In macrophages, HIV-1 assembles into an intracellular plasma membrane domain containing the tetraspanins CD81, CD9, and CD53

In macrophages, HIV-1 has been shown to bud into intracellular structures that contain the late endosome marker CD63. We show that these organelles are not endosomes, but an internally sequestered plasma membrane domain. Using immunofluorescence microscopy and immunoelectron microscopy, we find that HIV-1 buds into a compartment that contains the tetraspanins CD81, CD9, and CD53. On uninfected macrophages, these proteins are seen at the cell surface and in intracellular vacuole-like structures with a complex content of vesicles and interconnected membranes that lack endosome markers, including CD63. Significantly, these structures are accessible to small tracers (horseradish peroxidase or ruthenium red) applied to cells at 4 degrees C, indicating that they are connected to the cell surface. HIV assembles on, and accumulates within, these intracellular compartments. Furthermore, CD63 is recruited to the virus-containing structures and incorporated into virions. These results indicate that, in macrophages, HIV-1 exploits a previously undescribed intracellular plasma membrane domain to assemble infectious particles.     

Assembly of infectious HIV-1 in human epithelial and T-lymphoblastic cell lines

The canonical view of the ultimate steps of HIV-1 replication is that virus assembly and budding are taking place at the plasma membrane of infected cells. Surprisingly, recent studies revealed that these steps also occur on endosomal membranes in the interior of infected cells, such as macrophages. This prompted us to revisit the site of HIV-1 assembly in human epithelial-like cells and in infected human T-lymphoblastic cells. To address this question, we investigated the intracellular location of the major viral structural components of HIV-1, namely Gag, Env and the genomic RNA. Using a sub-cellular fractionation method, as well as immuno-confocal and electron microscopy, we show that Gag, the Env glycoproteins and the genomic RNA accumulate in late endosomes that contain infectious HIV-1 particles. In epithelial-like 293T cells, HIV-1 assembles and buds both at the plasma membrane and in endosomes, while in chronically infected human T lymphocytes, viral assembly mostly occurs within the cell where large amounts of infectious virions accumulate in endosomal compartments. In addition, HIV-1 release could be enhanced by ionomycin, a drug stimulating calcium-dependent exocytosis. These results favour the view that newly made Gag molecules associate with the genomic RNA in the cytosol, then viral core complexes can be targeted to late endosomes together with Env, where infectious HIV-1 are made and subsequently released by exocytosis.     

Infectious HIV-1 assembles in late endosomes in primary macrophages

Although human immunodeficiency virus type 1 (HIV-1) is generally thought to assemble at the plasma membrane of infected cells, virions have been observed in intracellular compartments in macrophages. Here, we investigated virus assembly in HIV-1-infected primary human monocyte-derived macrophages (MDM). Electron microscopy of cryosections showed virus particles, identified by their morphology and positive labeling with antibodies to the viral p17, p24, and envelope proteins, in intracellular vacuoles. Immunolabeling demonstrated that these compartments contained the late endosomal marker CD63, which was enriched on vesicles within these structures and incorporated into the envelope of budding virions. The virus-containing vacuoles were also labeled with antibodies against LAMP-1, CD81, and CD82, which were also incorporated into the viral envelope. To assess the cellular source of infectious viruses derived from MDM, virus-containing media from infected cells were precipitated with specific antibodies. Only antibodies against antigens found in late endosomes precipitated infectious virus, whereas antibodies against proteins located primarily on the cell surface did not. Our data indicate that most of the infectious HIV produced by primary macrophages is assembled on late endocytic membranes and acquires antigens characteristic of this compartment. This notion has significant implications for understanding the biology of HIV and its cell-cell transmission.     

Dendritic cell-mediated HIV-1 transmission to T cells of LAD-1 patients is impaired due to the defect in LFA-1

Background

Murine Leukemia Virus (MLV) assembly has been long thought to occur exclusively at the plasma membrane. Current models of retroviral particle assembly describe the recruitment of the host vacuolar protein sorting machinery to the cell surface to induce the budding of new particles. Previous fluorescence microscopy study reported the vesicular traffic of the MLV components (Gag, Env and RNA). Here, electron microscopy (EM) associated with immunolabeling approaches were used to go deeply into the assembly of the "prototypic" MLV in chronically infected NIH3T3 cells.

Results

Beside the virus budding events seen at the cell surface of infected cells, we observed that intracellular budding events could also occur inside the intracellular vacuoles in which many VLPs accumulated. EM in situ hybridization and immunolabeling analyses confirmed that these latter were MLV particles. Similar intracellular particles were detected in cells expressing MLV Gag alone. Compartments containing the MLV particles were identified as late endosomes using Lamp1 endosomal/lysosomal marker and BSA-gold pulse-chase experiments. In addition, infectious activity was detected in lysates of infected cells.

Conclusion

Altogether, our results showed that assembly of MLV could occur in part in intracellular compartments of infected murine cells and participate in the production of infectious viruses. These observations suggested that MLV budding could present similarities with the particular intracellular budding of HIV in infected macrophages.     

A novel sorting strategy of trichosanthin for hijacking human immunodeficiency virus type 1

Trichosanthin (TCS) is a type I ribosome-inactivating protein that plays dual role of plant toxin and anti-viral peptide. The sorting mechanism of such an exogenous protein is in long pursuit. Here, we examined TCS trafficking in cells expressing the HIV-1 scaffold protein Gag, and we found that TCS preferentially targets the Gag budding sites at plasma membrane or late endosomes depending on cell types. Lipid raft membrane but not the Gag protein mediates the association of TCS with viral components. After Gag budding, TCS is then released in association with the virus-like particles to generate TCS-enriched virions. The resulting TCS-enriched HIV-1 exhibits severely impaired infectivity. Overall, the observations indicate the existence of a unique and elaborate sorting strategy for hijacking HIV-1.     

Human macrophages accumulate HIV-1 particles in MHC II compartments

Macrophages are important targets for HIV-1 infection and harbor the virions in an as yet unidentified organelle. To determine the location of HIV-1 in these cells, an extensive analysis of primary human macrophages infected in vitro with HIV-1 was carried out by immuno-electron microscopy. Virus particles were found to accumulate in intracellular multivesicular compartments which were enriched in major histocompatibility complex class II molecules and CD63. These features are characteristics of major histocompatibility complex class II compartments where maturing class II molecules acquire their peptide cargo. The membrane-delimited, electron-dense virus particles of 100–110 nm diameter labeled strongly for HIV-1 p24 antigen, major histocompatibility complex class II molecules, CD63 and, to a lesser extent for HIV-1 gp120 envelope protein and Lamp 1. Our data suggest that virus particles may access the lumen of the major histocompatibility complex class II compartment by budding from the limiting membrane, thus acquiring proteins of this membrane such as class II and CD63. Viral assembly and budding would therefore occur in macrophages by a process similar to the formation of the internal vesicles in multivesicular bodies and at the same location. This could account for the particular content in lipids and proteins previously found in the membrane wrapping HIV particles. Our observations also suggest direct fusion of the virus containing major histocompatibility complex class II compartment with the plasma membrane, leading to massive release of viral particles into the extracellular medium.     

Ultrastructural analysis of ESCRT proteins suggests a role for endosome-associated tubular-vesicular membranes in ESCRT function

The endosomal sorting complex required for transport (ESCRT) is thought to support the formation of intralumenal vesicles of multivesicular bodies (MVBs). The ESCRT is also required for the budding of HIV and has been proposed to be recruited to the HIV-budding site, the plasma membrane of T cells and MVBs in macrophages. Despite increasing data on the function of ESCRT, the ultrastructural localization of its components has not been determined. We therefore localized four proteins of the ESCRT machinery in human T cells and macrophages by quantitative electron microscopy. All the proteins were found throughout the endocytic pathway, including the plasma membrane, with only around 10 and 3% of the total labeling in the cytoplasm and on the MVBs, respectively. The majority of the labeling (45%) was unexpectedly found on tubular-vesicular endosomal membranes rather than on endosomes themselves. The ESCRT labeling was twice as concentrated on early and late endosomes/lysosomes in macrophages compared with that in T cells, where it was twice more abundant at the plasma membrane. The ESCRT proteins were not redistributed on HIV infection, suggesting that the amount of ESCRT proteins located at the budding site suffices for HIV release. These results represent the first systematic ultrastructural localization of ESCRT and provide insights into its role in uninfected and HIV-infected cells.     

On the origin of the FcRIII (CD16)-containing vesicle population in human neutrophil granulocytes.

Human blood neutrophils bear two types of Fc receptors that recognize the Fc portion of immunoglobulin G: FcRII and FcRIII. In earlier studies we found that neutrophils not only express FcRIII on their plasma membrane but also contain a large population of FcRIII-containing vesicles mainly located in the juxtanuclear area. To find out whether these vesicles derive from the plasma membrane, we used electron microscopic techniques to study compartments involved in ligand-independent endocytosis in human neutrophil granulocytes. The endocytic compartments were labeled with BSA-gold. This marker entered the cell through non-coated invaginations of the plasma membrane as well as via coated pits. After internalization, BSA-gold was present in numerous electron-lucent vesicles in the juxtanuclear area and in the trans-Golgi reticulum, endosomes, and lysosome-like structures. FcRIII also occurred in the BSA-gold-containing electron-lucent vesicles in the juxtanuclear area, as shown by postembedding immunocytochemical labeling of FcRIII in cells already containing BSA-gold. Quantification showed that 29% of all FcRIII-containing vesicles also bear BSA-gold while the remaining 71% contain only the receptor. In sum, our findings show that one third of the FcRIII-containing electron-lucent vesicles in neutrophil granulocytes derive from the plasma membrane and are involved in ligand-independent endocytosis of FcRIII. The majority of these vesicles, however, are not of an endocytic origin and might constitute an "internal pool" of receptors in these cells.     

Receptor-mediated pinocytosis of IgG and immune complex in rat peritoneal macrophages: an electron microscopic study

The uptake mechanism of homologous IgG and immune complex, and the participation of coated vesicles in this process were studied in rat peritoneal macrophages. Peroxidase-antiperoxidase (PAP) immune complex produced in rat, and purified rat IgG adsorbed to gold particles (IgG-Au) were used as ligands. Freshly collected peritoneal macrophages were preincubated with the ligands at 4 degrees C, washed, warmed up to 37 degrees C, maintained in a serum-free culture medium for 5 sec to 30 min and subsequently fixed for electron microscopy. In the IgG-Au experiments, acid phosphatase reaction was also applied to identify lysosomes, and ruthenium red to trace membranes exposed to the extracellular space. At the end of the preincubation period PAP and IgG were found randomly distributed on the external surface of the plasma membrane. After warming up the cells to 37 degrees C, the ligands bound to the plasma membrane showed a tendency to move towards deep labyrinthic invaginations of the cell surface from where they were internalized via coated pits and coated vesicles. In the initial period, these structures seemed to be the primary carriers of the ligands. In the period between 5 and 10 min, ligands were concentrated in vacuoles (endosomes) located in the deeper cytoplasm, while after 30 min, they were present in large lysosome-like or multivesicular bodies, which were found to be acid phosphatase positive.     

Human immunodeficiency virus type 1 assembly, budding, and cell-cell spread in T cells take place in tetraspanin-enriched plasma membrane domains

Human immunodeficiency virus type-1 (HIV-1) egress from infected CD4+ T cells is thought to be via assembly and budding at the plasma membrane and may involve components of the T-cell secretory apparatus, including tetraspanins. However, many studies on HIV-1 assembly have examined the trafficking of viral proteins in isolation, and most have used immortalized epithelial, fibroblastic, or hematopoietic cell lines that may not necessarily reflect natural infection of susceptible T cells. Here we have used immunofluorescence and cryoimmunoelectron microscopy (CEM) to examine protein transport during HIV-1 assembly in productively infected Jurkat CD4+ T cells and primary CD4+ T cells. The HIV-1 envelope glycoprotein (Env) and the core protein (Gag) colocalize strongly with CD63 and CD81 and less strongly with CD9, whereas no colocalization was seen between Env or Gag and the late endosome/lysosomal marker Lamp2. CEM revealed incorporation of CD63 and CD81 but not Lamp2 into virions budding at the plasma membrane, and this was supported by immunoprecipitation studies, confirming that HIV-1 egress in T cells is trafficked via tetraspanin-enriched membrane domains (TEMs) that are distinct from lysosomal compartments. CD63, CD81, and, to a lesser extent, CD9 were recruited to the virological synapse (VS), and antibodies against these tetraspanins reduced VS formation. We propose that HIV-1 promotes virus assembly and cell-cell transfer in T cells by targeting plasma membrane TEMs.     

AP-3 directs the intracellular trafficking of HIV-1 Gag and plays a key role in particle assembly

Gag proteins direct the process of retroviral particle assembly and form the major protein constituents of the viral core. The matrix region of the HIV-1 Gag polyprotein plays a critical role in the transport of Gag to the plasma membrane assembly site. Recent evidence indicates that Gag trafficking to late endosomal compartments, including multivesicular bodies, occurs prior to viral particle budding from the plasma membrane. Here we demonstrate that the matrix region of HIV-1 Gag interacts directly with the delta subunit of the AP-3 complex, and that this interaction plays an important functional role in particle assembly. Disruption of this interaction eliminated Gag trafficking to multivesicular bodies and diminished HIV particle formation. These studies illuminate an early step in retroviral particle assembly and provide evidence that the trafficking of Gag to late endosomes is part of a productive particle assembly pathway.     

The Role of Cellular Factors in Promoting HIV Budding

Human immunodeficiency virus type 1 (HIV-1) becomes enveloped while budding through the plasma membrane, and the release of nascent virions requires a membrane fission event that separates the viral envelope from the cell surface. To facilitate this crucial step in its life cycle, HIV-1 exploits a complex cellular membrane remodeling and fission machinery known as the endosomal sorting complex required for transport (ESCRT) pathway. HIV-1 Gag directly interacts with early-acting components of this pathway, which ultimately triggers the assembly of the ESCRT-III membrane fission complex at viral budding sites. Surprisingly, HIV-1 requires only a subset of ESCRT-III components, indicating that the membrane fission reaction that occurs during HIV-1 budding differs in crucial aspects from topologically related cellular abscission events.     

Real-time visualization of HIV-1 GAG trafficking in infected macrophages

HIV-1 particle production is driven by the Gag precursor protein Pr55(Gag). Despite significant progress in defining both the viral and cellular determinants of HIV-1 assembly and release, the trafficking pathway used by Gag to reach its site of assembly in the infected cell remains to be elucidated. The Gag trafficking itinerary in primary monocyte-derived macrophages is especially poorly understood. To define the site of assembly and characterize the Gag trafficking pathway in this physiologically relevant cell type, we have made use of the biarsenical-tetracysteine system. A small tetracysteine tag was introduced near the C-terminus of the matrix domain of Gag. The insertion of the tag at this position did not interfere with Gag trafficking, virus assembly or release, particle infectivity, or the kinetics of virus replication. By using this in vivo detection system to visualize Gag trafficking in living macrophages, Gag was observed to accumulate both at the plasma membrane and in an apparently internal compartment that bears markers characteristic of late endosomes or multivesicular bodies. Significantly, the internal Gag rapidly translocated to the junction between the infected macrophages and uninfected T cells following macrophage/T-cell synapse formation. These data indicate that a population of Gag in infected macrophages remains sequestered internally and is presented to uninfected target cells at a virological synapse.     

Human immunodeficiency virus type 1 Gag contains a dileucine-like motif that regulates association with multivesicular bodies

Multivesicular bodies (MVBs) are cholesterol-enriched organelles formed by the endocytic pathway. The topology of vesicle formation in MVBs is identical to that of retroviral budding from the plasma membrane, and budding of human immunodeficiency virus type 1 (HIV-1) into MVBs in macrophages has recently been visualized. The Gag proteins from HIV-1, as well as many other retroviruses, contain short motifs that mediate interactions with MVBs and other endocytic components, suggesting that Gag proteins directly interface with the endocytic pathway. Here, we show that HIV-1 Gag contains an internalization signal that promotes endocytosis of a chimeric transmembrane fusion protein. Mutation of this motif within Gag strongly inhibits virus-like particle production. Moreover, wild-type Gag, but not the internalization-defective mutation, can be induced to accumulate within CD63-positive MVBs by treatment of cells with U18666A, a drug that redistributes cholesterol from the plasma membrane to MVBs. We propose that HIV-1 Gag contains a signal that promotes interaction with the cellular endocytic machinery and that the site of particle production is regulated by the subcellular distribution of cholesterol.     

SCAMP1 function in endocytosis

Secretory carrier membrane proteins (SCAMPs) are ubiquitous components of recycling vesicles that shuttle between the plasma membrane, endosomes, and the trans-Golgi complex. SCAMPs contain multiple N-terminal NPF repeats and four highly conserved transmembrane regions. NPF repeats often interact with EH domain proteins that function in budding of transport vesicles from the plasma membrane or the Golgi complex. We now show that the NPF repeats of SCAMP1 bind to two EH domain proteins, intersectin 1, which is involved in endocytic budding at the plasma membrane, and gamma-synergin, which may mediate the budding of vesicles in the trans-Golgi complex. Expression of SCAMP1 lacking the N-terminal NPF repeats potently inhibited transferrin uptake by endocytosis. Our data suggest that one of the functions of SCAMPs is to participate in endocytosis via a mechanism which may involve the recruitment of clathrin coats to the plasma membrane and the trans-Golgi network.     

Hrs function: viruses provide the clue

The endosomal protein Hrs plays a central role in the downregulation of receptors. A set of recent studies reveals a link between Hrs and the multiprotein complex ESCRT (endosome-associated complex required for transport) machinery that promotes inward vesiculation at the limiting membrane of the sorting endosome. A conserved sequence motif, PT/(S)AP, found in structural proteins of several RNA viruses (e.g. HIV Gag) promotes release of virus from the cell by recruiting the ESCRT machinery to the viral budding sites at the plasma membrane. The same motif is also found in Hrs and recruits the ESCRT I complex to endosomes through direct interaction with one of its components called TSG101. Fusion of Hrs with the gag gene of HIV-1 lacking this motif can complement a defect in virus budding. Further challenging data indicate a wider role for Hrs in the regulation of endosome dynamics.     

AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding

HIV-1 and other retroviruses exit infected cells by budding from the plasma membrane, a process requiring membrane fission. The primary late assembly (L) domain in the p6 region of HIV-1 Gag mediates the detachment of the virion by recruiting host Tsg101, a component of the class E vacuolar protein sorting (Vps) machinery. We now show that HIV Gag p6 contains a second region involved in L domain function that binds AIP1, a homolog of the yeast class E Vps protein Bro1. Further, AIP1 interacts with Tsg101 and homologs of a subunit of the yeast class E Vps protein complex ESCRT-III. AIP1 also binds to the L domain in EIAV p9, and this binding correlates perfectly with L domain function. These observations identify AIP1 as a component of the viral budding machinery, which serves to link a distinct region in the L domain of HIV-1 p6 and EIAV p9 to ESCRT-III.     

Macrophage bridging conduit trafficking of HIV-1 through the endoplasmic reticulum and Golgi network

Bridging conduits (BC) are tubular protrusions that facilitate cytoplasm and membrane exchanges between tethered cells. We now report that the human immunodeficiency virus type I (HIV-1) exploits these conduits to accelerate its spread and to shield it from immune surveillance. Endosome transport through BC drives HIV-1 intercellular transfers. How this occurs was studied in human monocyte-derived macrophages using proteomic, biochemical, and imaging techniques. Endosome, endoplasmic reticulum (ER), Golgi markers, and HIV-1 proteins were identified by proteomic assays in isolated conduits. Both the ER and Golgi showed elongated and tubular morphologies that extended into the conduits of polarized macrophages. Env and Gag antigen and fluorescent HIV-1 tracking demonstrated that these viral constituents were sequestered into endocytic and ER-Golgi organelles. Sequestered infectious viral components targeted the Golgi and ER by retrograde transport from early and Rab9 late endosomes. Disruption of the ER-Golgi network impaired HIV-1 trafficking in the conduit endosomes. This study provides, for the first time, mechanisms for how BC Golgi and ER direct cell-cell viral transfer.     

Human immunodeficiency virus type 1 entry into macrophages mediated by macropinocytosis

Whereas human immunodeficiency virus (HIV) infects various cell types by fusion at the plasma membrane, we observed a different entry route in human primary macrophages, in which macropinocytosis is active. Shortly after exposure of macrophages to HIV-1 and irrespective of viral envelope-receptor interactions, particles were visible in intracellular vesicles, which were identified as macropinosomes. Most virions appeared subsequently degraded. However, fusion leading to capsid release in the cytosol and productive infection could take place inside vesicles when particles were properly enveloped. These observations provide new insights into HIV-1 interactions with a cell target relevant to pathogenesis. They may have implications for the design of soluble inhibitors aimed at interfering with the fusion or entry processes.