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 Autographa californica Multiple Nucleopolyhedrovirus orf13 is Required for Efficient Nuclear Egress of Nucleocapsids

2020 ◽  
Author(s):  
Xingang Chen ◽  
Xiaoqin Yang ◽  
Chengfeng Lei ◽  
Fujun Qin ◽  
Jia Hu ◽  
...  
Keyword(s):  
Autographa Californica ◽  
Wild Type Virus ◽  
Nuclear Egress ◽  
Multiple Nucleopolyhedrovirus ◽  
Localization Signal ◽  
Replication Gene ◽  
Infected Cells ◽  
Nuclear Localization Signal Motif ◽  
Conserved Gene ◽  
Autographa Californica Multiple Nucleopolyhedrovirus

ABSTRACTAutographa californica multiple nucleopolyhedrovirus (AcMNPV) orf13 (ac13) is a conserved gene in all sequenced alphabaculoviruses. However, its function in the viral life cycle remains unknown. In this study we found that ac13 was a late gene and that the encoded protein, bearing a putative nuclear localization signal motif in the DUF3627 domain, colocalized with the nuclear membrane. Deletion of ac13 did not affect viral DNA replication, gene transcription, nucleocapsid assembly or occlusion body (OB) formation, but reduced virion budding from infected cells by approximately 400-fold compared with the wild-type virus. Deletion of ac13 substantially impaired the egress of nucleocapsids from the nucleus to the cytoplasm, while the number of occlusion-derived viruses embedded within OBs was unaffected. Taken together, our results indicated that ac13 was required for efficient nuclear egress of nucleocapsids during virion budding, but was dispensable for OB formation.IMPORTANCEEgress of baculovirus nucleocapsids from the nucleus is an essential process for morphogenesis of mature budded viruses, which is required to spread infection within susceptible cells and tissues. Although many viral and host proteins are required for nucleocapsid egress, the specific mechanisms underlying this process in baculoviruses remain somewhat enigmatic. In the present study, we found that the ac13 gene, in addition to ac11, ac51, ac66, ac75, ac78, gp41, ac93, p48, exon0 and ac142, was required for efficient nuclear egress of nucleocapsids. Our results contribute to a better understanding of nucleocapsid egress in baculoviruses.

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 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 Trichoplusia ni Kinesin-1 Associates with Autographa californica Multiple Nucleopolyhedrovirus Nucleocapsid Proteins and Is Required for Production of Budded Virus

2016 ◽  
Vol 90 (7) ◽  
pp. 3480-3495 ◽  
Author(s):  
Siddhartha Biswas ◽  
Gary W. Blissard ◽  
David A. Theilmann
Keyword(s):  
Plasma Membrane ◽  
Trichoplusia Ni ◽  
Autographa Californica ◽  
Nucleocapsid Proteins ◽  
Content Type ◽  
Multiple Nucleopolyhedrovirus ◽  
Microtubule Transport ◽  
Infected Cells ◽  
Budded Virus

ABSTRACTThe mechanism by which nucleocapsids ofAutographa californicamultiple nucleopolyhedrovirus (AcMNPV) egress from the nucleus to the plasma membrane, leading to the formation of budded virus (BV), is not known. AC141 is a nucleocapsid-associated protein required for BV egress and has previously been shown to be associated with β-tubulin. In addition, AC141 and VP39 were previously shown by fluorescence resonance energy transfer by fluorescence lifetime imaging to interact directly with theDrosophila melanogasterkinesin-1 light chain (KLC) tetratricopeptide repeat (TPR) domain. These results suggested that microtubule transport systems may be involved in baculovirus nucleocapsid egress and BV formation. In this study, we investigated the role of lepidopteran microtubule transport using coimmunoprecipitation, colocalization, yeast two-hybrid, and small interfering RNA (siRNA) analyses. We show that nucleocapsid AC141 associates with the lepidopteranTrichoplusia niKLC and kinesin-1 heavy chain (KHC) by coimmunoprecipitation and colocalization. Kinesin-1, AC141, and microtubules colocalized predominantly at the plasma membrane. In addition, the nucleocapsid proteins VP39, FP25, and BV/ODV-C42 were also coimmunoprecipitated withT. niKLC. Direct analysis of the role ofT. nikinesin-1 by downregulation of KLC by siRNA resulted in a significant decrease in BV production. Nucleocapsids labeled with VP39 fused with three copies of the mCherry fluorescent protein also colocalized with microtubules. Yeast two-hybrid analysis showed no evidence of a direct interaction between kinesin-1 and AC141 or VP39, suggesting that either other nucleocapsid proteins or adaptor proteins may be required. These results further support the conclusion that microtubule transport is required for AcMNPV BV formation.IMPORTANCEIn two key processes of the replication cycle of the baculovirusAutographa californicamultiple nucleopolyhedrovirus (AcMNPV), nucleocapsids are transported through the cell. These include (i) entry of budded virus (BV) into the host cell and (ii) egress and budding of nucleocapsids newly produced from the plasma membrane. Prior studies have shown that the entry of nucleocapsids involves the polymerization of actin to propel nucleocapsids to nuclear pores and entry into the nucleus. For the spread of infection, progeny viruses must rapidly exit the infected cells, but the mechanism by which AcMNPV nucleocapsids traverse the cytoplasm is unknown. In this study, we examined whether nucleocapsids interact with lepidopteran kinesin-1 motor molecules and are potentially carried as cargo on microtubules to the plasma membrane in AcMNPV-infected cells. This study indicates that microtubule transport is utilized for the production of budded virus.

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