{'fraction_non_alphanumeric': 0.020959290608625555, 'fraction_numerical': 0.0048367593712212815, 'mean_word_length': 4.995169082125604, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Furin cleavage of the Moloney murine leukemia virus Env precursor reorganizes the spike structure

Significance Viral membrane fusion proteins or spikes, like those of influenza, paramyxo, and retroviruses, mature by furin cleavage in the infected cell into a form that can be activated by receptor binding and/or low pH. Although the cleavage of the precursor releases the fusion peptide at the end of the transmembrane subunit, structural studies have shown that this causes only a local change in spike organization. Here we have studied the effect of furin cleavage on the fusion protein of a γ-retrovirus, the murine leukemia virus, by cryoelectron microscopy. We found that this caused a major reorganization of the spike structure. This might explain the activation of the intersubunit disulfide isomerase, which is unique for the spike of these retroviruses. The trimeric Moloney murine leukemia virus Env protein matures by two proteolytic cleavages. First, furin cleaves the Env precursor into the surface (SU) and transmembrane (TM) subunits in the cell and then the viral protease cleaves the R-peptide from TM in new virus. Here we analyzed the structure of the furin precursor, by cryoelectron microscopy. We transfected 293T cells with a furin cleavage site provirus mutant, R466G/K468G, and produced the virus in the presence of amprenavir to also inhibit the R-peptide cleavage. Although Env incorporation into particles was inhibited, enough precursor could be isolated and analyzed by cryoelectron microscopy to yield a 3D structure at 22 Å resolution. This showed an open cage-like structure like that of the R-peptide precursor and the mature Env described before. However, the middle protrusion of the protomeric unit, so prominently pointing out from the side of the more mature forms of the Env, was absent. Instead, there was extra density in the top protrusion. This suggested that the C-terminal SU domain was associated alongside the receptor binding N-terminal SU domain in the furin precursor. This was supported by mapping with a SU C-terminal domain-specific antigen binding fragment. We concluded that furin cleavage not only separates the subunits and liberates the fusion peptide at the end of TM but also allows the C-terminal domain to relocate into a peripheral position. This conformational change might explain how the C-terminal domain of SU gains the potential to undergo disulfide isomerization, an event that facilitates membrane fusion.