Melikian, G. | bioRxiv Science

bioRxiv2023

Human Immunodeficiency Virus 1 Capsid Uncoating in the Nucleus Progresses Through Defect Formation in the Capsid Lattice

The HIV-1 core consists of a cone-shaped capsid shell made of [~]250 capsid protein (CA) hexamers and 12 pentamers encapsulating the viral genome. HIV-1 capsid disassembly, referred to as uncoating, is a highly regulated process that is important for productive infection, however, the location, timing, and regulation of uncoating remain controversial. Here, we employ amber codon suppression to directly label CA and visualize capsid trafficking and uncoating in live cells. In addition to direct CA labeling, a fluid phase fluorescent probe is incorporated into the viral core to detect the formation of small defects in the capsid lattice. This double-labeling strategy does not significantly impact HIV-1 infectivity, maturation, nuclear import, or capsid stability. Single virus tracking reveals nuclear import of intact cores defined as complexes containing both the fluid phase marker and robust CA signal. Subsequent uncoating of HIV-1 cores in the nucleus is manifested by a sequential loss of both fluorescent markers. This two-step uncoating - release of the core content marker followed by loss of CA - is observed in different cells, including a macrophage line. Importantly, the lag between the two steps of uncoating ([~]30 min) appears independent of the cell type and is much longer than upon uncoating of cell-free viruses. These data suggest that HIV- 1 uncoating in the nucleus is initiated through a localized defect in the capsid lattice that precedes a global loss of CA. Our results imply that intact HIV-1 cores enter the cell nucleus and uncoat in a stepwise fashion, before integrating into the host genome.

microbiology↗

bioRxiv2021

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

Arenavirus 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.

microbiology↗