Dual mutations in the Autographa californica nucleopolyhedrovirus FP-25 and p35 genes result in plasma-membrane blebbing in Trichoplusia ni cells

Spodoptera frugiperda cells infected with Autographa californica nucleopolyhedrovirus (AcMNPV) lacking a functional anti-apoptotic p35 protein undergo apoptosis. However, such mutants replicate normally in Trichoplusia ni (TN-368) cells. An AcMNPV plaque isolate (AcdefrT) was identified during propagation of a virus deficient in p35 in TN-368 cells. This virus exhibited enhanced budded-particle formation in TN-368 cells, but was partially defective for polyhedra production in the same cells. Virus replication in AcdefrT-infected TN-368 cells was accompanied by extensive plasma-membrane blebbing and caspase activation late in infection, both features of apoptosis. Rescue of the p35 locus of AcdefrT continued to result in a reduction in polyhedra and increase in budded virus production in TN-368 cells, but no plasma-membrane blebbing was observed. The mutation was mapped to the FP-25 gene locus. This gene mutation combined with the non-functional p35 was found to be responsible for the cell-blebbing effect observed in AcdefrT-infected TN-368 cells.

Download Full-text

Characterization of Two Autographa californica Nucleopolyhedrovirus Proteins, Ac145 and Ac150, Which Affect Oral Infectivity in a Host-Dependent Manner

Journal of Virology ◽

10.1128/jvi.78.12.6439-6448.2004 ◽

2004 ◽

Vol 78 (12) ◽

pp. 6439-6448 ◽

Cited By ~ 36

Author(s):

Renee Lapointe ◽

Holly J. R. Popham ◽

Ursula Straschil ◽

David Goulding ◽

David R. O'Reilly ◽

...

Keyword(s):

Trichoplusia Ni ◽

Polyclonal Antibodies ◽

Heliothis Armigera ◽

Autographa Californica ◽

Dependent Manner ◽

Significant Drop ◽

Double Deletion ◽

Autographa Californica Nucleopolyhedrovirus ◽

Budded Virus ◽

Nuclear Polyhedrosis

ABSTRACT The genome of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) contains two homologues, orf145 and orf150, of the Heliothis armigera Entomopoxvirus (HaEPV) 11,000-kDa gene. Polyclonal antibodies raised against the Ac145 or Ac150 protein were utilized to demonstrate that they are expressed from late to very late times of infection and are within the nuclei of infected Sf-21 cells. Transmission electron microscopy coupled with immunogold labeling of Ac145 found this protein within the nucleus in areas of nucleocapsid assembly and maturation, along with some association with the enveloped bundles of virions within the developing occlusion bodies (OBs). Ac150 was found to be mainly associated with enveloped bundles of virions within OBs and also with those not yet occluded. Both Ac145 and Ac150 were found to be present in budded virus as well as OBs. Both orf145 and orf150 were deleted from the AcMNPV genome, singly or together, and these deletion mutants were assessed for oral infectivity both in Trichoplusia ni and Heliothis virescens larvae. Deletion of Ac145 led to a small but significant drop in infectivity (sixfold) compared to wild-type (wt) AcMNPV for T. ni but not for H. virescens. Deletion of Ac150 alone had no effect on infectivity of the virus for either host. However, deletion of both Ac145 and Ac150 gave a recombinant virus with a drastic (39-fold) reduction in infectivity compared to wt virus for H. virescens. Intrahemocoelic injection of budded virus from the double-deletion virus into H. virescens larvae is as infectious to this host as wt budded virus, indicating that Ac145 and Ac150 play a role in primary oral infection of AcMNPV, the extent of which is host dependent.

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.02912-15 ◽

2016 ◽

Vol 90 (7) ◽

pp. 3480-3495 ◽

Cited By ~ 14

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.

Download Full-text

Characterization of AcMNPV with a deletion of ac69 gene

Microbiology Research ◽

10.4081/mr.2011.e4 ◽

2011 ◽

Vol 2 (1) ◽

pp. 4

Author(s):

Jianhao Ke ◽

Jinwen Wang ◽

Riqiang Deng ◽

Lin Lin ◽

Bei Jinlong ◽

...

Keyword(s):

Spodoptera Frugiperda ◽

Trichoplusia Ni ◽

Virus Production ◽

Viral Infectivity ◽

Methyltransferase Activity ◽

Budded Virus

ORF69 (Ac69) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is conserved in some baculovirus genomes. Although it has been shown that Ac69 has cap 0-dependent methyltransferase activity and is not required for budded virus production in Spodoptera frugiperda Sf-9 cells, its role in occlusion-derived virus synthesis and virus oral infectivity is not known. This paper describes generation of an ac69 knockout AcMNPV bacmid mutant and analyses of the influence of ac69 deletion on the viral infectivity in Sf-9 cells and Trichoplusia ni larvae so as to investigate the role of ac69 in the viral life cycle. Results indicated that ac69 deletion has little effect on the production rates and morphogenesis of budded virus and occlusion-derived virus in Sf-9 cells. In addition, animal experiment revealed that the deletion mutant did not affect AcMNPV infectivity for Trichoplusia ni larvae in LD50 and LT50 bioassay when administered orally. These results suggest that ac69 may be dispensable for viral infectivity both in vitro and in vivo.

Download Full-text

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.

Download Full-text

Autographa californica Nucleopolyhedrovirus AC141 (Exon0), a Potential E3 Ubiquitin Ligase, Interacts with Viral Ubiquitin and AC66 To Facilitate Nucleocapsid Egress

Journal of Virology ◽

10.1128/jvi.01713-17 ◽

2017 ◽

Vol 92 (3) ◽

Cited By ~ 4

Author(s):

Siddhartha Biswas ◽

Leslie G. Willis ◽

Minggang Fang ◽

Yingchao Nie ◽

David A. Theilmann

Keyword(s):

Ubiquitin Ligase ◽

Nuclear Export ◽

Molecular Mechanisms ◽

Nuclear Periphery ◽

E3 Ubiquitin Ligase ◽

Autographa Californica ◽

Nucleocapsid Proteins ◽

Systemic Spread ◽

Autographa Californica Nucleopolyhedrovirus ◽

Budded Virus

ABSTRACTDuring the infection cycle of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), two forms of virions are produced, budded virus (BV) and occlusion-derived virus (ODV). Nucleocapsids that form BV have to egress from the nucleus, whereas nucleocapsids that form ODV remain inside the nucleus. The molecular mechanism that determines whether nucleocapsids remain inside or egress from the nucleus is unknown. AC141 (a predicted E3 ubiquitin ligase) and viral ubiquitin (vUbi) have both been shown to be required for efficient BV production. In this study, it was hypothesized that vUbi interacts with AC141, and in addition, that this interaction was required for BV production. Deletion of bothac141andvubirestricted viral infection to a single cell, and BV production was completely eliminated. AC141 was ubiquitinated by either vUbi or cellular Ubi, and this interaction was required for optimal BV production. Nucleocapsids in BV, but not ODV, were shown to be specifically ubiquitinated by vUbi, including a 100-kDa protein, as well as high-molecular-weight conjugates. The viral ubiquitinated 100-kDa BV-specific nucleocapsid protein was identified as AC66, which is known to be required for BV production and was shown by coimmunoprecipitation and mass spectrometry to interact with AC141. Confocal microscopy also showed that AC141, AC66, and vUbi interact at the nuclear periphery. These results suggest that ubiquitination of nucleocapsid proteins by vUbi functions as a signal to determine if a nucleocapsid will egress from the nucleus and form BV or remain in the nucleus to form ODV.IMPORTANCEBaculoviruses produce two types of virions called occlusion-derived virus (ODV) and budded virus (BV). ODVs are required for oral infection, whereas BV enables the systemic spread of virus to all host tissues, which is critical for killing insects. One of the important steps for BV production is the export of nucleocapsids out of the nucleus. This study investigated the molecular mechanisms that enable the selection of nucleocapsids for nuclear export instead of being retained within the nucleus, where they would become ODV. Our data show that ubiquitination, a universal cellular process, specifically tags nucleocapsids of BV, but not those found in ODV, using a virus-encoded ubiquitin (vUbi). Therefore, ubiquitination may be the molecular signal that determines if a nucleocapsid is destined to form a BV, thus ensuring lethal infection of the host.

Download Full-text

Proteotoxic stress induced by Autographa californica nucleopolyhedrovirus infection of Spodoptera frugiperda Sf9 cells

Virology ◽

10.1016/j.virol.2012.10.018 ◽

2013 ◽

Vol 436 (1) ◽

pp. 49-58 ◽

Cited By ~ 11

Author(s):

Yulia V. Lyupina ◽

Svetlana B. Abaturova ◽

Pavel A. Erokhov ◽

Olga V. Orlova ◽

Svetlana N. Beljelarskaya ◽

...

Keyword(s):

Spodoptera Frugiperda ◽

Autographa Californica ◽

Sf9 Cells ◽

Proteotoxic Stress ◽

Autographa Californica Nucleopolyhedrovirus

Download Full-text

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

Journal of General Virology ◽

10.1099/vir.0.80930-0 ◽

2005 ◽

Vol 86 (6) ◽

pp. 1619-1627 ◽

Cited By ~ 24

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.

Download Full-text

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.

Download Full-text

A Few-Polyhedra Mutant and Wild-Type Nucleopolyhedrovirus Remain as a Stable Polymorphism during Serial Coinfection in Trichoplusia ni

Applied and Environmental Microbiology ◽

10.1128/aem.69.4.2052-2057.2003 ◽

2003 ◽

Vol 69 (4) ◽

pp. 2052-2057 ◽

Cited By ~ 23

Author(s):

James C. Bull ◽

H. C. J. Godfray ◽

David R. O'Reilly

Keyword(s):

Cell Culture ◽

Population Genetics ◽

Trichoplusia Ni ◽

Serial Passage ◽

Autographa Californica ◽

Wild Type ◽

Cell Infection ◽

Occlusion Bodies ◽

Budded Virus

ABSTRACT Few-polyhedra (FP) mutants of nucleopolyhedroviruses (NPVs) are a well-known phenomenon during serial passage of virus in cell culture. Under these circumstances such mutants produce low yields of occlusion bodies (OBs) and poorly occlude virions, but they are selected for through advantageous rates of budded virus replication. Spontaneous insertion of transposable elements originating from host cell DNA into the viral fp25 gene has been shown to be a common cause of the phenotype. A model of NPV population genetics predicts that mutants with these characteristics might persist within stable polymorphisms in viral populations during serial passage of virus in vivo. However, this hypothesis was previously untested, and FP mutants have not been recovered from field isolates of NPVs. We isolated and characterized an FP mutant that arose during routine passage of Autographa californica multinucleocapsid NPV (AcMNPV) in cell culture and identified a transposable element within the fp25 gene. We tracked the fates of coinfecting wild-type and FP mutant AcMNPV strains through serial passage in fifth-instar Trichoplusia ni larvae. The levels of both strains remained stable during successive rounds of infection. We applied the data obtained to a model of NPV population genetics in order to derive the frequency distribution of the multiplicity of cell infection in infected insects and estimated that 4.3 baculovirus genomes per OB-producing cell would account for this equilibrium.

Download Full-text