The entry of the Picornaviridae virus family in resident macrophages

For viruses, the following mechanisms of penetration into cells are typical: clathrin- or dinaminmediated endocytosis, the formation of caveolae, local lysis of cell membranes, and macropinocytosis. It is accepted that (those nonenveloped viruses in the Picornaviridae family) enter cells mostly through the local lysis of their membranes. The purpose of the present study is to research the mechanisms of penetration into resident macrophages of viruses of the indicated family, including poliovirus, Echol1 and Coxsackie B1 viruses, and Type 71 enterovirus. It has been detected that, at the adhesion sites of the Coxsackie B1 virus and Type 71 enterovirus on a macrophage surface, invaginations of the plasma membranes of cells appear, followed by the consequent formation of endocytoplasmatic vesicles, i.e., caveolae. The penetration of poliovirus into macrophages occurs both through the formation of caveolae and the local lysis of the plasmolemma of cells; during the later terms (after 45 min), macropinocytosis is observed in the viral particles during the first 15 min after the Echol1 virus penetrated the cytoplasm of macrophages through the local lysis of their plasmolemma. Thereafter, the formation of endocytic vacuolae including viral particles was observed in the cytoplasm of infected macrophages. The exit of the Echol1 virus from endocytic vacuoles was performed by the local lysis of cell membrane.     

Evidence for the recycling nature of the fibronectin receptor of macrophages

Plasma fibronectin (pFN) has been shown to mediate phagocytosis of several types of artificial particles and tissue debris by macrophages. In the present investigation some of the dynamic aspects of this receptor-mediated cellular process have been studied. Plasma fibronectin did not bind specifically to fibronectin (FN)-receptors of rat peritoneal macrophages at either 4 degrees C or 37 degrees C. On the other hand, pFN aggregated on the surface of gelatin-coated latex beads (gLtx) and 125I-labeled pFN covalently coupled to latex beads (pFN-Ltx) bound strongly to macrophages at both temperatures. Both of these particles were also internalized at 37 degrees C. Treatment of macrophages by chymotrypsin, thermolysin, or trypsin in a protein-free tissue culture medium did not affect either of the above reactions; however, pronase treatment strongly reduced both the binding and internalization of the pFN-coated particles. The pronase-treated macrophage monolayers in time regained their ability to bind and internalize pFN-gLtx when incubated in fresh tissue culture medium. Such recovery, however, did not take place when the medium contained cycloheximide. On the other hand, phagocytosis of pFN-gLtx was not affected directly by cycloheximide with untreated macrophages; this suggests that the FN-receptor recycles during sustained phagocytosis. This assumption was substantiated by the observations that some of the established lysosomotropic amines--i.e., chloroquine, dansylcadaverine, and dimethyldansylcadaverine--caused total inhibition of internalization without affecting the binding of particles to macrophages. Furthermore, chloroquine protected the FN-receptors against destruction by pronase. Together these results suggest that macrophage receptors for FN are protein, present both on the cell surface and intracellularly, and recycle between the plasma membrane and intracellular sites during phagocytosis.     

Regeneration of plasmalemma and surface properties in macrophages

The regeneration of surface anionic groups in mouse peritoneal macrophages was investigated by electron microscopy, using cationized ferritin (CF) as a tool for the localization and evaluation of negative charge density on the cell surface. In vitro interaction of living macrophages with CF resulted in removal of most anionic groups, either by concentration of their receptor sites to a part of the membrane which is subsequently internalized, or by detachment of the aggregated label from the surface. After incubation of macrophages lacking surface anionic groups in tissue culture medium without the ligand, regeneration of the binding capacity for CF took place within 3 h. The first regenerated parts of the membrane can be visualized within 1 h on the upper part of the adherent cells; there is a discontinuous coating of ferritin, with the lateral regions of the plasmalemma free of label. The attached CF particles on the regenerated membrane are closer to the membrane and their density is considerably higher than on the normal control macrophages. The results indicate that the turnover of the plasmalemma is regional and not dispersed; the mechanism involved is insertion of membrane patches into the pre-existing plasma membrane.     

Modeling the step of endosomal escape during cell infection by a nonenveloped virus

Widely disparate viruses enter the host cell through an endocytic pathway and travel the cytoplasm inside an endosome. For the viral genetic material to be delivered into the cytoplasm, these viruses have to escape the endosomal compartment, an event triggered by the conformational changes of viral endosomolytic proteins. We focus here on small nonenveloped viruses such as adeno-associated viruses, which contain few penetration proteins. The first time a penetration protein changes conformation defines the slowest timescale responsible for the escape. To evaluate this time, we construct what to our knowledge is a novel biophysical model based on a stochastic approach that accounts for the small number of proteins, the endosomal maturation, and the protease activation dynamics. We show that the escape time increases with the endosomal size, whereas decreasing with the number of viral particles inside the endosome. We predict that the optimal escape probability is achieved when the number of proteases in the endosome is in the range of 250-350, achieved for three viral particles.     

Role of low CD4 levels in the influence of human immunodeficiency virus type 1 envelope V1 and V2 regions on entry and spread in macrophages

Human immunodeficiency virus type 1 (HIV-1) isolates vary in their ability to infect macrophages. Previous experiments have mapped viral determinants of macrophage infectivity to the V3 hypervariable region of the HIV-1 envelope glycoprotein. In our earlier studies, V1 and V2 sequences of HIV-1 were also shown to alter the ability of virus to spread in macrophage cultures, whereas no effect was seen in lymphocyte cultures. In the present study, determinants that allowed certain HIV-1 clones to infect and spread in macrophages were primarily mapped to the V2 region and were found to act by influencing early events of viral infection. By an assay of viral entry into macrophages, it was shown that viruses with the V2 region from the Ba-L strain of HIV-1 had >10-fold-higher entry efficiency than viruses with the V2 region derived from the NL4-3 strain. V1 region differences between these groups caused a twofold difference in entry. The known low expression of CD4 on macrophages appeared to be important in this process. In entry assays conducted with HeLa cell lines expressing various levels of CD4 and CCR5, low levels of CD4 influenced the efficiency of entry and fusion which were dependent on viral V1 and V2 envelope sequences. In contrast, no effect of V1 or V2 was seen in HeLa cells expressing high levels of CD4. Thus, the limited expression of CD4 on macrophages or other cell types could serve as a selective factor for V1 and V2 envelope sequences, and this selection could in turn influence many aspects of AIDS pathogenesis in vivo.     

Phagocytic response to fully controlled plural stimulation of antigens on macrophage using on-chip microcultivation system

To understand the control mechanism of innate immune response in macrophages, a series of phagocytic responses to plural stimulation of antigens on identical cells was observed. Two zymosan particles, which were used as antigens, were put on different surfaces of a macrophage using optical tweezers in an on-chip single-cell cultivation system, which maintains isolated conditions of each macrophage during their cultivation. When the two zymosan particles were attached to the macrophage simultaneously, the macrophage responded and phagocytosed both of the antigens simultaneously. In contrast, when the second antigen was attached to the surface after the first phagocytosis had started, the macrophage did not respond to the second stimulation during the first phagocytosis; the second phagocytosis started only after the first process had finished. These results indicate that (i) phagocytosis in a macrophage is not an independent process when there are plural stimulations; (ii) the response of the macrophage to the second stimulation is related to the time" delay from the first stimulation. Stimulations that occur at short time intervals resulted in simultaneous phagocytosis, while a second stimulation that is delayed long enough might be neglected until the completion of the first phagocytic process.     

Viral proteomics: global evaluation of viruses and their interaction with the host

Viruses constantly adapt to and modulate the host environment during replication and propagation. Both DNA and RNA viruses encode multifunctional proteins that interact with and modify host cell proteins. While viral genomes were the first complete sequences known, the corresponding proteomes are only now elucidated, with some surprising results. Even more daunting is the task to globally monitor the impact of viral infection on the proteome of the host cell and many technical hurdles must still be overcome in order to facilitate robust and reproducible measurements. Further complicating the picture is the dynamic nature of proteins, including post-translational modifications, enzymatic cleavage and activation or destruction by proteolytic events. Nevertheless, several promising studies have been published using high-throughput methods directly measuring protein abundance. Particularly, quantitative or semiquantitative mass spectrometry-based analysis of viral and cellular proteomes are now being used to characterize viruses and their host interaction. In addition, the full set of interactions between viral and host proteins, the interactome, is beginning to emerge, with often unexpected interactions that need to be carefully validated. In this review, we will discuss two major areas of viral proteomics: first, virion proteomics (such as the protein characterization of viral particles) and second, proteoviromics, including the viral protein interactomics and the quantitative analysis of host cell proteome during viral infection.     

Viral proteomics: global evaluation of viruses and their interaction with the host

Viruses constantly adapt to and modulate the host environment during replication and propagation. Both DNA and RNA viruses encode multifunctional proteins that interact with and modify host cell proteins. While viral genomes were the first complete sequences known, the corresponding proteomes are only now elucidated, with some surprising results. Even more daunting is the task to globally monitor the impact of viral infection on the proteome of the host cell and many technical hurdles must still be overcome in order to facilitate robust and reproducible measurements. Further complicating the picture is the dynamic nature of proteins, including post-translational modifications, enzymatic cleavage and activation or destruction by proteolytic events. Nevertheless, several promising studies have been published using high-throughput methods directly measuring protein abundance. Particularly, quantitative or semiquantitative mass spectrometry-based analysis of viral and cellular proteomes are now being used to characterize viruses and their host interaction. In addition, the full set of interactions between viral and host proteins, the interactome, is beginning to emerge, with often unexpected interactions that need to be carefully validated. In this review, we will discuss two major areas of viral proteomics: first, virion proteomics (such as the protein characterization of viral particles) and second, proteoviromics, including the viral protein interactomics and the quantitative analysis of host cell proteome during viral infection.     

Structural modifications of macrophages initiated by tick-borne encephalitis virus

Macrophages are cells of natural immunity and play a key role in pathogenesis of viral infections. Results of ultrastructural research on macrophages infected with tick-borne encephalitis virus (TBEV), an agent that causes dangerous infections affecting nervous system in human beings and belongs to the Flaviviridae family, were shown here. Using virology methods, it was ascertained that the TBEV is consumed by macrophages and multiplies in them. Ultrastructural research showed that the virus penetrates into the cytoplasm by means of local plasmalemma lysis and newly synthesized virus particles escape from the cell by the same path. At the same time, induration of the perinuclear space of cytoplasm was observed, where ribosomes, microfilaments, ribonucleoprotein threats, and virus-specific structures, namely, nucleocapsids, tube formations, and viroplasts, were found in large quantities. On the surface of viroplasts, newly synthesized virus particles were visualized. Thus, evidence was presented that microphages can play certain role in spreading of TBEV and are the target of the virus. Like active antigen-presenting cells, such macrophages can modulate the protective response of an organism and affect the pathogenesis of tick-borne encephalitis.     

Lymphatic dissemination and comparative pathology of recombinant measles viruses in genetically modified mice

The dissemination of the Edmonston measles virus (Ed-MV) vaccine strain was studied with genetically modified mice defective for the alpha/beta interferon receptor and expressing human CD46 with human-like tissue specificity and efficiency. A few days after intranasal infection, macrophages expressing Ed-MV RNA were detected in the lungs, in draining lymph nodes, and in the thymus. In lymph nodes, large syncytia which stained positive for viral RNA and for macrophage surface marker proteins were found and apoptotic cell death was monitored. In the thymus, smaller syncytia which stained positive for macrophage and dendritic cell markers were detected. Thus, macrophages appear to be the main vectors for dissemination of MV infection in these mice; human macrophages may have a similar function in the natural host. We then compared the pathogenicities of two recombinant viruses lacking the C or V nonstructural proteins to that of the parental strain, Ed-MV. These viruses were less effective in spreading through the lymphatic system and, unlike Ed-MV, were not detected in the liver. After intracerebral inoculation the recombinant viruses caused lethal disease less often than Ed-MV and induced distinctive patterns of gliosis and inflammation. Ed-MV was reisolated from brain tissue, but its derivatives were not. C- and V-defective viruses should be considered as more-attenuated MV vaccine candidates.     

Development of surface membrane characteristics of "premedullary" macrophages in organ cultures of embryonic rat and hamster lungs

A replicating population of non-monocyte-derived free cells appears in organ-cultured embryonic rat lungs, indistinguishable from alveolar macrophages by classical criteria such as ultrastructure, lysosomal enzyme cytochemistry, and phagocytic behavior. We demonstrate similar events in cultured embryonic hamster lungs and development of macrophage-associated properties on the plasmalemma of these cells in both species. Immunoperoxidase localizations were obtained using monoclonal antibodies against alveolar macrophage antigen (HAM1) in hamsters, and rat macrophage antigen (ED1) and leukocyte-common antigen (OX1) in rats. Fc and C3b receptors were identified in both species by immune rosetting. HAM1 staining, perinuclear in rare cells at explantation, gains definitive surface localization 3-4 days later as cells prepare to emerge through the pleura. ED1 and OX1 cytoplasmic staining first occurs after 24 hr, increases as macrophages multiply and congregate beneath the pleura, and translocates to the plasmalemma of emerged cells. Some glass-adherent cells from lung explants have Fc receptors. The proportion rises sharply for 24 hr and equals fully emerged cells (90-95%) by days 3-4. At first phagocytosis is slow to follow Fc receptor binding, but ingestion time decreases to 3-10 min as macrophages mature. A minority of emerged macrophages bind complement-opsonized erythrocytes, which are rarely taken up. These properties are shared by alveolar macrophages of adults.     

Visualization of reactive oxygen species formation by phagocytizing macrophages

Activated peritoneal macrophages exhibiting phagocytosing capacity produced an electron-dense precipitate of formazan in contact sites of macrophage plasmalemma and phagocytosed yeast cells. No production of formazan occurred, when non-opsonized latex particles were ingested by macrophages. Formazan precipitation could be prevented by anaerobiosis but not by addition of cyanide.     

Direct observation of the budding and fusion of an enveloped virus by video microscopy of viable cells

Video-enhanced microscopy and digital image processing were used to observe the assembly, budding, and fusion of Respiratory Syncytial virus. Viral filaments were seen to bud from the plasma membrane of viable infected cells to a final length of 5-10 micron with an average speed of elongation of 110-250 nm/s. The rapidity of viral assembly and its synchronous occurrence (leading to the production of several viral particles per minute from the same surface domain) suggests a directed process of recruitment of viral components to an area selected for virus maturation. Virions were also seen to adsorb to the cell surface, and to fuse with the plasma membrane. These are the first real time observations of viral morphogenesis and penetration which are crucial events in the infectious cycle of enveloped viruses.     

Biochemical markers of virus cytopathogenicity in macrophages

The results of macrophage metabolism studies at their infection by viruses differing in the level of virulence are presented. With the purpose of optimizing the estimation of viral cytopathogenic effects on macrophages, an index of cell reactions, which allows one to reveal the degree of virus influence in standard units, is offered. Generally, the application of high-sensitivity methods for functional activity determination and identification of the correlative communication between its changes and morphological features of cells can be prescribed to objective identification methods of not only viral reproduction, but also differentiation of types and the degree of their cytopathogenic effects.     

Candida albicans infection inhibits macrophage cell division and proliferation

The pathogenicity of the opportunistic human fungal pathogen Candida albicans depends on its ability to inhibit effective destruction by host phagocytes. Using live cell video microscopy, we show here for the first time that C. albicans inhibits cell division in macrophages undergoing mitosis. Inhibition of macrophage cell division is dependent on the ability of C. albicans to form hyphae, as it is rarely observed following phagocytosis of UV-killed or morphogenesis-defective mutant Candida. Interestingly, failed cell division following phagocytosis of hyphal C. albicans is surprisingly common, and leads to the formation of large multinuclear macrophages. This raises question as to whether inhibition of macrophage cell division is another virulence attribute of C. albicans or enables host macrophages to contain the pathogen.     

Endoplasmic reticulum-mediated phagocytosis is a mechanism of entry into macrophages

Phagocytosis is a key aspect of our innate ability to fight infectious diseases. In this study, we have found that fusion of the endoplasmic reticulum (ER) with the macrophage plasmalemma, underneath phagocytic cups, is a source of membrane for phagosome formation in macrophages. Successive waves of ER become associated with maturing phagosomes during phagolysosome biogenesis. Thus, the ER appears to possess unexpectedly pluripotent fusion properties. ER-mediated phagocytosis is regulated in part by phosphatidylinositol 3-kinase and used for the internalization of inert particles and intracellular pathogens, regardless of their final trafficking in the host. In neutrophils, where pathogens are rapidly killed, the ER is not used as a major source of membrane for phagocytosis. We propose that intracellular pathogens have evolved to adapt and exploit ER-mediated phagocytosis to avoid destruction in host cells.     

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.     

Application of freeze-drying intact cells to studies of murine oncornavirus morphogenesis.

Using a method for freeze-drying intact cells, uninfected and murine leukemia virus (MuLV)-infected JLSV9 cell surfaces, as well as murine mammary tumor virus (MuMTV)-infected cell surfaces, were examined by electron microscopy. The 10-nm knobs of MuLV and the 5-nm spikes of MuMTV were clearly revealed on the surfaces of budding viruses and were also found dispersed over the cell surface. The MuLV knobs are randomly arranged on the virus surface, whereas the MuMTV spikes are much more ordered. Because freeze-fractured budding viral envelopes are devoid of intramembranous particles, the observed surface particles do not appear to be merely accentuated intramembranous particles. This technique should permit further analysis of the morphogenesis of viral envelopes without the need for externally applied labels.     

Embryonic macrophages of early rat yolk sac: Immunohistochemistry and ultrastructure with reference to endodermal cell layer

Macrophages are multifunctional cells that participate in numerous biological processes; they actively phagocytose foreign particles and cell debris. Embryonic tissue macrophages are present at early stages of mammalian development; their ontogeny and function is still under investigation. Our study used immunohistochemistry and electron microscopy to investigate early rat yolk sac macrophages using mouse antirat macrophage monoclonal antibodies (mAb) Mar 1 and Mar 3 produced by our laboratory. Mar 3 mAb revealed the first emergence of immature macrophages in the rat yolk sac at fetal day nine coinciding with the beginning of yolk sac haemopoiesis that consisted mainly of erythropoiesis, while Mar 1 mAb detected specifically rat yolk sac macrophages at about the 13th to 14th day of gestation. Immunoreactivity against Mar mAbs was mainly located in the yolk sac endodermal cell layer, which may signify endodermal origin of the yolk sac macrophages. Ultrastructurally mature yolk sac macrophages contained numerous endocytic vesicles or vacuoles, well-developed Golgi saccules and many electron dense granules in their cytoplasm and a number of microvillous projections from the cell surface. After establishment of the circulation between yolk sac and embryo, Mar 3 positive cells were also demonstrated inside fetal undifferentiated mesenchymal tissue at fetal day 12. The study demonstrated the first emergence of immature yolk sac macrophages being among the earliest haemopoietic cells formed in mammalian development. Thus, Mar mAbs managed to detect macrophage differentiation antigens through their development early in the rat yolk sac.     

Filovirus-induced endothelial leakage triggered by infected monocytes/macrophages.

The pathogenetic mechanisms underlying hemorrhagic fevers are not fully understood, but hemorrhage, activation of coagulation, and shock suggest vascular instability. Here, we demonstrate that Marburg virus (MBG), a filovirus causing a severe form of hemorrhagic fever in humans, replicates in human monocytes/macrophages, resulting in cytolytic infection and release of infectious virus particles. Replication also led to intracellular budding and accumulation of viral particles in vacuoles, thus providing a mechanism by which the virus may escape immune surveillance. Monocytes/macrophages were activated by MBG infection as indicated by tumor necrosis factor alpha (TNF-alpha) release. Supernatants of monocyte/macrophage cultures infected with MBG increased the permeability of cultured human endothelial cell monolayers. The increase in endothelial permeability correlated with the time course of TNF-alpha release and was inhibited by a TNF-alpha specific monoclonal antibody. Furthermore, recombinant TNF-alpha added at concentrations present in supernatants of virus-infected macrophage cultures increased endothelial permeability in the presence of 10 micron H2O2. These results indicate that TNF-alpha plays a critical role in mediating increased permeability, which was identified as a paraendothelial route shown by formation of interendothelial gaps. The combination of viral replication in endothelial cells (H.-J. Schnittler, F. Mahner, D. Drenckhahn, H.-D. Klenk, and H. Feldmann, J. Clin. Invest. 19:1301-1309, 1993) and monocytes/macrophages and the permeability-increasing effect of virus-induced cytokine release provide the first experimental data for a novel concept in the pathogenesis of viral hemorrhagic fever.