(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
