Expression Of gB Research Articles

Combinatorial expression of ebony and tan generates body color variation from nymph through adult stages in the cricket, Gryllus bimaculatus.

Insect body colors and patterns change markedly during development in some species as they adapt to their surroundings. The contribution of melanin and sclerotin pigments, both of which are synthesized from dopamine, to cuticle tanning has been well studied. Nevertheless, little is known about how insects alter their body color patterns. To investigate this mechanism, the cricket Gryllus bimaculatus, whose body color patterns change during postembryonic development, was used as a model in this study. We focused on the ebony and tan genes, which encode enzymes that catalyze the synthesis and degradation, respectively, of the precursor of yellow sclerotin N-β-alanyl dopamine (NBAD). Expression of the G. bimaculatus (Gb) ebony and tan transcripts tended to be elevated just after hatching and the molting period. We found that dynamic alterations in the combined expression levels of Gb'ebony and Gb'tan correlated with the body color transition from the nymphal stages to the adult. The body color of Gb'ebony knockout mutants generated by CRISPR/Cas9 systemically darkened. Meanwhile, Gb'tan knockout mutants displayed a yellow color in certain areas and stages. The phenotypes of the Gb'ebony and Gb'tan mutants probably result from an over-production of melanin and yellow sclerotin NBAD, respectively. Overall, stage-specific body color patterns in the postembryonic stages of the cricket are governed by the combinatorial expression of Gb'ebony and Gb'tan. Our findings provide insights into the mechanism by which insects evolve adaptive body coloration at each developmental stage.

Growth and flavonol accumulation of Ginkgo biloba leaves affected by red and blue light

Ginkgo biloba L. is an economically important tree species in China rich in resources. Flavonoids in their leaves have a high medicinal value. Research on improving the flavonoid yield of G. biloba leaves by regulating environmental factors has been widely conducted. However, the effect of light quality on G. biloba growth and flavonoid accumulation is not clear, and its molecular mechanism has not been reported. In this study, one-year-old ginkgo seedlings were used as experimental material. Three treatments of red, mixed (red:blue = 1:1), and blue light were set up using LED lamps. Using white light as the control, differences of G. biloba growth and flavonol accumulation under different light qualities were explored. The results showed that under red, mixed, and blue light, the petiole of G. biloba elongated, the leaf area decreased, and the leaves tended to be slender, which was not conducive to the accumulation of leaves. The blue light significantly increased the height of G. biloba seedlings. The net photosynthetic rate under blue light was relatively lower, the transpiration rate was lower in the treatment groups, the stomatal conductance decreased significantly under mixed light, and the change in intercellular CO2 concentration was not significant. RNA sequencing (RNA-seq) combined with quantitative real-time polymerase chain reaction (qRT-PCR) showed that the transcription factors (TFs) HY5 (Gb_12012) and MYB (Gb_39081) and structural genes CHS (Gb_20355), F3H (Gb_29563), and FLS (Gb_14028, Gb_14029, and Gb_14030) positively regulated the biosynthesis of flavonols, whereas MYB-related TFs (Gb_27262 and Gb_34639) played a negative regulatory role. Combined with the existing research and the results of this experiment, we preliminarily speculated the mechanism of G. biloba response to red and blue light quality. After G. biloba leaves were stimulated by red and blue light, the expression of Gb_12012 changed the abundance of HY5 and affected the expression levels of downstream MYB TFs. MYB directly binds to the promoters of the structural genes CHS, F3H, and FLS to regulate its expression to promote (or inhibit) the biosynthesis of flavonols. Finally, Mixed and blue light significantly increased the flavonol content in G. biloba leaves, while red light was the opposite. Blue light significantly increased the flavone yield of G. biloba leaves per plant (increased by 0.76 times) and its antioxidant capacity. This lays a foundation for applying light quality in the cultivation of G. biloba for leaf use.

Gb_ANR-47 Enhances the Resistance of Gossypium barbadense to Fusarium oxysporum f. sp. vasinfectum (FOV) by Regulating the Content of Proanthocyanidins.

Anthocyanidin reductase (ANR) is an important regulator of flavonoid metabolism, and proanthocyanidins, the secondary metabolites of flavonoids, play an important role in the response of plants to pathogenic stress. Therefore, in this study, the expression analysis of the ANR gene family of Gossypium barbadense after inoculation with Fusarium oxysporum f. sp. vasinfectum (FOV) was performed at different time points. It was found that Gb_ANR-47 showed significant differences in the disease-resistant cultivar 06-146 and the susceptible cultivar Xinhai 14, as well as in the highest root expression. It was found that the expression of Gb_ANR-47 in the resistant cultivar was significantly higher than that in the susceptible cultivar by MeJA and SA, and different amounts of methyl jasmonate (MeJA) and salicylic acid (SA) response elements were found in the promoter region of Gb_ANR-47. After silencing GbANR-47 in 06-146 material by VIGS technology, its resistance to FOV decreased significantly. The disease severity index (DSI) was significantly increased, and the anthocyanin content was significantly decreased in silenced plants, compared to controls. Our findings suggest that GbANR-47 is a positive regulator of FOV resistance in Gossypium barbadense. The research results provide an important theoretical basis for in-depth analysis of the molecular mechanism of GbANR-47 and improving the anti-FOV of Gossypium barbadense.

Quantitative Trait Locus Mapping and Identification of Candidate Genes for Resistance to Fusarium Wilt Race 7 Using a Resequencing-Based High Density Genetic Bin Map in a Recombinant Inbred Line Population of Gossypium barbadense.

Fusarium wilt caused by Fusarium oxysporum f. sp. vasinfectum (FOV) is one of the most destructive diseases in cotton (Gossypium spp.) production, and use of resistant cultivars is the most cost-effective method managing the disease. To understand the genetic basis of cotton resistance to FOV race 7 (FOV7), this study evaluated a recombinant inbred line (RIL) population of 110 lines of G. barbadense from a cross between susceptible Xinhai 14 and resistant 06-146 in eight tests and constructed a high-density genetic linkage map with resequencing-based 933,845 single-nucleotide polymorphism (SNP) markers covering a total genetic distance of 2483.17 cM. Nine quantitative trait loci (QTLs) for FOV7 resistance were identified, including qFOV7-D03-1 on chromosome D03 in two tests. Through a comparative analysis of gene expression and DNA sequence for predicted genes within the QTL region between the two parents and selected lines inoculated with FOV7, GB_D03G0217 encoding for a calmodulin (CaM)-like (CML) protein was identified as a candidate gene. A further analysis confirmed that the expression of GB_D03G0217 was suppressed, leading to increased disease severity in plants of the resistant parent with virus induced gene silencing (VIGS).

Varicella-zoster virus: molecular controls of cell fusion-dependent pathogenesis.

Varicella-zoster virus (VZV) is the causative agent of chicken pox (varicella) and shingles (zoster). Although considered benign diseases, both varicella and zoster can cause complications. Zoster is painful and can lead to post herpetic neuralgia. VZV has also been linked to stroke, related to giant cell arteritis in some cases. Vaccines are available but the attenuated vaccine is not recommended in immunocompromised individuals and the efficacy of the glycoprotein E (gE) based subunit vaccine has not been evaluated for the prevention of varicella. A hallmark of VZV pathology is the formation of multinucleated cells termed polykaryocytes in skin lesions. This cell-cell fusion (abbreviated as cell fusion) is mediated by the VZV glycoproteins gB, gH and gL, which constitute the fusion complex of VZV, also needed for virion entry. Expression of gB, gH and gL during VZV infection and trafficking to the cell surface enables cell fusion. Recent evidence supports the concept that cellular processes are required for regulating cell fusion induced by gB/gH-gL. Mutations within the carboxyl domains of either gB or gH have profound effects on fusion regulation and dramatically restrict the ability of VZV to replicate in human skin. This loss of regulation modifies the transcriptome of VZV infected cells. Furthermore, cellular proteins have significant effects on the regulation of gB/gH-gL-mediated cell fusion and the replication of VZV, exemplified by the cellular phosphatase, calcineurin. This review provides the current state-of-the-art knowledge about the molecular controls of cell fusion-dependent pathogenesis caused by VZV.

Functional analysis of Pdp1 and vrille in the circadian system of a cricket

Circadian rhythms are generated by a circadian clock for which oscillations are based on the rhythmic expression of the so-called clock genes. The present study investigated the role of Gryllus bimaculatus vrille (Gb’vri) and Par domain protein 1 (Gb’Pdp1) in the circadian clock of the cricket Gryllus bimaculatus. Structural analysis of Gb’vri and Gb’Pdp1 cDNAs revealed that they are a member of the bZIP transcription factors. Under light/dark cycles (LD) both genes were rhythmically expressed in the clock tissue, the optic lobes, whereas the rhythm diminished under constant darkness (DD). Gb’vri and Gb’Pdp1 mRNA levels were significantly reduced by RNA interference (RNAi) of Gb’Clk and Gb’cyc, suggesting they are controlled by Gb’CLK/Gb’CYC. RNAi of Gb’vri and Gb’Pdp1 had little effect on locomotor rhythms, although their effects became visible when treated together with Gb’cycRNAi. The average free-running period of Gb’vriRNAi/Gb’cycRNAi crickets was significantly shorter than that of Gb’cycRNAi crickets. A similar period shortening was observed also when treated with Gb’Pdp1RNAi/Gb’cycRNAi. Some Gb’Pdp1RNAi/Gb’cycRNAi crickets showed rhythm splitting into two free-running components with different periods. Gb’vriRNAi and Gb’Pdp1RNAi treatments significantly altered the expression of Gb’Clk, Gb’cyc, and Gb’tim in LD. These results suggest that Gb’vri and Gb’Pdp1 play important roles in cricket circadian clocks.

Autophagic degradation of PML promotes susceptibility to HSV-1 by stress-induced corticosterone.

Rationale: Herpes simplex virus type 1 (HSV-1) is a neurotropic virus that can cause a variety of clinical syndromes including mucocutaneous disease and HSV-1 encephalitis (HSE). Here, we characterize the molecular mechanisms underlying the susceptibility to HSV-1 under stressful conditions.Methods: Restraint stress and corticosterone (CORT, a primary stress hormone) were respectively used to establish HSV-1 susceptible model in vivo and in vitro. Viral titers were determined by plaque assay. Western blotting, immunofluorescence, transmission electron microscopy (TEM), qRT-PCR, H&E staining, IHC staining and flow cytometry were employed to evaluate virus-related protein expressions and detect the activation of autophagy. Loss- and gain-function assays, co-immunoprecipitation (co-IP) technique and autophagy agonist/antagonist treatments were applied in mechanistic experiments.Results: Restraint stress increased the susceptibility of mouse brain to HSV-1. Similarly, CORT treatment enhanced the susceptibility of neural cells to HSV-1. Furthermore, PML protein level in HSV-1 infected brain tissues and neural cells was remarkably decreased by stress treatment in vivo or CORT treatment in vitro, while its transcriptional level was not affected. Notably, a striking decline in protein expressions of ICP27 and gB was observed in PML-overexpressing cells, which was reversed by CORT treatment. By contrast, protein expression of gB was increased by knockdown with si-PML in virus-infected SH-SY5Y cells. We further discovered that CORT-driven PML degradation was dependent on the activation of autophagy in a ULK1-independent manner, rather than proteasome pathway. Bafilomycin A1 (BaF1) attenuated the augmentation effect of CORT on HSV-1 infection. The expressions of viral proteins were reduced in LC3-depleted cells, and the degradation of PML by CORT-induced autophagy was prevented in cells with LC3 knockdown by RNAi. Interestingly, PML was revealed to interact with the autophagic cargo receptor P62 and the autophagic effector protein LC3. Additionally, CORT failed to increase gB protein level when PML was silenced, providing direct evidence linking autophagic degradation of PML and CORT-induced virus susceptibility.Conclusion: Our results revealed that restraint stress/CORT increased HSV-1 susceptibility by delivering PML into autolysosomes for degradation. The results obtained from in vitro and in vivo models not only demonstrated the adverse effects of stress on HSV-1 infection, but also systematically investigated the underlying molecular mechanisms. These discoveries broaden our understanding of the interplay between host and viruses, and a comprehensive understanding of the role of autophagy in viral infection will provide information for future development of innovative drugs against viral infection.

The Absence of DHHC3 Affects Primary and Latent Herpes Simplex Virus 1 Infection

UL20, an essential herpes simplex virus 1 (HSV-1) protein, is involved in cytoplasmic envelopment of virions and virus egress. We reported recently that UL20 can bind to a host protein encoded by the zinc finger DHHC-type containing 3 (ZDHHC3) gene (also known as Golgi-specific DHHC zinc finger protein [GODZ]). Here, we show for the first time that HSV-1 replication is compromised in murine embryonic fibroblasts (MEFs) isolated from GODZ-/- mice. The absence of GODZ resulted in blocking palmitoylation of UL20 and altered localization and expression of UL20 and glycoprotein K (gK); the expression of gB and gC; and the localization and expression of tegument and capsid proteins within HSV-1-infected MEFs. Electron microscopy revealed that the absence of GODZ limited the maturation of virions at multiple steps and affected the localization of virus and endoplasmic reticulum morphology. Virus replication in the eyes of ocularly HSV-1-infected GODZ-/- mice was significantly lower than in HSV-1-infected wild-type (WT) mice. The levels of UL20, gK, and gB transcripts in the corneas of HSV-1-infected GODZ-/- mice on day 5 postinfection were markedly lower than in WT mice, whereas only UL20 transcripts were reduced in trigeminal ganglia (TG). In addition, HSV-1-infected GODZ-/- mice showed notably lower levels of corneal scarring, and HSV-1 latency reactivation was also reduced. Thus, normal HSV-1 infectivity and viral pathogenesis are critically dependent on GODZ-mediated palmitoylation of viral UL20.IMPORTANCE HSV-1 infection is widespread. Ocular infection can cause corneal blindness; however, approximately 70 to 90% of American adults exposed to the virus show no clinical symptoms. In this study, we show for the first time that the absence of a zinc finger protein called GODZ affects primary and latent infection, as well as reactivation, in ocularly infected mice. The reduced virus infectivity is due to the absence of the GODZ interaction with HSV-1 UL20. These results strongly suggest that binding of UL20 to GODZ promotes virus infectivity in vitro and viral pathogenesis in vivo.

Use of CMV gB/pp65 eVLPs to harness the immunogenicity of foreign viral antigens to target solid tumors.

165 Background: Human cytomegalovirus (CMV) is a ubiquitous, generally asymptomatic virus that is present in over 90% of GBM and breast cancer tumors which have comparatively low mutational burdens and predicted neoantigens relative to melanoma or lung cancer. Memory CD4+ and CD8+T cells are most frequently directed against the gB and pp65 antigens, respectively. Thus, CMV gB and pp65 represent highly immunogenic “foreign” antigen components of a vaccine against GBM and breast cancer. Methods: Enveloped virus-like particles (eVLPs) are produced after transfection of HEK 293 cells with a plasmid encoding murine leukemia virus Gag plasmid fused in-frame with CMV pp65 antigen, which gives rise to particles. Co-transfected CMV gB plasmid enables particles budding from the cell surface to incorporate the gB protein into the lipid bilayer. Surface expression of gB and internal expression of pp65 have been confirmed by CryoEM and immunogold labelling. Production and purification of multiple batches at a GMP-compliant manufacturer has demonstrated consistent purity and yields that meet regulatory requirements. Results: eVLPs restimulate IFN-g secreting CD4+ and CD8+ T cells in ex vivo PBMCs from healthy subjects (n=8) at mean frequencies of 0.27% and 1.28%, respectively. eVLP formulation with GM-CSF augments IFN-g and CCL3 secretion in stimulated PBMCs from healthy subjects (n=5), GBM patients (n=5), and breast cancer (n=1) patients to comparable levels. This vaccine candidate also induces de novo CD4+ and CD8+responses in mice. The vaccine induces high titer antibody responses against CMV gB, and the potential for ADCC activity against gB-expressing GBM tumor cells is currently being investigated. Conclusions: CMV gB/pp65 eVLPs address several shortcomings of past theraeputic vaccines, including: 1) the poor immunogenicity of "self" tumor-associated antigens, 2) failure to induce robust CD4+ responses that support CD8+ tumor-specific responses, 3) inclusion of only one or a few epitopes that rapidly encourage tumor cell immunoselection of variants that no longer express the target(s). An IND submission to FDA for a phase I/IIa trial in GBM patients is planned for H1 2017.

Misdirection of endosomal trafficking mediated by herpes simplex virus-encoded glycoprotein B.

Herpes simplex virus (HSV)-encoded glycoprotein B (gB) is the most abundant protein in the viral envelope and promotes fusion of the virus with the cellular membrane. In the present study, we found that gB impacts on the major histocompatibility complex (MHC)-II pathway of antigen presentation by fostering homotypic fusion of early endosomes and trapping MHC-II molecules in these altered endosomes. By using an overexpression approach, we demonstrated that transient expression of gB induces giant vesicles of early endosomal origin, which contained Rab5, early endosomal antigen 1 (EEA1), and large amounts of MHC-II molecules [human leukocyte antigen (HLA)-DR, and HLA-DM], but no CD63. In HSV-1-infected and stably transfected cell lines that expressed lower amounts of gB, giant endosomes were not observed, but strongly increased amounts of HLA-DR and HLA-DM were found in EEA1+ early endosomes. We used these giant vesicles as a model system and revealed that gB interacts with Rab5 and EEA1, and that gB-induced homotypic fusion of early endosomes to giant endosomes requires phosphatidylinositol 3-phosphate, the activity of soluble N-ethylmaleimide-sensitive factor attachment protein receptors, and the cytosolic gB sequence 889YTQVPN894 We conclude that gB expression alters trafficking of molecules of the HLA-II processing pathway, which leads to increased retention of MHC-II molecules in early endosomal compartments, thereby intercepting antigen presentation.-Niazy, N., Temme, S., Bocuk, D., Giesen, C., König, A., Temme, N., Ziegfeld, A., Gregers, T. F., Bakke, O., Lang, T., Eis-Hübinger, A. M., Koch, N. Misdirection of endosomal trafficking mediated by herpes simplex virus-encoded glycoprotein B.

The clock gene cycle plays an important role in the circadian clock of the cricket Gryllus bimaculatus

To dissect the molecular oscillatory mechanism of the circadian clock in the cricket Gryllus bimaculatus, we have cloned a cDNA of the clock gene cycle (Gb’cyc) and analyzed its structure and function. Gb’cyc contains four functional domains, i.e. bHLH, PAS-A, PAS-B and BCTR domains, and is expressed rhythmically in light dark cycles, peaking at mid night. The RNA interference (RNAi) of Clock (Gb’Clk) and period (Gb’per) reduced the Gb’cyc mRNA levels and abolished the rhythmic expression, suggesting that the rhythmic expression of Gb’cyc is regulated by a mechanism including Gb’Clk and Gb’per. These features are more similar to those of mammalian orthologue of cyc (Bmal1) than those of Drosophila cyc. A single treatment with double-stranded RNA (dsRNA) of Gb’cyc effectively knocked down the Gb’cyc mRNA level and abolished its rhythmic expression. The cyc RNAi failed to disrupt the locomotor rhythm, but lengthened its free-running period in constant darkness (DD). It is thus likely that Gb’cyc is involved in the circadian clock machinery of the cricket. The cyc RNAi crickets showed a rhythmic expression of Gb’per and timeless (Gb’tim) in the optic lobe in DD, explaining the persistence of the locomotor rhythm. Surprisingly, cyc RNAi revealed a rhythmic expression of Gb’Clk in DD which is otherwise rather constitutively expressed in the optic lobe. These facts suggest that the cricket might have a unique clock oscillatory mechanism in which both Gb’cyc and Gb’Clk are rhythmically controlled and that under abundant expression of Gb’cyc the rhythmic expression of Gb’Clk may be concealed.

Analysis of RNA-Seq data reveals involvement of JAK/STAT signalling during leg regeneration in the cricket Gryllus bimaculatus

In the cricket Gryllus bimaculatus, missing distal parts of the amputated leg are regenerated from the blastema, a population of dedifferentiated proliferating cells that forms at the distal tip of the leg stump. To identify molecules involved in blastema formation, comparative transcriptome analysis was performed between regenerating and normal unamputated legs. Components of JAK/STAT signalling were upregulated more than twofold in regenerating legs. To verify their involvement, Gryllus homologues of the interleukin receptor Domeless (Gb'dome), the Janus kinase Hopscotch (Gb'hop) and the transcription factor STAT (Gb'Stat) were cloned, and RNAi was performed against these genes. Gb'dome(RNAi), Gb'hop(RNAi) and Gb'Stat(RNAi) crickets showed defects in leg regeneration. Blastema expression of Gb'cyclinE was decreased in the Gb'Stat(RNAi) cricket compared with that in the control. Hyperproliferation of blastema cells caused by Gb'fat(RNAi) or Gb'warts(RNAi) was suppressed by RNAi against Gb'Stat. The results suggest that JAK/STAT signalling regulates blastema cell proliferation during leg regeneration.

Modulation of Epstein-Barr Virus Glycoprotein B (gB) Fusion Activity by the gB Cytoplasmic Tail Domain

ABSTRACTEpstein-Barr virus (EBV), along with other members of the herpesvirus family, requires a set of viral glycoproteins to mediate host cell attachment and entry. Viral glycoprotein B (gB), a highly conserved glycoprotein within the herpesvirus family, is thought to be the viral fusogen based on structural comparison of EBV gB and herpes simplex virus (HSV) gB with the postfusion crystal structure of vesicular stomatitis virus fusion protein glycoprotein G (VSV-G). In addition, mutational studies indicate that gB plays an important role in fusion function. In the current study, we constructed a comprehensive library of mutants with truncations of the C-terminal cytoplasmic tail domain (CTD) of EBV gB. Our studies indicate that the gB CTD is important in the cellular localization, expression, and fusion function of EBV gB. However, in line with observations from other studies, we conclude that the degree of cell surface expression of gB is not directly proportional to observed fusion phenotypes. Rather, we conclude that other biochemical or biophysical properties of EBV gB must be altered to explain the different fusion phenotypes observed.

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.

Delaying the Expression of Herpes Simplex Virus Type 1 Glycoprotein B (gB) to a True Late Gene Alters Neurovirulence and Inhibits the gB-CD8 + T-Cell Response in the Trigeminal Ganglion

Following herpes simplex virus type 1 (HSV-1) ocular infection of C57BL/6 mice, activated CD8(+) T cells specific for an immunodominant epitope on HSV-1 glycoprotein B (gB-CD8 cells) establish a stable memory population in HSV-1 latently infected trigeminal ganglia (TG), whereas non-HSV-specific CD8(+) T cells are lost over time. The retention and activation of gB-CD8 cells appear to be influenced by persistent viral antigenic exposure within the latently infected TG. We hypothesized that the low-level expression of gB from its native promoter before viral DNA synthesis is critical for the retention and activation of gB-CD8 cells in the TG during HSV-1 latency and for their ability to block HSV-1 reactivation from latency. To test this, we created a recombinant HSV-1 in which gB is expressed only after viral DNA synthesis from the true late gC promoter (gCp-gB). Despite minor growth differences compared to its rescuant in infected corneas, gCp-gB was significantly growth impaired in the TG and produced a reduced latent genome load. The gCp-gB- and rescuant-infected mice mounted similar gB-CD8 effector responses, but the size and activation phenotypes of the memory gB-CD8 cells were diminished in gCp-gB latently infected TG, suggesting that the stimulation of gB-CD8 cells requires gB expression before viral DNA synthesis. Surprisingly, late gB expression did not compromise the capacity of gB-CD8 cells to inhibit HSV-1 reactivation from latency in ex vivo TG cultures, suggesting that gB-CD8 cells can block HSV-1 reactivation at a very late stage in the viral life cycle. These data have implications for designing better immunogens for vaccines to prevent HSV-1 reactivation.

Intracerebroventricular Shiga toxin 2 increases the expression of its receptor globotriaosylceramide and causes dendritic abnormalities

Neurological damage caused by intoxication with Shiga toxin (Stx) from enterohemorrhagic Escherichia coli is the most unrepairable and untreatable outcome of Hemolytic Uremic Syndrome, and occurs in 30% of affected infants. In this work intracerebroventricular administration of Stx2 in rat brains significantly increased the expression of its receptor globotriaosylceramide (Gb 3) in neuronal populations from striatum, hippocampus and cortex. Stx2 was immunodetected in neurons that expressed Gb 3 after intracerebroventricular administration of the toxin. Confocal immunofluorescence of microtubule-associated protein 2 showed aberrant dendrites in neurons expressing increased Gb 3. The pro-apoptotic Bax protein was concomitantly immunodetected in neurons and other cell populations from the same described areas including the hypothalamus. Confocal immunofluorescence showed that Gb 3 colocalized also with glial fibrillary acidic protein only in reactive astrocytic processes, and not in vehicle-treated normal ones. Rats showed weight variation and motor deficits as compared to controls. We thus suggest that Stx2 induces the expression of Gb 3 in neurons and triggers neuronal dysfunctions.

The Herpes Simplex Virus-1 Encoded Glycoprotein B Diverts HLA-DR into the Exosome Pathway

Neutralizing Abs play an important role for immunity against HSV-1 infection. This branch of the immune response is initiated by MHC class II Ag presentation and activation of T cell help. In this study, we show that the HSV-1 encoded glycoprotein B (gB) manipulates the class II processing pathway by perturbing endosomal sorting and trafficking of HLA-DR (DR) molecules. Expression of gB in the human melanoma cell line Mel JuSo results in formation of enlarged DR(+) intracellular vesicles. Costaining of the vesicles revealed the presence of DR, gB, and the late endosomal marker CD63. The lumen of these late endosomal membranes shows a variable content, containing either gB or CD63, or both CD63 and gB. gB targets DR molecules on their biosynthetic route, after the MHC class II invariant chain is released from the DR heterodimer. gB-DR complexes were detected in a post-Golgi compartment and in exosomes, but not on the cell surface. Interestingly, increasing expression of gB strongly elevated the amount of DR and CD63 released into the exosome pathway. In conclusion, this is a previously undescribed mode of viral immune evasion involving hijacking of DR from its normal transport route to the cell surface, followed by viral-mediated release of DR into the exosome pathway.

Herpes Simplex Virus 1 Protein Kinase Us3 Phosphorylates Viral Envelope Glycoprotein B and Regulates Its Expression on the Cell Surface

Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). As reported here, we attempted to identify the previously unreported physiological substrate of Us3 in HSV-1-infected cells. Our results were as follows. (i) Bioinformatics analysis predicted two putative Us3 phosphorylation sites in the viral envelope glycoprotein B (gB) at codons 557 to 562 (RRVSAR) and codons 884 to 889 (RRNTNY). (ii) In in vitro kinase assays, the threonine residue at position 887 (Thr-887) in the gB domain was specifically phosphorylated by Us3, while the serine residue at position 560 was not. (iii) The phosphorylation of gB Thr-887 in Vero cells infected with wild-type HSV-1 was specifically detected using an antibody that recognized phosphorylated serine or threonine residues with arginine at the -3 and -2 positions. (iv) The phosphorylation of gB Thr-887 in infected cells was dependent on the kinase activity of Us3. (v) The replacement of Thr-887 with alanine markedly upregulated the cell surface expression of gB in infected cells, whereas replacement with aspartic acid, which sometimes mimics constitutive phosphorylation, restored the wild-type phenotype. The upregulation of gB expression on the cell surface also was observed in cells infected with a recombinant HSV-1 encoding catalytically inactive Us3. These results supported the hypothesis that Us3 phosphorylates gB and downregulates the cell surface expression of gB in HSV-1-infected cells.

Human Cytomegalovirus Glycoproteins gB and gH/gL Mediate Epithelial Cell-Cell Fusion When Expressed either in cis or in trans

Herpesviruses use a cascade of interactions with different cell surface molecules to gain entry into cells. In many cases, this involves binding to abundant glycosaminoglycans or integrins followed by interactions with more limited cell surface proteins, leading to fusion with cellular membranes. Human cytomegalovirus (HCMV) has the ability to infect a wide variety of human cell types in vivo. However, very little is known about which HCMV glycoproteins mediate entry into various cell types, including relevant epithelial and endothelial cells. For other herpesviruses, studies of cell-cell fusion induced by viral proteins have provided substantial information about late stages of entry. In this report, we describe the fusion of epithelial, endothelial, microglial, and fibroblast cells in which HCMV gB and gH/gL were expressed from nonreplicating adenovirus vectors. Fusion frequently involved the majority of cells, and gB and gH/gL were both necessary and sufficient for fusion, whereas no fusion occurred when either glycoprotein was omitted. Coexpression of UL128, UL130, and UL131 did not enhance fusion. We concluded that the HCMV core fusion machinery consists of gB and gH/gL. Coimmunoprecipitation indicated that HCMV gB and gH/gL can interact. Importantly, expression of gB and gH/gL in trans (gB-expressing cells mixed with other gH/gL-expressing cells) resulted in substantial fusion. We believe that this is the first description of a multicomponent viral fusion machine that can be split between cells. If gB and gH/gL must interact for fusion, then these molecules must reach across the space between apposing cells. Expression of gB and gH/gL in trans with different cell types revealed surface molecules that are required for fusion on HCMV-permissive cells but not on nonpermissive cells.

Induction of HIV-1 resistance: cell susceptibility to infection is an inverse function of globotriaosyl ceramide levels

To examine the role of the glycosphingolipid (GSL), globotriaosylceramide (Gb(3), CD77, p(k) blood group antigen) in HIV-1 infection, we have pharmacologically modulated Gb(3) metabolism in an X4 HIV-1 infectable monocytic cell line (THP-1) that naturally expresses Gb(3) and in a Gb(3)-expressing glioblastoma cell line (U87) transfected to express both CD4 and CCR5 to permit R5 HIV-1 infection. THP-1 and U87 cells were treated with either a competitive inhibitor of alpha-galactosidase A, 1-deoxygalactonojirimycin (DGJ) to induce Gb(3) accumulation, or a glucosylceramide synthase inhibitor, phenyl-2-palmitylamino-3-pyrrolidino-1-propanol (P4) to deplete cells of Gb(3). HIV susceptibility was determined via measurement of p24(gag) antigen production by ELISA. In addition, total cellular Gb(3) content was determined using thin layer chromatography followed by Verotoxin1 overlay binding. The cell surface expression of Gb(3) was verified by FACS analysis. We found that DGJ significantly decreased THP-1 and U87 cell susceptibility to HIV-1(IIIB) and HIV-1(BaL) infection, respectively, at a concentration of approximately 100 microM. In contrast, P4 (2 microM) substantially increased cellular susceptibility to HIV-1 infection. Total cellular GSL analysis verified increased Gb(3) expression in cells treated with DGJ and considerable reduction of Gb(3) in P4-treated cells as compared to controls. These results show a reciprocal relationship between Gb(3) expression and infection with either X4 HIV-1(IIIB) or R5 HIV-1(Ba-L). These results support previous studies that Gb(3) provides resistance to HIV infection. Variable Gb(3) expression may provide a natural HIV resistance factor in the general population, and pharmacological manipulation of Gb(3) levels may provide an approach to induction of HIV resistance.