CLN7/MFSD8 may be an important factor for SARS-CoV-2 cell entry.

The SARS-CoV-2 virus has triggered a worldwide pandemic. According to the BioGrid database, CLN7 (MFSD8) is thought to interact with several viral proteins. The aim of this work was to investigate a possible involvement of CLN7 in the infection process. Experiments on a CLN7-deficient HEK293T cell line exhibited a 90% reduced viral load compared to wild-type cells. This observation may be linked to the finding that CLN7 ko cells have a significantly reduced GM1 content in their cell membrane. GM1 is found highly enriched in lipid rafts, which are thought to play an important role in SARS-CoV-2 infection. In contrast, overexpression of CLN7 led to an increase in viral load. This study provides evidence that CLN7 is involved in SARS-CoV-2 infection. This makes it a potential pharmacological target for drug development against COVID-19. Furthermore, it provides insights into the physiological function of CLN7 where still only little is known about.

Analysis of the structure-activity relationship of four herpesviral UL97 subfamily protein kinases reveals partial but not full functional conservation.

Herpesviral protein kinases of the UL97 subfamily are expressed by all known herpesviruses but the degree of functional conservation is unclear. A selection of representative members was investigated by a comparative structural and functional analysis. The coding sequences of human cytomegalovirus (HCMV) pUL97, rat CMV pR97, Epstein-Barr virus BGLF4, and herpes simplex virus UL13 showed a low degree of amino acid identity. A computational approach employing fold recognition techniques revealed structural similarity to the cellular kinase Cdk2 with a high level of conservation of the functionally important residues in ATP binding sites and the catalytic centers. Analyses of in vitro activities of these herpesviral protein kinases, including measurements of phosphorylation of cellular substrates, trans-complementation experiments with a UL97-deleted HCMV mutant, and sensitivity profiles toward protein kinase inhibitors, demonstrated marked similarities between pUL97 and pR97 and to a lesser extent between pUL97 and BGLF4 or UL13. Thus, the structure-activity analysis of pUL97-like herpesviral protein kinases indicates a partial but not a full conservation of their functional properties among the herpesviruses.

Oral brush biopsy analysis by MALDI-ToF Mass Spectrometry for early cancer diagnosis.

OBJECTIVES: Intact cell peptidome profiling (ICPP) with MALDI-ToF Mass Spectrometry holds promise as a non-invasive method to detect head and neck squamous cell carcinoma (HNSCC) objectively, which may significantly improve the early diagnosis of oral cancer. The present study was designed to discriminate between tumour samples and non-cancer controls (healthy mucosa and oral lesions) by analysing complete spectral patterns of intact cells using MALDI-ToF MS. MATERIALS AND METHODS: In the first step, a database consisting of 26 patients suffering from HNSCC was established by taking brush biopsy samples of the diseased area and of the healthy buccal mucosa of the respective contralateral area. After performing MALDI-ToF MS on these samples, classification analysis was used as the basis for further classification of an additional 26 blinded samples including HNSCC, oral lesions and healthy mucosa. RESULTS: By analysing spectral patterns of the blinded samples, all cancerous lesions were defined accurately. One incorrect evaluation (false positive) occurred in the lesion cohort, leading to a sensitivity of 100%, a specificity of 93% and an overall accuracy of 96.5%. CONCLUSION: ICPP using MALDI-ToF MS is able to distinguish between healthy and cancerous mucosa and between oral lesions and oral cancer with excellent sensitivity and specificity, which may lead to more accurate early diagnosis of HNSCC.

HIV-1 mRNA electroporation of PBMC: a simple and efficient method to monitor T-cell responses against autologous HIV-1 in HIV-1-infected patients.

Efficient monitoring of HIV-1-specific T-cells is crucial for the development of HIV-1 vaccines and immunotherapies. Currently, mainly peptides and vaccinia vectors are used for detection of HIV-1-specific cytotoxic T-lymphocytes (CTL), however, as HIV-1 is a variable virus, it is unknown to what extent the T-cell response against the autologous virus is under- or overestimated by using antigens from heterologous viral strains. Therefore, we established a new method for immunomonitoring of CTL using electroporation of peripheral blood mononuclear cells (PBMC) with mRNA derived from autologous viral strains. From six HIV-1-infected patients virus derived mRNA was produced after PCR-based cloning of autologous gag (n=5) and/or nef genes (n=3) from plasma and electroporated into PBMC from patients and healthy donors. Electroporation of PBMC with mRNA resulted in efficient protein expression with good induction of γ-interferon (γ-IFN) release by specific T-cells comparable to peptide pools and better than recombinant vaccinia viruses. Three mRNA encoded autologous Gag proteins and one autologous mRNA encoded Nef protein were better recognized by autologous PBMC in comparison to heterologous mRNA encoded Gag or Nef proteins (SF2 or HXB2). However, in one case each, mRNA encoded autologous Gag or Nef, respectively, was recognized less efficiently due to the presence of CTL escape mutations. In summary, electroporation of PBMC with mRNA is a very efficient, easy and rapid method for immunomonitoring of HIV-1-specific T-cell responses against autologous viral strains. Our data demonstrate that patients' CTL responses against autologous viral strains may be under- or overestimated by using antigens from heterologous viral strains.

Oral brush biopsy analysis by matrix assisted laser desorption/ionisation-time of flight mass spectrometry profiling--a pilot study.

Oral squamous cell carcinomas (OSCCs) often present as advanced tumours requiring aggressive local and regional therapy and result in significant functional impairment. The objective is to develop pre-symptomatic screening detection of OSCC by a brush biopsy method which is less invasive than the conventional biopsy for histology. Given the molecular heterogeneity of oral cancer, it is unlikely that even a panel of tumour markers would provide accurate diagnosis. Therefore, approaches such as the matrix-assisted-laser-desorption/ionisation-time-of-flight-mass-spectrometry (MALDI-TOF-MS) allow several biomarkers or peptide profile patterns to be simultaneously assessed. Brush biopsies from 27 patients with histology-proven OSCCs plus 40 biopsies from 10 healthy controls were collected. MALDI-TOF-MS profiling was performed and additional statistical analysis of the data was used to classify the disease status according to the biological behaviour of the lesion. For classification a support vector machine algorithm was trained using spectra of brush biopsy samples to distinguish healthy control patients from patients with histology-proven OSCC. MALDI-TOF-MS was able to distinguish between healthy patients and OSCC patients with a sensitivity of 100% and specificity of 93%. In summary, MALDI-TOF-MS in combination with sophisticated bioinformatic methods can distinguish OSCC patients from non-cancer controls with excellent sensitivity and specificity. Further improvement and validation of this approach is necessary to determine its feasibility to assist the pre-symptomatic detection of head and neck cancer screening in routine daily practice.

Role of cytotoxic T-lymphocyte-mediated immune selection in a dominant human leukocyte antigen-B8-restricted cytotoxic T-lymphocyte epitope in Nef.

OBJECTIVES: To study the role of cytotoxic T-lymphocyte (CTL) escape for disease progression in HIV-1 infection, we analyzed the CTL response to the dominant human leukocyte antigen (HLA)-B8-restricted CTL epitope FLKEKGGL (FL8) in HIV-1 Nef. METHODS: HIV-1 nef genes derived from 56 patients were analyzed by polymerase chain reaction (PCR)-based sequencing. T-cell responses against FL8 and mutated FL8 variants were detected by gamma-interferon (gamma-IFN) enzyme linked immunospot (ELISPOT) assay. RESULTS: The longitudinal analysis of an HIV-1-infected patient with good control of HIV-1 viremia for several years demonstrated an association of rising viremia with the emergence of CTL escape mutations within the HLA-B8-restricted Nef-specific CTL epitopes FLKEKGGL and WPAIRERM. Analysis of nef genes in 56 HIV-1-infected patients demonstrated a significant correlation between the occurrence of mutations in the FL8 epitope and the presence of HLA-B8. The mutations within the FL8 epitope could decrease CTL recognition; however, there was strong variation regarding the recognition of viral variants between individual donors. The presence of FL8 mutations was associated with lower CD4 cell counts and higher viral loads. CONCLUSIONS: Our data demonstrate a strong CTL selection pressure on the immunodominant HLA-B8-restricted CTL epitope FL8 in HIV-1 Nef. The association of FL8 mutations with lower CD4 cell counts indicates an important role of CTL escape mutations for disease progression.

Generation of HIV-1-specific T cells by electroporation of T-cell receptor RNA.

BACKGROUND: HIV-1-specific cytotoxic T lymphocytes, which recognize conserved epitopes of the virus, are correlated with prolonged survival of infected individuals. Unfortunately, most HIV-1-infected patients are unable to generate such an immune response. Antigen-specific cytotoxic T lymphocytes can be generated by T-cell receptor transfer. This is commonly done by retroviral transduction, which is complicated and poses the threat of stable genetic alteration of autologous cells. METHODS: We reprogrammed primary CD8+ T cells by electroporation of RNA, which encoded an HIV-1-pol- and an HIV-1-gag-specific T-cell receptor recognizing the human leukocyte antigen-A2 restricted epitopes ILKEPVHGV and SLYNTVATL, respectively. RESULTS: These reprogrammed cells specifically produced the proinflammatory cytokines interleukin-2, tumor necrosis factor-alpha, and interferon-gamma after stimulation with target cells that presented the corresponding peptides, and were able to lyse these targets efficiently and specifically. The lytic avidities of the HIV-1-pol- and HIV-1-gag-TCR-RNA-electroporated CD8+ T cells were within the same range than those of the parental cytotoxic T lymphocytes. Most importantly, HIV-1-gag-reprogrammed T cells recognized target cells that presented endogenously processed antigen, which resulted in cytokine production and lysis. CONCLUSION: It is shown here for the first time that functional transfer of virus-specific T-cell receptors by RNA electroporation is feasible, and represents an innovative, safe, and easy method to generate virus-specific T cells, avoiding the risks of retroviral transduction.

Mapping of a self-interaction domain of the cytomegalovirus protein kinase pUL97.

The human cytomegalovirus-encoded protein kinase pUL97 is a determinant of efficient virus replication and fulfils several regulatory functions. In particular, pUL97 interacts with and phosphorylates viral and cellular proteins. Substrate phosphorylation has regulatory consequences on viral replicative stages such as DNA synthesis, transcription and nuclear capsid egress. pUL97, in accordance with related herpesviral protein kinases, possesses strong autophosphorylation activity. Here, we demonstrate that pUL97 shows a pronounced potential to self-interact. Self-interaction of pUL97 is not dependent on its kinase activity, as seen with a catalytically inactive point mutant. The property of self-interaction maps to the amino acid region 231-280 which is separable from the postulated kinase domain. The detection of high-molecular-mass complexes of pUL97 suggests the formation of dimers and oligomers. Importantly, the analysis of pUL97 mutants by in vitro kinase assays demonstrated a correlation between self-interaction and protein kinase activity, i.e. all mutants lacking the ability to self-interact were negative or reduced in their kinase activity. Thus, our findings provide novel insights into the pUL97 structure-activity relationship suggesting an importance of self-interaction for pUL97 functionality.