Surface Of Infected Cells Research Articles

Combination of Antiretroviral Drugs and Radioimmunotherapy Specifically Kills Infected Cells from HIV-Infected Individuals

Eliminating virally infected cells is an essential component of any HIV eradication strategy. Radioimmunotherapy (RIT), a clinically established method for killing cells using radiolabeled antibodies, was recently applied to target HIV-1 gp41 antigen expressed on the surface of infected cells. Since gp41 expression by infected cells is likely downregulated in patients on antiretroviral therapy (ART), we evaluated the ability of RIT to kill ART-treated infected cells using both in vitro models and lymphocytes isolated from HIV-infected subjects. Human peripheral blood mononuclear cells (PBMCs) were infected with HIV and cultured in the presence of two clinically relevant ART combinations. Scatchard analysis of the 2556 human monoclonal antibody to HIV gp41 binding to the infected and ART-treated cells demonstrated sufficient residual expression of gp41 on the cell surface to warrant subsequent RIT. This is the first time the quantification of gp41 post-ART is being reported. Cells were then treated with Bismuth-213-labeled 2556 antibody. Cell survival was quantified by Trypan blue and residual viremia by p24 ELISA. Cell surface gp41 expression was assessed by Scatchard analysis. The experiments were repeated using PBMCs isolated from blood specimens obtained from 15 HIV-infected individuals: 10 on ART and 5 ART-naïve. We found that 213Bi-2556 killed ART-treated infected PBMCs and reduced viral production to undetectable levels. ART and RIT co-treatment was more effective at reducing viral load in vitro than either therapy alone, indicating that gp41 expression under ART was sufficient to allow 213Bi-2556 to deliver cytocidal doses of radiation to infected cells. This study provides proof of concept that 213Bi-2556 may represent an innovative and effective targeting method for killing HIV-infected cells treated with ART and supports continued development of 213Bi-2556 for co-administration with ART toward an HIV eradication strategy.

Cross-protective potential of anti-nucleoprotein human monoclonal antibodies against lethal influenza A virus infection.

The nucleoprotein (NP) possesses regions that are highly conserved among influenza A viruses, and has therefore been one of the target viral proteins for development of a universal influenza vaccine. It has been expected that human or humanized antibodies will be made available for the prophylaxis, pre-emptive and acute treatment of viral infection. However, it is still unclear whether anti-NP human antibody can confer protection against influenza virus infection. In this study, we generated transgenic mice expressing anti-NP human mAbs derived from lymphocytes of a patient infected with H5N1 highly pathogenic avian influenza (HPAI) virus, and experimental infections were conducted to examine antiviral effects of the anti-NP antibodies against H5N1 HPAI viral infections with a high fatality rate in mammals. Transgenic mouse lines expressing the anti-NP human mAbs at more than 1 mg ml-1 showed marked resistance to H5N1 virus infections. In addition, resistance to infection with an H1N1 subtype that shows strong pathogenicity to mice was also confirmed. Although the anti-NP mAbs expressed in the transgenic mice did not neutralize the virus, the mAbs could bind to NP located on the surface of infected cells. These results suggested a possibility that the non-neutralizing anti-NP human mAbs could induce indirect antiviral effects, such as antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity. Taken together, these results demonstrated that anti-NP human mAbs play an important role in heterosubtypic protection against lethal influenza virus infections in vivo.

Novel Acylguanidine-Based Inhibitor of HIV-1.

The emergence of transmissible HIV-1 strains with resistance to antiretroviral drugs highlights a continual need for new therapies. Here we describe a novel acylguanidine-containing compound, 1-(2-(azepan-1-yl)nicotinoyl)guanidine (or SM111), that inhibits in vitro replication of HIV-1, including strains resistant to licensed protease, reverse transcriptase, and integrase inhibitors, without major cellular toxicity. At inhibitory concentrations, intracellular p24(Gag) production was unaffected, but virion release (measured as extracellular p24(Gag)) was reduced and virion infectivity was substantially impaired, suggesting that SM111 acts at a late stage of viral replication. SM111-mediated inhibition of HIV-1 was partially overcome by a Vpu I17R mutation alone or a Vpu W22* truncation in combination with Env N136Y. These mutations enhanced virion infectivity and Env expression on the surface of infected cells in the absence and presence of SM111 but also impaired Vpu's ability to downregulate CD4 and BST2/tetherin. Taken together, our results support acylguanidines as a class of HIV-1 inhibitors with a distinct mechanism of action compared to that of licensed antiretrovirals. Further research on SM111 and similar compounds may help to elucidate knowledge gaps related to Vpu's role in promoting viral egress and infectivity. New inhibitors of HIV-1 replication may be useful as therapeutics to counteract drug resistance and as reagents to perform more detailed studies of viral pathogenesis. SM111 is a small molecule that blocks the replication of wild-type and drug-resistant HIV-1 strains by impairing viral release and substantially reducing virion infectivity, most likely through its ability to prevent Env expression at the infected cell surface. Partial resistance to SM111 is mediated by mutations in Vpu and/or Env, suggesting that the compound affects host/viral protein interactions that are important during viral egress. Further characterization of SM111 and similar compounds may allow more detailed pharmacological studies of HIV-1 egress and provide opportunities to develop new treatments for HIV-1.

Origins and Evolution of tetherin, an Orphan Antiviral Gene.

Tetherin encodes an interferon-inducible antiviral protein that traps a broad spectrum of enveloped viruses at infected cell surfaces. Despite the absence of any clearly related gene or activity, we describe possible scenarios by which tetherin arose that exemplify how protein modularity, evolvability, and robustness can create and preserve new functions. We find that tetherin genes in various organisms exhibit no sequence similarity and share only a common architecture and location in modern genomes. Moreover, tetherin is part of a cluster of three potential sister genes encoding proteins of similar architecture, some variants of which exhibit antiviral activity while others can be endowed with antiviral activity by a simple modification. Only in slowly evolving species (e.g., coelacanths) does tetherin exhibit sequence similarity to one potential sister gene. Neofunctionalization, drift, and genetic conflict appear to have driven a near complete loss of sequence similarity among modern tetherin genes and their sister genes.

Analysis of contributions of herpes simplex virus type 1 UL43 protein to induction of cell-cell fusion

Purpose : To investigate whether UL43 protein, which is highly conserved in alpha- and gamma herpes viruses, and a non-glycosylated transmembrane protein, is involved in virus entry and virus-induced cell fusion. Methods : Mutagenesis was accomplished by a markerless two-step Red recombination mutagenesis system implemented on the Herpes simplex virus 1 (HSV-1) bacterial artificial chromosome (BAC). Growth properties of HSV-1 UL43 mutants were analyzed using plaque morphology and one-step growth kinetics. SDS-PAGE and Western blot was employed to assay the synthesis of the viral glycoproteins. Virus-penetration was assayed to determine if UL43 protein is required for efficient virus entry. Results : Lack of UL43 expression resulted in significantly reduced plaque sizes of syncytial mutant viruses and inhibited cell fusion induced by gBΔ28 or gKsyn20 (p < 0.05). Deletion of UL43 did not affect overall expression levels of viral glycoproteins gB, gC, gD, and gH on HSV-1(F) BAC infected cell surfaces. Moreover, mutant viruses lacking UL43 gene exhibited slower kinetics of entry into Vero cells than the parental HSV-1(F) BAC. Conclusion : Thus, these results suggest an important role for UL43 protein in mediating virus-induced membrane fusion and efficient entry of virion into target cells. Keywords : Herpes simplex virus type 1, UL43 protein, Membrane fusion, Mutant viruses, Virion, Mutagenesis

Paramyxovirus Glycoproteins and the Membrane Fusion Process.

The family Paramyxoviridae includes many viruses that significantly affect human and animal health. An essential step in the paramyxovirus life cycle is viral entry into host cells, mediated by virus-cell membrane fusion. Upon viral entry, infection results in expression of the paramyxoviral glycoproteins on the infected cell surface. This can lead to cell-cell fusion (syncytia formation), often linked to pathogenesis. Thus membrane fusion is essential for both viral entry and cell-cell fusion and an attractive target for therapeutic development. While there are important differences between viral-cell and cell-cell membrane fusion, many aspects are conserved. The paramyxoviruses generally utilize two envelope glycoproteins to orchestrate membrane fusion. Here, we discuss the roles of these glycoproteins in distinct steps of the membrane fusion process. These findings can offer insights into evolutionary relationships among Paramyxoviridae genera and offer future targets for prophylactic and therapeutic development.

Cytomegalovirus-Infected Cells Resist T Cell Mediated Killing in an HLA-Recognition Independent Manner.

In order to explore the potential of HLA-independent T cell therapy for human cytomegalovirus (HCMV) infections, we developed a chimeric antigen receptor (CAR) directed against the HCMV encoded glycoprotein B (gB), which is expressed at high levels on the surface of infected cells. T cells engineered with this anti-gB CAR recognized HCMV-infected cells and released cytokines and cytotoxic granules. Unexpectedly, and in contrast to analogous approaches for HIV, Hepatitis B or Hepatitis C virus, we found that HCMV-infected cells were resistant to killing by the CAR-modified T cells. In order to elucidate whether this phenomenon was restricted to the use of CARs, we extended our experiments to T cell receptor (TCR)-mediated recognition of infected cells. To this end we infected fibroblasts with HCMV-strains deficient in viral inhibitors of antigenic peptide presentation and targeted these HLA-class I expressing peptide-loaded infected cells with peptide-specific cytotoxic T cells (CTLs). Despite strong degranulation and cytokine production by the T cells, we again found significant inhibition of lysis of HCMV-infected cells. Impairment of cell lysis became detectable 1 day after HCMV infection and gradually increased during the following 3 days. We thus postulate that viral anti-apoptotic factors, known to inhibit suicide of infected host cells, have evolved additional functions to directly abrogate T cell cytotoxicity. In line with this hypothesis, CAR-T cell cytotoxicity was strongly inhibited in non-infected fibroblasts by expression of the HCMV-protein UL37x1, and even more so by additional expression of UL36. Our data extend the current knowledge on Betaherpesviral evasion from T cell immunity and show for the first time that, beyond impaired antigen presentation, infected cells are efficiently protected by direct blockade of cytotoxic effector functions through viral proteins.

Characterization of the Antigen Processing Machinery and Endogenous Peptide Presentation of a Bat MHC Class I Molecule.

Bats are a major reservoir of emerging and re-emerging infectious diseases, including severe acute respiratory syndrome-like coronaviruses, henipaviruses, and Ebola virus. Although highly pathogenic to their spillover hosts, bats harbor these viruses, and a large number of other viruses, with little or no clinical signs of disease. How bats asymptomatically coexist with these viruses is unknown. In particular, little is known about bat adaptive immunity, and the presence of functional MHC molecules is mostly inferred from recently described genomes. In this study, we used an affinity purification/mass spectrometry approach to demonstrate that a bat MHC class I molecule, Ptal-N*01:01, binds antigenic peptides and associates with peptide-loading complex components. We identified several bat MHC class I-binding partners, including calnexin, calreticulin, protein disulfide isomerase A3, tapasin, TAP1, and TAP2. Additionally, endogenous peptide ligands isolated from Ptal-N*01:01 displayed a relatively broad length distribution and an unusual preference for a C-terminal proline residue. Finally, we demonstrate that this preference for C-terminal proline residues was observed in Hendra virus-derived peptides presented by Ptal-N*01:01 on the surface of infected cells. To our knowledge, this is the first study to identify endogenous and viral MHC class I ligands for any bat species and, as such, provides an important avenue for monitoring and development of vaccines against major bat-borne viruses both in the reservoir and spillover hosts. Additionally, it will provide a foundation to understand the role of adaptive immunity in bat antiviral responses.

The Epstein-Barr Virus Glycoprotein gp150 Forms an Immune-Evasive Glycan Shield at the Surface of Infected Cells

Cell-mediated immunity plays a key role in host control of viral infection. This is exemplified by life-threatening reactivations of e.g. herpesviruses in individuals with impaired T-cell and/or iNKT cell responses. To allow lifelong persistence and virus production in the face of primed immunity, herpesviruses exploit immune evasion strategies. These include a reduction in viral antigen expression during latency and a number of escape mechanisms that target antigen presentation pathways. Given the plethora of foreign antigens expressed in virus-producing cells, herpesviruses are conceivably most vulnerable to elimination by cell-mediated immunity during the replicative phase of infection. Here, we show that a prototypic herpesvirus, Epstein-Barr virus (EBV), encodes a novel, broadly acting immunoevasin, gp150, that is expressed during the late phase of viral replication. In particular, EBV gp150 inhibits antigen presentation by HLA class I, HLA class II, and the non-classical, lipid-presenting CD1d molecules. The mechanism of gp150-mediated T-cell escape does not depend on degradation of the antigen-presenting molecules nor does it require gp150’s cytoplasmic tail. Through its abundant glycosylation, gp150 creates a shield that impedes surface presentation of antigen. This is an unprecedented immune evasion mechanism for herpesviruses. In view of its likely broader target range, gp150 could additionally have an impact beyond escape of T cell activation. Importantly, B cells infected with a gp150-null mutant EBV displayed rescued levels of surface antigen presentation by HLA class I, HLA class II, and CD1d, supporting an important role for iNKT cells next to classical T cells in fighting EBV infection. At the same time, our results indicate that EBV gp150 prolongs the timespan for producing viral offspring at the most vulnerable stage of the viral life cycle.

The viral G protein-coupled receptor ORF74 unmasks phospholipase C signaling of the receptor tyrosine kinase IGF-1R

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes the constitutively active G protein-coupled receptor ORF74, which is expressed on the surface of infected host cells and has been linked to the development of the angioproliferative tumor Kaposi's sarcoma. Furthermore, the insulin-like growth factor (IGF)-1 receptor, a receptor tyrosine kinase, also plays an essential role in Kaposi's sarcoma growth and survival.In this study we examined the effect of the constitutively active viral receptor ORF74 on human IGF-1R signaling. Constitutive and CXCL1-induced ORF74 signaling did not transactivate IGF-1R. In contrast, IGF-1 stimulated phospholipase C (PLC) activation in an ORF74-dependent manner without affecting chemokine binding to ORF74. Inhibition of constitutive ORF74 activity by mutagenesis or the inverse agonist CXCL10, or neutralizing IGF-1R with an antibody or silencing IGF-1R expression using siRNA inhibited PLC activation by IGF-1. Transactivation of ORF74 in response to IGF-1 occurred independently of Src, PI3K, and secreted ORF74 ligands. Furthermore, tyrosine residues in the carboxyl-terminus and intracellular loop 2 of ORF74 are not essential for IGF-1-induced PLC activation. Interestingly, PLC activation in response to IGF-1 is specific for ORF74 as IGF-1 was unable to activate PLC in cells expressing the constitutively active human cytomegalovirus (HCMV)-encoded GPCR US28. Interestingly, IGF-1 does not induce β-arrestin recruitment to ORF74. The proximity ligation assay revealed close proximity between ORF74 and IGF-1R on the cell surface, but a physical interaction was not confirmed by co-immunoprecipitation. Unmasking IGF-1R signaling to PLC in response to IGF-1 is a previously unrecognized action of ORF74.

A microRNA from infectious spleen and kidney necrosis virus modulates expression of the virus-mock basement membrane component VP08R

Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus, family Iridoviridae. Infection of ISKNV is characterized by a unique pathological phenomenon in that the infected cells are attached by lymphatic endothelial cells (LECs). ISKNV mediates the formation of a virus-mock basement membrane (VMBM) structure on the surface of infected cells to provide attaching sites for LECs. The viral protein VP08R is an important component of VMBM. In this study, a novel ISKNV-encoded microRNA, temporarily named ISKNV-miR-1, was identified. ISKNV-miR-1 is complementary to the VP08R-coding sequence and can modulate VP08R expression through reducing its mRNA level. This suggests that formation of VMBM may be under fine regulation by ISKNV.

Filamin A Is Involved in HIV-1 Vpu-mediated Evasion of Host Restriction by Modulating Tetherin Expression

Tetherin, also known as bone marrow stromal antigen 2 (BST-2), inhibits the release of a wide range of enveloped viruses, including human immunodeficiency virus, type 1 (HIV-1) by directly tethering nascent virions to the surface of infected cells. The HIV-1 accessary protein Vpu counteracts tetherin restriction via sequestration, down-regulation, and/or displacement mechanisms to remove tetherin from sites of virus budding. However, the exact mechanism of Vpu-mediated antagonism of tetherin restriction remains to be fully understood. Here we report a novel role for the actin cross-linking regulator filamin A (FLNa) in Vpu anti-tetherin activities. We demonstrate that FLNa associates with tetherin and that FLNa modulates tetherin turnover. FLNa deficiency was found to enhance cell surface and steady-state levels of tetherin expression. In contrast, we observed that overexpression of FLNa reduced tetherin expression levels both on the plasma membrane and in intracellular compartments. Although FLNb shows high amino acid sequence similarity with FLNa, we reveal that only FLNa, but not FLNb, plays an essential role in tetherin turnover. We further showed that FLNa deficiency inhibited Vpu-mediated enhancement of virus release through interfering with the activity of Vpu to down-regulate cellular tetherin. Taken together, our studies suggest that Vpu hijacks the FLNa function in the modulation of tetherin to neutralize the antiviral factor tetherin. These findings may provide novel strategies for the treatment of HIV-1 infection.

Targets and Mechanisms Associated with Protection from Severe Plasmodium falciparum Malaria in Kenyan Children.

Severe malaria (SM) is a life-threatening complication of infection with Plasmodium falciparum. Epidemiological observations have long indicated that immunity against SM is acquired relatively rapidly, but prospective studies to investigate its immunological basis are logistically challenging and have rarely been undertaken. We investigated the merozoite targets and antibody-mediated mechanisms associated with protection against SM in Kenyan children aged 0 to 2 years. We designed a unique prospective matched case-control study of well-characterized SM clinical phenotypes nested within a longitudinal birth cohort of children (n = 5,949) monitored over the first 2 years of life. We quantified immunological parameters in sera collected before the SM event in cases and their individually matched controls to evaluate the prospective odds of developing SM in the first 2 years of life. Anti-AMA1 antibodies were associated with a significant reduction in the odds of developing SM (odds ratio [OR] = 0.37; 95% confidence interval [CI] = 0.15 to 0.90; P = 0.029) after adjustment for responses to all other merozoite antigens tested, while those against MSP-2, MSP-3, Plasmodium falciparum Rh2 [PfRh2], MSP-119, and the infected red blood cell surface antigens were not. The combined ability of total IgG to inhibit parasite growth and mediate the release of reactive oxygen species from neutrophils was associated with a marked reduction in the odds of developing SM (OR = 0.07; 95% CI = 0.006 to 0.82; P = 0.03). Assays of these two functional mechanisms were poorly correlated (Spearman rank correlation coefficient [rs] = 0.12; P = 0.07). Our data provide epidemiological evidence that multiple antibody-dependent mechanisms contribute to protective immunity via distinct targets whose identification could accelerate the development of vaccines to protect against SM.

Cell fusion assay by expression of respiratory syncytial virus (RSV) fusion protein to analyze the mutation of palivizumab-resistant strains

Respiratory syncytial virus (RSV) consists of fusion (F), glyco (G), and small hydrophobic (SH) proteins as envelope proteins, and infects through cell fusion. F protein is expressed on the surface of infected cells, and induces cell fusion. In the present report, expression plasmids of the F, G and SH proteins were constructed and cell fusion activity was investigated under T7 RNA polymerase. F protein alone induced cell fusion at a lower concentration than previously reported, and co-expression of F and SH proteins induced cell fusion more efficiently than F protein alone. Palivizumab is the only prophylactic agent against RSV infection. Palivizumab-resistant strains having mutations of the F protein of K272E and S275F were reported. These mutations were introduced into an F-expression plasmid, and exhibited no inhibition of cell fusion with palivizumab. Among the RSV F protein mutants, N276S has been reported to have partial resistance against palivizumab, but the F expression plasmid with the N276S mutation showed a reduction in cell fusion in the presence of palivizumab, showing no resistance to palivizumab. The present expression system was useful for investigating the mechanisms of RSV cell fusion.

Immune response in virus model structured by cell infection-age.

This paper concerns modeling the coupled within-host population dynamics of virus and CTL (Cytotoxic T Lymphocyte) immune response. There is substantial evidence that the CTL immune response plays a crucial role in controlling HIV in infected patients. Recent experimental studies have demonstrated that certain CTL variants can recognize HIV infected cells early in the infected cell lifecycle before viral production, while other CTLs only detect viral proteins (epitopes) presented on the surface of infected cells after viral production. The kinetics of epitope presentation and immune recognition can impact the efficacy of the immune response. We extend previous virus models to include cell infection-age structure in the infected cell compartment and immune response killing/activation rates of a PDE-ODE system. We characterize solutions to our system utilizing semigroup theory, determine equilibria and reproduction numbers, and prove stability and persistence results. Numerical simulations show that ' early immune recognition' precipitates both enhanced viral control and sustained oscillations via a Hopf bifurcation. In addition to inducing oscillatory dynamics, considering immune process rates to be functions of cell infection-age can also lead to coexistence of multiple distinct immune effector populations.

CD4 downregulation by the HIV-1 protein Nef reveals distinct roles for the γ1 and γ2 subunits of the AP-1 complex in protein trafficking.

The HIV accessory protein Nef is a major determinant of viral pathogenesis that facilitates viral particle release, prevents viral antigen presentation and increases infectivity of new virus particles. These functions of Nef involve its ability to remove specific host proteins from the surface of infected cells, including the CD4 receptor. Nef binds to the adaptor protein 2 (AP-2) and CD4 in clathrin-coated pits, forcing CD4 internalization and its subsequent targeting to lysosomes. Herein, we report that this lysosomal targeting requires a variant of AP-1 containing isoform 2 of γ-adaptin (AP1G2, hereafter γ2). Depletion of the γ2 or μ1A (AP1M1) subunits of AP-1, but not of γ1 (AP1G1), precludes Nef-mediated lysosomal degradation of CD4. In γ2-depleted cells, CD4 internalized by Nef accumulates in early endosomes and this alleviates CD4 removal from the cell surface. Depletion of γ2 also hinders EGFR-EGF-complex targeting to lysosomes, an effect that is not observed upon γ1 depletion. Taken together, our data provide evidence that the presence of γ1 or γ2 subunits delineates two distinct variants of AP-1 complexes, with different functions in protein sorting.

Substrate Specificity of Equine and Human Influenza A Virus Sialidase to Molecular Species of Sialic Acid.

Most equine influenza A viruses (IAVs) show strong binding to glycoconjugates containing N-glycolylneuraminic acid (Neu5Gc) as well as N-acetylneuraminic acid (Neu5Ac). Therefore, the progeny of equine IAV is thought to be released from the infected cell surface through removal of sialic acids by the viral sialidase. In the present study, equine IAV sialidases showed significantly lower substrate affinity than that of human IAV sialidases to artificial and natural Neu5Gc-conjugated substrates. The substrate specificity of equine IAV sialidases is in disagreement with their binding specificity to molecular species of sialic acid. The results suggest that substrate specificity of equine IAV sialidase for Neu5Ac, rather than for Neu5Gc, is important for an advantage at the early infection stage and the process of progeny virus release from the surface of infected cells.

The Yin and Yang of ADCC-Mediating Antibodies

Antibodies mediating antibody-dependent cellular cytotoxicity (ADCC) have been of increasing interest since their identification as a protective immune correlate in the RV144 clinical vaccine efficacy trial (Haynes et al., 2012). Earlier studies associated these non-neutralizing antibodies with protection against HIV infection and disease progression in both humans and non-human primates (for review see Vargas-Inchaustegui and Robert-Guroff, 2013). Therefore it is somewhat surprising to find them now linked to HIV pathogenesis. HIV disease progression is marked by a gradual loss of CD4+ T cells during which a small proportion of HIV-infected CD4+ T cells and a much larger proportion of uninfected “bystander” CD4+ T cells are killed. While numerous mechanisms have been described for infected cell killing, the mechanism of bystander killing has remained controversial. In this issue, Richard et al. (2016) attribute bystander killing largely to antibodies mediating ADCC. They further suggest that a small CD4 mimetic developed by the Sodroski group might be used to alter this pathogenic mechanism for therapeutic benefit. How can the conflicting findings regarding ADCC antibodies be explained? And what caveats should be considered in using CD4 mimetics? As reported by Richard et al. in this issue of EBioMedicine, the external envelope of HIV is labile and continually sheds the gp120 component which then binds the primary viral receptor, CD4, on bystander uninfected T cells. A subsequent conformational change opens the co-receptor binding site exposing an epitope recognized by cluster A antibodies, including the prototypic ADCC-mediating monoclonal antibody, A32. Killing of the bystander CD4+ T cells follows. Concurrently, the virus subverts killing of infected cells by down-regulating CD4, inhibiting tetherin, and internalizing Env (as cited in von Bredow et al., 2015). Consequently, less Env is expressed on the infected-cell surface, less CD4 is available to complex with gp120, and less expression of Env in the open conformation occurs. Thus ADCC killing of the infected cell is diminished in favor of bystander killing. CD4 mimetics were previously shown to interact with the viral envelope, inducing conformational changes, exposing the co-receptor binding site, and rendering the virus susceptible to neutralization by CD4-induced antibodies (Madani et al., 2008, Madani et al., 2014). The Finzi laboratory also previously reported that gp120/CD4 binding within the same infected cell led to exposure of ADCC epitopes (Veillette et al., 2014) and that the CD4 mimetic sensitized infected cells to ADCC killing (Richard et al., 2015). In the current paper they show that the mimetic decreases binding of shed gp120 to bystander CD4 cells, inferring that less ADCC-mediated killing of these uninfected cells will result. They also suggest that binding of the mimetic to envelope remaining on the infected cell surface should “redirect” ADCC-mediating antibodies to the infected cell away from bystanders, as stated above. Recent support of this hypothesis showed ADCC killing of primary HIV-infected T cells was enhanced in vitro by co-culturing with the CD4 mimetic (Lee et al., 2015). The current study was conducted primarily with the A32 antibody, together with a small number of sera from HIV-infected individuals. ADCC antibodies are among the first elicited following infection, as the gp120–CD4 interaction leads to exposure of cluster A epitopes. Not all such CD4-induced epitopes elicit equivalent ADCC activities, due to subtle differences in antibody-epitope recognition and/or Fc differences (Gohain et al., 2015). Moreover, antibodies targeting other Env sites, including some with neutralizing activity such as 2G12 and b12, also mediate ADCC. The relative abundance of all these antibodies in the human sera tested was not determined. Further, the degree of gp120 shedding and levels of CD4 and Env present on the cells tested in vitro compared to uncultured, primary HIV-infected cells was not reported. It will be critical to confirm the observations made with in vivo studies using the SHIV model. Similarly, the in vivo effects of CD4 mimetic administration must be determined. It is an obvious therapeutic candidate, however, the possibility that it might facilitate greater CD4-independent infection must be considered. Vaccine-elicited ADCC antibodies recognizing cluster A epitopes might well be protective, as they target an early epitope uncovered during virus transmission. Use of the CD4 mimetic in vaccine design might also stimulate development of such ADCC-mediating antibodies. However, if incomplete protection and break-through infection occurred following administration of such a vaccine, accelerated CD4 T cell loss and disease progression might occur by the mechanism reported by Richard et al.. To circumvent this possibility, vaccine design should include several immune targets to achieve balanced protection against HIV infection.

Characterization of Molecular Mimicry Between UL18 Glycoprotein of Human Cytomegalovirus [HCMV] and Class-I MHC Molecule through Pattern-based Analysis: An In-silico Approach

Viral replication occurs using the host cell synthesis mechanisms and they are able to exploit the mechanisms of nucleic acid replication and protein translation machinery. Viral proteins require a target to fit subcellular compartments of the host cell and localized in different cellular compartments including the nucleus. Human Cytomegalovirus downregulates expression of traditional class-I MHC [Major Histocompatibility Complex] molecules at the infected cell surface and allows the infected cells to avoid acknowledgment by cytotoxic T-cells. In the present study, we have focused on generally accessible online and offline computational tools to examine practical and fundamental characteristics of the most conserved domain [CD] of UL18 gene along with the viral class-I MHC molecule. Six open reading frames [ORF] were reanalyzed by selecting start codon. Site-specific homology was determined to the MHC class-I molecule [19 to 197 residual position; ID: pfam00129, E-value: 3.26e-14]. The predicted protein architecture contained about 28.90% helices [107 residues] and 10.32% strands [38 residues]. The tertiary structure represented that 276 residues [75% of sequence] were modeled by the single highest scoring template with 100% confidence and the structure represents a peptide-binding viral MHC mimic, apprenticed to a host inhibitory receptor [pdb code-3d2u]. Thus, our analysis suggests that the homologous sequence corresponding to MHC class-I gene is situated between 19 and 200th residues of UL18 ORF and the recognition domain was identified with significant E-value. Our study also demonstrated that the ORF18 is homologous to the Ig-superfamily in 229-289th position. Therefore, these domains were found to date homologous in HCMV proteins suggesting a particular functional role during infection. In light of the above, further experimental steps are needed to elucidate the exact role of the UL18 during infections.

Pharmacologic Inhibition of Nedd8 Activation Enzyme Exposes CD4-Induced Epitopes within Env on Cells Expressing HIV-1.

HIV-1 Vpu decreases the exposure of epitopes within the viral envelope glycoprotein (Env) on the surface of infected cells by downregulating both BST2 and CD4. To test the hypothesis that inhibiting Vpu activity would increase the exposure of these epitopes and sensitize infected cells to antibody-dependent cellular cytotoxicity (ADCC), we treated cells with the Nedd8 activation enzyme (NAE) inhibitor MLN4924, which inhibits the cullin1-based ubiquitin ligase complex coopted by Vpu to degrade cellular targets. Treatment of HeLa cells with MLN4924 or expression of a dominant negative mutant of cullin1 inhibited the Vpu-mediated downregulation of CD4 but not the downregulation of BST2. NAE inhibition also increased the surface exposure of CD4-induced epitopes within Env on HEK293 cells containing an inducible HIV genome, on infected CEM T cells, and on infected primary T cells. In contrast, the Vpu-mediated downregulation of BST2 was substantially inhibited by MLN4924 only when T cells were treated with alpha interferon (IFN-α) to induce high levels of BST2 expression. As reported previously, the absence of vpu or nef and even more so the combined absence of these two genes sensitized infected cells to ADCC. However, NAE inhibition affected ADCC minimally. Paradoxically, even in infected, IFN-treated cells in which NAE inhibition substantially rescued the surface level of BST2, the surface level of Env detected with an antibody recognizing a CD4-independent epitope (2G12) was minimally increased. Mutation of the C-terminal Vpu residue W76, which supports the ability of Vpu to stimulate virion release by displacing BST2 from assembly sites on the plasma membrane by a cullin1-independent mechanism, increased the exposure of Env detected by 2G12 on infected T cells. Thus, inhibiting the displacement function of Vpu together with its ability to degrade CD4 and BST2 may be required to sensitize infected cells to ADCC. Pathogenic viruses encode gene products that enable evasion of host immune surveillance mechanisms. One such mechanism is antibody-dependent cellular cytotoxicity (ADCC), whereby host antibodies bind envelope glycoproteins of the virus that are inserted into the cellular membrane and direct the destruction of infected cells. Targeting pharmacologically the activity of HIV-1 Vpu, which contributes to evasion of ADCC, could potentially sensitize infected cells to this immune surveillance mechanism, an outcome that would have therapeutic implications with respect to the goal of curing HIV-1 infection. The Nedd8 activation enzyme inhibitor MLN4924 blocks the activity of the host ubiquitin ligase that Vpu coopts to direct the degradation of CD4 and BST2. We observed that while MLN4924 partially reverses the activity of Vpu and could become part of a therapeutic approach by virtue of CD4-induced epitope exposure, sufficient Vpu activity as an antagonist of BST2 persists despite this drug to allow escape from ADCC.