Expression of Autographa californica multiple nucleopolyhedrovirus genes in mammalian cells and upregulation of the host beta-actin gene

Abstract

The gene expression of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was examined in two types of mammalian cells, human HeLa14 and hamster BHK cells. DNA microarray analysis followed by reverse transcription-PCR identified at least 12 viral genes transcribed in both HeLa14 cells and BHK cells inoculated with AcMNPV. 5' rapid amplification of cDNA ends was carried out to examine the transcriptional fidelity of these genes in HeLa14 cells. The transcription of ie-1, ie-0 and gp64 was initiated at a baculovirus early gene motif, CAGT, accompanied by a TATA motif. In addition, the same splicing observed for ie-0 mRNA in Sf9 cells occurred in HeLa14 cells. While the transcription initiation sites for pe38 and p6.9 were not located in the CAGT motif, most of them were in a typical eukaryotic RNA polymerase II promoter structure (a conventional TATA motif and/or an initiator). Interestingly, the expression of beta-actin was upregulated in the mammalian cells inoculated with AcMNPV. Subsequent experiments using UV-inactivated virus confirmed the upregulation, suggesting that de novo synthesis of viral products is not required for the event. These results indicated that the AcMNPV genome acts as a template for transcription in mammalian cells through the usual infection pathway, though there is no evidence for the functional expression of viral genes at present.

FIG. 1.

DNA microarray analysis using Cy5-labeled cDNA probes. The cDNA probes were synthesized from total RNA extracted from mock-infected cells (left) or AcMNPV-infected BHK cells (at a MOI of 30) (right). Daggers indicate the positions of the spots for β-actin. Asterisks indicate the internal control, λpolyA.

FIG. 2.

Mapping of the 5′ ends of the transcripts for ie-1 and ie-0, gp64, and pe38 in Sf9 and HeLa14 cells at 48 hpi. (A) Schematic representation of the gene structure for ie-1 and ie-0, gp64, and pe38. The open box shows the open reading frame. Arrows on the diagram for each gene indicate the initiation sites in Sf9 cells at the early (E) or late (L) stage and in HeLa14 cells (M), respectively. (B) Sequences around the transcription initiation sites in Sf9 cells and HeLa14 cells. The arrow shows the transcription initiation site and the number in parenthesis indicates the nucleotide position relative to the transcription initiation site in Sf9 cells (+1) at the early stage (for ie-0 [16]), ie-1 (25), gp64 (9), and pe38 (20) or the late stage (for p6.9 [this paper]). The CAGT early motif and the TAAG late motif are underlined. Boldface type indicates TATA-box like and BRE-like sequences.

FIG. 3.

Northern hybridization assays using ³²P-labeled β-actin and 18S rRNA cDNA probes. (A) Northern hybridization was performed using total RNA purified from AcMNPV-infected HeLa14 cells at the times shown in the panel. Kinetics of the expression of β-actin is presented as a sequential line graph below the autoradiograph and the values are shown as the ratio of β-actin to 18S rRNA on the basis of the signal intensities obtained from the Northern hybridization. Circles and boxes in the graph indicate the kinetics for AcMNPV-infected and mock-infected cells, respectively. (B) Northern hybridization assay using total RNA from mock-infected (1), AcMNPV-infected (MOI of 30) (2) and UV-inactivated virus-infected (3) cells. The bar graph below the autoradiograph shows the ratio of hybridization signal for β-actin to that for 18S rRNA.