Recombinant CD200 protein does not bind activating proteins closely related to CD200 receptor

CD200 (OX2) is a cell surface glycoprotein that interacts with a structurally related receptor (CD200R) expressed mainly on myeloid cells and is involved in regulation of macrophage and mast cell function. In mouse there are up to five genes related to CD200R with conflicting data as to whether they bind CD200. We show that mouse CD200 binds the inhibitory receptor CD200R with a comparable affinity (Kd = 4 microM) to those found for the rat and human CD200 CD200R interactions. CD200 gave negligible binding to the activating receptors, CD200RLa, CD200RLb, and CD200RLc, by direct analysis at the protein level using recombinant monomeric and dimeric fusion proteins or to CD200RLa and CD200RLb when expressed at the cell surface. An additional potential activating gene, CD200RLe, found in only some mouse strains also did not bind CD200. Thus, the CD200 receptor family consists of both activatory and inhibitory members like several other paired ligand receptors, such as signal regulatory protein, killer cell Ig-like receptor/KAR, LY49, dendritic cell immunoreceptor/dendritic cell immunoactivating receptor, and paired Ig-like type 2 receptor. Although the ligand for the inhibitory product is a widely distributed host protein, the ligands of the activating forms remain to be identified, and one possibility is that they are pathogen components.     

Myxoma virus M141R expresses a viral CD200 (vOX-2) that is responsible for down-regulation of macrophage and T-cell activation in vivo

M141R is a myxoma virus gene that encodes a cell surface protein with significant amino acid similarity to the family of cellular CD200 (OX-2) proteins implicated in the regulation of myeloid lineage cell activation. The creation of an M141R deletion mutant myxoma virus strain (vMyx141KO) and its subsequent infection of European rabbits demonstrated that M141R is required for the full development of a lethal infection in vivo but is not required for efficient virus replication in susceptible cell lines in vitro. Minor secondary sites of infection were detected in the majority of rabbits infected with the M141R deletion mutant, demonstrating that the M141R protein is not required for the dissemination of virus within the host. When compared to wild-type myxoma virus-infected rabbits, vMyx141KO-infected rabbits showed higher activation levels of both monocytes/macrophages and lymphocytes in situ through assessments of inducible nitric oxide synthase-positive and CD25(+) infiltrating cells in infected and lymphoid tissues. Purified peripheral blood mononuclear cells from vMyx141KO-infected rabbits demonstrated an increased ability to express gamma interferon upon activation by phorbol myristate acetate plus ionomycin compared to cells purified from wild-type myxoma virus-infected rabbits. We concluded that the M141R protein is a bona fide CD200-like immunomodulator protein which is required for the full pathogenesis of myxoma virus in the European rabbit and that its loss from the virus results in increased activation levels of macrophages in infected lesions and draining lymph nodes as well as an increased activation level of circulating T lymphocytes during infection. We propose a model whereby M141R transmits inhibitory signals to tissue macrophages, and possibly resident CD200R(+) dendritic cells, that reduce their ability to antigenically prime lymphocytes and possibly provides anergic signals to T cells directly.     

Human herpesvirus 8 K14 protein mimics CD200 in down-regulating macrophage activation through CD200 receptor

Many viral proteins limit host immune defenses, and their genes often originate from their hosts. CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a receptor on myeloid cells (CD200R) that is implicated in locally preventing macrophage activation. Distant, but recognizable, homologues of CD200 have been identified in many herpesviruses and poxviruses. Here, we show that the product of the K14 open reading frame from human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) interacts with human CD200R and is expressed at the surfaces of infected cells solely during the lytic cycle. Despite sharing only 40% primary sequence identity, K14 and CD200 interacted with CD200R with an almost identical and low affinity (K(D) = 0.5 microM), in contrast to other characterized viral homologue interactions. Cells expressing CD200 or K14 on the cell surface were able to inhibit secretion by activated macrophages of proinflammatory cytokines such as tumor necrosis factor alpha, an effect that could be specifically relieved by addition of monoclonal antibodies and soluble monomeric CD200 protein. We conclude that CD200 delivers local down-modulatory signals to myeloid cells through direct cell-cell contact and that the K14 viral homologue closely mimics this.     

Mast cells and basophils are selectively activated in vitro and in vivo through CD200R3 in an IgE-independent manner

Mast cells and basophils have been implicated in the host defense system against pathogens and in the development of allergic disorders. Although IgE-dependent responses via FcepsilonRI on these cells have been extensively studied, little is known about cell surface molecules that are selectively expressed by these cells and engaged in their activation via an IgE-independent mechanism. We have recently established two mAbs that reacted specifically with murine mast cells and basophils, and one of them selectively depleted basophils when administered in vivo. Biochemical and flow cytometric analyses revealed that both mAbs specifically recognized a CD200R-like protein, CD200R3, but not other CD200R family members. CD200R3 existed as a disulfide-linked dimer, unlike other CD200Rs, and was expressed on mast cells and basophils primarily in association with an ITAM-bearing adaptor DAP12. Cross-linking of CD200R3 with the mAbs induced degranulation in mast cells and production of the cytokine IL-4 in basophils in vitro. Administration of the nondepleting mAb in vivo elicited systemic and local anaphylaxis in a CD200R3-dependent manner. These results suggest that CD200R3 functions as an activating receptor on mast cells and basophils to regulate IgE-independent immune responses in cooperation with an inhibitory receptor CD200R, similar to the paired receptors expressed on NK cells.     

The vaccinia virus soluble alpha/beta interferon (IFN) receptor binds to the cell surface and protects cells from the antiviral effects of IFN

Poxviruses encode a broad range of proteins that interfere with host immune functions, such as soluble versions of receptors for the cytokines tumor necrosis factor, interleukin-1 beta, gamma interferon (IFN-gamma), IFN-alpha/beta, and chemokines. These virus-encoded cytokine receptors have a profound effect on virus pathogenesis and enable the study of the role of cytokines in virus infections. The vaccinia virus (VV) Western Reserve gene B18R encodes a secreted protein with 3 immunoglobulin domains that functions as a soluble receptor for IFN-alpha/beta. We have found that after secretion B18R binds to both uninfected and infected cells. The B18R protein present at the cell surface maintains the properties of the soluble receptor, binding IFN-alpha/beta with high affinity and with broad species specificity, and protects cells from the antiviral state induced by IFN-alpha/beta. VV strain Wyeth expressed a truncated B18R protein lacking the C-terminal immunoglobulin domain. This protein binds IFN with lower affinity and retains its ability to bind to cells, indicating that the C-terminal region of B18R contributes to IFN binding. The replication of a VV B18R deletion mutant in tissue culture was restricted in the presence of IFN-alpha, whereas the wild-type virus replicated normally. Binding of soluble recombinant B18R to cells protected the cultures from IFN and allowed VV replication. This represents a novel strategy of virus immune evasion in which secreted IFN-alpha/beta receptors not only bind the soluble cytokine but also bind to uninfected cells and protect them from the antiviral effects of IFN-alpha/beta, maintaining the cells' susceptibility to virus infections. The adaptation of this soluble receptor to block IFN-alpha/beta activity locally will help VV to replicate in the host and spread in tissues. This emphasizes the importance of local effects of IFN-alpha/beta against virus infections.     

Molecular mechanisms of CD200 inhibition of mast cell activation

CD200 and its receptor CD200R are both type I membrane glycoproteins that contain two Ig-like domains. Engagement of CD200R by CD200 inhibits activation of myeloid cells. Unlike the majority of immune inhibitory receptors, CD200R lacks an ITIM in the cytoplasmic domain. The molecular mechanism of CD200R inhibition of myeloid cell activation is unknown. In this study, we examined the CD200R signaling pathways that control degranulation of mouse bone marrow-derived mast cells. We found that upon ligand binding, CD200R is phosphorylated on tyrosine and subsequently binds to adapter proteins Dok1 and Dok2. Upon phosphorylation, Dok1 binds to SHIP and both Dok1 and Dok2 recruit RasGAP, which mediates the inhibition of the Ras/MAPK pathways. Activation of ERK, JNK, and p38 MAPK are all inhibited by CD200R engagement. The reduced activation of these MAPKs is responsible for the observed inhibition of mast cell degranulation and cytokine production. Similar signaling events were also observed upon CD200R engagement in mouse peritoneal cells. These data define a novel inhibitory pathway used by CD200R in modulating mast cell function and help to explain how engagement of this receptor in vivo regulates myeloid cell function.     

Distinct kinetics for binding of the CD46 and SLAM receptors to overlapping sites in the measles virus hemagglutinin protein

Measles virus (MV) is a human pathogen using two distinct cell surface receptors for entry into host cells. We present here a comparative analysis for binding of the MV receptors CD46 and SLAM to the measles virus hemagglutinin protein (MVH, Edmonston strain). Soluble monomeric and dimeric MVH variants were prepared in mammalian cells and their conformation assessed using a panel of monoclonal antibodies. The two receptor molecules specifically bound to the MVH protein with distinct binding modes. The association rate (k(a)) for SLAM binding to MVH was very low ( approximately 3000 m(-1)s(-1)), about 20 times lower that the k(a) determined for CD46 binding. However, SLAM bound tighter to the virus protein than CD46, as revealed by a 5-fold lower dissociation rate (k(d), approximately 1.5 x 10(-3) s(-1)). These data suggest that the SLAM receptor binds to a less accessible and more hydrophobic surface on MVH than the CD46 receptor, as illustrated in a binding model. Despite the differences in kinetics, receptor competition binding experiments revealed that they recognize overlapping sites in MVH. Indeed, a panel of anti-MVH monoclonal antibodies equally inhibited binding of both receptor molecules. The similar immune reactivity of the two receptor binding sites suggests that the shift in receptor usage by MV may not be driven by immune responses.     

Suppression of antigen-specific T cell responses by the Kaposi's sarcoma-associated herpesvirus viral OX2 protein and its cellular orthologue, CD200

Regulating appropriate activation of the immune response in the healthy host despite continual immune surveillance dictates that immune responses must be either self-limiting and therefore negatively regulated following their activation or prevented from developing inappropriately. In the case of antigen-specific T cells, their response is attenuated by several mechanisms, including ligation of CTLA-4 and PD-1. Through the study of the viral OX2 (vOX2) immunoregulator encoded by Kaposi's sarcoma-associated herpesvirus (KSHV), we have identified a T cell-attenuating role both for this protein and for CD200, a cellular orthologue of the viral vOX2 protein. In vitro, antigen-presenting cells (APC) expressing either native vOX2 or CD200 suppressed two functions of cognate antigen-specific T cell clones: gamma interferon (IFN-γ) production and mobilization of CD107a, a cytolytic granule component and measure of target cell killing ability. Mechanistically, vOX2 and CD200 expression on APC suppressed the phosphorylation of ERK1/2 mitogen-activated protein kinase in responding T cells. These data provide the first evidence for a role of both KSHV vOX2 and cellular CD200 in the negative regulation of antigen-specific T cell responses. They suggest that KSHV has evolved to harness the host CD200-based mechanism of attenuation of T cell responses to facilitate virus persistence and dissemination within the infected individual. Moreover, our studies define a new paradigm in immune modulation by viruses: the provision of a negative costimulatory signal to T cells by a virus-encoded orthologue of CD200.     

Mechanism of CD150 (SLAM) down regulation from the host cell surface by measles virus hemagglutinin protein

Measles virus has been reported to enter host cells via either of two cellular receptors, CD46 and CD150 (SLAM). CD46 is found on most cells of higher primates, while SLAM is expressed on activated B, T, and dendritic cells and is an important regulatory molecule of the immune system. Previous reports have shown that measles virus can down regulate expression of its two cellular receptors on the host cell surface during infection. In this study, the process of down regulation of SLAM by measles virus was investigated. We demonstrated that expression of the hemagglutinin (H) protein of measles virus was sufficient for down regulation. Our studies provided evidence that interactions between H and SLAM in the endoplasmic reticulum (ER) can promote the down regulation of SLAM but not CD46. In addition, we demonstrated that interactions between H and SLAM at the host cell surface can also contribute to SLAM down regulation. These results indicate that two mechanisms involving either intracellular interactions between H and SLAM in the ER or receptor-mediated binding to H at the surfaces of host cells can lead to the down regulation of SLAM during measles virus infection.     

A27L Protein Mediates Vaccinia Virus Interaction with Cell Surface Heparan Sulfate

Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.     

Suppression of a fusion defect by second site mutations in the ecotropic murine leukemia virus surface protein

Entry of ecotropic murine leukemia virus initiates when the envelope surface protein recognizes and binds to the virus receptor on host cells. The envelope transmembrane protein then mediates fusion of viral and host cell membranes and penetration into the cytoplasm. Using a genetic selection, we isolated an infectious retrovirus variant containing three changes in the surface protein-histidine 8 to arginine, glutamine 227 to arginine, and aspartate 243 to tyrosine. Single replacement of histidine 8 with arginine (H8R) resulted in almost complete loss of infectivity, even though the mutant envelope proteins were stable and efficiently incorporated into virions. Virions carrying H8R envelope were proficient at binding cells expressing receptor but failed to induce cell-cell fusion of XC cells, indicating that the histidine at position 8 plays an essential role in fusion during penetration of the host cell membrane. Thus, there is at least one domain in SU that is involved in fusion; the fusion functions do not reside exclusively in TM. In contrast, envelope with all three changes induced cell-cell fusion of XC cells and produced virions that were 10,000-fold more infectious than those containing only the H8R substitution, indicating that changes at positions 227 and 243 can suppress a fusion defect caused by loss of histidine 8 function. Moreover, the other two changes acted synergistically, indicating that both compensate for the loss of the same essential function of histidine 8. The ability of these changes to suppress this fusion defect might provide a means for overcoming postbinding defects found in targeted retroviral vectors for use in human gene therapy.     

Heparan sulfate targets the HIV-1 envelope glycoprotein gp120 coreceptor binding site

Human immunodeficiency virus (HIV) attachment to host cells is a multi-step process that involves interaction of the viral envelope gp120 with the primary receptor CD4 and coreceptors. HIV gp120 also binds to other cell surface components, including heparan sulfate (HS), a sulfated polysaccharide whose wide interactive properties are exploited by many pathogens for attachment and concentration at the cell surface. To analyze the structural features of gp120 binding to HS, we used soluble CD4 to constrain gp120 in a specific conformation. We first found that CD4 induced conformational change of gp120, dramatically increasing binding to HS. We then showed that HS binding interface on gp120 comprised, in addition to the well characterized V3 loop, a CD4-induced epitope. This epitope is efficiently targeted by nanomolar concentrations of size-defined heparin/HS-derived oligosaccharides. Because this domain of the protein also constitutes the binding site for the viral coreceptors, these results support an implication of HS at late stages of the virus-cell attachment process and suggest potential therapeutic applications.     

Characterization of the CD200 receptor family in mice and humans and their interactions with CD200

CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a structurally related receptor (CD200R) expressed on rodent myeloid cells and is involved in regulation of macrophage function. We report the first characterization of human CD200R (hCD200R) and define its binding characteristics to hCD200. We also report the identification of a closely related gene to hCD200R, designated hCD200RLa, and four mouse CD200R-related genes (termed mCD200RLa-d). CD200, CD200R, and CD200R-related genes were closely linked in humans and mice, suggesting that these genes arose by gene duplication. The distributions of the receptor genes were determined by quantitative RT-PCR, and protein expression was confirmed by a set of novel mAbs. The distribution of mouse and human CD200R was similar, with strongest labeling of macrophages and neutrophils, but also other leukocytes, including monocytes, mast cells, and T lymphocytes. Two mCD200 receptor-like family members, designated mCD200RLa and mCD200RLb, were shown to pair with the activatory adaptor protein, DAP12, suggesting that these receptors would transmit strong activating signals in contrast to the apparent inhibitory signal delivered by triggering the CD200R. Despite substantial sequence homology with mCD200R, mCD200RLa and mCD200RLb did not bind mCD200, and presently have unknown ligands. The CD200 receptor gene family resembles the signal regulatory proteins and killer Ig-related receptors in having receptor family members with potential activatory and inhibitory functions that may play important roles in immune regulation and balance. Because manipulation of the CD200-CD200R interaction affects the outcome of rodent disease models, targeting of this pathway may have therapeutic utility.     

CD200 Receptor Controls Sex-Specific TLR7 Responses to Viral Infection

Immunological checkpoints, such as the inhibitory CD200 receptor (CD200R), play a dual role in balancing the immune system during microbial infection. On the one hand these inhibitory signals prevent excessive immune mediated pathology but on the other hand they may impair clearance of the pathogen. We studied the influence of the inhibitory CD200-CD200R axis on clearance and pathology in two different virus infection models. We find that lack of CD200R signaling strongly enhances type I interferon (IFN) production and viral clearance and improves the outcome of mouse hepatitis corona virus (MHV) infection, particularly in female mice. MHV clearance is known to be dependent on Toll like receptor 7 (TLR7)-mediated type I IFN production and sex differences in TLR7 responses previously have been reported for humans. We therefore hypothesize that CD200R ligation suppresses TLR7 responses and that release of this inhibition enlarges sex differences in TLR7 signaling. This hypothesis is supported by our findings that in vivo administration of synthetic TLR7 ligand leads to enhanced type I IFN production, particularly in female Cd200^−/− mice and that CD200R ligation inhibits TLR7 signaling in vitro. In influenza A virus infection we show that viral clearance is determined by sex but not by CD200R signaling. However, absence of CD200R in influenza A virus infection results in enhanced lung neutrophil influx and pathology in females. Thus, CD200-CD200R and sex are host factors that together determine the outcome of viral infection. Our data predict a sex bias in both beneficial and pathological immune responses to virus infection upon therapeutic targeting of CD200-CD200R.     

Phagocytosis, a potential mechanism for myeloid-derived suppressor cell regulation of CD8+ T cell function mediated through programmed cell death-1 and programmed cell death-1 ligand interaction

CD8(+) T cells become exhausted, inducing cell surface protein programmed cell death-1 (PD-1) as chronic virus diseases or tumors progress, but underlying mechanisms of this are unclear. We previously showed that M-CSF is important for developing tolerogenic dendritic cells (DCs) from human CD14(+) monocytes. In this article, we identify M-CSF-derived DCs (M-DCs) after stimulation with IL-10 as myeloid-derived suppressor cells with additional tolerogenic activities to CD8(+) T cells. IL-10 increased PD-1 ligand expression on M-DC, and IL-10-stimulated M-DCs (M-DC/IL-10) induced expression of PD-1 on, and apoptosis of, CD8(+) T cells and phagocytosed CD8(+) T cells. Enhanced phagocytic activity of M-DC/IL-10 required IFN-γ, which further increased PD-1 ligand and PD-2 ligand expression on M-DC/IL-10. IFN-γ-stimulated M-DC/IL-10 cells were phenotypically macrophage-like cells with little or no expression of CD86, a costimulatory molecule, but with high expression levels of CD14, CD200R, and CD80. No phagocytic activity was detected with GM-CSF-derived DCs. We propose that phagocytosis by IFN-γ-stimulated M-DC/IL-10 cells, which may be DCs or, alternatively, a unique subset of macrophages, may be a mechanism by which IFN-γ-producing CD8(+) T cells are tolerized after type 1 immune responses to chronic virus or tumor, and that IFN-γ links effector CD8(+) T cells to their phagocytic clearance.     

Depletion of glycoprotein gp85 from virosomes made with Epstein-Barr virus proteins abolishes their ability to fuse with virus receptor-bearing cells.

Entry of an enveloped virus such as Epstein-Barr virus (EBV) into host cells involves fusion of the virion envelope with host cell membranes either at the surface of the cell or within endocytic vesicles. Previous work has indirectly implicated the EBV glycoprotein gp85 in this fusion process. A neutralizing monoclonal antibody to gp85, F-2-1, failed to inhibit binding of EBV to its receptor but interfered with virus fusion as measured with the self-quenching fluorophore octadecyl rhodamine B chloride (R18) (N. Miller and L. M. Hutt-Fletcher, J. Virol. 62:2366-2372, 1988). To test further the hypothesis that gp85 functions as a fusion protein, EBV virion proteins including or depleted of gp85 were incorporated into lipid vesicles to form virosomes. Virosomes were labeled with R18, and those that were made with undepleted protein were shown to behave in a manner similar to that of R18-labeled virus. They bound to receptor-positive but not to receptor-negative cells and fused with Raji cells but not with receptor-positive, fusion-incompetent Molt 4 cells; monoclonal antibodies that inhibited binding or fusion of virus inhibited binding and fusion of virosomes, and virus competed with virosomes for attachment to cells. In contrast, virosomes made from virus proteins depleted of gp85 by immunoaffinity chromatography remained capable of binding to receptor-positive cells but failed to fuse. These results are compatible with the hypothesis that gp85 is actively involved in the fusion of EBV with lymphoblatoid cell lines and suggest that the ability of antibody F-2-1 to neutralize infectivity of EBV represents a direct effect on the function of gp85 as a fusion protein.     

Insertion of host-derived costimulatory molecules CD80 (B7.1) and CD86 (B7.2) into human immunodeficiency virus type 1 affects the virus life cycle

Human immunodeficiency virus type 1 (HIV-1) carries virus-encoded and host-derived proteins. Recent advances in the functional characterization of host molecules inserted into mature virus particles have revealed that HIV-1 biology is influenced by the acquisition of host cell membrane components. The CD28/B7 receptor/ligand system is considered one of the fundamental elements of the normal immune response. Two major cell types that harbor HIV-1 in vivo, i.e., monocytes/macrophages and CD4+ T cells, express the costimulatory molecules CD80 (B7.1) and CD86 (B7.2). We investigated whether CD80 and CD86 are efficiently acquired by HIV-1, and if so, whether these host-encoded molecules can contribute to the virus life cycle. Here we provide the first evidence that the insertion of CD80 and CD86 into HIV-1 increases virus infectivity by facilitating the attachment and entry process due to interactions with their two natural ligands, CD28 and CTLA-4. Moreover, we demonstrate that NF-kappaB is induced by CD80- and CD86-bearing virions when they are combined with the engagement of the T-cell receptor/CD3 complex, an event that is inhibited upon surface expression of CTLA-4. Finally, both CD80 and CD86 were found to be efficiently incorporated into R5- and X4-tropic field strains of HIV-1 expanded in cytokine-treated macrophages. Thus, besides direct interactions between the virus envelope glycoproteins and cell surface constituents, such as CD4 and some specific chemokine coreceptors, HIV-1 may attach to target cells via interactions between cell-derived molecules incorporated into virions and their natural ligands. These findings support the theory that HIV-1-associated host proteins alter virus-host dynamics.     

THY-1 Cell Surface Antigen (CD90) Has an Important Role in the Initial Stage of Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) infects about 50% of the US population, is the leading infectious cause of birth defects, and is considered the most important infectious agent in transplant recipients. The virus infects many cell types in vivo and in vitro. While previous studies have identified several cellular proteins that may function at early steps of infection in a cell type dependent manner, the mechanism of virus entry is still poorly understood. Using a computational biology approach, correlating gene expression with virus infectivity in 54 cell lines, we identified THY-1 as a putative host determinant for HCMV infection in these cells. With a series of loss-of-function, gain-of-function and protein-protein interaction analyses, we found that THY-1 mediates HCMV infection at the entry step and is important for infection that occurs at a low m.o.i. THY-1 antibody that bound to the cell surface blocked HCMV during the initial 60 minutes of infection in a dose-dependent manner. Down-regulation of THY-1 with siRNA impaired infectivity occurred during the initial 60 minutes of inoculation. Both THY-1 antibody and siRNA inhibited HCMV-induced activation of the PI3-K/Akt pathway required for entry. Soluble THY-1 protein blocked HCMV infection during, but not after, virus internalization. Expression of exogenous THY-1 enhanced entry in cells expressing low levels of the protein. THY-1 interacted with HCMV gB and gH and may form a complex important for entry. However, since gB and gH have previously been shown to interact, it is uncertain if THY-1 directly binds to both of these proteins. Prior observations that THY-1 (a) interacts with αVβ3 integrin and recruits paxillin (implicated in HCMV entry), (b) regulates leukocyte extravasation (critical for HCMV viremia), and (c) is expressed on many cells targeted for HCMV infection including epithelial and endothelial cells, fibroblast, and CD34+/CD38- stem cells, all support a role for THY-1 as an HCMV entry mediator in a cell type dependent manner. THY-1 may function through a complex setting, that would include viral gB and gH, and other cellular factors, thus links virus entry with signaling in host cells that ultimately leads to virus infection.     

CD4-Negative cells bind human immunodeficiency virus type 1 and efficiently transfer virus to T cells

The ability of human immunodeficiency virus strain MN (HIV(MN)), a T-cell line-adapted strain of HIV, and X4 and R5 primary isolates to bind to various cell types was investigated. In general, HIV(MN) bound to cells at higher levels than did the primary isolates. Virus bound to both CD4-positive (CD4(+)) and CD4-negative (CD4(-)) cells, including neutrophils, Raji cells, tonsil mononuclear cells, erythrocytes, platelets, and peripheral blood mononuclear cells (PBMC), although virus bound at significantly higher levels to PBMC. However, there was no difference in the amount of HIV that bound to CD4-enriched or CD4-depleted PBMC. Virus bound to CD4(-) cells was up to 17 times more infectious for T cells in cocultures than was the same amount of cell-free virus. Virus bound to nucleated cells was significantly more infectious than virus bound to erythrocytes or platelets. The enhanced infection of T cells by virus bound to CD4(-) cells was not due to stimulatory signals provided by CD4(-) cells or infection of CD4(-) cells. However, anti-CD18 antibody substantially reduced the enhanced virus replication in T cells, suggesting that virus that bound to the surface of CD4(-) cells is efficiently passed to CD4(+) T cells during cell-cell adhesion. These studies show that HIV binds at relatively high levels to CD4(-) cells and, once bound, is highly infectious for T cells. This suggests that virus binding to the surface of CD4(-) cells is an important route for infection of T cells in vivo.