Novel insights into gene regulation of the rudivirus SIRV2 infecting Sulfolobus cells - PubMed (original) (raw)

Novel insights into gene regulation of the rudivirus SIRV2 infecting Sulfolobus cells

Ebru Okutan et al. RNA Biol. 2013 May.

Abstract

Microarray analysis of infection by a lytic Sulfolobus rudivirus, SIRV2, revealed both the temporal expression of viral genes and the differential regulation of host genes. A highly susceptible strain derived from Sulfolobus solfataricus P2 with a large genomic deletion spanning CRISPR clusters A to D was infected with SIRV2, and subjected to a microarray analysis. Transcripts from a few viral genes were detected at 15 min post-infection and all except one were expressed within 2 h. The earliest expressed genes were located mainly at the termini of the linear viral genome while later expressed genes were concentrated in the central region. Timing of the expression correlated with the known or predicted functions of the viral gene products and, thus, should facilitate functional characterization of many hypothetical viral genes. Evaluation of the microarray data with quantitative reverse-transcription PCR analyses of a few selected viral genes revealed a good correlation between the two methods. Expression of about 3,000 host genes was examined. Seventy-two were downregulated>2-fold that were mainly associated with stress response and vesicle formation, as well as chromosome structure maintenance, which appears to contribute to host chromosome degradation and cellular collapse. A further 76 host genes were upregulated>2-fold and they were dominated by genes associated with metabolism and membrane transport, including phosphate transport and DNA precursor synthesis. The altered transcriptional patterns suggest that the virus reprograms the host cellular machinery to facilitate its own DNA replication and to inhibit cellular processes required for defense against viruses.

Keywords: archaea; lytic virus; microarray; stress response; temporal regulation.

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Figures

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Figure 1. Susceptibility of S. solfataricus 5E6 to SIRV2 infection. (A) OD600 was measured after 1 ml fresh cells was diluted to 50 ml fresh medium with (filled squares) or without (open circles) the addition of SIRV2 (m.o.i. about 30). Cells were incubated at 78°C. (B) DNA content distributions from uninfected (left) and infected (right, m.o.i. 30) S.solfataricus 5E6 cultures were analyzed by flow cytometry time-course analysis. (C) One-step growth curve of SIRV2 infection of S. solfataricus 5E6. SIRV2 was added at an m.o.i. of about 30. The plaque forming units (PFU) are plotted against time (h p.i.)

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Figure 2. Temporal expression of SIRV2 genes. (A) Heat map of hierarchical clustered transcriptional changes generated from microarray data for SIRV2 genes. Genes belonging to the same group are clustered and indicated at left as early, middle and late, respectively. ORF and gp numbers for each gene are presented at the right side of the map with known or predicted functions indicated in red. hjc, Holiday junction resolvase; TMD, protein containing trans-membrane domain;sp, structure protein; pp, virus-associated pyramid protein; cp, coat protein; mt, SAM-dependent (RNA) methyltransferase; tr, putative transcription regulator. (B) qRT-PCR analysis of a selected early (ORF131a), middle (ORF56b) and late (ORF134) gene, respectively. The host RNA polymerase subunit C gene, sso0223, was equally expressed in virus infected and uninfected cells as revealed by microarray analysis and, therefore used as a reference gene. The log2 values (mean ± SD) of relative abundance of viral transcripts in relation to that of the reference gene are plotted against time (hours) p.i. (C) SIRV2 genome map showing the classification of viral genes according to results from microarray analyses (A).

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