Conserved motifs in the invertebrate iridescent virus 6 (IIV6) genome regulate virus transcription

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  • Temporal classification of all Invertebrate iridescent virus 6 (IIV6) genes was performed.

  • Using MEME Suite Software conserved motifs were identified in the upstream region of all viral ORFs.

  • Identified motifs are crucial for promoter activity of IIV6 genes.

  • A conserved motif for late genes showed decrease at promoter activity.

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.

Introduction

Invertebrate iridescent viruses (IIVs, family Iridoviridae, subfamily Betairidovirinae, genus Iridovirus) form icosahedral particles of 120–180 nm in diameter (Chinchar et al., 2017). Virions comprise a DNA/protein core surrounded by an internal lipid membrane, a protein capsid and in the case of those particles that bud out of cells, an outer viral envelope. IIVs have been reported to infect over 100 species of arthropods (Williams et al., 2017). Invertebrate iridescent virus 6 (IIV-6), also known as Chilo iridescent virus (CIV), is the type species of the Iridovirus genus. The IIV6 genome consists of 212,482 bp of linear dsDNA (Jakob and Darai, 2002) with 215 non-overlapping and putative protein-encoding ORFs selected from the 468 computationally predicted ORFs (Eaton et al., 2007). Proteomic analysis has shown that IIV6 particles contain 54 structural, viral-encoded proteins (Ince et al., 2010). The replication of the IIV6 genome is presumed to be essentially similar to that of Frog virus 3 (FV3), the type species of the genus Ranavirus, in the subfamily Alphairidovirinae (Granoff, 1984, Williams and Ward, 2010). Viral genome replication starts in the nucleus and is followed by genome concatamerization and subsequent cleavage, particle assembly and maturation in the cytoplasm (Goorha, 1982). Since purified IIV6 DNA is not infectious, one or more virion-associated proteins are needed for the initiation of IIV gene transcription (Cerutti et al., 1989).

A previous study on IIV6 mRNAs detectable by northern blot analysis revealed three temporal transcript classes in IIV6 infections: immediate-early (IE), delayed-early (DE) and late (L) (D'Costa et al., 2001). Thirty eight of the detected transcripts were synthesized in the presence of protein synthesis inhibitors and were classified in the IE class; thirty four transcripts were produced in the presence of DNA synthesis inhibitors and were classified in the DE class, while 65 five transcripts were detected only in the absence of inhibitors and were classified in the L class. However, as the transcripts were classified prior to genome sequencing, the relationship between the ORFs and their temporal classification was not previously established. In a later study, the 54 IIV6 structural virion protein genes were analyzed for their temporal expression, showing that the majority of these were expressed as IE genes (Ince et al., 2013).

It is known that IIV6 transcripts possess generally short 5′ untranslated regions and lack poly A tails (Nalcacioglu et al., 2003). On the other hand, information regarding the promoter elements of IIV6 genes is rather limited. So far, potential promoter regions of only three IIV6 genes, exonuclease (012L, IE), DNApol (037L, DE) and major capsid protein gene (mcp) (274L, L), have been characterized in detail (Nalcacioglu et al., 2003, Nalcacioglu et al., 2007, Dizman et al., 2012). 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, we 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. Therefore, the upstream regions of all genes in a particular class (classified in this paper and in previous studies) were compared and analyzed for conserved sequence motifs. The identified conserved sequences were examined for promoter activity in insect cells using the luciferase reporter assay.

Section snippets

Cell line, virus and virus infections

Spodoptera frugiperda 9 (Sf9) cells were maintained in Sf-900 II SFM (Gibco) supplemented with 5% fetal bovine serum (FBS, Sigma) at 28 °C as monolayer. Invertebrate iridescent virus 6 (IIV6) was propagated in these cells and the virus titer was determined in End Point Dilution Assays (EPDAs) (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

Transcriptional classification of all IIV6 transcripts

To be able to categorize the whole set of genes in the IIV6 genome according to their transcriptional classes, we examined the expression of 170 IIV6 genes at the transcriptional level by RT-PCR. The other 45 genes in the IIV6 genome have previously been classified transcriptionally (Nalcacioglu et al., 2007, Ince et al., 2008; 2013; Dizman et al., 2012) and were not examined again, except for 012L (IE), 037L (DE) and 274L (L) that were used as positive controls in the current study. In order

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

Discussion

This study presents extensive information on the transcriptional regulation of invertebrate iridescent virus 6 (IIV6) genes. Transcriptional studies on iridovirids (members of the family Iridoviridae) have been reported previously for Frog virus 3 (Majji et al., 2009), Singapore grouper iridovirus (Chen et al., 2006, Teng et al., 2008), Red sea bream iridovirus (Lua et al., 2005, Dang et al., 2007, Dang et al., 2008), IIV6 (D'Costa et al., 2001, D'Costa et al., 2004, Ince et al., 2008, Ince et

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

This study was supported by a 2214/a scholarship (Project no: 214Z172) to Aydin Yesilyurt from the Scientific and Technological Research Council of Turkey (TUBITAK) allowing him to do part of his studies at Wageningen University, the Netherlands and a research grant from the Karadeniz Technical University (Project no. 5839).

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