scholarly journals Autographa californica multiple nucleopolyhedrovirus core gene ac92 (p33) is required for efficient budded virus production

Virology ◽  
2011 ◽  
Vol 409 (1) ◽  
pp. 38-45 ◽  
Author(s):  
Yingchao Nie ◽  
Minggang Fang ◽  
David A. Theilmann
Keyword(s):  
Virus Production ◽  
Core Gene ◽  
Autographa Californica ◽  
Multiple Nucleopolyhedrovirus ◽  
Budded Virus ◽  
Autographa Californica Multiple Nucleopolyhedrovirus

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

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.

scholarly journals Nuclear localization of actin requires AC102 in Autographa californica multiple nucleopolyhedrovirus-infected cells

2012 ◽  
Vol 93 (8) ◽  
pp. 1795-1803 ◽  
Author(s):  
Kamal M. Gandhi ◽  
Taro Ohkawa ◽  
Matthew D. Welch ◽  
Loy E. Volkman
Keyword(s):  
Virus Production ◽  
Autographa Californica ◽  
Current Evidence ◽  
Progeny Virus ◽  
Wild Type ◽  
Multiple Nucleopolyhedrovirus ◽  
Infected Cells ◽  
Budded Virus ◽  
Autographa Californica Multiple Nucleopolyhedrovirus ◽  
Type Infection

Autographa californica multiple nucleopolyhedrovirus requires nuclear actin for progeny virus production and thereby encodes viral products that ensure actin’s translocation to and retention within the nucleus. Current evidence suggests that the ie0–ie1 gene complex along with five nuclear localization of actin (NLA) genes are sufficient for NLA in transient transfection experiments. Here we report that, during infection, only one of the five NLA genes, Ac102, was essential for NLA, and that AC102 had at least one other activity critical for budded virus (BV) production. Viral deletion mutants in the other four NLA genes were viable, with only two having replication phenotypes different from that of the wild type. Infection with AcΔpe38 revealed a delay in both BV production and NLA. Infection with AcΔ152 revealed a delay in BV production, but no corresponding delay in NLA. Infection with either AcΔpe38 or AcΔ152 resulted in slightly reduced BV titres. Deletion of Ac004 or he65 had no impact on actin translocation kinetics, timing of BV production or BV titres. These results implicate AC102 as a key player in baculovirus manipulation of actin.

scholarly journals No single homologous repeat region is essential for DNA replication of the baculovirus Autographa californica multiple nucleopolyhedrovirus

2007 ◽  
Vol 88 (1) ◽  
pp. 114-122 ◽  
Author(s):  
Eric B. Carstens ◽  
Yuntao Wu
Keyword(s):  
Dna Replication ◽  
Virus Production ◽  
Autographa Californica ◽  
Viral Dna ◽  
Multiple Nucleopolyhedrovirus ◽  
Single Region ◽  
Budded Virus ◽  
Virus Expression ◽  
Autographa Californica Multiple Nucleopolyhedrovirus

The presence of homologous repeat (hr) regions in multiple locations within baculovirus genomes has led to the hypothesis that they represent origins of DNA replication. This hypothesis has been supported by transient replication assays where plasmids carrying hrs replicated in the presence of virus DNA replication. This study investigated whether any specific hr region was essential for viral DNA replication in vivo, by generating a series of recombinant Autographa californica multiple nucleopolyhedrovirus where the lacZ gene replaced hr1, hr1a, hr2, hr3, hr4a or hr4b. In addition, a double-hr knockout virus was constructed where both hr2 and hr3 were deleted. The successful construction of these knockout viruses indicated that no specific region was essential for virus production. These recombinant viruses were characterized by titrations of budded virus, expression of a variety of virus-specific proteins and the synthesis of viral DNA at various times after infection. The results demonstrated that each hr was dispensable for all of these properties and that no single region was absolutely essential for virus replication in cell culture. The functional significance of multiple origin regions is still unclear.

scholarly journals Autographa californica Multiple Nucleopolyhedrovirus Core Gene ac96 Encodes a Per Os Infectivity Factor (pif-4)

2009 ◽  
Vol 83 (23) ◽  
pp. 12569-12578 ◽  
Author(s):  
Minggang Fang ◽  
Yingchao Nie ◽  
Stephanie Harris ◽  
Martin A. Erlandson ◽  
David A. Theilmann
Keyword(s):  
Trichoplusia Ni ◽  
Core Gene ◽  
Autographa Californica ◽  
Immunofluorescence Analysis ◽  
Viral Dna Replication ◽  
Multiple Nucleopolyhedrovirus ◽  
Confocal Immunofluorescence ◽  
Budded Virus ◽  
And Control ◽  
Autographa Californica Multiple Nucleopolyhedrovirus

ABSTRACT Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac96 is a core gene, but its role in virus replication is still unknown. To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac96-null virus (vAc 96 null). Our analyses showed that the absence of ac96 does not affect budded virus (BV) production or viral DNA replication in infected Sf9 cells. Western blotting and confocal immunofluorescence analysis showed that AC96 is expressed in both the cytoplasm and the nucleus throughout infection. In addition, AC96 was detected in the envelope fractions of both BV and occlusion-derived virus. Injection of vAc 96 null BV into the hemocoel killed Trichoplusia ni larvae as efficiently as repaired and control viruses; however, vAc 96 null was unable to infect the midgut tissue of Trichoplusia ni larvae when inoculated per os. Therefore, the results of this study show that ac96 encodes a new per os infectivity factor (PIF-4).

scholarly journals Effects of Ac150 on virulence and pathogenesis of Autographa californica multiple nucleopolyhedrovirus in noctuid hosts

2005 ◽  
Vol 86 (6) ◽  
pp. 1619-1627 ◽  
Author(s):  
Ji-Hong Zhang ◽  
Taro Ohkawa ◽  
Jan O. Washburn ◽  
Loy E. Volkman
Keyword(s):  
Trichoplusia Ni ◽  
Autographa Californica ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Lepidopteran Insect ◽  
Charged Amino Acids ◽  
Multiple Nucleopolyhedrovirus ◽  
Budded Virus ◽  
Autographa Californica Multiple Nucleopolyhedrovirus

Ac150 is expressed late during infection of cultured lepidopteran insect cells by Autographa californica multiple nucleopolyhedrovirus. The Ac150 gene product is predicted to have a molecular mass of 11 161 Da and consists of a hydrophobic N terminus and a single ‘peritrophin-A’-like domain, connected by a short region of charged amino acids. An Ac150 deletion mutant and its parental wild-type virus were compared for differences in virulence by both oral and intrahaemocoelic routes of infection. It was found that the mutant was significantly less virulent in larvae of all three host species tested (Heliothis virescens, Spodoptera exigua and Trichoplusia ni) when occlusions were administered orally, but not when isolated occlusion-derived virus (ODV) was administered orally or budded virus was administered intrahaemocoelically. ODV yields were the same from equal numbers of mutant and wild-type occlusions, and nucleocapsid-distribution frequencies within the two ODV populations were the same, eliminating these features as explanations for the observed differences in virulence. Comparison of pathogenesis, as revealed by lacZ expression from identical reporter-gene cassettes in the mutant and wild-type virus, indicated that the mutant was less efficient at establishing primary infection in midgut cells; otherwise, it exhibited infection kinetics identical to those of wild-type virus. Ac150, therefore, can be considered a per os infection factor that mediates, but is not essential for, oral infection.

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

2009 ◽  
Vol 83 (15) ◽  
pp. 7440-7448 ◽  
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.

scholarly journals Autographa californica Multiple Nucleopolyhedrovirus AC83 is a Per Os Infectivity Factor (PIF) Protein Required for Occlusion-Derived Virus (ODV) and Budded Virus Nucleocapsid Assembly as well as Assembly of the PIF Complex in ODV Envelopes

2016 ◽  
Vol 91 (5) ◽  
Author(s):  
Muhammad Afzal Javed ◽  
Siddhartha Biswas ◽  
Leslie G. Willis ◽  
Stephanie Harris ◽  
Caitlin Pritchard ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Core Gene ◽  
Infection Process ◽  
Binding Domain ◽  
Autographa Californica ◽  
Zinc Finger Domain ◽  
Chitin Binding Domain ◽  
New Model ◽  
Multiple Nucleopolyhedrovirus ◽  
Autographa Californica Multiple Nucleopolyhedrovirus

ABSTRACT Baculovirus occlusion-derived virus (ODV) initiates infection of lepidopteran larval hosts by binding to the midgut epithelia, which is mediated by per os infectivity factors (PIFs). Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes seven PIF proteins, of which PIF1 to PIF4 form a core complex in ODV envelopes to which PIF0 and PIF6 loosely associate. Deletion of any pif gene results in ODV being unable to bind or enter midgut cells. AC83 also associates with the PIF complex, and this study further analyzed its role in oral infectivity to determine if it is a PIF protein. It had been proposed that AC83 possesses a chitin binding domain that enables transit through the peritrophic matrix; however, no chitin binding activity has ever been demonstrated. AC83 has been reported to be found only in the ODV envelopes, but in contrast, the Orgyia pseudotsugata MNPV AC83 homolog is associated with both ODV nucleocapsids and envelopes. In addition, unlike known pif genes, deletion of ac83 eliminates nucleocapsid formation. We propose a new model for AC83 function and show AC83 is associated with both ODV nucleocapsids and envelopes. We also further define the domain required for nucleocapsid assembly. The cysteine-rich region of AC83 is also shown not to be a chitin binding domain but a zinc finger domain required for the recruitment or assembly of the PIF complex to ODV envelopes. As such, AC83 has all the properties of a PIF protein and should be considered PIF8. In addition, pif7 (ac110) is reported as the 38th baculovirus core gene. IMPORTANCE ODV is essential for the per os infectivity of the baculovirus AcMNPV. To initiate infection, ODV binds to microvilli of lepidopteran midgut cells, a process which requires a group of seven virion envelope proteins called PIFs. In this study, we reexamined the function of AC83, a protein that copurifies with the ODV PIFs, to determine its role in the oral infection process. A zinc finger domain was identified and a new model for AC83 function was proposed. In contrast to previous studies, AC83 was found to be physically located in both the envelope and nucleocapsid of ODV. By deletion analysis, the AC83 domain required for nucleocapsid assembly was more finely delineated. We show that AC83 is required for PIF complex formation and conclude that it is a true per os infectivity factor and should be called PIF8.

scholarly journals 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

2012 ◽  
Vol 93 (2) ◽  
pp. 364-373 ◽  
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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