Mark Marsh

Virus Entry: Open Sesame

Detailed information about the replication cycle of viruses and their interactions with host organisms is required to develop strategies to stop them. Cell biology studies, live-cell imaging, and systems biology have started to illuminate the multiple and subtly different pathways that animal viruses use to enter host cells. These insights are revolutionizing our understanding of endocytosis and the movement of vesicles within cells. In addition, such insights reveal new targets for attacking viruses before they can usurp the host-cell machinery for replication.

CD4-independent infection by HIV-2 is mediated by Fusin/CXCR4

Several members of the chemokine receptor family have been shown to function in association with CD4 to permit HIV-1 entry and infection. However, the mechanism by which these molecules serve as CD4-associated cofactors is unclear. In the present report, we show that one member of this family, termed Fusin/ CXCR4, is able to function as an alternative receptor for some isolates of HIV-2 in the absence of CD4. This conclusion is supported by the finding that (1) CD4-independent infection by these viruses is inhibited by an anti-Fusin monoclonal antibody, (2) Fusin expression renders human and nonhuman CD4-negative cell lines sensitive to HIV-2-induced syncytium induction and/or infection, and (3) Fusin is selectively down-regulated from the cell surface following HIV-2 infection. The finding that one chemokine receptor can function as a primary viral receptor strongly suggests that the HIV envelope glycoprotein contains a binding site for these proteins and that differences in the affinity and/or the availability of this site can extend the host range of these viruses to include a number of CD4-negative cell types.

Virus entry into animal cells.

Publisher Summary In addition to its many other functions, the plasma membrane of eukaryotic cells serves as a barrier against invading parasites and viruses. It is not permeable to ions and to low molecular weight solutes, let alone to proteins and polynucleotides. Yet it is clear that viruses are capable of transferring their genome and accessory proteins into the cytosol or into the nucleus, and thus infect the cell. While the detailed mechanisms remain unclear for most animal viruses, a general theme is apparent like other stages in the replication cycle; their entry depends on the activities of the host cell. In order to take up nutrients, to communicate with other cells, to control the intracellular ion balance, and to secrete substances, cells have a variety of mechanisms for bypassing and modifying the barrier properties imposed by their plasma membrane. It is these mechanisms, and the molecules involved in them, that viruses exploit.

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.

Penetration of Semliki Forest virus from acidic prelysosomal vacuoles.

To identify and characterize the intracellular site from which the penetration of Semliki Forest virus (SFV) to the cytosolic compartment of the host cell occurs, we determined the time course and temperature dependence of nucleocapsid uncoating and infection in BHK-21 cells. At 37 degrees C the genome release to the cytosol was detected within 5-7 min after virus endocytosis, whereas delivery of the virus particles to secondary lysosomes occurred within 15-20 min. At temperatures of 15 degrees -20 degrees C virus particles were internalized by endocytosis, but they were not delivered to the secondary lysosomes. Nevertheless, at 20 degrees C nucleocapsid uncoating and infection occurred, indicating that secondary lysosomes are not required for SFV penetration. We conclude that the penetration reaction occurs in prelysosomal endocytic vacuoles (endosomes). As SFV penetration by membrane fusion requires a pH less than 6 and the presence of cholesterol in the target membrane, the data indicate that endosomes are acidic and contain cholesterol.

Disordered macrophage cytokine secretion underlies impaired acute inflammation and bacterial clearance in Crohn's disease

The cause of Crohns disease (CD) remains poorly understood. Counterintuitively, these patients possess an impaired acute inflammatory response, which could result in delayed clearance of bacteria penetrating the lining of the bowel and predispose to granuloma formation and chronicity. We tested this hypothesis in human subjects by monitoring responses to killed Escherichia coli injected subcutaneously into the forearm. Accumulation of 111In-labeled neutrophils at these sites and clearance of 32P-labeled bacteria from them were markedly impaired in CD. Locally increased blood flow and bacterial clearance were dependent on the numbers of bacteria injected. Secretion of proinflammatory cytokines by CD macrophages was grossly impaired in response to E. coli or specific Toll-like receptor agonists. Despite normal levels and stability of cytokine messenger RNA, intracellular levels of tumor necrosis factor (TNF) were abnormally low in CD macrophages. Coupled with reduced secretion, these findings indicate accelerated intracellular breakdown. Differential transcription profiles identified disease-specific genes, notably including those encoding proteins involved in vesicle trafficking. Intracellular destruction of TNF was decreased by inhibitors of lysosomal function. Together, our findings suggest that in CD macrophages, an abnormal proportion of cytokines are routed to lysosomes and degraded rather than being released through the normal secretory pathway.

Adsorptive Endocytosis of Semliki Forest Virus

Abstract Endocytosis of Semliki Forest virus into BHK-21 cells has been studied at multiplicities varying between one virus and 4 × 10 6 viruses per cell. Uptake requires attachment to the cell surface, it is very rapid (half-life 10 to 35 min), it occurs in coated vesicles, it is inhibited at temperatures below 15 °C and it does not elevate the fluid-phase uptake of the cells. Inhibitor studies using colcemid, cytochalasin B, sodium azide, dinitrophenol, 2-deoxy- d -glucose and lysosomotropic weak bases show that the uptake is largely independent of cytoskeletal and lysosomal function, but partially dependent on oxidative phosphorylation. Together the biochemical and morphological results indicate that SFV † uptake occurs by adsorptive endocytosis in a manner very similar to the receptor-mediated endocytosis of serum low-density lipoprotein and other physiologically important extracellular proteins (Goldstein et al. , 1979). SFV thus appears to exploit a facultative cellular process in gaining entry into cells. The uptake of SFV has been used to study quantitative aspects of the adsorptive endocytosis process. At high multiplicities of viruses to cells, 1500 to 3000 virus particles can be internalized per minute per cell. Electron microscopy indicated an average of 1.3 viruses per virus-containing coated vesicle.

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

Deneka et al. 2007. J. Cell Biol. doi:10.1083/jcb.200609050 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1083%252Fjcb.200609050%26rft_id%253Dinfo%253Apmid%252F17438075%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%

Human immunodeficiency virus infection of CD4-bearing cells occurs by a pH-independent mechanism.

The effect of weak bases (NH4Cl and amantadine) and carboxylic ionophores (monensin) on the infection of CD4 (T4) positive human cell lines by HIV‐1 is examined. These reagents, which raise the pH of acidic intracellular organelles, fail to inhibit HIV‐1 entry and the events leading to viral protein synthesis at concentrations inhibitory for low pH‐dependent fusogenic enveloped viruses. The infectivity of VSV (HIV‐1) pseudotypes is unaffected by weak bases at concentrations causing 95% plaque reduction of VSV in its own envelope. HIV‐1 dependent cell–cell fusion (syncytium formation) occurs in medium maintained at pH 7.4‐7.6, and virions are not irreversibly inactivated by incubation in acid medium. Our results show that HIV‐1 entry and membrane fusion do not require exposure to low pH. The production of infectious HIV‐1 particles, however, is inhibited in cells treated with NH4Cl.

HIV-1 trafficking to the dendritic cell-T-cell infectious synapse uses a pathway of tetraspanin sorting to the immunological synapse

Dendritic cells (DCs) are essential components of the early events of HIV infection. Here, we characterized the trafficking pathways that HIV‐1 follows during its capture by DCs and its subsequent presentation to CD4+ T cells via an infectious synapse. Immunofluorescence microscopy indicates that the virus‐containing compartment in mature DCs (mDCs) co‐labels for the tetraspanins CD81, CD82, and CD9 but contains little CD63 or LAMP‐1. Using ratio imaging of pH‐reporting fluorescent virions in live DCs, we show that HIV‐1 is internalized in an intracellular endocytic compartment with a pH of 6.2. Significantly, we demonstrate that the infectivity of cell‐free virus is more stable at mildly acidic pH than at neutral pH. Using electron microscopy, we confirm that HIV‐1 accumulates in intracellular vacuoles that contain CD81 positive internal membranes but overlaps only partially with CD63. When allowed to contact T cells, HIV‐1‐loaded DCs redistribute CD81, and CD9, as well as internalized HIV‐1, but not the immunological synapse markers MHC‐II and T‐cell receptor to the infectious synapse. Together, our results indicate that HIV‐1 is internalized into a non‐conventional, non‐lysosomal, endocytic compartment in mDCs and further suggest that HIV‐1 is able to selectively subvert components of the intracellular trafficking machinery required for formation of the DC–T‐cell immunological synapse to facilitate its own cell‐to‐cell transfer and propagation.