Particles that bud off from the cell surface, including viruses and microvesicles, typically have a unique membrane protein composition distinct from that of the originating plasma membrane. This selective protein composition enables viruses to evade the immune response and infect other cells. But how membrane proteins sort into budding viruses such as human immunodeficiency virus (HIV) remains unclear. Proteins could passively distribute into HIV-assembly-site membranes producing compositions resembling pre-existing plasma-membrane domains. Here, we demonstrate that proteins instead sort actively into HIV-assembly-site membranes, generating compositions enriched in cholesterol and sphingolipids that undergo continuous remodelling. Proteins are recruited into and removed from the HIV assembly site through lipid-based partitioning, initiated by oligomerization of the HIV structural protein Gag. Changes in membrane curvature at the assembly site further amplify this sorting process. Thus, a lipid-based sorting mechanism, aided by increasing membrane curvature, generates the unique membrane composition of the HIV surface.

A lipid-based partitioning mechanism for selective incorporation of proteins into membranes of HIV particles.

Phosphoinositides play a crucial role in regulating many cellular functions, such as actin dynamics, signaling, intracellular trafficking, membrane dynamics, and cell-matrix adhesion. Central to this process is phosphatidylinositol bisphosphate (PIP2). The levels of PIP2 in the membrane are rapidly altered by the activity of phosphoinositide-directed kinases and phosphatases, and it binds to dozens of different intracellular proteins. Despite the vast literature dedicated to understanding the regulation of PIP2 in cells over past 30 years, much remains to be learned about its cellular functions. In this review, we focus on past and recent exciting results on different molecular mechanisms that regulate cellular functions by binding of specific proteins to PIP2 or by stabilizing phosphoinositide pools in different cellular compartments. Moreover, this review summarizes recent findings that implicate dysregulation of PIP2 in many diseases.

Review of PIP2 in Cellular Signaling, Functions and Diseases.

Viruses are highly dependent on the host they infect. Their dependence triggers processes of virus–host co-adaptation, enabling viruses to explore host resources whilst escaping immunity. Scientists have tackled viral–host interplay at differing levels of complexity—in individual hosts, organs, tissues and cells—and seminal studies advanced our understanding about viral lifecycles, intra- or inter-species transmission, and means to control infections. Recently, it emerged as important to address the physical properties of the materials in biological systems; membrane-bound organelles are only one of many ways to separate molecules from the cellular milieu. By achieving a type of compartmentalization lacking membranes known as biomolecular condensates, biological systems developed alternative mechanisms of controlling reactions. The identification that many biological condensates display liquid properties led to the proposal that liquid–liquid phase separation (LLPS) drives their formation. The concept of LLPS is a paradigm shift in cellular structure and organization. There is an unprecedented momentum to revisit long-standing questions in virology and to explore novel antiviral strategies. In the first part of this review, we focus on the state-of-the-art about biomolecular condensates. In the second part, we capture what is known about RNA virus-phase biology and discuss future perspectives of this emerging field in virology.

Liquid Biomolecular Condensates and Viral Lifecycles: Review and Perspectives

HIV-1 Gag protein assembles at the plasma membrane of infected cells for viral particle formation. Gag targets lipids, mainly PI(4,5)P2, at the inner leaflet of this membrane. Here, we address the question whether Gag is able to trap specifically PI(4,5)P2 or other lipids during HIV-1 assembly in the host CD4+ T lymphocytes. Lipid dynamics within and away from HIV-1 assembly sites were determined using super-resolution microscopy coupled with scanning fluorescence correlation spectroscopy in living cells. Analysis of HIV-1–infected cells revealed that, upon assembly, HIV-1 is able to specifically trap PI(4,5)P2 and cholesterol, but not phosphatidylethanolamine or sphingomyelin. Furthermore, our data showed that Gag is the main driving force to restrict the mobility of PI(4,5)P2 and cholesterol at the cell plasma membrane. This is the first direct evidence highlighting that HIV-1 creates its own specific lipid environment by selectively recruiting PI(4,5)P2 and cholesterol as a membrane nanoplatform for virus assembly.

https://advances.sciencemag.org/content/advances/5/10/eaaw8651.full.pdf

HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly.

Gag, the primary structural protein of HIV-1, is recruited to the plasma membrane for virus assembly by its matrix (MA) domain. Gag is subsequently cleaved into its component domains, causing structural maturation to repurpose the virion for cell entry. We determined the structure and arrangement of MA within immature and mature HIV-1 through cryo-electron tomography. We found that MA rearranges between two different hexameric lattices upon maturation. In mature HIV-1, a lipid extends out of the membrane to bind with a pocket in MA. Our data suggest that proteolytic maturation of HIV-1 not only assembles the viral capsid surrounding the genome but also repurposes the membrane-bound MA lattice for an entry or postentry function and results in the partial removal of up to 2500 lipids from the viral membrane.

Maturation of the matrix and viral membrane of HIV-1.

Viruses are parasites that must infect the living cell of a host in order to grow and replicate To do so, the virus attaches to the host’s cell and transfers its genetic material to the inside The virus then hijacks the cell and forces it to build proteins and more genetic material that will assemble into new copies of the virus, and the completed virus particles are released to infect new cells The HIV-1 virus encodes a protein called Gag that coordinates the assembly of new copies of this virus Thousands of Gag proteins accumulate into a lattice at the inside of the host cell membrane by an unknown mechanism, where they gather all essential components to form a new infectious virus Once completed, the virus particle detaches itself from the host cell and Gag gives the virus its structure Previous research by several groups has shown that a lipid molecule found in cell membranes called PIP2 can bind to Gag and helps the virus to assemble However, until now, it was unclear if PIP2 anchors Gag to the cell membrane of living cells and if it plays any other roles in the later stages of assembly Now, Mucksch et al have developed a new approach to study this question by rapidly manipulating the levels of PIP2 in living cells and monitoring Gag assembly through a fluorescent marker The experiments showed that PIP2 was needed to initiate the assembly process, but also to maintain the partially assembled Gag lattice at the membrane When PIP2 was removed, Gag proteins could not gather at the cell membrane and the already assembled Gag lattices broke down When PIP2 levels were increased again, the Gag proteins that had disappeared from the membrane formed new lattices This suggests that PIP2 has a much broader role in HIV-1 particle formation than previously assumed A next step will be to use this new experimental approach to study how particles of HIV are assembled and released This knowledge may help scientists to develop antiviral drugs that interfere with the assembly step of the virus that could be used to prevent or treat HIV infections

Synchronized HIV assembly by tunable PIP2 changes reveals PIP2 requirement for stable Gag anchoring

Human immunodeficiency virus type 1 (HIV-1) acquires its lipid envelope during budding from the plasma membrane of the host cell. Various studies indicated that HIV-1 membranes differ from producer cell plasma membranes, suggesting budding from specialized membrane microdomains. The phosphoinositide PI(4,5)P2 has been of particular interest since PI(4,5)P2 is needed to recruit the viral structural polyprotein Gag to the plasma membrane and thus facilitates viral morphogenesis. While there is evidence for an enrichment of PIP2 in HIV-1, fully quantitative analysis of all phosphoinositides remains technically challenging and therefore has not been reported, yet. Here, we present a comprehensive analysis of the lipid content of HIV-1 and of plasma membranes from infected and non-infected producer cells, resulting in a total of 478 quantified lipid compounds, including molecular species distribution of 25 different lipid classes. Quantitative analyses of phosphoinositides revealed strong enrichment of PIP2, but also of PIP3, in the viral compared to the producer cell plasma membrane. We calculated an average of ca. 8,000 PIP2 molecules per HIV-1 particle, three times more than Gag. We speculate that the high density of PIP2 at the HIV-1 assembly site is mediated by transient interactions with viral Gag polyproteins, facilitating PIP2 concentration in this microdomain. These results are consistent with our previous observation that PIP2 is not only required for recruiting, but also for stably maintaining Gag at the plasma membrane. We believe that this quantitative analysis of the molecular anatomy of the HIV-1 lipid envelope may serve as standard reference for future investigations.

/pdf/quantification-of-phosphoinositides-reveals-strong-49d7phndhk.pdf

Quantification of phosphoinositides reveals strong enrichment of PIP 2 in HIV-1 compared to producer cell membranes

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Maturation of the matrix and viral membrane of HIV-1.

Synchronized HIV assembly by tunable PIP2 changes reveals PIP2 requirement for stable Gag anchoring

Quantification of phosphoinositides reveals strong enrichment of PIP 2 in HIV-1 compared to producer cell membranes