ORF4 of the Temperate Archaeal Virus SNJ1 Governs the Lysis-Lysogeny Switch and Superinfection Immunity

Abstract

Recent environmental and metagenomic studies have considerably increased the repertoire of archaeal viruses and suggested that they play important roles in nutrient cycling in the biosphere. However, very little is known about how they regulate their life cycles and interact with their hosts. Here, we report that the life cycle of the temperate haloarchaeal virus SNJ1 is controlled by the product ORF4, a small protein belonging to the antitoxin MazE superfamily. We show that ORF4 controls the lysis-lysogeny switch of SNJ1 and mediates superinfection immunity by repression of genomic DNA replication of the superinfecting viruses. Bioinformatic analysis shows that ORF4 is highly conserved in two SNJ1-like proviruses, suggesting that the mechanisms for lysis-lysogeny switch and superinfection immunity are conserved in this group of viruses. As the lysis-lysogeny switch and superinfection immunity of archaeal viruses have been poorly studied, we suggest that SNJ1 could serve as a model system to study these processes.IMPORTANCE Archaeal viruses are important parts of the virosphere. Understanding how they regulate their life cycles and interact with host cells provide crucial insights into their biological functions and the evolutionary histories of viruses. However, mechanistic studies of the life cycle of archaeal viruses are scarce due to a lack of genetic tools and demanding cultivation conditions. Here, we discover that the temperate haloarchaeal virus SNJ1, which infects Natrinema sp. strain J7, employs a lysis-lysogeny switch and establishes superinfection immunity like bacteriophages. We show that its ORF4 is critical for both processes and acts as a repressor of the replication of SNJ1. These results establish ORF4 as a master regulator of SNJ1 life cycle and provides novel insights on the regulation of life cycles by temperate archaeal viruses and on their interactions with host cells.

Keywords: Archaea; SNJ1; lysis-lysogeny switch; superinfection immunity; temperate virus.

FIG 1

Discovery of clear-plaque mutants of SNJ1. (a) Viruses obtained from CJ7/pYC-S culture formed both clear and turbid plaques on lawns of CJ7. A late-exponential-phase culture of CJ7 was infected with SNJ1 viruses or viruses generated using CJ7/pYC-S. SNJ1 formed turbid plaques, while viruses generated using pYC-S formed two kinds of plaques. Arrows represent turbid plaques, and triangles represent clear plaques. Viruses purified from the turbid or clear plaques were propagated and used to infect CJ7 as above, the morphotypes of the plaques were maintained for either virus. (b) Ability of different viruses to lyse cells. The same amount of viruses was added into CJ7 culture (OD₆₀₀ ≈ 0.1). Growth of the culture was monitored for 49 h postinfection by measuring OD₆₀₀. SNJ1-Ca/Cb and SNJ1-Ta/Tb represented two clear-plaque viruses and two turbid-plaque viruses isolated form lawns of CJ7 infected with viruses from CJ7/pYC-S culture, respectively. CJ7 without virus infection was used as a control. The experiment was repeated twice, and error bars indicate the standard deviations.

FIG 2

Schematic diagrams of the genomic deletion of 20 turbid and clear plaque viral genomes. (a) Scheme of SNJ1 genome from 1 to 1,134 bp and locations of genomic deletion of turbid/clear plaque viruses of SNJ1. The putative ORFs of SNJ1 were indicated by numbered arrows, while the end site of genomic deletion in turbid/clear plaque viruses were marked by T1-T20 and C1-C20. The red dotted arrow represents the region of genomic deletion. Detailed deletion locations are shown in panels b (turbid plaque viruses) and c (clear plaque viruses). The pUC19-mev fragment and flanking sequences of 20 turbid and clear-plaque viruses were amplified by the HJ-F/R primer pair and sequenced. Black solid lines represent genomes packaged in viral particles, while black dotted lines represent deletions. The putative ORFs were indicated by numbered arrows and the SacI site, where foreigner DNA was inserted, was indicated. The numbers on the right side represent the start (x) and end (y) locations of the genomic deletion. “z” represents the genome size of mutant SNJ1 viruses, and the smaller genomes are indicated in boldface compared to the wild-type SNJ1 (16,492 bp).

FIG 3

Identification of orf4 as a critical factor for SNJ1 lysis-lysogeny switch. (a) orf4 disrupted viruses formed only clear plaques on lawns of CJ7. A late-exponential-phase culture of CJ7 was infected with SNJ1^{orf4 mut} and SNJ1^Δorf4 viruses (generated from CJ7/pYC-S-4M and CJ7/pYC-SΔ1-575 cultures, respectively), and only clear plaques were observed. (b) Expression of ORF4 in trans restored turbid plaque formation of clear-plaque SNJ1 mutant viruses and inhibited plaque formation dramatically. Tenfold serial dilutions of SNJ1 viruses or its clear-plaque mutants were spotted onto the lawns of CJ7-F/pFJ6-MCS, or CJ7-F/pFJ6-1-656 (with orf4). Plates were incubated at 37°C for 48 h and photographed. The maximum dilution for observed plaques is highlighted by asterisk.

FIG 4

ORF4 is critical for stability and copy number control of SNJ1-based plasmids. (a) Relative copy numbers of SNJ1-based plasmids pYC-SHS (with orf4) and pYC-SHS-4M (without orf4) in CJ7 cells with or without MMC treatment. “WT” represents wild-type pYC-SHS plasmid, “Δorf4” represents pYC-SHS-4M (start codon mutation of orf4), and “Δorf4+orf4” represents pFJ6-1-656 complemented pYC-SHS-4M. These plasmids were transformed to CJ7 strain and cultured to late exponential phase (24 h) in 18% MGM+Mev and treated with MMC (1 μg ml⁻¹) for 30 min, while cultures not treated with MMC were set as controls. Cells were collected by centrifugation and washed twice in the same volume of 18% MGM to remove MMC. Cell pellets were resuspended in 18% MGM+Mev and cultured for 24 h. Samples were taken for qPCR analysis using the primer pairs vector-F/vector-R, orf14-F/orf14-R, and radA-F/radA-R to determine the plasmid copy numbers relative to the chromosome. Three independent experiments were performed, and the error bars indicate the standard deviations. (b) Plasmid stability of pYC-SHS and pYC-SHS-4M during passage. Portions (100 μl) of stationary-phase cultures of CJ7/pYC-SHS, CJ7/pYC-SHS-4M, and CJ7/pYC-SHS-4M+pFJ6-1-656 were inoculated into 5 ml of Halo-2 medium every day. Samples were taken and measured by qPCR using the primer pairs vector-F/vector-R, orf14-F/orf14-R, and radA-F/radA-R for 5 days. Three independent experiments were performed, and error bars indicate the standard deviations.

FIG 5

ORF4 mediates superinfection immunity of SNJ1. (a) Expression of ORF4 confers resistance to SNJ1 infection in CJ7. To test the immunity to against SNJ1, 10-fold serial dilutions of SNJ1 virus stocks were spotted onto lawns of CJ7, J7-1, and CJ7-F strains carrying pFJ6 plasmids with or without orf3 and orf4. The maximum dilution for observed plaques was highlighted by asterisk. (b) Sequence alignment of ORF4 with other MazE/SpoVT family members. C68 protein from hybrid virus-plasmid pSSVx (residues 1 to 68, PDB code 3O27) (32), the N-terminal domains of MazE (residues 1 to 53, PDB code 1UB4) (33), AbrB (residues 1 to 51, PDB code 1YSF) (34), and SpoVT (residues 1 to 55, PDB code 2W1T) (35) were aligned with ORF4 using Clustal Omega. The secondary structure of ORF4 was predicted by PSIPRED in MPI Bioinformatics Toolkit. ORF4 contains five β-strands (light-gray arrows) and one α-helix (dark-gray cylinder). The same color is used for other proteins. (c) The N-terminal 33 amino acids of SNJ1 were necessary and sufficient for immunity against SNJ1. Tenfold serial dilutions of SNJ1 were spotted onto lawns of CJ7-F harboring plasmid pFJ6-MCS or its derivatives carrying different portions of ORF4; superscript denotes the amino acid residues of ORF4. Hpro stands for the promoter of heat shock protein 70 from Haloferax volcanii DS52. The plates were incubated at 37°C for 48 h and photographed.

FIG 6

ORF4 blocks SNJ1 infection by inhibiting viral gDNA replication. (a) ORF4 did not affect SNJ1 adsorption to host cells CJ7. SNJ1 was incubated with early-exponential-phase cultures of CJ7, J7-1, CJ7-F/pFJ6-MCS (− orf4), and CJ7-F/pFJ6-Hpro-orf4 (+ orf4) at different MOIs (0.1, 1, and 10) for about 1 h at 45°C. After absorption, the culture was centrifuged, and the titers of unbound viruses in the supernatant were measured by the double-layer method. The adsorption efficiency was determined by comparing the titer before and after virus adsorption. Three independent experiments were performed, and error bars indicate the standard deviations. (b) Relative copy number of viral gDNA intercellular 1 h postinfection. Indicated strains were infected with SNJ1 as in panel a. After 1 h of adsorption, the cells were centrifuged, and the relative copy numbers of viral gDNA intercellular were measured by qPCR using the primer pairs orf14-F/orf14-R and radA-F/radA-R, which represented proviral genome and host chromosome, respectively. The bars represent the means and standard deviations of three independent experiments. Significance testing against CJ7-F/pFJ6-Hpro-orf4 was performed using a one-sample t test (***, P < 0.001; **, P < 0.01). (c) ORF4 repressed SNJ1 genome replication. SNJ1 was incubated with CJ7 or CJ7-F/pFJ6-MCS at an MOI of 0.1 and CJ7-F/pFJ6-Hpro-orf4 at an MOI of 0.5 for 1 h. The cells were collected and washed in fresh Halo-2 medium twice to eliminate free viruses. The cells were then cultivated for 8 h, and samples were taken every hour postinfection. qPCR analyses were performed using the same primer pairs as in panel b. The bars represent the means and standard deviations of three independent experiments. Significance testing against CJ7-F/pFJ6-Hpro-orf4 was performed using a one-sample t test (***, P < 0.001; **, P < 0.01; *, P < 0.05). (d) The genomic integrity of provirus was not affected by ORF4. Experiments were performed as in panel c expect that SNJ1 was incubated at an MOI of 5, with all strains and samples for Southern blot analysis taken every 2 h after 1 h of infection. The DNA samples were electrophoresed on agarose gels and transferred onto positively charged nylon membranes with alkaline transfer buffer. A specific probe that recognized nucleotides 1483 to 2537 of SNJ1 was used for Southern blot analysis. The DNA marker were shown on the left. Lanes: C, CJ7 strain; –, CJ7-F/pFJ6-MCS strain; +, CJ7-F/pFJ6-Hpro-orf4 strain.

FIG 7

Alignment of SNJ1 with two SNJ1-like plasmids. Genomic alignment of SNJ1, Natrinema versiforme strain BOL5-4 plasmid pNVE19 (GenBank accession no. NZ_CP040333, starting from 8,613 bp) and Haloterrigena jeotgali strain A29 plasmid unnamed5 (GenBank accession no. CP031302, starting from 9,455 bp). ORFs of SNJ1 are noted in the arrows. Proteins predicted to be regulators or related to DNA replication are indicated by red or yellow arrows. Capsid proteins defined by mass spectrometric analysis previously (9) are colored orange. Characteristic conserved proteins in family Sphaerolipoviridae, including the packaging ATPase, the small major capsid protein, and the large major capsid protein, are marked in pink, light blue, and dark blue (bottom legend). Homologous ORFs in the other two plasmids are shown in the same color, and the percentages of protein identities were shown.

FIG 8

Schematic view of the life cycle of SNJ1. (i) SNJ1 proviral genome resides in J7-1 cytoplasm as plasmid pHH205 with relative copy numbers 1 to 3. ORF4 represses the expression of the lytic pathway genes, presumably by binding to the viral DNA as a dimer, thus maintains SNJ1 in the lysogenic state. (ii to v) Upon MMC treatment, ORF4 was inactivated by an unknown mechanism, resulting in expression of the lytic pathway genes and replication of viral genome by the RepA protein. Host cells are lysed, and assembled progeny viruses are released into supernatant. (vi to x) Released SNJ1 viruses infect CJ7 either by entering the lysogenic state as a plasmid or by replicating actively in a process controlled by ORF4. (xi) A lysogen of SNJ1 (J7-1) was immune to superinfection of SNJ1 because ORF4 represses replication of the ejected gDNA.