Abstract
(1) Background: Haloarchaea comprise extremely halophilic organisms of the Archaea domain. They are single-cell organisms with distinctive membrane lipids and a protein-based cell wall or surface layer (S-layer) formed by a glycoprotein array. Pleolipoviruses, which infect haloarchaeal cells, have an envelope analogous to eukaryotic enveloped viruses. One such member, Halorubrum pleomorphic virus 6 (HRPV-6), has been shown to enter host cells through virus-cell membrane fusion. The HRPV-6 fusion activity was attributed to its VP4-like spike protein, but the physiological trigger required to induce membrane fusion remains yet unknown. (2) Methods: We used SDS-PAGE mass spectroscopy to characterize the S-layer extract, established a proteoliposome system, and used R18-fluorescence dequenching to measure membrane fusion. (3) Results: We show that the S-layer extraction by Mg2+ chelating from the HRPV-6 host, Halorubrum sp. SS7-4, abrogates HRPV-6 membrane fusion. When we in turn reconstituted the S-layer extract from Hrr. sp. SS7-4 onto liposomes in the presence of Mg2+, HRPV-6 membrane fusion with the proteoliposomes could be readily observed. This was not the case with liposomes alone or with proteoliposomes carrying the S-layer extract from other haloarchaea, such as Haloferax volcanii. (4) Conclusions: The S-layer extract from the host, Hrr. sp. SS7-4, corresponds to the physiological fusion trigger of HRPV-6.
Highlights
Archaeal viruses are highly diverse in shape [1,2,3] and several are enveloped, including members of the Pleolipoviridae family [4,5,6,7]
SS7-4NP by EDTA treatment, generating spheroplasts [47]. These spheroplasts were mixed with R18-labeled Halorubrum pleomorphic virus 6 (HRPV-6) particles, and the dequenching of the dye by lipid mixing was monitored over time at different temperatures (Figure 1A)
This data confirms that, as with liposom can be triggered with spheroplasts at 55 °C and that at the optimal growth temp 37 °C, an intact surface layer (S-layer) seems to play a critical role in triggering HRPV-6 membra and inf3e7c◦tiCo, nan. intact S-layer seems to play a critical role in triggering HRPV-6 membrane fusion and infection
Summary
Archaeal viruses are highly diverse in shape [1,2,3] and several are enveloped, including members of the Pleolipoviridae family [4,5,6,7]. The entry mechanisms of archaeal viruses are in general poorly characterized, but it is likely that archaeal viruses, like their bacterial counterparts, recognize and attach to cell wall components, such as S-layer proteins, sugar moieties, or filamentous surface structures, such as pili [9,10,11,12,13,14]. The sole constituent of the cell wall is the proteinaceous surface layer (S-layer) that encloses the cellular membrane and functions as a protective coat that covers the whole cell [15,16,17,18]. The S-layer is organized into a hexameric array of protein subunits consisting of six highly conserved immunoglobulin-like domains; these hexamers cover the whole cell with pentameric defects [21].
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