Unravelling the Role of the F55 Regulator in the Transition from Lysogeny to UV Induction of Sulfolobus Spindle-Shaped Virus 1

Abstract

Sulfolobus spindle-shaped virus 1 represents a model for studying virus-host interaction in harsh environments, and it is so far the only member of the family Fuselloviridae that shows a UV-inducible life cycle. Although the virus has been extensively studied, mechanisms underpinning the maintenance of lysogeny as well as those regulating the UV induction have received little attention. Recently, a novel SSV1 transcription factor, F55, was identified. This factor was able to bind in vitro to several sequences derived from the early and UV-inducible promoters of the SSV1 genome. The location of these binding sites together with the differential affinity of F55 for these sequences led to the hypothesis that this protein might be involved in the maintenance of the SSV1 lysogeny. Here, we report an in vivo survey of the molecular events occurring at the UV-inducible region of the SSV1 genome, with a focus on the binding profile of F55 before and after the UV irradiation. The binding of F55 to the target promoters correlates with transcription repression, whereas its dissociation is paralleled by transcription activation. Therefore, we propose that F55 acts as a molecular switch for the transcriptional regulation of the early viral genes.

Importance: Functional genomic studies of SSV1 proteins have been hindered by the lack of similarity with other characterized proteins. As a result, few insights into their in vivo roles have been gained throughout the last 3 decades. Here, we report the first in vivo investigation of an SSV1 transcription regulator, F55, that plays a key role in the transition from the lysogenic to the induced state of SSV1. We show that F55 regulates the expression of the UV-inducible as well as the early genes. Moreover, the differential affinity of this transcription factor for these targets allows a fine-tuned and temporal coordinated regulation of transcription of viral genes.

FIG 1

Growth curves of mock- and UV-treated SSV1-InF1 cultures. The OD₆₀₀ values were measured every 2 h and plotted versus the incubation time. Before being mock or UV treated, cells were grown exponentially to an OD₆₀₀ of 0.5 (21st hour of incubation). Afterwards, the culture was split in two and incubated to 75°C to estimate the effect of the treatment. Error bars show standard deviations (n = 3). *, P < 0.05.

FIG 2

Time course of the viral replication induction after UV irradiation by semiquantitative PCR. Molecular size markers are indicated on the left; host (orc1, 108 bp) and viral (vp2, 155 bp) PCR products are indicated on the right. Total DNA samples were prepared from mock-treated cells (control) and UV-treated cultures collected at 2, 4, 6, 8, and 10 h postirradiation. The maximum amount of SSV1 DNA is detectable for the sample collected 8 h posttreatment (dashed white box).

FIG 3

Detection of the viral DNA after UV irradiation by EcoRI restriction analysis. The restriction profiles of total DNA samples from control cultures and SSV1-InF1-irradiated cells and of SSV1 episomal DNA digested with EcoRI are shown. Molecular size markers are indicated on the left; SSV1-derived fragments (7.9, 2.9, 2.4, and 2.1 kbp) are indicated on the right. The intensity of the SSV1 fragments is higher for the sample collected 8 to 10 h postirradiation.

FIG 4

Transcription analysis of T_lys and T_ind. (A) Schematic representation of the head-to-head-oriented transcripts T_ind and T_lys. (B) SSV1-InF1 total RNAs isolated from mock- and UV-treated cells at different time points were analyzed by Northern hybridization to detect T_lys and T_ind transcripts. The hybridization signals were normalized using the 16S housekeeping gene. The T_ind transcript was expressed over a short time and only in UV-irradiated cells, whereas T_lys was detectable in both mock-treated and UV-irradiated cells. Degradation occurred around the 8th to 10th hour only for the T_lys transcript.

FIG 5

Western blot analysis of F55 in mock- and UV-treated InF1-SSV1 cells. (A) Western blot of cell extracts from control, mock-treated, and UV-treated samples. (B) F55 quantification was performed using a standard curve. (C) Quantitative data are reported as histograms. Error bars show the standard deviations (n = 3), and a control sample (gray bar) is used as a reference. The amount of F55 in the UV-treated culture is significantly lower than that in the mock one at every time point analyzed (*, P < 0.05).

FIG 6

Chromatin immunoprecipitation (ChIPs) on the UV-inducible region of SSV1 using F55 specific antibodies. (A) Scheme of the UV-inducible region of SSV1 genome. The amplified regions of the target promoters that are recognized by F55 are in blue, yellow, green, and red. All the promoters except that of T_lys contain two F55 binding sites. (B) Total DNA samples prepared from control and UV-treated cultures and collected at 2 and 4 h postirradiation were used as the templates for semiquantitative PCRs. Two negative controls, vp2 (viral) and orc1 (host), were included to assess method specificity. N is the negative control for PCRs.

FIG 7

A suboptimal concentration of F55 allows the derepression of the target genes. Schematic representations of the infected cell and of the UV-inducible region of SSV1 genome are presented. The operators recognized by F55 are in green, yellow, and blue. Bent arrows indicate the transcription start sites, and dashed lines represent transcripts. Dimers of F55 are represented by purple ovals. In the lysogenic cell, the amount of F55 is suitable to saturate most of its binding sites and to keep SSV1 in a steady carrier state. At 2 h postirradiation, a decrease of about 50% of the F55 concentration and a concurrent increase of the viral copy number led to the dissociation from the lower-affinity operators in the promoter of T_ind and T_lys. Later, at 4 h postirradiation, the dilution effect is enhanced by a further accumulation of the viral DNA, which results in the release of the early promoters (i.e., those of T₅ and T₆), thus allowing transcription derepression.