Dataset Information

Cloning and Characterization of Sf9 Cell Lamin and the Lamin Conformational Changes during Autographa californica multiple nucleopolyhedrovirus Infection.

ABSTRACT: At present, the details of lamina alterations after baculovirus infection remain elusive. In this study, a lamin gene in the Sf9 cell line of Spodoptera frugiperda was cloned. The open reading frame (orf) of the Sf9 lamin was 1860 bp and encoded a protein with a molecular weight of 70 kDa. A transfection assay with a red fluorescence protein (rfp)-lamin fusion protein indicated that Sf9 lamin was localized in the nuclear rim. Transmission electron microscopy observations indicated that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) nucleocapsids may pass through the nuclear envelope. Immunofluorescence assay indicated that the lamina showed a ruffled staining pattern with the formation of invaginations in the Sf9 cells infected with AcMNPV, while it was evenly distributed at the nuclear periphery of mock-infected cells. Western blotting results indicated that the total amount of lamin in the baculovirus-infected Sf9 cells was significantly decreased compared with the mock-infected cells. These results imply that AcMNPV infection induces structural and biochemical rearrangements of lamina of Sf9 cells.

SUBMITTER: Wei W

PROVIDER: S-EPMC4885081 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Cloning and Characterization of Sf9 Cell Lamin and the Lamin Conformational Changes during Autographa californica multiple nucleopolyhedrovirus Infection.

Wei Wenqiang W Wang Hongju H Li Xiaoya X Fang Na N Yang Shili S Liu Hongyan H Kang Xiaonan X Sun Xiulian X Ji Shaoping S

Viruses 20160507 5

At present, the details of lamina alterations after baculovirus infection remain elusive. In this study, a lamin gene in the Sf9 cell line of Spodoptera frugiperda was cloned. The open reading frame (orf) of the Sf9 lamin was 1860 bp and encoded a protein with a molecular weight of 70 kDa. A transfection assay with a red fluorescence protein (rfp)-lamin fusion protein indicated that Sf9 lamin was localized in the nuclear rim. Transmission electron microscopy observations indicated that Autograph ...[more]

PMID: 27164127

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Project description:Multiplication of the invertebrate DNA baculoviruses activates the host DNA damage response (DDR), which promotes virus DNA replication. DDR signaling is initiated by the host insect's phosphatidylinositol-3 kinase-related kinases (PIKKs), including ataxia telangiectasia-mutated kinase (ATM). Like other PIKKs, ATM phosphorylates an array of host DDR proteins at serine/threonine glutamine (S/TQ) motifs, the result of which leads to cell cycle arrest, DNA repair, or apoptosis. To define the role of host PIKKs in baculovirus replication, we compared replication levels of the baculovirus prototype species Autographa californica multiple nucleopolyhedrovirus in permissive Spodoptera frugiperda (SF21) cells with and without ATM function. Caffeine, which inhibits multiple DDR kinases, and the ATM-specific inhibitors KU-55933 and KU-60019 each prevented phosphorylation of Spodoptera histone H2AX (SfH2AX), a recognized indicator of ATM activity. However, only caffeine reduced autographa californica multiple nucleopolyhedrovirus (AcMNPV)-induced bulk phosphorylation of S/TQ protein motifs. Furthermore, only caffeine, not KU-55933 or KU-60019, reduced AcMNPV yields, suggesting a limited role for ATM. To investigate further, we identified and edited the Spodoptera ATM gene (sfatm). Consistent with ATM's known functions, CRISPR/Cas9-mediated knockout of sfatm eliminated DNA damage-induced phosphorylation of DDR marker SfH2AX in SF21 cells. However, loss of sfatm failed to affect the levels of AcMNPV multiplication. These findings suggested that in the absence of the kinase SfATM, another caffeine-sensitive host DDR kinase promotes S/TQ phosphorylation and baculovirus multiplication. Thus, baculoviruses activate and utilize the host insect DDR in an ATM-independent manner. IMPORTANCE The DDR, while necessary for the maintenance and fidelity of the host genome, represents an important cellular response to viral infection. The prolific DNA baculoviruses activate and manipulate the invertebrate DDR by using mechanisms that positively impact virus multiplication, including virus DNA replication. As the key DDR initiator kinase, ATM was suspected to play a critical role in this host response. However, we show here that baculovirus AcMNPV activates an ATM-independent DDR. By identifying the insect host ATM ortholog (Spodoptera frugiperda SfATM) and evaluating genetic knockouts, we show that SfATM is dispensable for AcMNPV activation of the DDR and for virus replication. Thus, another PIKK, possibly the closely related kinase ATR (ATM- and Rad3-related kinase), is responsible for efficient baculovirus multiplication. These findings better define the host pathways used by invertebrates to engage viral pathogens, including DNA viruses.

Project description:Baculoviruses encode a variety of auxiliary proteins that are not essential for viral replication but provide them with a selective advantage in nature. P10 is a 10-kDa auxiliary protein produced in the very late phase of gene transcription by Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The P10 protein forms cytoskeleton-like structures in the host cell that associate with microtubules varying from filamentous forms in the cytoplasm to aggregated perinuclear tubules that form a cage-like structure around the nucleus. These P10 structures may have a role in the release of occlusion bodies (OBs) and thus mediate the horizontal transmission of the virus between insect hosts. Here, using mass spectrometric analysis, it is demonstrated that the C terminus of P10 is phosphorylated during virus infection of cells in culture. Analysis of P10 mutants encoded by recombinant baculoviruses in which putative phosphorylation residues were mutated to alanine showed that serine 93 is a site of phosphorylation. Confocal microscopy examination of the serine 93 mutant structures revealed aberrant formation of the perinuclear tubules. Thus, the phosphorylation of serine 93 may induce the aggregation of filaments to form tubules. Together, these data suggest that the phosphorylation of serine 93 affects the structural conformation of P10.IMPORTANCE The baculovirus P10 protein has been researched intensively since it was first observed in 1969, but its role during viral infection remains unclear. It is conserved in the alphabaculoviruses and expressed at high levels during virus infection. Producing large amounts of a protein is wasteful for the virus unless it is advantageous for the survival of its progeny, and therefore, P10 presents an enigma. As P10 polymerizes to form organized cytoskeletal structures that colocalize with host cell microtubules, the structural relationship of the protein with the host cell may present a key to help understand the function and importance of this protein. This study addresses the importance of the structural changes in P10 during infection and how they may be governed by phosphorylation. The P10 structures affected by phosphorylation are closely associated with the viral progeny and thus may potentially be responsible for its dissemination and survival.

Dataset Information

Cloning and Characterization of Sf9 Cell Lamin and the Lamin Conformational Changes during Autographa californica multiple nucleopolyhedrovirus Infection.

Publications

Cloning and Characterization of Sf9 Cell Lamin and the Lamin Conformational Changes during Autographa californica multiple nucleopolyhedrovirus Infection.

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