Enhancement of enteric adenovirus cultivation by viral transactivator proteins

Abstract

Human enteric adenoviruses (HAdVs; serotypes 40 and 41) are important waterborne and food-borne pathogens. However, HAdVs are fastidious, are difficult to cultivate, and do not produce a clear cytopathic effect during cell culture within a reasonable time. Thus, we examined whether the viral transactivator proteins cytomegalovirus (CMV) IE1 and hepatitis B virus (HBV) X promoted the multiplication of HAdVs. Additionally, we constructed a new 293 cell line expressing CMV IE1 protein for cultivation assays. We analyzed the nucleic acid sequences of the promoter regions of both E1A and hexon genes, which are considered to be the most important regions for HAdV replication. Expression of either HBV X or CMV IE1 protein significantly increased the promoter activities of E1A and hexon genes of HAdVs by as much as 14-fold during cell cultivation. The promotion of HAdV expression was confirmed by increased levels of both adenoviral DNA and mRNA expression. Finally, the newly developed 293 cell line expressing CMV IE1 protein showed an increase in viral DNA ranging from 574% to 619% compared with the conventional 293 cell line. These results suggest that the newly constructed cell line could be useful for efficient cultivation and research of fastidious HAdVs.

FIG. 1.

(a) Schematic diagram for subcloning the promoter regions of HAdV-41. Both hexon and E1A amplicons were cloned into the pGL2-basic vector (Promega) and directly controlled luciferase expression. Hexon (b) and E1A (c) genes of HAdV F group (ATCC VR-930) were compared with L19443 using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

FIG. 2.

Transcriptional activation by HBV X and CMV IE1 expression vector in 293 cells. Transcriptional levels of HAdV genes were monitored by luciferase enzyme activity. Data indicate the percent luciferase activity for HBV X and CMV IE1 compared with the pcDNA control vector. Both hexon and E1A promoter showed increased transcriptional levels relative to the control DNA (pcDNA 3 vector) by as much as 307 to 632% and 437 to 2,078%, respectively. The level of luciferase activity with control DNA was set at 100%, and others are shown relative to this level. Each experiment was repeated three times in duplicate. Error bars indicate the standard error. *, P < 0.05 compared to pcDNA vector control.

FIG. 3.

Increments of viral RNA and DNA levels by CMV IE1 in 293 cells. The 293 cells were transiently transfected with either pcDNA3 (control) or CMV IE1 expression vector and then infected with HAdV-41. After 3 days of inoculation, 293 cells were harvested, and total RNA and DNA were extracted and quantified by real-time TaqMan RT-PCR assays. The copy number with control DNA was set at 100%, and others values are shown relative to this level. These mRNA and DNA experiments were repeated three and two times in duplicate, respectively. Error bars indicate the standard error.

FIG. 4.

Confirmation of protein expression, DNA incorporation, and mRNA expression of the CMV IE1 vector in the 293 cell line. (a) Protein expression of CMV IE1 from the transiently transfected 293 cell line. Lane M, size marker; lane 1, mock-treated 293 cells; lane 2, transiently transfected 293 cell line. (b) PCR amplification of genomic DNA of the new cell line. (c) PCR and RT-PCR amplification of the CMV IE1 gene using mRNA extraction solution from the new cell line. Lane M, 100-bp size marker; lane 1, 293 cell line stably expressing IE1; lane 2, 293 cell line; lane 3, negative control (distilled water); lane 4, positive control (CMV IE1 expression vector).

FIG. 5.

HAdV multiplication in both 293 and 293-CMV cell lines. Cell morphology by inverted microscope is shown as follows (magnification, ×100): 293 cell line without infection (a), 293-CMV cell line without infection (b), 293 cell line at 6 days p.i. with HAdV-41 (c), and 293-CMV cell line at 6 days p.i. with HAdV-41 (d). Transmission electronic microscope sections are shown as follows: 293 cell line at 3 days p.i. with HAdV-41 (magnification, ×40,000; bar, 500 nm) (e) and 293-CMV cell line at 3 days p.i. with HAdV-41 (magnification, ×50,000; bar, 500 nm) (f).

FIG. 6.

Confirmation of increased viral replication in the newly constructed cell line. Three different HAdVs were inoculated into both 293 cells and the new cell line. Viral DNA was amplified using real-time TaqMan PCR at 3 days p.i. The experiments were repeated three times in duplicate for laboratory-adapted HAdV and twice in duplicate for clinical stool samples. Error bars indicate the standard error. *, P < 0.05 compared to the 293 control cell line.