Contribution of endocytic motifs in the cytoplasmic tail of herpes simplex virus type 1 glycoprotein B to virus replication and cell-cell fusion

The use of endocytic pathways by viral glycoproteins is thought to play various functions during viral infection. We previously showed in transfection assays that herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is transported from the cell surface back to the trans-Golgi network (TGN) and that two motifs of gB cytoplasmic tail, YTQV and LL, function distinctly in this process. To investigate the role of each of these gB trafficking signals in HSV-1 infection, we constructed recombinant viruses in which each motif was rendered nonfunctional by alanine mutagenesis. In infected cells, wild-type gB was internalized from the cell surface and concentrated in the TGN. Disruption of YTQV abolished internalization of gB during infection, whereas disruption of LL induced accumulation of internalized gB in early recycling endosomes and impaired its return to the TGN. The growth of both recombinants was moderately diminished. Moreover, the fusion phenotype of cells infected with the gB recombinants differed from that of cells infected with the wild-type virus. Cells infected with the YTQV-mutated virus displayed reduced cell-cell fusion, whereas giant syncytia were observed in cells infected with the LL-mutated virus. Furthermore, blocking gB internalization or impairing gB recycling to the cell surface, using drugs or a transdominant negative form of Rab11, significantly reduced cell-cell fusion. These results favor a role for endocytosis in virus replication and suggest that gB intracellular trafficking is involved in the regulation of cell-cell fusion.     

NKT cells are not critical for HSV-1 disease resolution

NKT cells are a minor subset of T cells that have important roles in controlling immune responses in disease states including cancer, autoimmunity and pathogenic infections. In contrast to conventional T cells, NKT cells express an invariant TCR and respond to glycolipids presented by CD1d. In this study, we sought to investigate the role of NKT cells in regulating the response to infection with HSV-1, and the mechanism involved, in well-established mouse models. Previous studies of HSV-1 disease in mice have shown clear roles for CD4+ and CD8+ T cells. The role of NKT cells in the resolution of HSV-1 (KOS strain) infection was investigated through flank zosteriform or footpad infection in wild-type versus CD1d-deficient mice, by measurement of viral plaque-forming units at different sites after infection, lesion severity and HSV-1-specific T-cell responses. In contrast to a previous study using a more virulent strain of HSV-1 (SC16 strain), no differences were observed in disease magnitude or resolution, and furthermore, the T-cell response to HSV-1 (KOS strain) was unaltered in the absence of NKT cells. In conclusion, this study shows that NKT cells do not play a general role in controlling the resolution or severity of HSV-1 infection. Instead, the resolution or severity of the infection may depend on the HSV-1 strain under investigation.     

Role of the herpes simplex virus 1 Us3 kinase phosphorylation site and endocytosis motifs in the intracellular transport and neurovirulence of envelope glycoprotein B

Herpes simplex virus 1 (HSV-1) Us3 protein kinase phosphorylates threonine at position 887 (Thr-887) in the cytoplasmic tail of envelope glycoprotein B (gB) in infected cells. This phosphorylation downregulates cell surface expression of gB and plays a role in viral pathogenesis in the mouse herpes stromal keratitis model. In the present study, we demonstrated that Us3 phosphorylation of gB Thr-887 upregulated the accumulation of endocytosed gB from the surfaces of infected cells. We also showed that two motifs in the cytoplasmic tail of gB, tyrosine at position 889 (Tyr-889) and dileucines at positions 871 and 872, were required for efficient downregulation of gB cell surface expression and upregulation of accumulation of endocytosed gB in infected cells. A systematic analysis of mutations in these three sequences in gB suggested that the expression of gB on the surfaces of infected cells was downregulated in part by the increase in the accumulation of endocytosed gB, which was coordinately and tightly regulated by the three gB trafficking signals. Tyr-889 appeared to be of predominant importance in regulating the intracellular transport of gB and was linked to HSV-1 neurovirulence in mice following intracerebral infection. These observations support the hypothesis that HSV-1 evolved the three gB sequences for proper regulation of gB intracellular transport and that this regulation plays a critical role in diverse aspects of HSV-1 pathogenesis.     

Natural killer T cells restricted by the monomorphic MHC class 1b CD1d1 molecules behave like inflammatory cells.

Murine Valpha14(inv)T cells (NKT cells), restricted by the CD1d1 MHC 1b molecules, are a distinctive subset of T cells endowed with pleiotropic functions. CD1d1-restricted NKT cells infiltrate the granulomas induced by the s.c. injection of mycobacterial phosphatidylinositoldimannoside (PIM(2)) but not of its deacylated derivative. NKT cells are detectable as early as 6 hours following the injection. Although the molecular structure of PIM(2) meets the requirements for presentation by CD1d1, Ab blocking and adoptive transfer experiments of wild-type NKT cells into CD1d1(-/-) mice show that CD1d1 expression is not required for the early recruitment of NKT cells to the injection site. This conclusion was confirmed by the finding that IL-12Rbeta(-/-) and CD40(-/-) mice were able to recruit NKT cells after PIM(2) challenge. Moreover, the injection of alpha-galactosylceramide, an NKT cell ligand that is recognized in the context of CD1d1, promoted only a minor recruitment of NKT cells. By contrast, injection of beta-galactosylceramide, a synthetic glycolipid that binds to CD1d1 but does not activate the CD1d/TCR pathway, resulted in the development of large granulomas rich in NKT cells. Finally, local injection of TNF-alpha mimics the effect of glycolipids. It is concluded that NKT cells migrate to and accumulate at inflammatory sites in the same way as other cells of the innate immune system and that migration to and accumulation at inflammatory sites are processes independent of the CD1d1 molecule.     

Differential rates of processing and transport of herpes simplex virus type 1 glycoproteins gB and gC.

The kinetics of processing and transport of herpes simplex virus type 1 (HSV-1) glycoproteins gB and gC was investigated. The conversion of precursor to mature forms and the appearance of the glycoproteins at the infected-cell surface at different times postinfection (p.i.) were studied. gB, synthesized at 4 h p.i., was converted to the mature form with a half-time (t1/2) of 120 min and appeared at the plasma membrane with a t1/2 of 270 min. The gB synthesized at later times p.i. (6, 8, and 10.5 h) was transported less efficiently. Less than 50% of gB synthesized at later times p.i. was processed and transported to the cell surface. gB synthesized in transfected cells was transported to the plasma membrane with kinetics similar to that for gB synthesized at early times p.i. gC was processed efficiently when synthesized at both 8 and 10.5 h p.i., with t1/2 of conversion of pgC to gC of 40 and 60 min, respectively. Approximately 90 to 95% of the gC synthesized was converted to the mature form. The gC synthesized at 8 h p.i. was also transported rapidly to the cell surface, compared with the transport of gB synthesized at the same time, with a t1/2 of 240 min. Greater than 70% of the gC synthesized at 8 h p.i. appeared at the cell surface. The gC synthesized at 10.5 h was transported less efficiently to the cells surface during a 6-h chase.     

Human CD1d molecules are resistant to human cytomegalovirus US2- and US11-mediated degradation

Natural killer T (NKT) cells may play a crucial role in controlling viral infection by bridging the innate and adaptive immune systems. These cells are activated by lipids presented by CD1d molecules, which are structurally homologous to major histocompatibility complex class I (MHC-I) molecules. Although human cytomegalovirus (HCMV) can avoid T cell recognition by down-regulating MHC-I-mediated antigen presentation, it remains unknown whether it can also interfere with CD1d-mediated lipid presentation. Here, we show that CD1d is resistant to rapid degradation induced by the HCMV gene products US2 and US11, which cause dislocation of MHC-I molecules from the endoplasmic reticulum (ER) to the cytosol for destruction by proteasomes. The resistance of CD1d to US11 is mainly due to the short cytosolic tail of CD1d; a hybrid CD1d protein, whose cytosolic tail was replaced with that of HLA-A2.1, was efficiently degraded by US11. Finally, we found that HCMV infection did not significantly influence the cell surface expression of CD1d. Thus, these results suggest that antigen presentation by CD1d is largely unaffected by the multiple immune-modulating functions of HCMV.     

Human NKT cells mediate antitumor cytotoxicity directly by recognizing target cell CD1d with bound ligand or indirectly by producing IL-2 to activate NK cells.

alpha-Galactosylceramide (alphaGalCer) stimulates NKT cells and has antitumor activity in mice. Murine NKT cells may directly kill tumor cells and induce NK cell cytotoxicity, but the mechanisms are not well defined. Newly developed human CD1d/alphaGalCer tetrameric complexes were used to obtain highly purified human alphaGalCer-reactive NKT cell lines (>99%), and the mechanisms of NKT cell cytotoxicity and activation of NK cells were investigated. Human NKT cells were cytotoxic against CD1d(-) neuroblastoma cells only when they were rendered CD1d(+) by transfection and pulsed with alphaGalCer. Four other CD1d(-) tumor cell lines of diverse origin were resistant to NKT cells, whereas Jurkat and U937 leukemia cell lines, which are constitutively CD1d(+), were killed. Killing of the latter was greatly augmented in the presence of alphaGalCer. Upon human CD1d/alphaGalCer recognition, NKT cells induced potent cytotoxicity of NK cells against CD1d(-) neuroblastoma cell lines that were not killed directly by NKT cells. NK cell activation depended upon NKT cell production of IL-2, and was enhanced by secretion of IFN-gamma. These data demonstrate that cytotoxicity of human NKT cells can be CD1d and ligand dependent, and that TCR-stimulated NKT cells produce IL-2 that is required to induce NK cell cytotoxicity. Thus, NKT cells can mediate potent antitumor activity both directly by targeting CD1d and indirectly by activating NK cells.     

Defining the herpes simplex virus-specific CD8+ T cell repertoire in C57BL/6 mice

HSV type 1 (HSV-1) expresses its genes sequentially as immediate early (α), early (β), leaky late (γ1), and true late (γ2), where viral DNA synthesis is an absolute prerequisite only for γ2 gene expression. The γ1 protein glycoprotein B (gB) contains a strongly immunodominant CD8(+) T cell epitope (gB(498-505)) that is recognized by 50% of both the CD8(+) effector T cells in acutely infected trigeminal ganglia (TG) and the CD8(+) memory T cells in latently infected TG. Of 376 predicted HSV-1 CD8(+) T cell epitopes in C57BL/6 mice, 19 (gB(498-505) and 18 subdominant epitopes) stimulated CD8(+) T cells in the spleens and TG of HSV-1 acutely infected mice. These 19 epitopes identified virtually all CD8(+) T cells in the infected TG that represent all or the vast majority of the HSV-specific CD8(+) TCR repertoire. Only 11 of ～84 HSV-1 proteins are recognized by CD8(+) T cells, and most (～80%) are expressed before viral DNA synthesis. Neither the immunodominance of gB(498-505) nor the dominance hierarchy of the subdominant epitopes is due solely to MHC or TCR affinity. We conclude that the vast majority of CD8(+) T cells in HSV-1 acutely infected TG are HSV specific, that HSV-1 β and γ1 proteins that are expressed before viral DNA synthesis are favored targets of CD8(+) T cells, and that dominance within the TCR repertoire is likely due to the frequency or expansion and survival characteristics of CD8(+) T cell precursors.     

Antibody responses to glycolipid-borne carbohydrates require CD4+ T cells but not CD1 or NKT cells

Naturally occurring anti-carbohydrate antibodies play a major role in both the innate and adaptive immune responses. To elicit an anti-carbohydrate immune response, glycoproteins can be processed to glycopeptides and presented by the classical antigen-presenting molecules, major histocompatibility complex (MHC) Class I and II. In contrast, much less is known about the mechanism(s) for anti-carbohydrate responses to glycolipids, although it is generally considered that the CD1 family of cell surface proteins presents glycolipids to T cells or natural killer T (NKT) cells. Using model carbohydrate systems (isogloboside 3 and B blood group antigen), we examined the anti-carbohydrate response on glycolipids using both antibody neutralisation and knockout mouse-based experiments. These studies showed that CD4(+) T cells were required to generate antibodies to the carbohydrates expressed on glycolipids, and unexpectedly, these antibody responses were CD1d and NKT cell independent. They also did not require peptide help. These data provide new insight into glycolipid antigen recognition by the immune system and indicate the existence of a previously unrecognised population of glycolipid antigen-specific, CD1-independent, CD4(+) T cells.     

Herpes simplex virus type 1 gK is required for gB-mediated virus-induced cell fusion, while neither gB and gK nor gB and UL20p function redundantly in virion de-envelopment

Multiple amino acid changes within herpes simplex virus type 1 (HSV-1) gB and gK cause extensive virus-induced cell fusion and the formation of multinucleated cells (syncytia). Early reports established that syncytial mutations in gK could not cause cell-to-cell fusion in the absence of gB. To investigate the interdependence of gB, gK, and UL20p in virus-induced cell fusion and virion de-envelopment from perinuclear spaces as well as to compare the ultrastructural phenotypes of the different mutant viruses in a syngeneic HSV-1 (F) genetic background, gB-null, gK-null, UL20-null, gB/gK double-null, and gB/UL20 double-null viruses were constructed with the HSV-1 (F) bacterial artificial chromosome pYEBac102. The gK/gB double-null virus YEbacDeltagBDeltagK was used to isolate the recombinant viruses gBsyn3DeltagK and gBamb1511DeltagK, which lack the gK gene and carry the gBsyn3 or gBamb1511 syncytial mutation, respectively. Both viruses formed small nonsyncytial plaques on noncomplementing Vero cells and large syncytial plaques on gK-complementing cells, indicating that gK expression was necessary for gBsyn3- and gBamb1511-induced cell fusion. Lack of virus-induced cell fusion was not due to defects in virion egress, since recombinant viruses specifying the gBsyn3 or gKsyn20 mutation in the UL19/UL20 double-null genetic background caused extensive cell fusion on UL20-complementing cells. As expected, the gB-null virus failed to produce infectious virus, but enveloped virion particles egressed efficiently out of infected cells. The gK-null and UL20-null viruses exhibited cytoplasmic defects in virion morphogenesis like those of the corresponding HSV-1 (KOS) mutant viruses. Similarly, the gB/gK double-null and gB/UL20 double-null viruses accumulated capsids in the cytoplasm, indicating that gB, gK, and UL20p do not function redundantly in membrane fusion during virion de-envelopment at the outer nuclear lamellae.     

Herpes simplex virus type 1 targets the MHC class II processing pathway for immune evasion

HSV type 1 (HSV-1) has evolved numerous strategies for modifying immune responses that protect against infection. Important targets of HSV-1 infection are the MHC-encoded peptide receptors. Previous studies have shown that a helper T cell response and Ab production play important roles in controlling HSV-1 infection. The reduced capacity of infected B cells to stimulate CD4(+) T cells is beneficial for HSV-1 to evade immune defenses. We investigated the impact of HSV-1 infection on the MHCII processing pathway, which is critical to generate CD4(+) T cell help. HSV-1 infection targets the molecular coplayers of MHC class II processing, HLA-DR (DR), HLA-DM (DM), and invariant chain (Ii). HSV-1 infection strongly reduces expression of Ii, which impairs formation of SDS-resistant DR-peptide complexes. Residual activity of the MHC class II processing pathway is diminished by viral envelope glycoprotein B (gB). Binding of gB to DR competes with binding to Ii. In addition, we found gB associated with DM molecules. Both, gB-associated DR and DM heterodimers are exported from the endoplasmic reticulum, as indicated by carbohydrate maturation. Evaluation of DR, DM, and gB subcellular localization revealed abundant changes in intracellular distribution. DR-gB complexes are localized in subcellular vesicles and restrained from cell surface expression.     

Mutation of a positively charged cytoplasmic motif within CD1d results in multiple defects in antigen presentation to NKT cells

CD1d is an MHC class I-like molecule that presents glycolipid Ags to types I and II NKT cells. The YxxI motif in the cytoplasmic tail of CD1d contributes to its intracellular localization to the endolysosomal compartment and is important for Ag presentation to type I NKT cells. In this study, we identified the (327-329)RRR motif in CD1d and showed that it is critical for the control of CD1d intracellular trafficking and Ag presentation. The replacement of the arginines in this motif with alanines resulted in the extensive accumulation of CD1d in lysosomes but did not affect the cell surface expression. The defect in its cellular localization was accompanied by defects in Ag presentation to both type I and type II NKT cells. These results demonstrated that the (327-329)RRR motif of CD1d is required for proper cellular distribution of CD1d and optimal Ag presentation to both type I and type II NKT cells.     

Effects of mutations in the cytoplasmic domain of herpes simplex virus type 1 glycoprotein B on intracellular transport and infectivity

Herpes simplex virus type 1 (HSV-1) is a human pathogen of the alphaherpesvirus family which infects and spreads in the nervous system. Glycoproteins play a key role in the process of assembly and maturation of herpesviruses, which is essential for neuroinvasion and transneuronal spread. Glycoprotein B (gB) is a main component of the HSV-1 envelope and is necessary for the production of infectious particles. The cytoplasmic domain of gB, the longest one among HSV-1 glycoproteins, contains several highly conserved peptide sequences homologous to motifs involved in intracellular sorting. To determine the specific roles of these motifs in processing, subcellular localization, and the capacity of HSV-1 gB to complement a gB-null virus, we generated truncated or point mutated forms of a green fluorescent protein (GFP)-tagged gB. GFP-gB with a deletion in the acidic cluster DGDADEDDL (amino acids [aa] 896 to 904) behaved the same as the parental form. Deletion or disruption of the YTQV motif (aa 889 to 892) abolished internalization and reduced complementation by 60%. Disruption of the LL motif (aa 871 to 872) impaired the return of the protein to the trans-Golgi network (TGN) while enhancing its recycling to the plasma membrane. Truncations from residue E 857 abolished transport and processing of the truncated proteins, which had null complementation activity, through the Golgi complex. Altogether, our results favor a model in which HSV-1 gets its final envelope in the TGN, and they suggest that endocytosis, albeit not necessary, might play a role in infectivity.     

Potent neutralizing anti-CD1d antibody reduces lung cytokine release in primate asthma model

CD1d is a receptor on antigen-presenting cells involved in triggering cell populations, particularly natural killer T (NKT) cells, to release high levels of cytokines. NKT cells are implicated in asthma pathology and blockade of the CD1d/NKT cell pathway may have therapeutic potential. We developed a potent anti-human CD1d antibody (NIB.2) that possesses high affinity for human and cynomolgus macaque CD1d (K_D ～100 pM) and strong neutralizing activity in human primary cell-based assays (IC₅₀ typically <100 pM). By epitope mapping experiments, we showed that NIB.2 binds to CD1d in close proximity to the interface of CD1d and the Type 1 NKT cell receptor β-chain. Together with data showing that NIB.2 inhibited stimulation via CD1d loaded with different glycolipids, this supports a mechanism whereby NIB.2 inhibits NKT cell activation by inhibiting Type 1 NKT cell receptor β-chain interactions with CD1d, independent of the lipid antigen in the CD1d antigen-binding cleft. The strong in vitro potency of NIB.2 was reflected in vivo in an Ascaris suum cynomolgus macaque asthma model. Compared with vehicle control, NIB.2 treatment significantly reduced bronchoalveolar lavage (BAL) levels of Ascaris-induced cytokines IL-5, IL-8 and IL-1 receptor antagonist, and significantly reduced baseline levels of GM-CSF, IL-6, IL-15, IL-12/23p40, MIP-1α, MIP-1β, and VEGF. At a cellular population level NIB.2 also reduced numbers of BAL lymphocytes and macrophages, and blood eosinophils and basophils. We demonstrate that anti-CD1d antibody blockade of the CD1d/NKT pathway modulates inflammatory parameters in vivo in a primate inflammation model, with therapeutic potential for diseases where the local cytokine milieu is critical.     

Human cytomegalovirus (HCMV) US2 protein interacts with human CD1d (hCD1d) and down-regulates invariant NKT (iNKT) cell activity

To avoid host immune surveillance, human cytomegalovirus (HCMV) encoded endoplasmic reticulum (ER)-membrane glycoprotein US2, which interferes with antigen presenting mechanism of major histocompatibility complex (MHC) class Ia and class II molecules. However, not many attempts have been made to study the effect of HCMV US2 on the expression of MHC class Ib molecules. In this study, we examined the effect of HCMV US2 on the expression and function of human CD1d (hCD1d), which presents glycolipid antigens to invariant NKT (iNKT) cells. Our results clearly showed that the physiological interaction between ER lumenal domain of HCMV US2 and α3 domain of hCD1d was observed within ER. Compared with mature form of hCD1d, immature form of hCD1d is more susceptible to ubiquitin-dependent proteasomal degradation mediated by HCMV US2. Moreover, the ectopic expression of HCMV US2 leads to the down-modulation of iNKT cell activity without significant change of hCD1d expression. These results will advance our understanding of the function of HCMV US2 in immune evasive mechanisms against anti-viral immunity of iNKT cells.     

The bovine CD1 family contains group 1 CD1 proteins, but no functional CD1d

The CD1 family of proteins presents lipid Ags to T cells. Human CD1a, CD1b, and CD1c have been shown in humans to present mycobacterial lipid Ags. Cattle, like humans, are a natural host of several mycobacterial pathogens. In this study, we describe the CD1 family of genes in cattle (Bos taurus) and provide evidence that B. taurus expresses CD1a, CD1e, and multiple CD1b molecules, but no CD1c and CD1d molecules. In mice and humans, CD1d is known to present Ag to NKT cells, a T cell lineage that is characterized by a limited TCR repertoire, capable of rapidly secreting large amounts of IFN-gamma and IL-4. In cattle, two CD1D pseudogenes were found and no intact CD1D genes. Consistent with this, we found complete lack of reactivity to a potent, cross-reactive Ag for NKT cells in mice and humans, alpha-galactosylceramide. Our data suggest the absence of NKT cells in cattle. It remains open whether other cells with the NKT-like phenotype and functions are present in this species. With its functional CD1A and CD1B genes, B. taurus is well equipped to present Ags to CD1-restricted T cells other than NKT cells. Cattle can be used as a model to study group 1 CD1-restricted T cell immunity, including its role in the defense against mycobacterial infections that occur naturally in this species.     

Herpes simplex virus type 1 induces CD83 degradation in mature dendritic cells with immediate-early kinetics via the cellular proteasome

Mature dendritic cells (DCs) are the most potent antigen-presenting cells within the human immune system. However, Herpes simplex virus type 1 (HSV-1) is able to interfere with DC biology and to establish latency in infected individuals. In this study, we provide new insights into the mechanism by which HSV-1 disarms DCs by the manipulation of CD83, a functionally important molecule for DC activation. Fluorescence-activated cell sorter (FACS) analyses revealed a rapid downmodulation of CD83 surface expression within 6 to 8 h after HSV-1 infection, in a manner strictly dependent on viral gene expression. Soluble CD83 enzyme-linked immunosorbent assays, together with Western blot analysis, demonstrated that CD83 rapidly disappears from the cell surface after contact with HSV-1 by a mechanism that involves protein degradation rather than shedding of CD83 from the cell surface into the medium. Infection experiments with an ICP0 deletion mutant demonstrated an important role for this viral immediate-early protein during CD83 degradation, since this particular mutant strain leads to strongly reduced CD83 degradation. This hypothesis was further strengthened by cotransfection of plasmids expressing CD83 and ICP0 into 293T cells, which led to significantly reduced accumulation of CD83. In strong contrast, transfection of plasmids expressing CD83 and a mutant ICP0 defective in its RING finger-mediated E3 ubiquitin ligase function did not reduce CD83 expression. Inhibition of the proteasome, the cellular protein degradation machinery, almost completely restored CD83 surface expression during HSV-1 infection, indicating that proteasome-mediated degradation and HSV-1 ICP0 play crucial roles in this novel viral immune escape mechanism.     

Simultaneous tracking of capsid, tegument, and envelope protein localization in living cells infected with triply fluorescent herpes simplex virus 1

We report here the construction of a triply fluorescent-tagged herpes simplex virus 1 (HSV-1) expressing capsid protein VP26, tegument protein VP22, and envelope protein gB as fusion proteins with monomeric yellow, red, and cyan fluorescent proteins, respectively. The recombinant virus enabled us to monitor the dynamics of these capsid, tegument, and envelope proteins simultaneously in the same live HSV-1-infected cells and to visualize single extracellular virions with three different fluorescent emissions. In Vero cells infected by the triply fluorescent virus, multiple cytoplasmic compartments were found to be induced close to the basal surfaces of the infected cells (the adhesion surfaces of the infected cells on the solid growth substrate). Major capsid, tegument, and envelope proteins accumulated and colocalized in the compartments, as did marker proteins for the trans-Golgi network (TGN) which has been implicated to be the site of HSV-1 secondary envelopment. Moreover, formation of the compartments was correlated with the dynamic redistribution of the TGN proteins induced by HSV-1 infection. These results suggest that HSV-1 infection causes redistribution of TGN membranes to form multiple cytoplasmic compartments, possibly for optimal secondary envelopment. This is the first real evidence for the assembly of all three types of herpesvirus proteins-capsid, tegument, and envelope membrane proteins-in TGN.     

A method for production of recombinant mCD1d protein in insect cells

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.