Let’s phase it: viruses are master architects of biomolecular condensates

Viruses compartmentalize their replication and assembly machinery to both evade detection and concentrate the viral proteins and nucleic acids necessary for genome replication and virion production. Accumulating evidence suggests that diverse RNA and DNA viruses form replication organelles and nucleocapsid assembly sites using phase separation. In general, the biogenesis of these compartments is regulated by two types of viral protein, collectively known as antiterminators and nucleocapsid proteins, respectively. Herein, we discuss how RNA viruses establish replication organelles and nucleocapsid assembly sites, and the evidence that these compartments form through phase separation. While this review focuses on RNA viruses, accumulating evidence suggests that all viruses rely on phase separation and form biomolecular condensates important for completing the infectious cycle.     

HIV-1 buds and accumulates in "nonacidic" endosomes of macrophages

Macrophages represent viral reservoirs in HIV-1-infected patients and accumulate viral particles within an endosomal compartment where they remain infectious for long periods of time. To determine how HIV-1 survives in endocytic compartments that become highly acidic and proteolytic and to study the nature of these virus-containing compartments, we carried out an ultrastructural study on HIV-1-infected primary macrophages. The endosomal compartments contain newly formed virions rather than internalized ones. In contrast to endocytic compartments free of viral proteins within the same infected cells, the virus containing compartments do not acidify. The lack of acidification is associated with an inability to recruit the proton pump vacuolar ATPase into the viral assembly compartment. This may prevent its fusion with lysosomes, since acidification is required for the maturation of endosomes. Thus, HIV-1 has developed a strategy for survival within infected macrophages involving prevention of acidification within a devoted endocytic virus assembly compartment.     

Phosphatidylinositol 3-kinase-mediated effects of glucose on vacuolar H+-ATPase assembly, translocation, and acidification of intracellular compartments in renal epithelial cells

Vacuolar H+-ATPases (V-ATPases) are a family of ATP-driven proton pumps. They maintain pH gradients between intracellular compartments and are required for proton secretion out of the cytoplasm. Mechanisms of extrinsic control of V-ATPase are poorly understood. Previous studies showed that glucose is an important regulator of V-ATPase assembly in Saccharomyces cerevisiae. Human V-ATPase directly interacts with aldolase, providing a coupling mechanism for glucose metabolism and V-ATPase function. Here we show that glucose is a crucial regulator of V-ATPase in renal epithelial cells and that the effect of glucose is mediated by phosphatidylinositol 3-kinase (PI3K). Glucose stimulates V-ATPase-dependent acidification of the intracellular compartments in human proximal tubular cells HK-2 and porcine renal epithelial cells LLC-PK1. Glucose induces rapid ATP-independent assembly of the V1 and Vo domains of V-ATPase and extensive translocation of the V-ATPase V1 and Vo domains between different membrane pools and between membranes and the cytoplasm. In HK-2 cells, glucose stimulates polarized translocation of V-ATPase to the apical plasma membrane. The effects of glucose on V-ATPase trafficking and assembly can be abolished by pretreatment with the PI3K inhibitor LY294002 and can be reproduced in glucose-deprived cells by adenoviral expression of the constitutively active catalytic subunit p110alpha of PI3K. Taken together these data provide evidence that, in renal epithelial cells, glucose plays an important role in the control of V-ATPase-dependent acidification of intracellular compartments and V-ATPase assembly and trafficking and that the effects of glucose are mediated by PI3K-dependent signaling.     

The Protein Quality Control Machinery Regulates Its Misassembled Proteasome Subunits

Cellular toxicity introduced by protein misfolding threatens cell fitness and viability. Failure to eliminate these polypeptides is associated with various aggregation diseases. In eukaryotes, the ubiquitin proteasome system (UPS) plays a vital role in protein quality control (PQC), by selectively targeting misfolded proteins for degradation. While the assembly of the proteasome can be naturally impaired by many factors, the regulatory pathways that mediate the sorting and elimination of misassembled proteasomal subunits are poorly understood. Here, we reveal how the dysfunctional proteasome is controlled by the PQC machinery. We found that among the multilayered quality control mechanisms, UPS mediated degradation of its own misassembled subunits is the favored pathway. We also demonstrated that the Hsp42 chaperone mediates an alternative pathway, the accumulation of these subunits in cytoprotective compartments. Thus, we show that proteasome homeostasis is controlled through probing the level of proteasome assembly, and the interplay between UPS mediated degradation or their sorting into distinct cellular compartments.     

DNA Virus Replication Compartments

Viruses employ a variety of strategies to usurp and control cellular activities through the orchestrated recruitment of macromolecules to specific cytoplasmic or nuclear compartments. Formation of such specialized virus-induced cellular microenvironments, which have been termed viroplasms, virus factories, or virus replication centers, complexes, or compartments, depends on molecular interactions between viral and cellular factors that participate in viral genome expression and replication and are in some cases associated with sites of virion assembly. These virus-induced compartments function not only to recruit and concentrate factors required for essential steps of the viral replication cycle but also to control the cellular mechanisms of antiviral defense. In this review, we summarize characteristic features of viral replication compartments from different virus families and discuss similarities in the viral and cellular activities that are associated with their assembly and the functions they facilitate for viral replication.     

Microscale and nanoscale compartments for biotechnology

Compartmentalization is essential in the organization of biological systems, playing a fundamental role in modulating biochemical activity. An appreciation of the impact that biological compartments have on chemical reactions and an understanding of the physical and chemical phenomena that affect their assembly and function have inspired the development of synthetic compartments. Organic compartments assembled from amphiphilic molecules or derived from biological materials, have formed the basis of initial work in the field. However, inorganic and hybrid organic-inorganic compartments that capitalize on the optical and catalytic properties of metal and semiconductor materials are emerging. Methods for arraying these microcompartment and nanocompartment materials in higher order systems promise to enable the scaling and integration of these technologies for industrial and commercial applications.     

Traffic COPs of the early secretory pathway

Intracellular transport between the endoplasmic reticulum and Golgi compartments is mediated by coat protein complexes (COPI and COPII) that form transport vesicles and collect the desired set of cargo. Although the COPI and COPII coats are molecularly distinct, a number of mechanistic parallels appear to be emerging, most notably a general role for small guanine triphosphatases in co-ordinating coat assembly with cargo selection. A combination of morphological, biochemical, and genetic methods is revealing a very dynamic relationship between these compartments, and highlights a central role for COPs in directing traffic through the early secretory pathway. This review focuses on recent advances in molecular mechanisms underlying coated-vesicle assembly and connections with cellular structures.     

Biomolecular condensates in epithelial junctions

Epithelial junctions are transmembrane protein complexes that regulate cell adhesion, cell polarity, tissue permeability, and tissue mechanics. Most junctional complexes contain membrane attached cytoplasmic plaques that regulate junction assembly and are composed of multivalent scaffold proteins. In this review, we discuss phase separation of multivalent proteins as a general process that drives assembly of many membrane-less cellular compartments. And we summarise recent evidence that phase separation of junctional scaffold proteins is involved in the assembly of tight junctions and focal adhesions.     

β2 integrin adhesion complexes maintain the integrity of HIV-1 assembly compartments in primary macrophages

In human monocyte-derived macrophages (MDM), human immunodeficiency virus type 1 (HIV-1) assembly takes place primarily on complex intracellular plasma membrane domains connected to the cell surface by closely apposed membrane sheets or narrow channels. Some of the membranes associated with these compartments are decorated by thick (≈30 nm), electron-dense, cytoplasmic coats. Here we show by immunolabelling of ultrathin cryosections that the β2 integrin CD18, together with the αM and αX integrins (CD11b and CD11c), is clustered at these coated domains, and that the coats themselves contain the cytoskeletal linker proteins talin, vinculin and paxillin that connect the integrin complexes to the actin cytoskeleton. Intracellular plasma membrane-connected compartments (IPMC) with CD18-containing focal adhesion-like coats are also present in uninfected MDM. These compartments become more prominent as the cells mature in tissue culture and their appearance correlates with increased expression of CD18, CD11b/c and paxillin. Depletion of CD18 by RNA interference leads to parallel down-regulation of CD11b and CD11c, as well as of paxillin, and the disappearance of the adhesion-like coats. In addition, CD18 knockdown alters the appearance of virus-containing IPMC in HIV-infected MDM, indicating that the β2 integrin/focal adhesion-like coat structures are involved in the organization of these compartments.     

F-actin-dependent endocytosis of cell wall pectins in meristematic root cells. Insights from brefeldin A-induced compartments

Brefeldin A (BFA) inhibits exocytosis but allows endocytosis, making it a valuable agent to identify molecules that recycle at cell peripheries. In plants, formation of large intracellular compartments in response to BFA treatment is a unique feature of some, but not all, cells. Here, we have analyzed assembly and distribution of BFA compartments in development- and tissue-specific contexts of growing maize (Zea mays) root apices. Surprisingly, these unique compartments formed only in meristematic cells of the root body. On the other hand, BFA compartments were absent from secretory cells of root cap periphery, metaxylem cells, and most elongating cells, all of which are active in exocytosis. We report that cell wall pectin epitopes counting rhamnogalacturonan II dimers cross-linked by borate diol diester, partially esterified (up to 40%) homogalacturonan pectins, and (1-->4)-beta-D-galactan side chains of rhamnogalacturonan I were internalized into BFA compartments. In contrast, Golgi-derived secretory (esterified up to 80%) homogalacturonan pectins localized to the cytoplasm in control cells and did not accumulate within characteristic BFA compartments. Latrunculin B-mediated depolymerization of F-actin inhibited internalization and accumulation of cell wall pectins within intracellular BFA compartments. Importantly, cold treatment and protoplasting prevented internalization of wall pectins into root cells upon BFA treatment. These observations suggest that cell wall pectins of meristematic maize root cells undergo rapid endocytosis in an F-actin-dependent manner.     

Endosomes, exosomes and Trojan viruses

Retroviruses are enveloped viruses that are generally assumed to bud at the plasma membrane of infected cells. Recently it has become apparent that some of these viruses use the endocytic pathway to coordinate their assembly and release. In addition, these and some other enveloped viruses exploit the machinery that generates the internal membranes of multivesicular bodies (MVB). These observations and others have led to the suggestion that retroviruses be regarded as "viral exosomes". Here we discuss this concept and the emerging evidence that compartments of the endocytic pathway play important roles in the biogenesis of both the internal vesicles of MVB and viruses.     

Analysis of Rab GTPase-activating proteins indicates that Rab1a/b and Rab43 are important for herpes simplex virus 1 secondary envelopment

Assembly of herpes simplex virus 1 (HSV-1) occurs in the cytoplasm, where the capsid and tegument bud into host cell membranes. It is at this point that the viral glycoproteins are incorporated into the virion, as they are located at the assembly site. We investigated the role of the Rab GTPases in coordinating the assembly process by overexpressing 37 human Rab GTPase-activating proteins (GAPs) and assessing infectious titers. Rab GTPases are key cellular regulators of membrane trafficking events that, by their membrane association and binding of effector proteins, ensure the appropriate fusion of membranes. We identified that TBC1D20 and RN-tre and their partner Rabs, Rab1a/b and Rab43, respectively, are important for virion assembly. In the absence of Rab1a/b, the viral glycoproteins are unable to traffic from the endoplasmic reticulum to the assembly compartment, and thus unenveloped particles build up in the cytoplasm. The defect resulting from Rab43 depletion is somewhat more complex, but it appears that the fragmentation and dispersal of the trans-Golgi network and associated membranes render these compartments unable to support secondary envelopment.     

Signalling to and from tight junctions

Tight junctions have long been regarded as simple barriers that separate compartments of different compositions, but recent research indicates that different types of signalling proteins and transduction pathways are associated with these junctions. They receive and convert signals from the cell interior to regulate junction assembly and function, and transmit signals to the cell interior to modulate gene expression and cell behaviour.     

A dynamic machinery for import of mitochondrial precursor proteins

Mitochondria contain approximately 1000 different proteins, which are located in four different compartments, outer membrane, inner membrane, intermembrane space and matrix. The vast majority of these proteins has to be imported from the cytosol. Therefore, sophisticated molecular machineries have evolved that mediate protein translocation across or insertion into mitochondrial membranes and subsequent assembly into multi-subunit complexes. While the initial entry of virtually all mitochondrial proteins is mediated by the general import pore of the outer membrane, at least four different downstream pathways are dedicated to import and assembly of proteins into a specific compartment.     

Stochastic processes shape the biogeographic variations in core bacterial communities between aerial and belowground compartments of common bean

Although studies of biogeography in soil bacterial communities have attracted considerable attention, the generality of these patterns along with assembly processes and underlying drivers is poorly understood in the inner tissues of plants. Plant tissues provide unique ecological habitats for microorganisms, which play an essential role in plant performance. Here, we compared core bacterial communities among five soil–plant associated compartments of common bean across five sampling sites in China. Neutral and null modelling consistently suggested that stochastic processes dominated the core community assembly processes and escalated from the belowground compartments to the inner tissues of aerial plant parts. The multiple distance-decay relationships also varied and had flattened patterns in the stem endosphere, which were shaped by distinct environmental factors in each compartment. Coexistence patterns also varied in topological features, in addition with the sparsest networks in the stem endosphere resulted from the interaction with the stochastic processes. This study considerably expanded our understanding of various biogeographic patterns, assembly processes, and the underlying mechanisms of core bacterial communities between aerial and belowground compartments of common bean. That will provide a scientific basis for the reasonable regulation of core bacterial consortia to get better plant performance.     

Evidence that subcellular flagellin pools in Caulobacter crescentus are precursors in flagellum assembly

To study the assembly of the Caulobacter crescentus flagellar filament, we have devised a fractionation protocol that separates the cellular flagellin into three compartments: soluble, membrane, and assembled. Radioactive labeling in pulse-chase and pulse-labeling experiments has demonstrated for the first time that both soluble and membrane-associated flagellin pools are precursors in the assembly of the flagellar filament. The results of these experiments also indicate that flagellar filament assembly occurs via the translocation of newly synthesized flagellins from the soluble pool to the membrane pool to the assembled flagellar filaments. It is not possible to conclude whether the soluble flagellin fraction is synthesized cytoplasmically or as a loosely associated membrane intermediate which is released during lysis. It is clear, however, that the soluble and membrane flagellins are in physically and functionally distinct pools. The implications of these findings for the study of protein secretion from cells and the invariant targeting of flagellar proteins to the stalk-distal pole of the dividing cell during flagellum morphogenesis are discussed.     

Structure, dynamics and function of nuclear pore complexes

Nuclear pore complexes are large aqueous channels that penetrate the nuclear envelope, thereby connecting the nuclear interior with the cytoplasm. Until recently, these macromolecular complexes were viewed as static structures, the only function of which was to control the molecular trafficking between the two compartments. It has now become evident that this simplistic scenario is inaccurate and that nuclear pore complexes are highly dynamic multiprotein assemblies involved in diverse cellular processes ranging from the organization of the cytoskeleton to gene expression. In this review, we discuss the most recent developments in the nuclear-pore-complex field, focusing on the assembly, disassembly, maintenance and function of this macromolecular structure.     

Phosphoinositides direct equine infectious anemia virus gag trafficking and release

Phosphatidylinositol 4,5-biphosphate [PI(4,5)P(2) ], the predominant phosphoinositide (PI) on the plasma membrane, binds the matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV) with similar affinities in vitro. Interaction with PI(4,5)P(2) is critical for HIV-1 assembly on the plasma membrane. EIAV has been shown to localize in internal compartments; hence, the significance of its interaction with PI(4,5)P(2) is unclear. We therefore investigated the binding in vitro of other PIs to EIAV MA and whether intracellular association with compartments bearing these PIs was important for assembly and release of virus-like particles (VLPs) formed by Gag. In vitro, EIAV MA bound phosphatidylinositol 3-phosphate [PI(3)P] with higher affinity than PI(4,5)P(2) as revealed by nuclear magnetic resonance (NMR) spectra upon lipid titration. Gag was detected on the plasma membrane and in compartments enriched in phosphatidylinositol 3,5-biphosphate [PI(3,5)P(2) ]. Treatment of cells with YM201636, a kinase inhibitor that blocks production of PI(3,5)P(2) from PI(3)P, caused Gag to colocalize with aberrant compartments and inhibited VLP release. In contrast to HIV-1, release of EIAV VLPs was not significantly diminished by coexpression with 5-phosphatase IV, an enzyme that specifically depletes PI(4,5)P(2) from the plasma membrane. However, coexpression with synaptojanin 2, a phosphatase with broader specificity, diminished VLP production. PI-binding pocket mutations caused striking budding defects, as revealed by electron microscopy. One of the mutations also modified Gag-Gag interaction, as suggested by altered bimolecular fluorescence complementation. We conclude that PI-mediated targeting to peripheral and internal membranes is a critical factor in EIAV assembly and release.