion analysis of this domain reduces ORF 50-mediated transactivation of the DE ORF 6 and ORF 57 promoters by 100 and 90%, respectively. Furthermore, gel retardation experiments demonstrated that the AT hook motif is required for binding the ORF 50 response element in the promoters of DE genes. Single site-directed mutagenesis of the AT hook revealed that mutation of the glycine residue at position 408 to an alanine reduces ORF 50 transactivation of the ORF 57 promoter by 40%. Moreover, the mutation of multiple basic residues in conjunction with the glycine residue within the core element of the AT hook abolishes ORF 50-mediated transactivation. In addition, p50GFPΔAT-hook is capable of functioning as a trans-dominant mutant, leading to a reduction in virus production of approximately 50% compared to that for wild-type ORF 50.

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Characterization of an Autographa californica multiple nucleopolyhedrovirus dual mutant: ORF82 is required for budded virus production, and a point mutation in LEF-8 alters late and abolishes very late transcription

Journal of General Virology ◽

10.1099/vir.0.037028-0 ◽

2012 ◽

Vol 93 (2) ◽

pp. 364-373 ◽

Cited By ~ 1

Author(s):

David Gauthier ◽

Kannan Thirunavukkarasu ◽

Brian L. Faris ◽

Darcy L. Russell ◽

Robert F. Weaver

Keyword(s):

Virus Production ◽

Autographa Californica ◽

Host Protein ◽

Temperature Sensitive ◽

Multiple Nucleopolyhedrovirus ◽

Plaque Phenotype ◽

Late Transcription ◽

Conserved Region ◽

Budded Virus

A temperature-sensitive (ts) Autographa californica multiple nucleopolyhedrovirus dual mutant, ts42, was generated that displayed tiny-plaque and polyhedral inclusion body (PIB)-defective phenotypes at 33 °C. The mutation responsible for the tiny-plaque phenotype was mapped to orf82, which was characterized as a late gene. Its product was not studied. The mutation responsible for the PIB-defective phenotype was mapped to a highly conserved region of lef-8, which encodes the largest subunit of the viral RNA polymerase. These mutations did not cause a global defect in viral DNA replication or a defect in the shutoff of host protein synthesis. However, the mutation in orf82 caused a dramatic defect in the production of progeny budded virus (BV) but did not decrease the infectivity of those BVs that were released. Hence, ORF82 is required for BV production. The mutation in lef-8 affected a conserved residue that is part of a highly conserved region of LEF-8. This mutation abolished very late transcription whilst altering the transcript size and level of transcription of two late genes.

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A Conserved Phenylalanine Residue of Autographa Californica Multiple Nucleopolyhedrovirus AC75 Protein Is Required for Occlusion Body Formation

Frontiers in Microbiology ◽

10.3389/fmicb.2021.663506 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xingang Chen ◽

Jian Yang ◽

Xiaoqin Yang ◽

Chengfeng Lei ◽

Xiulian Sun ◽

...

Keyword(s):

Virus Production ◽

Autographa Californica ◽

Occlusion Body ◽

Body Formation ◽

Viral Propagation ◽

Multiple Nucleopolyhedrovirus ◽

Infected Cells ◽

Budded Virus ◽

Conserved Gene ◽

Autographa Californica Multiple Nucleopolyhedrovirus

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf75 (ac75) is a highly conserved gene that is essential for AcMNPV propagation. However, the key domains or residues of the AC75 protein that play a role in viral propagation have not been identified. In this study, sequence alignment revealed that residues Phe-54 and Gln-81 of AC75 were highly conserved among alphabaculoviruses and betabaculoviurses. Thus, Phe-54 and Gln-81 AC75 mutation bacmids were constructed. We found that Gln-81 was not required for viral propagation, whereas mutating Phe-54 reduced budded virus production by 10-fold and impaired occlusion body formation when compared with that of the wild-type AcMNPV. Electron microscopy observations showed that the Phe-54 mutation affected polyhedrin assembly and also occlusion-derived virus embedding, whereas western blot analysis revealed that mutating Phe-54 reduced the amount of AC75 but did not affect the localization of AC75 in infected cells. A protein stability assay showed that the Phe-54 mutation affected AC75 stability. Taken together, Phe-54 was identified as an important residue of AC75, and ac75 is a pivotal gene in budding virus production and occlusion body formation.

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The Immediate-Early Protein IE0 of the Autographa californica Nucleopolyhedrovirus Is Not Essential for Viral Replication

Journal of Virology ◽

10.1128/jvi.79.15.10077-10082.2005 ◽

2005 ◽

Vol 79 (15) ◽

pp. 10077-10082 ◽

Cited By ~ 4

Author(s):

Lu Liqun ◽

Hadassah Rivkin ◽

Nor Chejanovsky

Keyword(s):

Virus Production ◽

Autographa Californica ◽

Targeted Mutagenesis ◽

Immediate Early ◽

Published Data ◽

Sf9 Cells ◽

Early Protein ◽

Autographa Californica Nucleopolyhedrovirus ◽

Budded Virus

ABSTRACT The role of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) immediate-early protein IE0 in the baculoviral infection is not clear. In this study, we constructed the recombinant virus vAcΔie0 null for ie0 expression by targeted mutagenesis replacing exon0 with the cat gene. We found that vAcΔie0 replicated efficiently in Spodoptera littoralis SL2 cells, which are poorly permissive for AcMNPV. In contrast, in Spodoptera frugiperda SF9 cells, which are fully permissive for AcMNPV, vAcΔie0 DNA replication and budded virus production were delayed. These results and recently published data (X. Dai et al., J. Virol. 78:9633-9644, 2004) indicate that ie0 is not essential for AcMNPV replication but enhances it in permissive SF9 cells.

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