Surface Of Virus-infected Cells Research Articles

Antiviral NK cell responses in HIV infection: I. NK cell receptor genes as determinants of HIV resistance and progression to AIDS

NK cells play an important role in controlling viral infections. They can kill virus-infected cells directly as well as indirectly via antibody-dependent, cell-mediated cytotoxicity. They need no prior sensitization and expansion for this killing. NK cells are also considered as important regulators of antiviral immune responses. They do so by secreting a multitude of soluble mediators and by directly interacting with other immune cells, e.g., dendritic cells. NK cells do not possess a single well-defined receptor to recognize antigens on target cells. Instead, they express an array of inhibitory and activating receptors and coreceptors, which bind to their cognate ligands expressed on the surface of target cells. These ligands include classical and nonclassical MHC class I antigens, MHC-like proteins, and a variety of other self- and virus-derived molecules. They may be expressed constitutively and/or de novo on the surface of virus-infected cells. NK cell receptors (NKRs) of the killer-cell Ig-like receptor (KIR) family, like their MHC class I ligands, are highly polymorphic. Several recent studies suggest that epistatic interactions between certain KIR and MHC class I genes may determine innate resistance of the host to viral infections, including HIV. In the first part of this review article, we provide an overview of the current state of knowledge of NK cell immunobiology and describe how NKR genes, alone and in combination with HLA genes, may determine genetic resistance/susceptibilty to HIV infection and the development of AIDS in humans.

Elimination of Influenza Virus‐Infected Cells by Phagocytosis

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Elimination of Influenza Virus-infected Cells by Phagocytosis

Infection with a variety of viruses induces apoptosis in host cells. This phenomenon may be considered to be a self-defense mechanism to avoid viral propagation. However, the growth of influenza virus is completed before host cells become dysfunctional due to apoptosis. To clarify the physiologic consequences of influenza virus-induced apoptosis, the fate of influenza virus-infected cells was examined in vitro as well as in vivo. Influenza virus-infected cells were engulfed by macrophages in vitro, and virus propagation was almost completely inhibited. This phagocytosis was dependent on the specific recognition of the membrane phospholipid phosphatidylserine exposed on the surface of virus-infected apoptotic cells by macrophages. In addition, the activity of viral neuraminidase expressed at the surface of virus-infected cells was necessary for the maximal level of phagocytosis. When mice infected with influenza virus were administered phagocytosis inhibitors, the level of lethality and inflammation in the lung were augmented. These results show that apoptosis makes influenza virus-infected cells susceptible to phagocytosis by macrophages, and that this leads to a reduction in the extent of influenza pathology.

Influenza virus assembly and budding in raft-derived microdomains: A quantitative analysis of the surface distribution of HA, NA and M2 proteins

Influenza virus hemagglutinin (HA) and neuraminidase (NA) are known to associate with lipid rafts, membrane microdomains comprised of densely packed cholesterol and sphingolipids. These specialized membrane regions are believed to be involved in the budding of many enveloped viruses including influenza virus. Quantitative analysis of HA distribution on the surface of virus-infected cells by immunogold staining shows an organization into clusters that grow in size as the expression level of HA increases with time post-infection (p.i.) (∼325–500 nm at 4 h p.i. and ∼425–600 nm at 6 h p.i.). These HA-containing clusters are likely derived from lipid rafts as they contain a high density of the raft marker ganglioside GM1 and are dependent upon the presence of cholesterol. The clustering of HA is an intrinsic property of the HA protein and occurs in the absence of expression of other viral proteins. NA is also found sequestered within the same microdomains as HA, whereas the M2 ion channel protein does not concentrate within the raft-like microdomains. Quantification of the distribution of surface expressed HA by examining serial sections of virus-infected cells suggests that the HA-containing microdomains give rise to regions of influenza assembly and budding.

Natural Cytotoxicity Receptors: Pattern Recognition and Involvement of Carbohydrates

Natural cytotoxicity receptors (NCRs), expressed by natural killer (NK) cells, trigger NK lysis of tumor and virus-infected cells on interaction with cell-surface ligands of these target cells. We have determined that viral hemagglutinins expressed on the surface of virus-infected cells are involved in the recognition by the NCRs, NKp44 and NKp46. Recognition of tumor cells by the NCRs NKp30 and NKp46 involves heparan sulfate epitopes expressed on the tumor cell membrane. Our studies provide new evidence for the identity of the ligands for NCRs and indicate that a broader definition should be applied to pathological patterns recognized by innate immune receptors. Since nonmicrobial endogenous carbohydrate structures contribute significantly to this recognition, there is an imperative need to develop appropriate tools for the facile sequencing of carbohydrate moieties.

The cytomegalovirus m155 gene product subverts natural killer cell antiviral protection by disruption of H60-NKG2D interactions.

Natural killer (NK) cells are an important early mediator of host immunity to murine cytomegalovirus (MCMV) infection. However, MCMV has evolved mechanisms to elude recognition and clearance by NK cells. We have identified an MCMV immune evasion protein that impairs NKG2D-mediated NK cell antiviral activity. Infection of BALB/c 3T3 cells with the Smith strain of MCMV resulted in strong down-regulation of H60, a high affinity ligand for NKG2D, from the surface of virus-infected cells. The MCMV m155 protein specifically down-regulated H60 without affecting expression of the other known NKG2D ligands, RAE-1 and MULT-1. Treatment with the proteasome inhibitors lactacystin or epoxomicin reversed m155 down-regulation of H60. An MCMV mutant virus lacking m155 was severely attenuated in BALB/c mice; however, treatment with neutralizing anti-NKG2D monoclonal antibody or with NK-depleting anti-asialo GM1 antisera restored virulence of the mutant virus. Thus, down-regulation of H60 by m155 is a powerful mechanism of inhibiting NKG2D-mediated antiviral function.

Influenza A vaccine based on the extracellular domain of M2: weak protection mediated via antibody-dependent NK cell activity.

Vaccination of mice with a peptide corresponding to the extracellular part of M2 protein coupled to the immunodominant domain of hepatitis B core can protect mice from a lethal challenge with influenza A virus. As the extracellular part of M2 protein is highly conserved in all known human influenza A strains, such a vaccine may protect against all human influenza A strains, which would represent a major advantage over current vaccine strategies. The present study demonstrates that protection is mediated exclusively by Abs, a very important feature of a successful preventive vaccine. However, these Abs neither bind efficiently to the free virus nor neutralize virus infection, but bind to M2 protein expressed on the surface of virus-infected cells. The presence of NK cells is important for protection, whereas complement is not, supposing that protection is mediated via Ab-dependent, cell-mediated cytotoxicity. The absence of neutralizing Abs results in much weaker protection than that achieved by vaccination with UV-inactivated influenza virus. Specifically, whereas neutralizing Abs completely eliminate signs of disease even at high viral challenge doses, M2-specific Abs cannot prevent infection, but merely reduce disease at low challenge doses. M2-specific Abs fail to protect from high challenge doses, as vaccinated mice undergo lethal infection under these conditions. In conclusion, protection mediated by M2-hepatitis B core vaccine would be insufficient during the yearly epidemics, for which full protection is desirable, and overall is clearly inferior to protection achieved by immunization with classical inactivated viral preparations.

NKG2D-mediated natural killer cell protection against cytomegalovirus is impaired by viral gp40 modulation of retinoic acid early inducible 1 gene molecules.

Natural killer (NK) cells play a critical role in the innate immune response against cytomegalovirus (CMV) infections. Although CMV encodes several gene products committed to evasion of adaptive immunity, viral modulation of NK cell activity is only beginning to be appreciated. A previous study demonstrated that the mouse CMV m152-encoded gp40 glycoprotein diminished expression of ligands for the activating NK cell receptor NKG2D on the surface of virus-infected cells. Here we have defined the precise ligands that are affected and have directly implicated NKG2D in immune responses to CMV infection in vitro and in vivo. Murine CMV (MCMV) infection potently induced transcription of all five known retinoic acid early inducible 1 (RAE-1) genes (RAE-1α, RAE-1β, RAE-1δ, RAE-1ɛ, and RAE-1γ), but not H-60. gp40 specifically down-regulated the cell surface expression of all RAE-1 proteins, but not H-60, and diminished NK cell interferon γ production against CMV-infected cells. Consistent with previous findings, a m152 deletion mutant virus (Δm152) was less virulent in vivo than the wild-type Smith strain of MCMV. Treatment of BALB/c mice with a neutralizing anti-NKG2D antibody before infection increased titers of Δm152 virus in the spleen and liver to levels seen with wild-type virus. These experiments demonstrate that gp40 impairs NK cell recognition of virus-infected cells through disrupting the RAE-1–NKG2D interaction.

Varicella-zoster virus infection facilitates VZV glycoprotein E trafficking to the membrane surface of melanoma cells.

Varicella-zoster virus glycoprotein E (gE) is the most abundant VZV glycoprotein on the surface of virus-infected cells. VZV gE has targeting sequences for the trans-Golgi network (TGN) and is transported from the ER to the TGN in infected and gE-transfected cells. In this study, VZV gE expressing melanoma cell lines were generated. gE is expressed under the control of the reverse Tet repressor (Tet-On). gE induced by Tet-On is retained at the ER as well as in the cis Golgi by immunofluorescence confocal microscopy. To test whether other viral protein(s) may facilitate gE trafficking and surface localization, MSPgE-vOka virus that contains MSPgE in place of wt gE was made. MAb 3B3 anti-gE does not bind to MSPgE. This MAb was used to track the localization of gE in Met-gE cells post MSPgE-vOka infection. gE became detectable mostly at the TGN and on the cell surface after viral infection. These data indicate that viral proteins facilitate the trafficking and cell surface expression of gE.

Viral interference with antigen presentation

CD8+ T cells play an important role in immunity to viruses. Just how important these cells are is demonstrated by the evolution of viral strategies for blocking the generation or display of peptide-major histocompatibility complex class I complexes on the surfaces of virus-infected cells. Here, we focus on viral interference with antigen presentation; in particular we consider the importance (and difficulty) of establishing the evolutionary significance (that is, the ability to enhance viral transmission) of viral gene products that interfere with antigen presentation in vitro.

Role of Phosphatidylserine Exposure and Sugar Chain Desialylation at the Surface of Influenza Virus-infected Cells in Efficient Phagocytosis by Macrophages

HeLa cells infected with influenza A virus undergo typical caspase-dependent apoptosis and are efficiently phagocytosed by mouse peritoneal macrophages in a manner mediated by the membrane phospholipid phosphatidylserine, which is translocated to the surface of virus-infected cells during apoptosis. However, the extent of phagocytosis is not always parallel with the level of phosphatidylserine externalization. Here we examined the involvement of influenza virus neuraminidase (NA) in efficient phagocytosis of virus-infected cells. HeLa cells infected with an influenza virus strain expressing temperature-sensitive NA underwent apoptosis and produced viral proteins, including the defective NA, at a non-permissive temperature to almost the same extent as cells infected with the wild-type virus. The cells were, however, phagocytosed by macrophages with reduced efficiency. In addition, phagocytosis of cells infected with the wild-type virus was severely inhibited when the cells had been maintained in the presence of the NA inhibitor zanamivir. On the other hand, the binding of sialic acid-recognizing lectins to the cell surface declined after infection with the wild-type virus. The decrease in the extent of lectin binding was greatly attenuated when cells were infected with the mutant virus or when wild-type virus-infected cells were maintained in the presence of zanamivir. These results indicate that sugar chains are desialylated by NA at the surface of virus-infected cells. We conclude that the presence of both phosphatidylserine and asialoglycomoieties on the cell surface is required for efficient phagocytosis of influenza virus-infected cells by macrophages.

Caveolin-1 is incorporated into mature respiratory syncytial virus particles during virus assembly on the surface of virus-infected cells.

We have employed immunofluorescence microscopy and transmission electron microscopy to examine the assembly and maturation of respiratory syncytial virus (RSV) in the Vero cell line C1008. RSV matures at the apical cell surface in a filamentous form that extends from the plasma membrane. We observed that inclusion bodies containing viral ribonucleoprotein (RNP) cores predominantly appeared immediately below the plasma membrane, from where RSV filaments form during maturation at the cell surface. A comparison of mock-infected and RSV-infected cells by confocal microscopy revealed a significant change in the pattern of caveolin-1 (cav-1) fluorescence staining. Analysis by immuno-electron microscopy showed that RSV filaments formed in close proximity to cav-1 clusters at the cell surface membrane. In addition, immuno-electron microscopy showed that cav-1 was closely associated with early budding RSV. Further analysis by confocal microscopy showed that cav-1 was subsequently incorporated into the envelope of RSV filaments maturing on the host cell membrane, but was not associated with other virus structures such as the viral RNPs. Although cav-1 was incorporated into the mature virus, it was localized in clusters rather than being uniformly distributed along the length of the viral filaments. Furthermore, when RSV particles in the tissue culture medium from infected cells were examined by immuno-negative staining, the presence of cav-1 on the viral envelope was clearly demonstrated. Collectively, these findings show that cav-1 is incorporated into the envelope of mature RSV particles during egress.

Overview of the cellular immunity against JC virus in progressive multifocal leukoencephalopathy.

The human polyomavirus JC (JCV) infects most healthy adults without causing any disease. In the setting of severe deficit of cell-mediated immunity, such as in acquired immunodeficiency syndrome (AIDS), malignancies or in organ transplant recipients, JCV can reactivate and cause progressive multifocal leukoencephalopathy (PML), a deadly demyelinating disease of the central nervous system. The humoral immune response, measured by the presence of virus-specific immunoglobulin G (IgG) in the blood or by intrathecal synthesis of IgG in the cerebrospinal fluid (CSF), is unable to contain the progression of PML. CD4+ T lymphocytes recognize extracellular viral proteins that have been degraded into peptides through the exogenous pathway and presented on major histocompatibility complex (MHC) class II molecules at the surface of antigen-presenting cells. Consistent with their underlying immunosuppression, the proliferative response of CD4+ T lymphocytes to mitogens or JCV antigens is reduced in PML patients. CD8+ cytotoxic T lymphocytes recognize intracellularly synthesized viral proteins that have been degraded into peptides through the endogenous pathway, and presented on MHC class I molecules at the surface of virus-infected cells. One of such JCV peptide, the VP1(p100) ILMWEAVTL, has been characterized as a cytotoxic T lymphocyte (CTL) epitope in HLA-A *0201 + PML survivors. Staining with the corresponding A *0201/JCV VP1(p100) tetrameric complex showed that VP1(p100)-stimulated peripheral blood mononuclear cells (PBMCs) of 5/7 (71%) PML survivors had JCV-specific CTL, versus none of 6 PML progressors (P = .02). This cellular immune response may therefore be crucial in the prevention of PML disease progression and the tetramer staining assay may be used as a prognostic marker in the clinical management of these patients.

Furin cleavage of the respiratory syncytial virus fusion protein is not a requirement for its transport to the surface of virus-infected cells.

The intracellular cleavage of respiratory syncytial virus (RSV) fusion (F) protein by furin was examined. In RSV-infected LoVo cells, which express an inactive form of furin, and in RSV-infected Vero cells treated with the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (dec-RVKR-cmk), the F protein was expressed as a non-cleaved 73 kDa species. In both cases the F protein was initially expressed as an endoglycosidase H (Endo H)-sensitive precursor (F0(EHs)) which was modified approximately 40 min post-synthesis by the addition of complex carbohydrates to produce the Endo H-resistant form (F0(EHr)). The size and glycosylation state of F0(EHr) were identical to a transient intermediate form of non-cleaved F protein which was detected in RSV-infected Vero cells in the absence of inhibitor. Cell surface biotinylation and surface immunofluorescence staining showed that F0(EHr) was present on the surface of RSV-infected cells. RSV filaments have been shown to be the predominant form of the budding virus that is detected during virus replication. Analysis of the RSV-infected cells using scanning electron microscopy (SEM) showed that, in the presence of dec-RVKR-cmk, virus budding was impaired, producing fewer and much smaller viral filaments than in untreated cells. A comparison of immunofluorescence and SEM data showed that F0(EHr) was routed to the surface of virus-infected cells but not located in these smaller structures. Our findings suggest that activation of the F protein is required for the efficient formation of RSV filaments.

Recombinant rinderpest vaccines expressing membrane-anchored proteins as genetic markers: evidence of exclusion of marker protein from the virus envelope.

Rinderpest virus (RPV) causes a severe disease of cattle resulting in serious economic losses in parts of the developing world. Effective control and elimination of this disease require a genetically marked rinderpest vaccine that allows serological differentiation between animals that have been vaccinated against rinderpest and those which have recovered from natural infection. We have constructed two modified cDNA clones of the vaccine strain RNA genome of the virus, with the coding sequence of either a receptor site mutant form of the influenza virus hemagglutinin (HA) gene or a membrane-anchored form of the green fluorescent protein (GFP) gene (ANC-GFP), inserted as a potential genetic marker. Infectious recombinant virus was rescued in cell culture from both constructs. The RPVINS-HA and RPVANC-GFP viruses were designed to express either the HA or ANC-GFP protein on the surface of virus-infected cells with the aim of stimulating a strong humoral antibody response to the marker protein. In vitro studies showed that the marker proteins were expressed on the surface of virus-infected cells, although to different extents, but neither was incorporated into the envelope of the virus particles. RPVINS-HA- or RPVANC-GFP-vaccinated cattle produced normal levels of humoral anti-RPV antibodies and significant levels of anti-HA or anti-GFP antibodies, respectively. Both viruses were effective in stimulating protective immunity against RPV and antibody responses to the marker protein in all animals when tested in a cattle vaccination trial.

Phosphatidylserine-mediated phagocytosis of influenza A virus-infected cells by mouse peritoneal macrophages.

Influenza virus induces apoptosis in cultured cell lines as well as in animal tissues. HeLa cells were infected with influenza virus A/Udon/72 (H3N2) under conditions resulting in almost 100% infection. Such cells underwent typical caspase-dependent apoptosis and were efficiently phagocytosed by macrophages prepared from peritoneal fluids of thioglycolate-treated mice. The membrane phospholipid phosphatidylserine appeared on the surfaces of virus-infected cells at around the time efficient phagocytosis became detectable. In fact, the phagocytosis was almost completely inhibited in the presence of liposomes containing phosphatidylserine, which did not influence the antibody-dependent uptake of zymosan particles by the same macrophages. These results indicate that macrophages phagocytose influenza virus-infected HeLa cells in a manner mediated by phosphatidylserine that appears on the surfaces of infected cells during the process of apoptosis.

Complement and immunity to viruses.

Complement is one of the first lines of host defence to be faced and countered by viruses as they struggle to establish an infection. As an important arm of the humoral immune response, the complement system is immediately ready to target and eliminate virus particles and to interact with the surface of virus-infected cells to mark them for destruction by other branches of the immune response. Nevertheless, some viruses are still very successful human pathogens. This article will discuss the role of complement in antiviral immunity, the mechanisms by which complement may be activated by viruses or virus-infected cells, and explore some of the strategies which viruses have evolved to subvert the immune response, including mechanisms by which complement activation may be prevented or aborted.

Host-derived ICAM-1 glycoproteins incorporated on human immunodeficiency virus type 1 are biologically active and enhance viral infectivity

Human immunodeficiency virus type 1 (HIV-1) acquires several host cell membrane proteins when it buds from infected cells. To study the effect of virally incorporated host-derived ICAM-1 glycoproteins on the biology of HIV-1, we have developed a transient expression system that has enabled us to produce virus particles differing only in the absence or the presence of virion-bound ICAM-1. By using a single-round infection assay based on an ICAM-1-negative target T-cell line stably transfected with an HIV-1 long terminal repeat driven luciferase gene construct, we have been able to demonstrate that the acquisition of host-derived ICAM-1 by HIV-1 has functional significance, since it leads to a pronounced increase in viral infectivity (4.6- to 9.8-fold) in an ICAM-1/LFA-1-dependent fashion, as shown by blocking with anti-ICAM-1 and -LFA-1 antibodies. The same potentiating effect on viral infectivity was also observed with monocytoid cells. Studies of the kinetics of infection revealed that the positive effect mediated by virally embedded host cell membrane ICAM-1 is due to an increase in the efficiency of early steps in the viral life cycle. These results provide new insights into how incorporation of host proteins can modulate the biological properties of HIV-1. Our findings have direct clinical relevance, considering that ICAM-1 is expressed on the surface of virus-infected cells and, more importantly, that host-derived ICAM-1 has been shown to be acquired by clinical HIV-1 isolates grown on primary mononuclear cells. These data justify a more complete analysis of the other putative role(s) that virally incorporated ICAM-1 may play in the life cycle of HIV-1, for example, at the level of neutralization sensitivity.

In vitro cytocidal effects of human immunodeficiency virus type 1 Nef on unprimed human CD4+ T cells without MHC restriction.

We have previously shown that the C-terminal region of human immunodeficiency virus type 1 (HIV-1) Nef antigen present on the outer surface of virus-infected cells has an affinity for uninfected T cells and that the Nef protein is responsible for T cell death. To exclude completely the possibility of MHC restriction of this cytotoxic activity, the in vitro cytotoxic potential of HIV-1 Nef against various CD4+ T cell lines as well as naive T lymphocytes was investigated using a baculovirus expression system. Insect cells expressing myristoylated Nef on their cell surface were shown to kill a proportion of CD4+ T cells within 8 h. However, N-terminal truncated and unmyristoylated Nef proteins were not present on the outer surface of insect cell membranes and failed to show any killing activity. Monoclonal antibodies against the C-terminal region of Nef inhibited cytolysis. Thus, we conclude that specific Nef-mediated cytolysis is induced by contact with unprimed CD4+ T lymphocytes without MHC restriction.

Human immunodeficiency virus type 1 Nef protein on the cell surface is cytocidal for human CD4+ T cells

We have previously shown that the carboxyl-terminal region of human immunodeficiency virus type 1 (HIV-1) Nef antigen present on the outer surface of virus-infected cells has affinity for uninfected T cells. Here, the in vitro cytotoxic potential of HIV-1 Nef on the T cell surface against CD4+ T cells was investigated in detail. Human T cells expressing Nef on the cell surface by transfection with non-infectious mutant HIV-1 proviruses were demonstrated to kill CD4+ T cells effeciently. Furthermore, it was shown that the carboxyl-terminal portion of Nef was cytotoxic for CD4+ T cells and that monoclonal antibody against the carboxyl-terminal region of Nef inhibited Nef induced-cytolysis. Thus, we concluded that Nef protein on CD4+ T cells may play an important role in the specific loss of CD4+ T lymphocytes during HIV-1 infection.