Identification of six Autographa californica multicapsid nucleopolyhedrovirus early genes that mediate nuclear localization of G-actin

Abstract

Nuclear filamentous actin (F-actin) is required for nucleopolyhedrovirus (NPV) progeny production in NPV-infected, cultured lepidopteran cells. We have determined that monomeric G-actin is localized within the nuclei of host cells during the early stage of infection by Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). With a library of cloned AcMNPV genomic fragments, along with a plasmid engineered to express enhanced green fluorescent protein-Bombyx mori G-actin in transient transfection experiments, we identified six AcMNPV early genes that mediate nuclear localization of G-actin in TN-368 cells: ie-1, pe38, he65, Ac004, Ac102, and Ac152. Within this subset, ie-1 and pe38 encode immediate-early transcriptional transactivators, he65 encodes a delayed-early product, and the products encoded by Ac004, Ac102, and Ac152 have not been characterized. We found that when driven by foreign promoters, ie-1, pe38, and Ac004 had to be expressed prior to Ac102 or he65 for nuclear G-actin to accumulate and that expression of Ac152 was no longer required. These results and others suggested that the product of Ac152 was a transactivator (directly or indirectly) of both Ac102 and he65 and that recruitment of G-actin to the nucleus was a temporally regulated process. Determining the functions of each of the six AcMNPV gene products with respect to our assay should provide valuable clues to basic cellular mechanisms of actin regulation and how AcMNPV infection affects them.

FIG. 1.

Vector constructs used in transfection experiments. Common plasmid vector backbone details are omitted. pIE1/153A and pIE1actGA contain the ie-1 open reading frame driven by the ie-1 promoter (ie-1 p). hr3, B. mori NPV homologous region 3; act p, B. mori A3 actin promoter; MCS, multiple cloning site; poly A, polyadenylation signal; eGFP-actin, fused open reading frames of enhanced green fluorescent protein and actin.

FIG. 2.

Fluorescence microscopy images of Sf21 cells infected with AcMNPV at a multiplicity of infection of 10 in the presence of 5 μg of aphidicolin per ml and then transfected with pIE1actGA at 2 h postinfection. Cells were fixed and stained at 48 h postinfection. (A) DAPI-stained DNA. (B) eGFP-B. mori G-actin. (C) Tetramethylrhodamine isocyanate-phalloidin staining of filamentous actin.

FIG. 3.

Genomic locations of plasmid DNAs used in transfections with pIE1actGA. Three separate mixtures, represented on three separate lines, are shown beneath the EcoRI map of AcMNPV.

FIG. 4.

Fluorescence microscopy images of TN-368 cells transfected with cloned fragments of AcMNPV DNA, at 3 days posttransfection. (A) eGFP-B. mori G-actin in cells transfected with pIE1actGA and the PstI-C and HindIII-F fragments of AcMNPV. (B) DAPI staining of cells shown in panel A. (C) eGFP-B. mori G-actin in cells transfected with pIE1actGA and the EcoRI-A, EcoRI-J, and PstI-F′-E′ fragments of AcMNPV. (D) DAPI staining of cells shown in panel C.

FIG. 5.

Map showing subcloned fragments of PstI-C and HindIII-F. The locations of the ends of the fragments within the AcMNPV genome are indicated by the numbers flanking PstI-C and HindIII-F. Predicted open reading frames (2) are indicated below the PstI-C and HindIII-F fragments by number (and gene name if previously characterized), with the predicted direction of transcription indicated by arrowheads.

FIG. 6.

Confocal fluorescence microscopy images of TN-368 cells transfected with pIE1actGA alone or with cloned genes or genomic fragments of AcMNPV. Cells were fixed 3 days posttransfection and stained with DAPI. (A) PstI-C and HindIII-F fragments of AcMNPV. (B) Ac004, Ac152, and pe-38 and 24 h later PstI-C. (C) HindIII-F and 24 h later Ac102. (D) HindIII-F and 24 h later he65. (E) Ac004, Ac152, and pe-38 and 24 h later Ac102. (F and G) Ac004, Ac152, and pe-38 and 24 h later he65. (H) pIE1act GA alone.