Isolation of archaeal viruses with lipid membrane from Tengchong acidic hot springs

Abstract

Archaeal viruses are one of the most mysterious parts of the virosphere because of their diverse morphologies and unique genome contents. The crenarchaeal viruses are commonly found in high temperature and acidic hot springs, and the number of identified crenarchaeal viruses is being rapidly increased in recent two decades. Over fifty viruses infecting the members of the order Sulfolobales have been identified, most of which are from hot springs distributed in the United States, Russia, Iceland, Japan, and Italy. To further expand the reservoir of viruses infecting strains of Sulfolobaceae, we investigated virus diversity through cultivation-dependent approaches in hot springs in Tengchong, Yunnan, China. Eight different virus-like particles were detected in enrichment cultures, among which five new archaeal viruses were isolated and characterized. We showed that these viruses can infect acidophilic hyperthermophiles belonging to three different genera of the family Sulfolobaceae, namely, Saccharolobus, Sulfolobus, and Metallosphaera. We also compared the lipid compositions of the viral and cellular membranes and found that the lipid composition of some viral envelopes was very different from that of the host membrane. Collectively, our results showed that the Tengchong hot springs harbor highly diverse viruses, providing excellent models for archaeal virus-host studies.

Keywords: Sulfolobaceae; Tengchong; archaeal virus; hot springs; viral membrane lipid.

Figure 1

Location of Tengchong (red solid circle) in Yunnan Province, southwestern China, and the distribution of hot springs. Scale bar, 2 m.

Figure 2

Transmission electron micrographs of the VLPs observed in enrichment cultures. (A) filamentous VLPs (870 ± 10 × 25 ± 2 nm); (B) short rod-shaped VLPs (220 ± 10 × 45 ± 5 nm, probably structural variants of spindle-shaped viruses); (C) thin rod-shaped VLPs (920 ± 20 × 20 ± 5 nm). (D) rod-shaped VLPs (750 ± 50 × 25 ± 2 nm); (E) spherical VLPs (50 ± 10 nm); (F) spherical enveloped VLPs (100 ± 90 nm, probably membrane vesicles); (G) spindle-shaped VLPs (55 ± 10 × 30 ± 12 nm); (H) tailed spindle-shaped VLPs (143 ± 20 × 85 ± 10 nm, tail 98 ± 10 nm). Scale bar, 200 nm.

Figure 3

Transmission electron micrographs of the five isolated viruses in the study. (A,B) STSV3 and STSV4 (tailed spindle-shaped virions); (C) MTIV3 (spherical virions); (D) SIFV3 (filamentous virions); (E) STIV3 (spherical enveloped virions); (F) The morphology and size of these viruses. (G) Schematic representation of the five viruses with the lipid membrane (Reuter et al., 2005; Wagner et al., 2017; Han et al., 2022; Wang et al., 2022). The inside or outside membrane is illustrated as a yellow layer, and the protein capsid is depicted in purple. (A–E) Scale bar, 100 nm. The viral particles are not drawn in scale in (G).

Figure 4

Phylogenetic tree of archaeal viruses based on whole genome analysis at the amino acid level using VICTOR (Meier-Kolthoff and Göker, 2017). The tree is rooted at mid-point, and the branch length is scaled in terms of the distance formula D6. The branch supporting value >50% is shown. For each genome, the virus name and the GenBank accession number are indicated. The newly isolated viruses from this study are marked in bold font.

Figure 5

Distribution of lipid species identified in host cells and the highly purified virions. (A) The lipid compositions and the proportion of the virus envelope and host cellular membranes; (B) Chemical diagrams for the GDGT 0–8 (GDGT, glycerol dibiphytanyl glycerol tetraether) and archaeol. Host 1: Sulfolobus sp. 1,201–2/1201–4; Host 2: Metallosphaera sp. D4-4; Host 3: Saccharolobus sp. 1,201–1; Host 4: Sulfolobus sp. D4-HJ.