Conserved motifs in the invertebrate iridescent virus 6 (IIV6) genome regulate virus transcription.
TL;DR: The transcriptional class of all IIV6 genes that had not been classified until now is investigated and single nucleotide mutations in the highly conserved nucleotides at the end of the second motif showed that this motif acted as a repressor sequence for late genes in the IIV 6 genome.
Abstract: Invertebrate iridescent virus 6 (IIV6) is the type species of the Iridovirus genus in the Betairidovirinae subfamily of the Iridoviridae family. Transcription of the 215 predicted IIV6 genes is temporally regulated, dividing the genes into three kinetic classes: immediate-early (IE), delayed-early (DE), and late (L). So far, the transcriptional class has been determined for a selection of virion protein genes and only for three genes the potential promoter regions have been analyzed in detail. In this study, we investigated the transcriptional class of all IIV6 genes that had not been classified until now. RT-PCR analysis of total RNA isolated from virus-infected insect cells in the presence or absence of protein and DNA synthesis inhibitors, placed 113, 23 and 22 of the newly analyzed viral ORFs into the IE, DE and L gene classes, respectively. Afterwards, in silico analysis was performed to the upstream regions (200 bp) of all viral ORFs using the MEME Suite Software. The AA(A/T)(T/A)TG(A/G)A and (T/A/C)(T/G/C)T(T/A)ATGG motifs were identified in the upstream region of IE and DE genes, respectively. These motifs were validated by luciferase reporter assays as crucial sequences for promoter activity. For the L genes two conserved motifs were identified for all analyzed genes: (T/G)(C/T)(A/C)A(T/G/C)(T/C)T(T/C) and (C/G/T)(G/A/C)(T/A)(T/G) (G/T)(T/C). However, the presence of these two motifs did not influence promoter activity. Conversely, the presence of these two sequences upstream of the reporter decreased its expression. Single nucleotide mutations in the highly conserved nucleotides at the end of the second motif (TTGT) showed that this motif acted as a repressor sequence for late genes in the IIV6 genome. Next, upstream sequences of IIV6 L genes from which we removed this second motif in silico, were re-analyzed for the presence of potential conserved promoter sequences. Two additional motifs were identified in this way for L genes: (T/A)(A/T)(A/T/G)(A/T)(T/C)(A/G)(A/C)(A/C) and (C/G)(T/C)(T/A/C)C(A/T)(A/T)T(T/G) (T/G)(T/G/A). Independent mutations in either motif caused a severe decrease in luciferase expression. Information on temporal classes and upstream regulatory sequences will contribute to our understanding of the transcriptional mechanisms in IIV6.
Summary (3 min read)
- Invertebrate iridescent viruses (IIVs, family Iridoviridae, subfamily Betairidovirinae, genus Iridovirus) form icosahedral particles of 120–180 nm in diameter (Chinchar et al., 2017).
- Invertebrate iridescent virus 6 (IIV-6), also known as Chilo iridescent virus (CIV), is the type species of the Iridovirus genus.
- These promoters have been identified by means of a luciferase reporter assay in conjunction with deletion mutagenesis of the sequences in the 5′upstream region of the respective ORFs.
- In the current study, the authors investigated the transcriptional class of all as of yet unclassified IIV6 ORFs (170 transcripts) to complete the temporal classification and to be able to search for essential, conserved promoter motifs in IIV6 genes.
2.1. Cell line, virus and virus infections
- Invertebrate iridescent virus 6 (IIV6) was propagated in these cells and the virus titer was determined in End Point Dilution Assays (Cook et al., 1976).
- Virus infections were carried out with 2x106 Sf9 cells in 6-well plates, infected at a multiplicity of infection (MOI) of 2.
- For the temporal classification of the genes, cultures were pre-treated 1 h before infection with cytosine-1-β-D-arabinofuranoside (Ara-C, 100 µg/ml) and cycloheximide (CHX, 150 µg/ml) to inhibit DNA and protein synthesis, respectively.
- The inhibitors remained present during the infection.
2.2. Reverse transcription PCR (RT-PCR)
- To determine the temporal expression classes of IIV6 genes, RNA isolated as described below was subjected to RT–PCR.
- Total RNA was isolated from infected and mock-infected Sf9 cells at 12 h post infection (p.i.) using Trizol Reagent (Sigma, T9424) following the manufacturer’s instructions.
- Isolated RNA samples were treated with DNase I (Sigma, AMPD1-1KT) to remove any residual DNA and then extracted with phenol–chloroform.
- And the resulting cDNA mixture was then used as template for gene specific PCR amplifications with forward and reverse primers.
- PCR performed with cDNA, obtained from infected cells in absence of inhibitors, was used as positive control (PC).
2.3. Conserved sequence analyses
- MEME (multiple expectation maximization for motif elicitation) (Bailey et al., 2009) software was used to search for conserved sequences in IIV6 noncoding sequences in the 200 nt regions upstream of the translation initiation codons.
- To that aim, the upstream sequences were categorized based on experimental data (IE, DE or L) to be able to compare upstream sequences within each expression class.
- Parameters were set to zero or one occurrence per sequence and the authors searched only the provided strand.
2.4. Plasmid construction
- Upstream sequences of selected genes from each temporal group were investigated to determine whether conserved motifs, found with the MEME software, are indeed important for promoter activity.
- One gene was selected from each of the three groups.
- Upstream regions of these genes were tested for promoter activity in combination with a luciferase reporter system.
- Subsequently, upstream sequences of two additional L genes, 061R and 084L were also investigated with this system.
- These DNA fragments were amplified from the viral genome using two different forward primers and a common reverse primer, for each gene (Table 1).
2.5. Transfection and luciferase assay
- Sf9 cells (2.5 × 106 cells/well) in 6-well plates were infected with IIV6 for 2 h and then transfected using Cellfectin with plasmid DNA (1 µg) harboring the upstream sequences.
- The various putative promoter constructs were tested in parallel.
- Firefly luciferase activities were measured in cell extracts using the single luciferase reporter assay system following the manufacturer’s instructions.
- Transfections were conducted in triplicate, and average values are reported.
2.6. Site directed mutagenesis
- Highly conserved sequences, found in the upstream regions of the L gene 061R were mutated to understand the role of these sequences in determining promoter activity.
- Mutations were performed by PCR using primers specific for the upstream region of 061R, but carrying a number of mismatched nucleotides (Table 1) (Nalcacioglu et al., 2003).
- Amplified sequences were first cloned into the pJET1.2/blunt cloning vector and then transferred to the pSPLuc + vector, as described above.
3.1. Transcriptional classification of all IIV6 transcripts
- The other 45 genes in the IIV6 genome have previously been classified A. Yesilyurt et al.
- In order to classify the IIV6 genes, Sf9 cells were infected with IIV6 in the presence or absence of cycloheximide, which inhibits de novo polypeptide synthesis, and Ara-C, an inhibitor of DNA replication.
- In infected cells, a total of 113 newly analyzed transcripts was detected in the presence of protein or DNA synthesis inhibitors, which means that viral protein synthesis and DNA replication are not necessary for these transcripts and therefore they are classified in the IE class (Fig. 1).
- The number of the late transcripts among the 170 newly tested ORFs was 22 (Fig. 2B).
- With the previously classified IIV6 genes, the total number of IE, DE and L genes became 138, 35 and 30, respectively.
4. Conserved motifs in the upstream region of IIV6 genes
- After grouping the genes in the three temporal classes, sequences upstream of the translational start codon of each gene were investigated for the presence of conserved and potentially important motifs for promoter activity.
- For each classified group of genes, motifs were generated by the MEME Suite database (Fig. 3).
- The AA(A/T)(T/A)TG(A/G)A and (T/A/C)(T/G/C)T(T/A) ATGG sequences were identified with high probability as conserved motifs in the upstream regions of IE and DE genes, respectively (Fig. 3A-B).
- For the 35 scanned DE genes, the motif obtained was only observed in 20 genes.
- The locations of all these upstream motifs respective to the translation start site varies for each gene.
4.1. Investigating the motifs for promoter activity
- To analyze the influence of the conserved motifs on promoter activity, deletion mutagenesis was performed on the upstream regions of 193R, 126R and 259R ORFs, belonging to the IE, DE and L classes, respectively.
- Two fragments, one containing the motif and the other not, were prepared for each ORF.
- Reporter plasmids harboring the wild type (wt) or mutant sequence upstream of a firefly luciferase reporter ORF, were transfected into Sf9 cells.
- For L genes, the result was opposite.
- The plasmids containing both the L1 and L2 motifs produced a low luciferase activity (pSP259Rprom, pSP084Lprom, pSP061Rprom), but the plasmids without these two motifs (pSP259RdelL1 + L2, pSP084LdelL1 + L2, pSP061RdelL1 + L2) produced a high activity.
4.2. Site-directed mutations in conserved late gene motifs
- Reporter plasmids were prepared carrying both L1 and L2 motifs, but one unmutated sequence and the other mutated, to determine the impact of such changes on promoter activity.
- Mutation of motif L1 (mutation 1) did not affect the promoter activity, however mutation at motif L2 (mutation 2) increased promoter activity.
- The fact that the detected L motifs do not act as promoters led us to search for other conserved sequences that might have promoter activity.
- KpnI and HindIII are shown in italicized and underlined.
- Therefore, upstream sequences of all IIV6 L genes, from which the L1 and L2 motifs were in silico removed, were re-analyzed for the presence of potential promoter sequences using MEME Suite Software, resulting in two additional conserved sequences (motif L3 and motif L4) (Fig. 5).
- This study presents extensive information on the transcriptional regulation of invertebrate iridescent virus 6 (IIV6) genes.
- Other transcriptional studies on IIV6 genes included temporal classification of a few genes: DNA polymerase (037L), major capsid protein (274L), A. Yesilyurt et al.
- This result was therefore not conforming the defined temporal groups.
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Q1. What are the contributions in "Conserved motifs in the invertebrate iridescent virus 6 (iiv6) genome regulate virus transcription" ?
In this study, the authors investigated the transcriptional class of all IIV6 genes that had not been classified until now. Conversely, the presence of these two sequences upstream of the reporter decreased its expression. Next, upstream sequences of IIV6 L genes from which the authors removed this second motif in silico, were re-analyzed for the presence of potential conserved promoter sequences.