Synthetic virions reveal fatty acid-coupled adaptive immunogenicity of SARS-CoV-2 spike glycoprotein.

SARS-CoV-2 infection is a major global public health concern with incompletely understood pathogenesis. The SARS-CoV-2 spike (S) glycoprotein comprises a highly conserved free fatty acid binding pocket (FABP) with unknown function and evolutionary selection advantage1,2. Deciphering FABP impact on COVID-19 progression is challenged by the heterogenous nature and large molecular variability of live virus. Here we create synthetic minimal virions (MiniVs) of wild-type and mutant SARS-CoV-2 with precise molecular composition and programmable complexity by bottom-up assembly. MiniV-based systematic assessment of S free fatty acid (FFA) binding reveals that FABP functions as an allosteric regulatory site enabling adaptation of SARS-CoV-2 immunogenicity to inflammation states via binding of pro-inflammatory FFAs. This is achieved by regulation of the S open-to-close equilibrium and the exposure of both, the receptor binding domain (RBD) and the SARS-CoV-2 RGD motif that is responsible for integrin co-receptor engagement. We find that the FDA-approved drugs vitamin K and dexamethasone modulate S-based cell binding in an FABP-like manner. In inflammatory FFA environments, neutralizing immunoglobulins from human convalescent COVID-19 donors lose neutralization activity. Empowered by our MiniV technology, we suggest a conserved mechanism by which SARS-CoV-2 dynamically couples its immunogenicity to the host immune response.

SAR of novel benzothiazoles targeting an allosteric pocket of DENV and ZIKV NS2B/NS3 proteases.

In recent years, dengue virus (DENV) and Zika virus (ZIKV), both mosquito-borne members of the Flaviviridae family, have emerged as intercontinental health issues since their vectors have spread from their tropical origins to temperate climate zones due to climate change and increasing globalization. DENV and ZIKV are positive-sense, single-stranded RNA viruses, whose genomes consist of three structural (capsid, membrane precursor, envelope) and seven non-structural (NS) proteins, all of which are initially expressed as a single precursor polyprotein. For virus maturation, the polyprotein processing is accomplished by host proteases and the viral NS2B/NS3 protease complex, whose inhibitors have been shown to be effective antiviral agents with loss of viral pathogenicity. In this work, we elucidate new structure-activity relationships of benzo[d]thiazole-based allosteric NS2B/NS3 inhibitors. We developed a new series of Y-shaped inhibitors, which, with its larger hydrophobic contact surface, should bind to previously unaddressed regions of the allosteric NS2B/NS3 binding pocket. By scaffold-hopping, we varied the benzo[d]thiazole core and identified benzofuran as a new lead scaffold shifting the selectivity of initially ZIKV-targeting inhibitors to higher activities towards the DENV protease. In addition, we were able to increase the ligand efficiency from 0.27 to 0.41 by subsequent inhibitor truncation and identified N-(5,6-dihydroxybenzo[d]thiazol-2-yl)-4-iodobenzamide as a novel sub-micromolar NS2B/NS3 inhibitor. Utilizing cell-based assays, we could prove the antiviral activity in cellulo. Overall, we report new series of sub-micromolar allosteric DENV and ZIKV inhibitors with good efficacy profile in terms of cytotoxicity and protease inhibition selectivity.

Chemical targeting of NEET proteins reveals their function in mitochondrial morphodynamics.

Several human pathologies including neurological, cardiac, infectious, cancerous, and metabolic diseases have been associated with altered mitochondria morphodynamics. Here, we identify a small organic molecule, which we named Mito-C. Mito-C is targeted to mitochondria and rapidly provokes mitochondrial network fragmentation. Biochemical analyses reveal that Mito-C is a member of a new class of heterocyclic compounds that target the NEET protein family, previously reported to regulate mitochondrial iron and ROS homeostasis. One of the NEET proteins, NAF-1, is identified as an important regulator of mitochondria morphodynamics that facilitates recruitment of DRP1 to the ER-mitochondria interface. Consistent with the observation that certain viruses modulate mitochondrial morphogenesis as a necessary part of their replication cycle, Mito-C counteracts dengue virus-induced mitochondrial network hyperfusion and represses viral replication. The newly identified chemical class including Mito-C is of therapeutic relevance for pathologies where altered mitochondria dynamics is part of disease etiology and NEET proteins are highlighted as important therapeutic targets in anti-viral research.

CDK1 couples proliferation with protein synthesis.

Cell proliferation exerts a high demand on protein synthesis, yet the mechanisms coupling the two processes are not fully understood. A kinase and phosphatase screen for activators of translation, based on the formation of stress granules in human cells, revealed cell cycle-associated kinases as major candidates. CDK1 was identified as a positive regulator of global translation, and cell synchronization experiments showed that this is an extramitotic function of CDK1. Different pathways including eIF2α, 4EBP, and S6K1 signaling contribute to controlling global translation downstream of CDK1. Moreover, Ribo-Seq analysis uncovered that CDK1 exerts a particularly strong effect on the translation of 5'TOP mRNAs, which includes mRNAs encoding ribosomal proteins and several translation factors. This effect requires the 5'TOP mRNA-binding protein LARP1, concurrent to our finding that LARP1 phosphorylation is strongly dependent on CDK1. Thus, CDK1 provides a direct means to couple cell proliferation with biosynthesis of the translation machinery and the rate of protein synthesis.

Spatiotemporal Coupling of the Hepatitis C Virus Replication Cycle by Creating a Lipid Droplet- Proximal Membranous Replication Compartment.

The hepatitis C virus (HCV) is a major cause of chronic liver disease, affecting around 71 million people worldwide. Viral RNA replication occurs in a membranous compartment composed of double-membrane vesicles (DMVs), whereas virus particles are thought to form by budding into the endoplasmic reticulum (ER). It is unknown how these steps are orchestrated in space and time. Here, we established an imaging system to visualize HCV structural and replicase proteins in live cells and with high resolution. We determined the conditions for the recruitment of viral proteins to putative assembly sites and studied the dynamics of this event and the underlying ultrastructure. Most notable was the selective recruitment of ER membranes around lipid droplets where structural proteins and the viral replicase colocalize. Moreover, ER membranes wrapping lipid droplets were decorated with double membrane vesicles, providing a topological map of how HCV might coordinate the steps of viral replication and virion assembly.

microRNA-122 amplifies hepatitis C virus translation by shaping the structure of the internal ribosomal entry site.

The liver-specific microRNA-122 (miR-122) recognizes two conserved sites at the 5' end of the hepatitis C virus (HCV) genome and contributes to stability, translation, and replication of the viral RNA. We show that stimulation of the HCV internal ribosome entry site (IRES) by miR-122 is essential for efficient viral replication. The mechanism relies on a dual function of the 5' terminal sequence in the complementary positive (translation) and negative strand (replication), requiring different secondary structures. Predictions and experimental evidence argue for several alternative folds involving the miR-binding region (MBR) adjacent to the IRES and interfering with its function. Mutations in the MBR, designed to suppress these dysfunctional structures indeed stimulate translation independently of miR-122. Conversely, MBR mutants favoring alternative folds show impaired IRES activity. Our results therefore suggest that miR-122 binding assists the folding of a functional IRES in an RNA chaperone-like manner by suppressing energetically favorable alternative secondary structures.

The host-cell restriction factor SERINC5 restricts HIV-1 infectivity without altering the lipid composition and organization of viral particles.

The host-cell restriction factor SERINC5 potently suppresses the infectivity of HIV, type 1 (HIV-1) particles, and is counteracted by the viral pathogenesis factor Nef. However, the molecular mechanism by which SERINC5 restricts HIV-1 particle infectivity is still unclear. Because SERINC proteins have been suggested to facilitate the incorporation of serine during the biosynthesis of membrane lipids and because lipid composition of HIV particles is a major determinant of the infectious potential of the particles, we tested whether SERINC5-mediated restriction of HIV particle infectivity involves alterations of membrane lipid composition. We produced and purified HIV-1 particles from SERINC5293T cells with very low endogenous SERINC5 levels under conditions in which ectopically expressed SERINC5 restricts HIV-1 infectivity and is antagonized by Nef and analyzed both virions and producer cells with quantitative lipid MS. SERINC5 restriction and Nef antagonism were not associated with significant alterations in steady-state lipid composition of producer cells and HIV particles. Sphingosine metabolism kinetics were also unaltered by SERINC5 expression. Moreover, the levels of phosphatidylserine on the surface of HIV-1 particles, which may trigger uptake into non-productive internalization pathways in target cells, did not change upon expression of SERINC5 or Nef. Finally, saturating the phosphatidylserine-binding sites on HIV target cells did not affect SERINC5 restriction or Nef antagonism. These results demonstrate that the restriction of HIV-1 particle infectivity by SERINC5 does not depend on alterations in lipid composition and organization of HIV-1 particles and suggest that channeling serine into lipid biosynthesis may not be a cardinal cellular function of SERINC5.

Flavivirus Infection Uncouples Translation Suppression from Cellular Stress Responses.

As obligate parasites, viruses strictly depend on host cell translation for the production of new progeny, yet infected cells also synthesize antiviral proteins to limit virus infection. Modulation of host cell translation therefore represents a frequent strategy by which viruses optimize their replication and spread. Here we sought to define how host cell translation is regulated during infection of human cells with dengue virus (DENV) and Zika virus (ZIKV), two positive-strand RNA flaviviruses. Polysome profiling and analysis of de novo protein synthesis revealed that flavivirus infection causes potent repression of host cell translation, while synthesis of viral proteins remains efficient. Selective repression of host cell translation was mediated by the DENV polyprotein at the level of translation initiation. In addition, DENV and ZIKV infection suppressed host cell stress responses such as the formation of stress granules and phosphorylation of the translation initiation factor eIF2α (α subunit of eukaryotic initiation factor 2). Mechanistic analyses revealed that translation repression was uncoupled from the disruption of stress granule formation and eIF2α signaling. Rather, DENV infection induced p38-Mnk1 signaling that resulted in the phosphorylation of the eukaryotic translation initiation factor eIF4E and was essential for the efficient production of virus particles. Together, these results identify the uncoupling of translation suppression from the cellular stress responses as a conserved strategy by which flaviviruses ensure efficient replication in human cells. IMPORTANCE: For efficient production of new progeny, viruses need to balance their dependency on the host cell translation machinery with potentially adverse effects of antiviral proteins produced by the infected cell. To achieve this, many viruses evolved mechanisms to manipulate host cell translation. Here we find that infection of human cells with two major human pathogens, dengue virus (DENV) and Zika virus (ZIKV), leads to the potent repression of host cell translation initiation, while the synthesis of viral protein remains unaffected. Unlike other RNA viruses, these flaviviruses concomitantly suppress host cell stress responses, thereby uncoupling translation suppression from stress granule formation. We identified that the p38-Mnk1 cascade regulating phosphorylation of eIF4E is a target of DENV infection and plays an important role in virus production. Our results define several molecular interfaces by which flaviviruses hijack host cell translation and interfere with stress responses to optimize the production of new virus particles.

DDX60L Is an Interferon-Stimulated Gene Product Restricting Hepatitis C Virus Replication in Cell Culture.

UNLABELLED: All major types of interferon (IFN) efficiently inhibit hepatitis C virus (HCV) replication in vitro and in vivo. Remarkably, HCV replication is not sensitive to IFN-γ in the hepatoma cell line Huh6, despite an intact signaling pathway. We performed transcriptome analyses between Huh6 and Huh-7 cells to identify effector genes of the IFN-γ response and thereby identified the DExD/H box helicase DEAD box polypeptide 60-like (DDX60L) as a restriction factor of HCV replication. DDX60L and its homolog DEAD box polypeptide 60 (DDX60) were both induced upon viral infection and IFN treatment in primary human hepatocytes. However, exclusively DDX60L knockdown increased HCV replication in Huh-7 cells and rescued HCV replication from type II IFN as well as type I and III IFN treatment, suggesting that DDX60L is an important effector protein of the innate immune response against HCV. In contrast, we found no impact of DDX60L on replication of hepatitis A virus. DDX60L protein was detectable only upon strong ectopic overexpression, displayed a broad cytoplasmic distribution, but caused cytopathic effects under these conditions. DDX60L knockdown did not alter interferon-stimulated gene (ISG) induction after IFN treatment but inhibited HCV replication upon ectopic expression, suggesting that it is a direct effector of the innate immune response. It most likely inhibits viral RNA replication, since we found neither impact of DDX60L on translation or stability of HCV subgenomic replicons nor additional impact on assembly of infectious virus. Similar to DDX60, DDX60L had a moderate impact on RIG-I dependent activation of innate immunity, suggesting additional functions in the sensing of viral RNA. IMPORTANCE: Interferons induce a plethora of interferon-stimulated genes (ISGs), which are our first line of defense against viral infections. In addition, IFNs have been used in antiviral therapy, in particular against the human pathogen hepatitis C virus (HCV); still, their mechanism of action is not well understood, since diverse, overlapping sets of antagonistic effector ISGs target viruses with different biologies. Our work identifies DDX60L as a novel factor that inhibits replication of HCV. DDX60L expression is regulated similarly to that of its homolog DDX60, but our data suggest that it has distinct functions, since we found no contribution of DDX60 in combatting HCV replication. The identification of novel components of the innate immune response contributes to a comprehensive understanding of the complex mechanisms governing antiviral defense.

Control of temporal activation of hepatitis C virus-induced interferon response by domain 2 of nonstructural protein 5A.

BACKGROUND & AIMS: Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a multifunctional protein playing a crucial role in diverse steps of the viral replication cycle and perturbing multiple host cell pathways. We showed previously that removal of a region in domain 2 (D2) of NS5A (mutant NS5A(D2Δ)) is dispensable for viral replication in hepatoma cell lines. By using a mouse model and immune-competent cell systems, we studied the role of D2 in controlling the innate immune response. METHODS: In vivo replication competence of NS5A(D2Δ) was studied in transgenic mice with human liver xenografts. Results were validated using primary human hepatocytes (PHHs) and mechanistic analyses were conducted in engineered Huh7 hepatoma cells with reconstituted innate signaling pathways. RESULTS: Although the deletion in NS5A removed most of the interferon (IFN) sensitivity determining-region, mutant NS5A(D2Δ) was as sensitive as the wild type to IFN-α and IFN-λ in vitro, but severely attenuated in vivo. This attenuation could be recapitulated in PHHs and was linked to higher activation of the IFN response, concomitant with reduced viral replication and virus production. Importantly, immune-reconstituted Huh7-derived cell lines revealed a sequential activation of the IFN-response via RIG-I (retinoic acid-inducible gene I) and MDA5 (Myeloma differentiation associated factor 5), respectively, that was significantly higher in the case of the mutant lacking most of NS5A D2. CONCLUSIONS: Our study reveals an important role of NS5A D2 for suppression of the IFN response that is activated by HCV via RIG-I and MDA5 in a sequential manner.

The interactomes of influenza virus NS1 and NS2 proteins identify new host factors and provide insights for ADAR1 playing a supportive role in virus replication.

Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.

Identification of type I and type II interferon-induced effectors controlling hepatitis C virus replication.

UNLABELLED: Persistent infection with hepatitis C virus (HCV) can lead to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. All current therapies of hepatitis C include interferon-alpha (IFN-α). Moreover, IFN-gamma (IFN-γ), the only type II IFN, strongly inhibits HCV replication in vitro and is the primary mediator of HCV-specific antiviral T-cell responses. However, for both cytokines the precise set of effector protein(s) responsible for replication inhibition is not known. The aim of this study was the identification of IFN-α and IFN-γ stimulated genes (ISGs) responsible for controlling HCV replication. We devised an RNA interference (RNAi)-based "gain of function" screen and identified, in addition to known ISGs earlier reported to suppress HCV replication, several new ones with proven antiviral activity. These include IFIT3 (IFN-induced protein with tetratricopeptide repeats 3), TRIM14 (tripartite motif containing 14), PLSCR1 (phospholipid scramblase 1), and NOS2 (nitric oxide synthase 2, inducible). All ISGs identified in this study were up-regulated both by IFN-α and IFN-γ, demonstrating a substantial overlap of HCV-specific effectors induced by either cytokine. Nevertheless, some ISGs were more specific for IFN-α or IFN-γ, which was most pronounced in case of PLSCR1 and NOS2 that were identified as main effectors of IFN-γ-mediated anti-HCV activity. Combinatorial knockdowns of ISGs suggest additive or synergistic effects demonstrating that with either IFN, inhibition of HCV replication is caused by the combined action of multiple ISGs. CONCLUSION: Our study identifies a number of novel ISGs contributing to the suppression of HCV replication by type I and type II IFN. We demonstrate a substantial overlap of antiviral programs triggered by either cytokine and show that suppression of HCV replication is mediated by the concerted action of multiple effectors.

GAS41 amplification results in overexpression of a new spindle pole protein.

Amplification is a hallmark of many human tumors but the role of most amplified genes in human tumor development is not yet understood. Previously, we identified a frequently amplified gene in glioma termed glioma-amplified sequence 41 (GAS41). Using the TCGA data portal and performing experiments on HeLa and TX3868, we analyzed the role of GAS41 amplification on GAS41 overexpression and the effect on the cell cycle. Here we show that GAS41 amplification is associated with overexpression in the majority of cases. Both induced and endogenous overexpression of GAS41 leads to an increase in multipolar spindles. We showed that GAS41 is specifically associated with pericentrosome material. As result of an increased GAS41 expression we found bipolar spindles with misaligned chromosomes. This number was even increased by a combined overexpression of GAS41 and a reduced expression of NuMA. We propose that GAS41 amplification may have an effect on the highly altered karyotype of glioblastoma via its role during spindle pole formation.

Persistence of HCV in quiescent hepatic cells under conditions of an interferon-induced antiviral response.

BACKGROUND & AIMS: Hepatitis C virus (HCV) is a common cause of chronic liver disease. Many patients do not clear the viral infection; little is known about the mechanisms of HCV persistence or the frequent failure of interferon (IFN) to eliminate it. Better culture systems are needed to study viral replication in quiescent liver cells. METHODS: We used human hepatoma (Huh7.5) cells and those that had undergone proliferation arrest and differentiation (Huh7.5(dif)) to study the persistence of HCV infection following exposure of the cells to IFN-α and to compare the antiviral effects of IFN-α and IFN-λ. We validated these results with primary human hepatocytes and Huh7 cells that expressed an IFN-inducible fluorophore. RESULTS: Following infection of Huh7.5(dif) cells, HCV replicated persistently and released infectious particles. Long-term exposure of the cells to IFN-α reduced HCV replication ∼1000-fold but did not eliminate the virus; viral replication rebounded after withdrawal of IFN, as it does in patients with chronic HCV infection. HCV replicated at higher levels, but not exclusively, in cells that had a low level of response to IFN-α. Following incubation of cells with equipotent concentrations of IFN-α or IFN-λ, Huh7.5(dif) cells expressed a wider pattern of IFN-stimulated genes than undifferentiated Huh7.5 cells or primary human hepatocytes, indicating that the antiviral response depends on the differentiation status of the cells. CONCLUSIONS: We developed a cell culture system using hepatoma cells to study persistent HCV infection during the type I or type III IFN-induced antiviral response. The level and range of the antiviral responses were associated with the differentiation status of the cells. We propose that HCV exploits the stochastic nature of the response of hepatocytes to IFN to sustain persistence.

Angiogenesis in oral lichen planus: an in vivo and immunohistological evaluation.

Oral lichen planus (OLP) is an autoimmune disease with an inflammatory pathogenesis. The angiogenetic phenomenon is a mechanism at the base of the pathogenesis of chronic inflammatory processes. The aim of this research is to evaluate the angiogenetic phenomenon, comparing an in vitro method with an in vivo one. Thirty OLP patients and 30 healthy subjects were enrolled in the study. Immunohistochemical analysis of the vascular-endothelial growth factor (VEGF) and vascular-endothelial adhesion molecules were carried out by the means of primary antibodies and anti-CD34, anti-VEGF, anti-CD106 antigen (VCAM-1) and anti-CD54 antigen (ICAM-1). Capillary density and others capillaroscopic parameters were tested in vivo using oral videocapillaroscopy. The results reveal the presence of a significant angiogenesis in OLP patients through the immunoexpression of VEGF, CD34, CD106 and CD54 (p < 0.001). Capillaroscopic analysis demonstrates significant value for the following parameters: density, tortuosity, loop diameter, afferent and efferent capillary loop diameter. The in vivo and in vitro investigation in OLP reveals a significant angiogenesis.

The YEATS family member GAS41 interacts with the general transcription factor TFIIF.

BACKGROUND: In eukaryotes the transcription initiation by RNA polymerase II requires numerous general and regulatory factors including general transcription factors. The general transcription factor TFIIF controls the activity of the RNA polymerase II both at the initiation and elongation stages. The glioma amplified sequence 41 (GAS41) has been associated with TFIIF via its YEATS domain. RESULTS: Using GST pull-down assays, we demonstrated that GAS41 binds to both, the small subunit (RAP30) and the large subunit (RAP74) of TFIIF in vitro. The in vivo interaction of GAS41 and endogenous RAP30 and RAP74 was confirmed by co-immunoprecipitation. GAS41 binds to two non-overlapping regions of the C-terminus of RAP30. There is also an ionic component to the binding between GAS41 and RAP30. There was no evidence for a direct interaction between GAS41 and TBP or between GAS41 and RNA polymerase II. CONCLUSIONS: Our results demonstrate binding between endogenous GAS41 and the endogenous TFIIF subunits (RAP30 and RAP74). Since we did not find evidence for a binding of GAS41 to TBP or RNA polymerase II, GAS41 seems to preferentially bind to TFIIF. GAS41 that does not contain a DNA-binding domain appears to be a co-factor of TFIIF.

Chronic hyperplastic candidosis: a pilot study of the efficacy of 0.18% isotretinoin.

Management of oral candidiasis depends on an accurate diagnosis, identification and elimination of predisposing factors, and, often, use of antifungal agents. Chronic hyperplastic candidosis (CHC) is considered a premalignant lesion of the oral mucosa, occurring as speckled or homogeneous white lesions. If the lesions are untreated, a minor proportion may become dysplastic and progress to carcinoma. The traditional treatment of this lesion is based on the use of antifungal agents. The aim of this study was to examine the efficacy of 0.18% isotretinoin for treatment of nystatin-resistant candidiasis. Isotretinoin was administered topically twice a day for one month to six patients affected by nystatin-resistant CHC. In all six patients, daily antimycotic topical therapy with nystatin for 30 days had failed to resolve the candidal stomatitis. After one month of isotretinoin treatment, five of the six patients were negative for Candida, whereas in untreated control patients the situation was unchanged. Only one patient with suspected sicca syndrome was found to have oral Candida 15 days after the last administration of isotretinoin. None of the patients had any complaints about the medication. These findings suggest that 0.18% isotretinoin applied twice a day for one month is able to suppress nystatin-resistant candidiasis.

Angiogenesis of oral lichen planus: a possible pathogenetic mechanism.

OBJECTIVE: Oral Lichen Planus (OLP) is a chronic inflammatory disease with an autoimmune inflammatory pathogenesis. The aim of the research is to compare the vascular endothelial growth factor (VEGF) and adhesion of molecules in the biopsy samples of patients affected by OLP, in order to research the presence of the angiogenetic phenomenon and to understand its pathogenetic mechanism. MATERIALS AND METHODS: Thirty OLP patients and thirty healthy subjects were enrolled in a study. The immunohistochemical analysis of the VEGF and vascular-endothelial adhesion molecules was carried out by means of primary antibodies and anti-CD34, anti-VEGF, anti-CD106 antigen (VCAM-1) and anti-CD54 antigen (ICAM-1). The statistical significance of the differences was checked with the Mann-Whitney test (MW test). The level of significance was set to P<0.001. Data analysis was carried out with StatView 5.0.1 (SAS Institute Inc., Cary, NC). RESULTS: The results reveal the presence of a significant angiogenesis in OLP patients for the VEGF, CD34, CD106 and CD54 (P < 0.001).. The number of vessels in the biopsies of the patients with OLP (mean +/-SD: 21.27+/-4.85), compared with the healthy subjects (mean +/-SD: 4.74+/-0.97) was significantly more (Mann-Whitney test, P < 0.001). The positive expression rate of VEGF, CD34, VCAM-1 and ICAM-1 in oral lichen samples was 64.2%, 54.3%, 32.5% and 29.7%, respectively. Isolated endothelial cells and newly-formed micro-vessels and endothelial cells with high-immune-positivity to the antibodies anti-ICAM-1 and anti-VCAM-1 were observed. CONCLUSIONS: The results of our immunohistochemical research show that a significant neoangiogenesis occurs in oral lichen planus.

Human endogenous retrovirus HERV-K(HML-2) encodes a stable signal peptide with biological properties distinct from Rec.

BACKGROUND: The human endogenous retrovirus HERV-K(HML-2) family is associated with testicular germ cell tumors (GCT). Various HML-2 proviruses encode viral proteins such as Env and Rec. RESULTS: We describe here that HML-2 Env gives rise to a 13 kDa signal peptide (SP) that harbors a different C-terminus compared to Rec. Subsequent to guiding Env to the endoplasmatic reticulum (ER), HML-2 SP is released into the cytosol. Biochemical analysis and confocal microscopy demonstrated that similar to Rec, SP efficiently translocates to the granular component of nucleoli. Unlike Rec, SP does not shuttle between nucleus and cytoplasm. SP is less stable than Rec as it is subjected to proteasomal degradation. Moreover, SP lacks export activity towards HML-2 genomic RNA, the main function of Rec in the original viral context, and SP does not interfere with Rec's RNA export activity. CONCLUSION: SP is a previously unrecognized HML-2 protein that, besides targeting and translocation of Env into the ER lumen, may exert biological functions distinct from Rec. HML-2 SP represents another functional similarity with the closely related Mouse Mammary Tumor Virus that encodes an Env-derived SP named p14. Our findings furthermore support the emerging concept of bioactive SPs as a conserved retroviral strategy to modulate their host cell environment, evidenced here by a "retroviral fossil". While the specific role of HML-2 SP remains to be elucidated in the context of human biology, we speculate that it may be involved in immune evasion of GCT cells or tumorigenesis.