Identification of BV/ODV-C42, an Autographa californica nucleopolyhedrovirus orf101-encoded structural protein detected in infected-cell complexes with ODV-EC27 and p78/83

Abstract

orf101 is a late gene of Autographa californica nucleopolyhedrovirus (AcMNPV). It encodes a protein of 42 kDa which is a component of the nucleocapsid of budded virus (BV) and occlusion-derived virus (ODV). To reflect this viral localization, the product of orf101 was named BV/ODV-C42 (C42). C42 is predominantly detected within the infected-cell nucleus: at 24 h postinfection (p.i.), it is coincident with the virogenic stroma, but by 72 h p.i., the stroma is minimally labeled while C42 is more uniformly located throughout the nucleus. Yeast two-hybrid screens indicate that C42 is capable of directly interacting with the viral proteins p78/83 (1629K) and ODV-EC27 (orf144). These interactions were confirmed using blue native gels and Western blot analyses. At 28 h p.i., C42 and p78/83 are detected in two complexes: one at approximately 180 kDa and a high-molecular-mass complex (500 to 600 kDa) which also contains EC27.

FIG. 1

Amino acid sequence comparison of orf101. Identical amino acids of the nucleopolyhedroviruses are shaded and outlined, while conservative changes are shown in shaded regions. Because it is the most divergent, Xc-n GV C42 was treated separately, and both identical and conserved amino acids are shaded. The clones that were identified as interacting with orf144 using yeast two-hybrid library screening are noted with arrows above the sequence. The location of the LxCxE motif (canonical binding sequence for pocket proteins) is underlined, while the nuclear localization signal (KRKK) is noted with asterisks. Rules used to assign conservation are as follows: A = G = S = T, V = L = I = M = F = Y = W, N = Q = D = E, and R = K = H. Accession numbers: AcMNPV, L22858 (nucleotides 88004 to 86921); BmMNPV (B. mori nucleopolyhedrovirus), L33180 (nucleotides 81679 to 80591); OpMNPV, U79530 (nucleotides 85649 to 85485); LdMNPV (L. dispar nucleopolyhedrovirus), U58676 (nucleotides 101348 to 100203); SeMNPV (S. exigua nucleopolyhedrovirus), AF169823 (nucleotides 61806 to 62972); HaSNPV (H. armigera single nucleopolyhedrovirus), AF271059 (nucleotides 82544 to 83653); and Xc-n GV, AF162221 (nucleotides 86182 to 87300).

FIG. 2

Primer extension analysis of orf101. Transcription initiation was mapped using 3 μg of mRNA isolated from infected-cell extracts. Two oligonucleotides were used to accurately assign nucleotide initiation sites, although only the results from oligonucleotide 1 are shown here. The initiation sites of the extension products are indicated to the right and in the sequence shown below. Because of the high degree of amino acid sequence conservation (Fig. 1), the first ATG is tentatively assigned as the start codon.

FIG. 3

Temporal analysis of appearance and accumulation of C42. Preparations of uninfected- and infected-cell extracts collected at various times p.i. (noted above lanes 1 to 9) and purified BV and ODV and respective envelope (ENV) and nucleocapsid (CAP) preparations were separated by SDS-polyacrylamide gel electrophoresis, transferred to a PVDF membrane, and tested with antisera to C42 (pAb 10910, 1:5,000). Sample concentrations are noted on the bottom (micrograms), and migration of molecular weight (MW; in thousands) markers is noted on the right. The purity of the envelope and nucleocapsid preparations was verified using antibodies to the marker proteins ODV-E66, gp67, and p39 (data not shown).

FIG. 4

Confocal analysis of C42 localization in infected Sf9 cells. Infected Sf9 cells were collected at 24, 48, and 72 h p.i. and reacted with primary antibody to C42 (pAb 10910) and secondary antibody labeled with Alexa-488 IgG (Alexa-488; column 1). DNA was stained using DAPI (column 2). The merged image includes Alexa-488 and DAPI (column 3), while the bright-field image shows the gross structure of the cell (column 4). Z-stack sections from representative cells are shown, and column 5 shows a single view of the three-dimensional reconstruction. The white arrow indicates the location of the virogenic stroma, while the yellow arrow (24 h p.i., columns 3 and 4) shows the cytoplasmic label of C42.

FIG. 5

IEM analyses of C42 localization in infected Sf9 cells. Infected Sf9 cells were collected at 24, 48, and 72 h p.i. and processed for IEM. Primary antibody was C42 (pAb 10910), and secondary antibody was anti-rabbit 30-nm-gold-labeled IgG. (A) Preimmune control serum (48 h p.i.). (B and C) Labeling of virogenic stroma (48 h p.i. [B] and 72 h p.i. [C]). (D to F) Labeling of C42 at the nucleocapsid of ODV (48 h p.i.). Size designations are in micrometers. Arrows show immunogold labeling of C42.

FIG. 6

Native gel electrophoresis. (A) Summary overview of purification protocol of 28-h-p.i. infected-cell extracts. (B) The samples of the soluble fraction were separated using blue native electrophoresis, blotted onto a PVDF membrane, and probed with the indicated antibody. Arrows point to a complex containing EC27, C42, and p78/83, while the circles show a complex containing only C42 and p78/83. U, uninfected-cell lysate; I, infected-cell lysate. Approximate molecular masses are indicated on the left (kilodaltons).