Isolation of a virus causing a chronic infection in the archaeal model organism Haloferax volcanii reveals antiviral activities of a provirus

Abstract

Viruses are important ecological, biogeochemical, and evolutionary drivers in every environment. Upon infection, they often cause the lysis of the host cell. However, some viruses exhibit alternative life cycles, such as chronic infections without cell lysis. The nature and the impact of chronic infections in prokaryotic host organisms remains largely unknown. Here, we characterize a novel haloarchaeal virus, Haloferax volcanii pleomorphic virus 1 (HFPV-1), which is currently the only virus infecting the model haloarchaeon Haloferax volcanii DS2, and demonstrate that HFPV-1 and H. volcanii are a great model system to study virus-host interactions in archaea. HFPV-1 is a pleomorphic virus that causes a chronic infection with continuous release of virus particles, but host and virus coexist without cell lysis or the appearance of resistant cells. Despite an only minor impact of the infection on host growth, we uncovered an extensive remodeling of the transcriptional program of the host (up to 1,049 differentially expressed genes). These changes are highlighted by a down-regulation of two endogenous provirus regions in the host genome, and we show that HFPV-1 infection is strongly influenced by a cross-talk between HFPV-1 and one of the proviruses mediated by a superinfection-like exclusion mechanism. Furthermore, HFPV-1 has a surprisingly wide host range among haloarchaea, and purified virus DNA can cause an infection after transformation into the host, making HFPV-1 a candidate for being developed into a genetic tool for a range of so far inaccessible haloarchaea.

Keywords: CRISPR; archaea; chronic infection; virus.

Fig. 1.

(A) Transmission electron micrographs of HFPV-1. Viral concentrates were sterile filtered (0.2 µm) and purified through CsCl density gradients. Particles were negatively stained with uranyl acetate and imaged at 200 kV on an FEI Tecnai TF20. (Scale bar, 100 nm.) (B) Analytic restriction digest of HFPV-1 genomic DNA with different restriction enzymes. Molecular weight size marker (L) is shown (gene ruler 1 kb; DNA ladder, Thermo Fisher Scientific). Untreated (-) and restriction digestion with enzymes HindIII, DpnI, and Bsp143I. DNA was separated on 1% agarose gels and stained with SYBR Safe (Invitrogen) at a final concentration of 1×. Data represent one sample of three biological replicates. The restriction digest patterns were observed in four repetitions.

Fig. 2.

Genome comparison of HFPV-1 with closest related pleolipoviruses. Homolog genes are indicated with the same colors. Vertical colored bars represent the percentage of identity between protein pairs. HRPV-10, HRPV-11, and HRPV-12: Halorubrum pleomorphic viruses; HGPV-1, Halogeometricum pleomorphic virus 1.

Fig. 3.

Virus life cycle: growth curve of uninfected (black circles) and infected (black triangles) H. volcanii DS2 cultures. Error bars represent the SD for biological triplicates on both treatments. Gray bars represent the number of free virus particles in the supernatant of liquid cultures, assessed through qPCR using specific probes targeting the HFPV-1 genome. Inverted gray triangles indicate time points chosen for transcriptomic analyses.

Fig. 4.

Functional profile of differentially expressed genes. Bars represent the number of differentially expressed genes assigned to each particular functional category at a given growth stage, that is, lag, exponential, and stationary phases. Functional classification of H. volcanii genome was performed using the COG database. The colors of the bars indicate whether genes are up-regulated (red) or down-regulated (blue). Genes that were not assigned to any functional category are not displayed on the plot (for detailed information, see Dataset S2).

Fig. 5.

Specific differential expression of prophage regions in the H. volcanii genome: heat map representation of gene expression profile of the host H. volcanii. The magnitudes of expression level changes are displayed as the log₂ fold change (log2 FC) for up-regulated genes (red) and down-regulated genes (blue). Each heat map ring corresponds to one of the growth phases studied, that is: lag phase, outer ring; exponential phase, middle ring; and stationary phase, inner ring. Prophage regions phylogenetically related to pleolipoviruses are zoomed in, and genes that are differentially expressed during at least one growth phase are highlighted in blue (for details, see Dataset S2).

Fig. 6.

Virus life cycle in provirus knockout strain. (A) Growth curve of uninfected (circle) and infected (black triangles) ΔHalfvol 3 mutant strains cultures. (B) VHR calculated by qPCR of virus and host gcn within cells for the parental (open circles) and the mutant strain (open squares). Error bars represent the SD for biological triplicates on both control and treatments.