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

2010 ◽  
Vol 84 (12) ◽  
pp. 6153-6162 ◽  
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.

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 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.

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