Temporal transcription program of recombinant Autographa californica multiple nucleopolyhedrosis virus

Abstract

Baculoviruses, a family of large, rod-shaped viruses that mainly infect lepidopteran insects, have been widely used to transduce various cells for exogenous gene expression. Nonetheless, how a virus controls its transcription program in cells is poorly understood. With a custom-made baculovirus DNA microarray, we investigated the recombinant Autographa californica multiple nucleopolyhedrosis virus (AcMNPV) gene expression program in lepidopteran Sf21 cells over the time course of infection. Our analysis of transcription kinetics in the cells uncovered sequential viral gene expression patterns possibly regulated by different mechanisms during different phases of infection. To gain further insight into the regulatory network, we investigated the transcription program of a mutant virus deficient in an early transactivator (pe38) and uncovered several pe38-dependent and pe38-independent genes. This study of baculovirus dynamic transcription programs in different virus genetic backgrounds provides new molecular insights into how gene expression in viruses is regulated.

FIG. 1.

AcMNPV microarray design and calibration method. (A) Illustration of a small region in an array with two out of four replicates of virus-specific and external control probes (represented by V1 to -18 and C1 to -23, respectively). (B) Array image of a mock infection experiment revealing virtually no cross hybridization to virus-specific probes. (C and D) Array assays of Sf21 cells infected with Bac-PH-EGFP at an MOI of 10 at 6 and 48 hpi, respectively. (E) Nonlinear calibration curve showing the relationship between signal intensities obtained from external control probes and predetermined quantities of spiked-in control targets.

FIG. 2.

Time course microarray analysis of recombinant AcMNPV gene expression patterns in Sf21 cells. (A) Sf21 cells infected with Bac-PH-EGFP at an MOI of 10 were harvested at the indicated time points for microarray analysis. The patterns of viral gene expression during the course of infection are depicted as a two-dimensional heat map. Black blocks represent zero expression, and yellow blocks symbolize different expression levels corresponding to the scale bar at the bottom. (B) Quantitative real-time PCR confirmation of microarray results for five randomly selected genes. For data comparison, results derived by both methods were adjusted to 10,000 (lef-3, orf-68, iap1, and gp37) or 1,000 (sod) arbitrary units. All PCR data are averages of duplicate tests, and microarray data are averages of four replicates.

FIG. 3.

Gene expression kinetics and classification. (A) T_on (time to onset of gene expression) and T_max (time to maximum expression level) were demonstrated by using chitinase (orf-126) as an example. (B) A classification methodology for AcMNPV genes based on the criteria of T_on and T_max kinetics. Selected known genes in each classified group are listed at the bottom.

FIG. 4.

Cluster analysis of gene expression patterns and colocalization in baculovirus genomic map. (A) Five gene clusters obtained by K means clustering analysis were arranged in an increasing order of the average T_ons. (B) Correlation between the frequency of promoter sequence motifs and gene clusters. (C) Genome map view of the five gene clusters color coded in the AcMNPV genome. Gene clusters that colocalized to nearby loci are indicated. A colocalized cluster is defined as a region that contains at least five consecutive genes from the same gene cluster where no more than one interruption occurs by a gene from other gene clusters in either the plus or the minus strand. ID, identification.

FIG. 5.

Differential gene expression profiling of a pe38 deletion mutant virus in strain Sf21 cells. (A to E) Scatter plot analysis for the dynamic differential gene expression delay of the Δpe38-E9/E9 mutant. Both wild-type and mutant viruses at an MOI of 10 were used to infect Sf21 cells. The gene expression levels shown here have been subjected to normalization. (F) Statistics of the delayed gene expression patterns at various times postinfection. An expressed gene is defined as one whose normalized expression level at the indicated time postinfection was greater than zero. (G) Temporal expression profiles of selective AcMNPV genes that were expressed in the wild-type virus in an earlier phase (<4.5 hpi) and their counterparts in the Δpe38-E9/E9 mutant virus. Genes in groups a and b are pe38 independent and pe38 dependent, respectively.