Macrophage internal HIV-1 is protected from neutralizing antibodies

In macrophages, HIV-1 accumulates in intracellular vesicles designated virus-containing compartments (VCCs). These might play an important role in the constitution of macrophages as viral reservoirs and allow HIV-1 to evade the immune system by sequestration in an internal niche, which is difficult to access from the exterior. However, until now, evidence of whether internal virus accumulations are protected from the host's humoral immune response is still lacking. In order to be able to study the formation and antibody accessibility of VCCs, we generated HIV-1 with green fluorescent protein (GFP)-tagged Gag replicating in primary macrophages. Live-cell observations revealed faint initial cytosolic Gag expression and subsequent large intracellular Gag accumulations which stayed stable over days. Taking advantage of the opportunity to study the accessibility of intracellular VCCs via the cell surface, we demonstrate that macrophage internal HIV-1-containing compartments cannot be targeted by neutralizing antibodies. Furthermore, HIV-1 was efficiently transferred from antibody-treated macrophages to T cells. Three-dimensional reconstruction of electron microscopic slices revealed that Gag accumulations correspond to viral particles within enclosed compartments and convoluted membranes. Thus, although some VCCs were connected to the plasma membrane, the complex membrane architecture of the HIV-1-containing compartment might shield viral particles from neutralizing antibodies. In sum, our study provides evidence that HIV-1 is sequestered into a macrophage internal membranous web, posing an obstacle for the elimination of this viral reservoir.     

The intracellular virus-containing compartments in primary human macrophages are largely inaccessible to antibodies and small molecules

HIV-1 assembly and release occurs at the plasma membrane of human T lymphocytes and model epithelial cell lines, whereas in macrophages intracellular sites of virus assembly or accumulation predominate. The origin of the intracellular virus-containing compartment (VCC) has been controversial. This compartment is enriched in markers of the multivesicular body, and has been described as a modified endosomal compartment. Several studies of this compartment have revealed the presence of small channels connecting to the plasma membrane, suggesting that instead of an endosomal origin the compartment is a modified plasma membrane compartment. If the compartment is accessible to the external environment, this would have important implications for antiviral immune responses and antiviral therapy. We performed a series of experiments designed to determine if the VCC in macrophages was open to the external environment and accessible to antibodies and small molecules. The majority of VCCs were found to be inaccessible to exogenously-applied antibodies to tetraspanins in the absence of membrane permeabilization, while tetraspanin staining was readily observed following membrane permeabilization. Cationized ferritin was utilized to stain the plasma membrane, and revealed that the majority of virus-containing compartments were inaccessible to ferritin. Low molecular weight dextrans could access only a very small percentage of VCCs, and these tended to be more peripheral compartments. We conclude that the VCCs in monocyte-derived human macrophages are heterogeneous, but the majority of VCCs are closed to the external environment.     

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.     

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.     

Plasma membrane signaling in HIV-1 infection

Plasma membrane is a multifunctional structure that acts as the initial barrier against infection by intracellular pathogens. The productive HIV-1 infection depends upon the initial interaction of virus and host plasma membrane. Immune cells such as CD4 + T cells and macrophages contain essential cell surface receptors and molecules such as CD4, CXCR4, CCR5 and lipid raft components that facilitate HIV-1 entry. From plasma membrane HIV-1 activates signaling pathways that prepare the grounds for viral replication. Through viral proteins HIV-1 hijacks host plasma membrane receptors such as Fas, TNFRs and DR4/DR5, which results in immune evasion and apoptosis both in infected and uninfected bystander cells. These events are hallmark in HIV-1 pathogenesis that leads towards AIDS. The interplay between HIV-1 and plasma membrane signaling has much to offer in terms of viral fitness and pathogenicity, and a better understanding of this interplay may lead to development of new therapeutic approaches. This article is part of a Special Issue entitled: Viral Membrane Proteins — Channels for Cellular Networking.     

Biochemical and biologic characterization of exosomes and microvesicles as facilitators of HIV-1 infection in macrophages

Exosomes and microvesicles (MV) are cell membranous sacs originating from multivesicular bodies and plasma membranes that facilitate long-distance intercellular communications. Their functional biology, however, remains incompletely understood. Macrophage exosomes and MV isolated by immunoaffinity and sucrose cushion centrifugation were characterized by morphologic, biochemical, and molecular assays. Lipidomic, proteomic, and cell biologic approaches uncovered novel processes by which exosomes and MV facilitate HIV-1 infection and dissemination. HIV-1 was "entrapped" in exosome aggregates. Robust HIV-1 replication followed infection with exosome-enhanced fractions isolated from infected cell supernatants. MV- and exosome-facilitated viral infections are affected by a range of cell surface receptors and adhesion proteins. HIV-1 containing exosomes readily completed its life cycle in human monocyte-derived macrophages but not in CD4(-) cells. The data support a significant role for exosomes as facilitators of viral infection.     

Differential HIV-1 Endocytosis and Susceptibility to Virus Infection in Human Macrophages Correlate with Cell Activation Status

HIV-1 is an enveloped virus that enters target cells by fusion either directly at the plasma membrane or at the endosomal membrane. The latter mechanism follows a rapid engulfment of HIV-1 after its receptor engagement at the cell surface, and its scale depends on cellular endocytosis/degradation rates and virus fusion kinetics. HIV-1 has recently been shown to exploit a novel Pak1-dependent macropinocytosis mechanism as a way to productively infect macrophages. However, macrophages are highly heterogeneous cells that can adapt functionally to their changing environment, and their endosomal/lysosomal pathway is highly regulated upon cell activation. These changes might impact the ability of HIV-1 to exploit endocytosis as a way to productively infect macrophages. In this study, we compared HIV-1 endocytosis/degradation rates in nonactivated, M1-activated, and M2a-activated monocyte-derived macrophages (MDMs). We found that the rate of HIV-1 endocytosis was increased in M1-activated but decreased in M2a-activated MDMs. However, both M1 and M2a activations of MDMs led specifically to a greater clathrin-mediated endocytosis of HIV-1, which was independent of CD4 and CCR5 binding. Furthermore, clathrin-mediated endocytosis is unlikely to result in productive HIV-1 infection, given that it leads to increased viral degradation. Therefore, we suggest that viral fusion following endocytosis is restricted in activated macrophages.     

TRAIL-mediated apoptosis in HIV-1-infected macrophages is dependent on the inhibition of Akt-1 phosphorylation

HIV-1 uses mononuclear phagocytes (monocytes, tissue macrophages, and dendritic cells) as a vehicle for its own dissemination and as a reservoir for continuous viral replication. The mechanism by which the host immune system clears HIV-1-infected macrophages is not understood. TRAIL may play a role in this process. TRAIL is expressed on the cell membrane of peripheral immune cells and can be cleaved into a soluble, secreted form. The plasma level of TRAIL is increased in HIV-1-infected patients, particularly those with high viral loads. To study the effect of elevated TRAIL on mononuclear phagocytes, we used recombinant human (rh) TRAIL and human monocyte-derived macrophages (MDM) as an in vitro model. Our results demonstrated rhTRAIL-induced apoptosis in HIV-1-infected MDM and inhibited viral replication, while having a reduced effect on uninfected MDM. HIV-1 infection significantly decreased Akt-1 phosphorylation; rhTRAIL exposure further decreased Akt-1 phosphorylation. Infection with a dominant-negative Akt-1 adenovirus potentiated rhTRAIL-induced apoptosis, while constitutively active Akt-1 blocked rhTRAIL-induced apoptosis in HIV-1-infected MDM. From this data we conclude the death ligand TRAIL preferentially provokes apoptosis of HIV-1-infected MDM, and the mechanism is reliant upon the inhibition of Akt-1 phosphorylation. Understanding this mechanism may facilitate the elimination of HIV-1-infected macrophages and lead to new therapeutic avenues for treatment of HIV-1 infection.     

Evolution of Envelope Sequences from the Genital Tract and Peripheral Blood of Women Infected with Clade A Human Immunodeficiency Virus Type 1

The development of viral diversity during the course of human immunodeficiency virus type 1 (HIV-1) infection may significantly influence viral pathogenesis. The paradigm for HIV-1 evolution is based primarily on studies of male cohorts in which individuals were presumably infected with a single virus variant of subtype B HIV-1. In this study, we evaluated virus evolution based on sequence information of the V1, V2, and V3 portions of HIV-1 clade A envelope genes obtained from peripheral blood and cervical secretions of three women with genetically heterogeneous viral populations near seroconversion. At the first sample following seroconversion, the number of nonsynonymous substitutions per potential nonsynonymous site (dn) significantly exceeded substitutions at potential synonymous sites (ds) in plasma viral sequences from all individuals. Generally, values of dn remained higher than values of ds as sequences from blood or mucosa evolved. Mutations affected each of the three variable regions of the envelope gene differently; insertions and deletions dominated changes in V1, substitutions involving charged amino acids occurred in V2, and sequential replacement of amino acids over time at a small subset of positions distinguished V3. The relationship among envelope nucleotide sequences obtained from peripheral blood mononuclear cells, plasma, and cervical secretions was evaluated for each individual by both phylogenetic and phenetic analyses. In all subjects, sequences from within each tissue compartment were more closely related to each other than to sequences from other tissues (phylogenetic tissue compartmentalization). At time points after seroconversion in two individuals, there was also greater genetic identity among sequences from the same tissue compartment than among sequences from different tissue compartments (phenetic tissue compartmentalization). Over time, temporal phylogenetic and phenetic structure was detectable in mucosal and plasma viral samples from all three women, suggesting a continual process of migration of one or a few infected cells into each compartment followed by localized expansion and evolution of that population.     

Lysosomal interconnections and horseradish peroxidase compartmentalization in three-dimensionally reconstructed bovine alveolar macrophages

Following a 1-h incubation of bovine alveolar macrophages in 1 to 2 mg/ml exogenous horseradish peroxidase (HRP), ultrathin sections revealed vacuolar interconnections among both labeled and unlabeled vacuoles constituting the lysosomal compartment. Four entire cells and their vacuolar components were subsequently computer resconstructed from serial transmission electron micrographs and measured using a morphometric technique. HRP-labeled and unlabeled vacuoles ranged in size from 0.5 micron to greater than or equal to 4.0 microns in diameter and occupied up to 25% of the cytoplasmic volume. HRP-containing vacuoles were distributed throughout each cell in a clumped distribution (P less than 0.05) and occupied up to 75% of the total vacuole compartment. Up to 60% of all vacuoles were interconnected through a series of openings formed by membrane fusions (average pore diameter 0.42 micron), which resulted in a labyrinth of vacuoles comprising up to 55% of the total volume of the lysosomal compartment. The area of open interconnections resulting from vacuolar fusions represented less than 1% of the total surface area of the lysosomal membrane. Rotation of a three-dimensionally reconstructed macrophage about the Y-axis revealed an interconnected vacuolar network of 75 fused vacuoles in a chain up to 21 microns in length. We have demonstrated that HRP-labeled vacuoles interconnect with each other as well as with preexisting unlabeled vacuoles. As a result of such interconnections, individual vacuoles become contributing members of a large, continuous, lysosomal compartment in bovine alveolar macrophages.     

Cell-type-dependent targeting of human immunodeficiency virus type 1 assembly to the plasma membrane and the multivesicular body

The human immunodeficiency virus type 1 (HIV-1) assembly-and-release pathway begins with the targeting of the Gag precursor to the site of virus assembly. The molecular mechanism by which Gag is targeted to the appropriate subcellular location remains poorly understood. Based on the analysis of mutant Gag proteins, we and others have previously demonstrated that a highly basic patch in the matrix (MA) domain of Gag is a major determinant of Gag transport to the plasma membrane. In this study, we determined that in HeLa and T cells, the MA mutant Gag proteins that are defective in plasma membrane targeting form virus particles in a CD63-positive compartment, defined as the late endosome or multivesicular body (MVB). Interestingly, we find that in primary human macrophages, both wild-type (WT) and MA mutant Gag proteins are targeted specifically to the MVB. Despite the fact that particle assembly in macrophages occurs at an intracellular site rather than at the plasma membrane, we observe that WT Gag expressed in this cell type is released as extracellular virions with high efficiency. These results demonstrate that Gag targeting to and assembly in the MVB are physiologically important steps in HIV-1 virus particle production in macrophages and that particle release in this cell type may follow an exosomal pathway. To determine whether Gag targeting to the MVB is the result of an interaction between the late domain in p6(Gag) and the MVB sorting machinery (e.g., TSG101), we examined the targeting and assembly of Gag mutants lacking p6. Significantly, the MVB localization of Gag was still observed in the absence of p6, suggesting that an interaction between Gag and TSG101 is not required for Gag targeting to the MVB. These data are consistent with a model for Gag targeting that postulates two different cellular binding partners for Gag, one on the plasma membrane and the other in the MVB.     

Interleukin-8 stimulates human immunodeficiency virus type 1 replication and is a potential new target for antiretroviral therapy

Production of the C-X-C chemokines interleukin-8 (IL-8) and growth-regulated oncogene alpha (GRO-alpha) in macrophages is stimulated by exposure to human immunodeficiency virus type 1 (HIV-1). We have demonstrated previously that GRO-alpha then stimulates HIV-1 replication in both T lymphocytes and macrophages. Here we demonstrate that IL-8 also stimulates HIV-1 replication in macrophages and T lymphocytes. We further show that increased levels of IL-8 are present in the lymphoid tissue of patients with AIDS. In addition, we demonstrate that compounds which inhibit the actions of IL-8 and GRO-alpha via their receptors, CXCR1 and CXCR2, also inhibit HIV-1 replication in both T lymphocytes and macrophages, indicating potential therapeutic uses for these compounds in HIV-1 infection and AIDS.     

HIV-1 buds predominantly at the plasma membrane of primary human macrophages

HIV-1 assembly and release are believed to occur at the plasma membrane in most host cells with the exception of primary macrophages, for which exclusive budding at late endosomes has been reported. Here, we applied a novel ultrastructural approach to assess HIV-1 budding in primary macrophages in an immunomarker-independent manner. Infected macrophages were fed with BSA-gold and stained with the membrane-impermeant dye ruthenium red to identify endosomes and the plasma membrane, respectively. Virus-filled vacuolar structures with a seemingly intracellular localization displayed intense staining with ruthenium red, but lacked endocytosed BSA-gold, defining them as plasma membrane. Moreover, HIV budding profiles were virtually excluded from gold-filled endosomes while frequently being detected on ruthenium red-positive membranes. The composition of cellular marker proteins incorporated into HIV-1 supported a plasma membrane-derived origin of the viral envelope. Thus, contrary to current opinion, the plasma membrane is the primary site of HIV-1 budding also in infected macrophages.     

The role of monocyte-lineage cells in human immuno-deficiency virus persistence: mechanisms and progress

Human immunodeficiency virus type 1(HIV-1) persistence is a major barrier to the successful treatment and eradication of acquired immunodeficiency syndrome(AIDS).In addition to resting CD4+ T cells,a significant long-lived compartment of HIV-1 infection in vivo includes blood monocytes and tissue macrophages.Studying HIV-1 persistence in monocyte-lineage cells is critical because these cells are important HIV-1 target cells in vivo.Monocyte-lineage cells,including monocytes,dendritic cells(DCs) and macrophages,play a significant role in HIV-1 infection and transmission.These cells have been implicated as viral reservoirs that facilitate HIV-1 latency and persistence.A better understanding of HIV-1 interactions with monocyte-lineage cells can potentially aid in the development of new approaches for intervention.This minireview highlights the latest advances in understanding the role of monocyte-lineage cells in HIV-1 persistence and emphasizes new insights into the mechanisms underlying viral persistence.     

Biological activity of human immunodeficiency virus type 1 Vif requires membrane targeting by C-terminal basic domains.

Human immunodeficiency virus type 1 (HIV-1) encodes a Vif protein which is important for virus replication and infectivity. Vif is a cytoplasmic protein which exists in both membrane-associated and soluble forms. The membrane-associated form is an extrinsic membrane protein which is tightly associated with the cytoplasmic side of membranes. We have analyzed the mechanism of membrane targeting of Vif and its role in HIV-1 replication. Mutagenesis studies demonstrate that C-terminal basic domains are required for membrane association. Vif mutations which disrupt membrane association also inhibit HIV-1 replication, indicating that membrane localization of Vif is likely to be required for its biological activity in vivo. Membrane binding of Vif is almost completely abolished by trypsin treatment of membranes. These results demonstrate that membrane localization of Vif requires C-terminal basic domains and interaction with a membrane-associated protein(s). This interaction may serve to direct Vif to a specific cellular site, since immunofluorescence staining and plasma membrane fractionation studies show that Vif is localized predominantly to an internal cytoplasmic compartment rather than to the plasma membrane. The mechanism of membrane targeting of Vif is different in some respects from that of other extrinsic membrane proteins, such as Ras, Src, and MARCKS, which utilize a basic domain together with a lipid modification for membrane targeting. Membrane targeting of Vif is likely to play an important role in HIV-1 replication and thus may be a therapeutic target.