Establishment of Subclones of the Severe Fever with Thrombocytopenia Syndrome Virus YG1 Strain Selected Using Low pH-Dependent Cell Fusion Activity

ABSTRACT Coronaviruses are the causative agents of respiratory disease in humans and animals, including severe acute respiratory syndrome. Fusion of coronaviruses is generally thought to occur at neutral pH, although there is also evidence for a role of acidic endosomes during entry of a variety of coronaviruses. Therefore, the molecular basis of coronavirus fusion during entry into host cells remains incompletely defined. Here, we examined coronavirus-cell fusion and entry employing the avian coronavirus infectious bronchitis virus (IBV). Virus entry into cells was inhibited by acidotropic bases and by other inhibitors of pH-dependent endocytosis. We carried out fluorescence-dequenching fusion assays of R18-labeled virions and show that for IBV, coronavirus-cell fusion occurs in a low-pH-dependent manner, with a half-maximal rate of fusion occurring at pH 5.5. Fusion was reduced, but still occurred, at lower temperatures (20°C). We observed no effect of inhibitors of endosomal proteases on the fusion event. These data are the first direct measure of virus-cell fusion for any coronavirus and demonstrate that the coronavirus IBV employs a direct, low-pH-dependent virus-cell fusion activation reaction. We further show that IBV was not inactivated, and fusion was unaffected, by prior exposure to pH 5.0 buffer. Virions also showed evidence of reversible conformational changes in their surface proteins, indicating that aspects of the fusion reaction may be reversible in nature.

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The late endosome-resident lipid bis(monoacylglycero)phosphate is a cofactor for Lassa virus fusion

10.1101/2021.03.22.436413 ◽

2021 ◽

Author(s):

Ruben M. Markosyan ◽

Mariana Marin ◽

You Zhang ◽

Fredric S. Cohen ◽

Gregory B. Melikyan

Keyword(s):

Cell Fusion ◽

New World ◽

Virus Entry ◽

Low Ph ◽

Host Cells ◽

Fusion Pore ◽

Lassa Virus ◽

Anionic Lipid ◽

Late Endosomes ◽

Ph Dependent

AbstractArenavirus entry into host cells occurs through a low pH-dependent fusion with late endosomes that is mediated by the viral glycoprotein complex (GPC). The mechanisms of GPC-mediated membrane fusion and of virus targeting to late endosomes are not well understood. To gain insights into arenavirus fusion, we examined cell-cell fusion induced by the Old World Lassa virus (LASV) GPC complex. LASV GPC-mediated cell fusion is more efficient and occurs at higher pH in cells expressing human LAMP1 compared to cells lacking this cognate receptor, but this receptor is not absolutely required for virus entry. GPC-induced fusion progresses through the same lipid intermediates as fusion mediated by other viral glycoproteins – a lipid curvature-sensitive intermediate upstream of hemifusion and a hemifusion intermediate downstream of acid-dependent steps that can be arrested in the cold. Importantly, GPC-mediated fusion is specifically augmented by an anionic lipid, bis(monoacylglycero)phosphate (BMP), which is highly enriched in late endosomes. We show that BMP promotes late steps of LASV fusion downstream of hemifusion – the formation and enlargement of fusion pores. This lipid also specifically promotes cell fusion mediated by GPC of the unrelated New World Junin arenavirus. The BMP-dependence of post-hemifusion stages of arenavirus fusion suggests that these viruses evolved to use this lipid as a cofactor to direct virus entry to late endosomes.Author SummaryPathogenic arenaviruses pose a serious health threat. The viral envelope glycoprotein GPC mediates attachment to host cells and drives virus entry via endocytosis and low pH-dependent fusion within late endosomes. Understanding the host factors and processes that are essential for arenavirus fusion may identify novel therapeutic targets. To delineate the mechanism of arenavirus entry, we examined cell-cell fusion induced by the Old World Lassa virus GPC proteins at low pH. Lassa virus fusion was augmented by the LAMP1 receptor and progressed through lipid curvature-sensitive intermediates, such as hemifusion (merger of contacting leaflets of viral and cell membrane without the formation of a fusion pore). We found that most GPC-mediated fusion events were off-path hemifusion structures and that the transition from hemifusion to full fusion and fusion pore enlargement were specifically promoted by an anionic lipid, bis(monoacylglycero)phosphate, which is highly enriched in late endosomes. This lipid also specifically promotes fusion of unrelated New World Junin arenavirus. Our results imply that arenaviruses evolved to use bis(monoacylglycero)phosphate to enter cells from late endosomes.

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Low-pH-Dependent Changes in the Conformation and Oligomeric State of the Prefusion Form of Herpes Simplex Virus Glycoprotein B Are Separable from Fusion Activity

Journal of Virology ◽

10.1128/jvi.05291-11 ◽

2011 ◽

Vol 85 (19) ◽

pp. 9964-9973 ◽

Cited By ~ 34

Author(s):

S. J. Dollery ◽

C. C. Wright ◽

D. C. Johnson ◽

A. V. Nicola

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Low Ph ◽

Glycoprotein B ◽

Fusion Activity ◽

Oligomeric State ◽

Virus Glycoprotein ◽

Herpes Simplex Virus Glycoprotein ◽

Ph Dependent ◽

Simplex Virus

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Fusogenicity of Jaagsiekte Sheep Retrovirus Envelope Protein Is Dependent on Low pH and Is Enhanced by Cytoplasmic Tail Truncations

Journal of Virology ◽

10.1128/jvi.01852-07 ◽

2007 ◽

Vol 82 (5) ◽

pp. 2543-2554 ◽

Cited By ~ 24

Author(s):

Marceline Côté ◽

Yi-Min Zheng ◽

Lorraine M. Albritton ◽

Shan-Lu Liu

Keyword(s):

Cell Fusion ◽

Conformational Changes ◽

Cultured Cells ◽

Cytoplasmic Tail ◽

Syncytium Formation ◽

Low Ph ◽

Fusion Activity ◽

Jaagsiekte Sheep Retrovirus ◽

Neutral Ph ◽

Membrane Spanning

ABSTRACT Jaagsiekte sheep retrovirus (JSRV) envelope (Env) is an active oncogene responsible for neoplastic transformation in animals and cultured cells. In this study, we used syncytium induction and fluorescence-based cell fusion assays to investigate JSRV Env fusion and its modulation by the cytoplasmic tail (CT). We found that JSRV Env induced syncytia in cells overexpressing the receptor for JSRV and that a low pH was required for this process to occur. Fusion kinetics studies revealed that cell-cell fusion by JSRV Env at neutral pH was poor, taking up to a day, in sharp contrast to fusion at low pH, which peaked within 2 min following a low-pH trigger. Deletion of the C-terminal 7 or 16 amino acids of the JSRV Env CT had no or little effect on fusion, yet additional truncation toward the membrane-spanning domain, resulting in mutants retaining as little as 1 amino acid of the CT, led to progressively increased syncytium formation at neutral pH that was further enhanced by low-pH treatment. Notably, the severely truncated mutants showed elevated levels of surface subunits in culture medium, suggesting that the CT truncations resulted in conformational changes in the ectodomain of Env that impaired surface subunit associations. Taken together, this study reveals for the first time that the fusion activity of the JSRV Env protein is dependent on a low pH and is modulated by the CT, whose truncation overcomes, at least partially, the low-pH requirement for fusion and enhances Env fusion activity and kinetics.

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