Modulation of DNA methyltransferases (DNMTs) in Spodoptera frugiperda (Sf9) cells following AcMNPV infection and its effects on the virus-cell interaction
Abstract
In this study, we examined the gene expression profiles of Sf9 insect cells following infection with baculovirus, utilizing reverse transcription quantitative PCR (RT-qPCR) to analyze how the host cellular machinery responds at the transcriptional level to viral invasion. Our findings highlight the intricate regulatory mechanisms in place during virus replication. To further understand the relationship between the host response and viral propagation, we also quantified the dynamics of baculovirus replication within the Sf9 cells using quantitative PCR (qPCR). This dual approach provides a comprehensive view of both the efficiency of viral replication and the accompanying host transcriptional changes.
Dataset DOI: 10.5061/dryad.pk0p2nh1j
Description of the data and file structure
File: data.txt
Description: Includes a text file of tables.
To analyze gene expression levels in AcMNPV-infected and control uninfected Sf9 cells, total RNA was extracted from cells at 4, 8, 16, 24 and 48 hpi. Also, to determine the effect of 5-AZA on the expression of immune-related genes, RNA samples were extracted at 24 and 48 h post AcMNPV infection. Total RNA was extracted by Tri- Reagent™ according to the manufacturer’s instructions (Molecular Research Center Inc.). RNA samples were treated with DNase I and incubated at 37 °C for 10 min followed by heat inactivation at 75 °C for 10 min. RNA concentrations were measured using an Epoch microplate spectrophotometer (BioTek) and integrity was ensured through analysis of RNA on a 1% (w/v) agarose gel. The first strand cDNA was synthetized using MMLV-reverse transcriptase enzyme (Promega) and an oligo dT primer using 1μg of total RNA as template at 42 °C for 60 min followed by heating at 65 °C for 5 min.
qPCR was performed by utilizing the SYBR Green Mix without ROX (Ampliqon) with a Mic real-time PCR (BMS) under the following conditions: 95 °C for 15min, followed by 40 cycles of 95 °C for 10s, 57 °C for 10 s, and 72 °C for 20 s, followed by the melting curve (60–95 °C). In all experiments, ribosomal protein L27 (Rpl27) was used as the reference gene and also three biological replicates with three technical replicates were analyzed for each experiment. QPCR data were analyzed using the relative expression ratio method [ratio = (Etarget)ΔCPtarget(control – sample)/(Eref)ΔCPref(control – sample)].
This data quantifies how host cell gene expression and viral replication change over time after baculovirus infection, and shows how treatment with a demethylating agent (Aza-5-azacytidine) alters expression of selected genes.
The data shown in each table represent normalized gene expression results (three replicates) at hours post-infection. The mean and standard deviation for each time point are also indicated in the table. Each table corresponds to the specified gene (DNMT1, DNMT2, Rel – Relish, Dor – Dorsal, Glo – Gloverin, Def – Defensin). The replication level of AcMNPV in Sf9 cells at hours post-infection is also indicated. In this table, the normalized virus replication results (three replicates) at hours post-infection are shown. The mean and standard deviation for each time point are also indicated in the table.