Alternative substitutions of N332 in HIV-1AD8 gp120 differentially affect envelope glycoprotein function and viral sensitivity to broadly neutralizing antibodies targeting the V3-glycan.

The envelope glycoprotein (Env) trimer on the surface of human immunodeficiency virus type I (HIV-1) mediates viral entry into host CD4+ T cells and is the sole target of neutralizing antibodies. Broadly neutralizing antibodies (bnAbs) that target gp120 V3-glycan of HIV-1 Env trimer are potent and block the entry of diverse HIV-1 strains. Most V3-glycan bnAbs interact, to a different extent, with a glycan attached to N332, but Asn at this position is not absolutely conserved or required for HIV-1 entry based on the prevalence of N332 in different circulating HIV-1 strains from diverse clades. Here, we studied the effects of amino acid changes at position 332 of HIV-1AD8 Envs on HIV-1 sensitivity to antibodies, cold exposure, and soluble CD4. We further investigated how these changes affect Env function and HIV-1 infectivity in vitro. Our results suggest robust tolerability of HIV-1AD8 Env N332 to changes, with specific changes that resulted in extended exposure of gp120 V3 loop, which is typically concealed in most primary HIV-1 isolates. Viral evolution leading to Asn at position 332 of HIVAD8 Envs is supported by the selection advantage of high levels of cell-cell fusion, transmission, and infectivity with high levels of cell surface expression and slightly higher gp120 shedding than most N332 variants. Thus, tolerance of HIV-1AD8 Envs to different amino acids at position 332 provides increased flexibility to respond to changing conditions/environments and evade the immune system. Modeling studies of the distance between N332 glycan and specific bnAbs were in agreement with N332 glycan dependency on bnAb neutralization. Overall, our studies provide insights into the contribution of specific amino acids at position 332 to Env antigenicity, stability on ice, and conformational states. IMPORTANCE: Glycan attached to amino acid asparagine at position 332 of HIV-1 envelope glycoproteins is a main target of a subset of broadly neutralizing antibodies that block HIV-1 infection. Here, we defined the contribution of different amino acids at this position to Env antigenicity, stability on ice, and conformational states.

Ultrasensitive quantification of HIV-1 cell-to-cell transmission in primary human CD4+ T cells measures viral sensitivity to broadly neutralizing antibodies.

IMPORTANCE: HIV-1 can efficiently transmit from one cell to another but accurate quantification of this mode of transmission is still challenging. Here, we developed an ultrasensitive assay to measure HIV-1 transmission between cells and to evaluate HIV-1 escape from broadly neutralizing antibodies in primary human T cells. This assay will contribute to understanding the fundamental mechanisms of HIV-1 cell-to-cell transmission, allow evaluation of pre-existing or acquired HIV-1 resistance in clinical trials, and can be adapted to study the biology of other retroviruses.

Alternative substitutions of N332 in HIV-1AD8 gp120 differentially affect envelope glycoprotein function and viral sensitivity to broadly neutralizing antibodies targeting the V3-glycan.

The envelope glycoprotein (Env) trimer on the surface of human immunodeficiency virus type I (HIV-1) mediates viral entry into host CD4+ T cells and is the sole target of neutralizing antibodies. Broadly neutralizing antibodies (bnAbs) that target gp120 V3-glycan of HIV-1 Env trimer are potent and block the entry of diverse HIV-1 strains. Most V3-glycan bnAbs interact, to a different extent, with a glycan attached to N332 but Asn at this position is not absolutely conserved or required for HIV-1 entry based on prevalence of N332 in different circulating HIV-1 strains from diverse clades. Here, we studied the effects of amino acid changes at position 332 of HIV-1AD8 Envs on HIV-1 sensitivity to antibodies, cold exposure, and soluble CD4. We further investigated how these changes affect Env function and HIV-1 infectivity in vitro. Our results suggest robust tolerability of HIV-1AD8 Env N332 to changes with specific changes that resulted in extended exposure of gp120 V3 loop, which is typically concealed in most primary HIV-1 isolates. Viral evolution leading to Asn at position 332 of HIVAD8 Envs is supported by the selection advantage of high levels of cell-cell fusion, transmission, and infectivity even though cell surface expression levels are lower than most N332 variants. Thus, tolerance of HIV-1AD8 Envs to different amino acids at position 332 provides increased flexibility to respond to changing conditions/environments and to evade the immune system. Modeling studies of the distance between N332 glycan and specific bnAbs was in agreement with N332 glycan dependency on bnAb neutralization. Overall, our studies provide insights into the contribution of specific amino acids at position 332 to Env antigenicity, stability on ice, and conformational states.

Packaging and Uncoating of CRISPR/Cas Ribonucleoproteins for Efficient Gene Editing with Viral and Non-Viral Extracellular Nanoparticles.

Rapid progress in gene editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) has revolutionized functional genomic studies and genetic disease correction. While numerous gene editing applications have been easily adapted by experimental science, the clinical utility of CRISPR/Cas remains very limited due to difficulty in delivery to primary cells and possible off-target effects. The use of CRISPR in the form of a ribonucleoprotein (RNP) complex substantially reduces the time of DNA exposure to the effector nuclease and minimizes its off-target activity. The traditional electroporation and lipofection methods lack the cell-type specificity of RNP delivery, can be toxic for cells, and are less efficient when compared to nanoparticle transporters. This review focuses on CRISPR/Cas RNP packaging and delivery using retro/lentiviral particles and exosomes. First, we briefly describe the natural stages of viral and exosomal particle formation, release and entry into the target cells. This helps us understand the mechanisms of CRISPR/Cas RNP packaging and uncoating utilized by the current delivery systems, which we discuss afterward. Much attention is given to the exosomes released during viral particle production that can be passively loaded with RNPs as well as the mechanisms necessary for particle fusion, RNP release, and transportation inside the target cells. Collectively, together with specific packaging mechanisms, all these factors can substantially influence the editing efficiency of the system. Finally, we discuss ways to improve CRISPR/Cas RNP delivery using extracellular nanoparticles.

Application of CRISPR/Cas Genomic Editing Tools for HIV Therapy: Toward Precise Modifications and Multilevel Protection.

Although highly active antiretroviral therapy (HAART) can robustly control human immunodeficiency virus (HIV) infection, the existence of latent HIV in a form of proviral DNA integrated into the host genome makes the virus insensitive to HAART. This requires patients to adhere to HAART for a lifetime, often leading to drug toxicity or viral resistance to therapy. Current genome-editing technologies offer different strategies to reduce the latent HIV reservoir in the body. In this review, we systematize the research on CRISPR/Cas-based anti-HIV therapeutic methods, discuss problems related to viral escape and gene editing, and try to focus on the technologies that effectively and precisely introduce genetic modifications and confer strong resistance to HIV infection. Particularly, knock-in (KI) approaches, such as mature B cells engineered to produce broadly neutralizing antibodies, T cells expressing fusion inhibitory peptides in the context of inactivated viral coreceptors, or provirus excision using base editors, look very promising. Current and future advancements in the precision of CRISPR/Cas editing and its delivery will help extend its applicability to clinical HIV therapy.

HIV-1 and HTLV-1 Transmission Modes: Mechanisms and Importance for Virus Spread.

So far, only two retroviruses, human immunodeficiency virus (HIV) (type 1 and 2) and human T-cell lymphotropic virus type 1 (HTLV-1), have been recognized as pathogenic for humans. Both viruses mainly infect CD4+ T lymphocytes. HIV replication induces the apoptosis of CD4 lymphocytes, leading to the development of acquired immunodeficiency syndrome (AIDS). After a long clinical latency period, HTLV-1 can transform lymphocytes, with subsequent uncontrolled proliferation and the manifestation of a disease called adult T-cell leukemia (ATLL). Certain infected patients develop neurological autoimmune disorder called HTLV-1-associated myelopathy, also known as tropical spastic paraparesis (HAM/TSP). Both viruses are transmitted between individuals via blood transfusion, tissue/organ transplantation, breastfeeding, and sexual intercourse. Within the host, these viruses can spread utilizing either cell-free or cell-to-cell modes of transmission. In this review, we discuss the mechanisms and importance of each mode of transmission for the biology of HIV-1 and HTLV-1.

Engineering T-Cell Resistance to HIV-1 Infection via Knock-In of Peptides from the Heptad Repeat 2 Domain of gp41.

Previous studies suggest that short peptides from the heptad repeat 2 (HR2) domain of gp41 expressed on the cell surface are more potent inhibitors of HIV-1 entry than soluble analogs. However, their therapeutic potential has only been examined using lentiviral vectors. Here, we aimed to develop CRISPR/Cas9-based fusion inhibitory peptide knock-in (KI) technology for the generation and selection of HIV-1-resistant T cells. First, we embedded a series of HIV-1 fusion inhibitory peptides in CD52, the shortest glycosylphosphatidylinositol (GPI)-anchored protein, which efficiently delivers epitope tags to the cell surface and maintains a sufficient level of KI. Among the seven peptides tested, MT-C34, HP-23L, and 2P23 exhibited significant activity against both cell-free and cell-to-cell HIV-1 infection. The shed variant of MT-C34 provided insufficient protection against HIV-1 due to its low concentration in the culture medium. Using Cas9 plasmids or ribonucleoprotein electroporation and peptide-specific antibodies, we sorted CEM/R5 cells with biallelic KI of MT-C34 and 2P23 peptides at the CXCR4 locus. In combination, these peptides provided a higher level of protection than individual KI. By extending homology arms and cloning donor DNA into a plasmid containing signals for nuclear localization, we achieved KI of MT-C34 into the CXCR4 locus and HIV-1 proviral DNA at levels of up to 35% in the T-cell line and up to 4 to 5% in primary CD4 lymphocytes. Compared to lentiviral delivery, KI resulted in the higher MT-C34 surface expression and stronger protection of lymphocytes from HIV-1. Thus, we demonstrate that KI is a viable strategy for peptide-based therapy of HIV infection. IMPORTANCE HIV is a human lentivirus that infects CD4-positive immune cells and, when left untreated, manifests in the fatal disease known as AIDS. Antiretroviral therapy (ART) does not lead to viral clearance, and HIV persists in the organism as a latent provirus. One way to control infection is to increase the population of HIV-resistant CD4 lymphocytes via entry molecule knockout or expression of different antiviral genes. Peptides from the heptad repeat (HR) domain of gp41 are potent inhibitors of HIV-1 fusion, especially when designed to express on the cell surface. Individual gp41 peptides encoded by therapeutic lentiviral vectors have been evaluated and some have entered clinical trials. However, a CRISPR/Cas9-based gp41 peptide delivery platform that operates through concomitant target gene modification has not yet been developed due to low knock-in (KI) rates in primary cells. Here, we systematically evaluated the antiviral activity of different HR2 peptides cloned into the shortest carrier molecule, CD52. The resulting small-size transgene constructs encoding selected peptides, in combination with improvements to enhance donor vector nuclear import, helped to overcome precise editing restrictions in CD4 lymphocytes. Using KI into CXCR4, we demonstrated different options for target gene modification, effectively protecting edited cells against HIV-1.

Different Neutralization Sensitivity of SARS-CoV-2 Cell-to-Cell and Cell-Free Modes of Infection to Convalescent Sera.

The COVID-19 pandemic caused by SARS-CoV-2 has posed a global threat to human lives and economics. One of the best ways to determine protection against the infection is to quantify the neutralizing activity of serum antibodies. Multiple assays have been developed to validate SARS-CoV-2 neutralization; most of them utilized lentiviral or vesicular stomatitis virus-based particles pseudotyped with the spike (S) protein, making them safe and acceptable to work with in many labs. However, these systems are only capable of measuring infection with purified particles. This study has developed a pseudoviral assay with replication-dependent reporter vectors that can accurately quantify the level of infection directly from the virus producing cell to the permissive target cell. Comparative analysis of cell-free and cell-to-cell infection revealed that the neutralizing activity of convalescent sera was more than tenfold lower in cell cocultures than in the cell-free mode of infection. As the pseudoviral system could not properly model the mechanisms of SARS-CoV-2 transmission, similar experiments were performed with replication-competent coronavirus, which detected nearly complete SARS-CoV-2 cell-to-cell infection resistance to neutralization by convalescent sera. These findings suggest that the cell-to-cell mode of SARS-CoV-2 transmission, for which the mechanisms are largely unknown, could be of great importance for treatment and prevention of COVID-19.

Pattern of circulating SARS-CoV-2-specific antibody-secreting and memory B-cell generation in patients with acute COVID-19.

OBJECTIVES: To predict the spread of coronavirus disease (COVID-19), information regarding the immunological memory for disease-specific antigens is necessary. The possibility of reinfection, as well as the efficacy of vaccines for COVID-19 that are currently under development, will largely depend on the quality and longevity of immunological memory in patients. To elucidate the process of humoral immunity development, we analysed the generation of plasmablasts and virus receptor-binding domain (RBD)-specific memory B (Bmem) cells in patients during the acute phase of COVID-19. METHODS: The frequencies of RBD-binding plasmablasts and RBD-specific antibody-secreting cells (ASCs) in the peripheral blood samples collected from patients with COVID-19 were measured using flow cytometry and the ELISpot assay. RESULTS: The acute phase of COVID-19 was characterised by the transient appearance of total as well as RBD-binding plasmablasts. ELISpot analysis indicated that most patients exhibited a spontaneous secretion of RBD-specific ASCs in the circulation with good correlation between the IgG and IgM subsets. IL-21/CD40L stimulation of purified B cells induced the activation and proliferation of Bmem cells, which led to the generation of plasmablast phenotypic cells as well as RBD-specific ASCs. No correlation was observed between the frequency of Bmem cell-derived and spontaneous ASCs, suggesting that the two types of ASCs were weakly associated with each other. CONCLUSION: Our findings reveal that SARS-CoV-2-specific Bmem cells are generated during the acute phase of COVID-19. These findings can serve as a basis for further studies on the longevity of SARS-CoV-2-specific B-cell memory.

Identification of a novel splice variant for mouse and human interleukin-5.

Expression of interleukins and their receptors is often regulated by alternative splicing. Alternative isoform of IL-5 receptor α-chain is well studied; however, no data on functional alternative splice variants of IL-5 has been reported up today. In the present study, we describe a novel splice variant for the mouse and human IL-5. The new form was found during analysis of PCR-products amplified from different mouse lymphoid tissues with a pair of primers designed to clone full-length mIL-5 ORF. A single short isoform of mIL-5 was detected along with the canonical full-length mRNA in ConA-stimulated lymphoid cells isolated from spleen, thymus, lymph nodes and blood. It was 30-40 nt shorter, and less abundant than classical form. The sequence analysis of an additional form of mIL-5 revealed that it lacks exon-2 (δ2). Using RT-PCR with the splice-specific primers we obtained an additional evidence for δ2 form expression. To verify whether mIL-5δ2 transcript is translated into protein, the coding sequences corresponding to full and δ2 forms of mIL-5 were cloned into an expression plasmid. After transfection into the human 293T cell line, we found that the short form of mIL-5 protein is expressed in cells and secreted into the supernatant, but at the reduced level than that detected for full isoform of mIL-5. Fluorescence microscopy examination revealed a partial translocation of mIL-5δ2 into cytoplasm, whereas mIL-5 resided mostly within endoplasmic reticulum. This can explain why the level of δ2 protein expression was reduced. Using a similar set of experimental approaches, we received the evidence that the human IL-5 mRNA has the δ2 splice form (hIL-5δ2) as well. It can be firmly detected by RT-PCR in PHA-activated mononuclear cells isolated from peripheral blood of healthy persons or patients with asthma. Altogether, our results showed that the human and mouse IL-5 have an alternative mRNA splice isoform, which loses exon-2, but nevertheless is expressed at protein level. However, more comprehensive studies will be required for evaluation of IL-5δ2 expression, regulation, biological function and clinical significance.

NHEJ pathway is involved in post-integrational DNA repair due to Ku70 binding to HIV-1 integrase.

BACKGROUND: HIV-1 integration results in genomic DNA gaps that are repaired by cellular DNA repair pathways. This step of the lentiviral life cycle remains poorly understood despite its crucial importance for successful replication. We and others reported that Ku70 protein of the non-homologous end joining pathway (NHEJ) directly binds HIV-1 integrase (IN). Here, we studied the importance of this interaction for post-integrational gap repair and the recruitment of NHEJ factors in this process. RESULTS: We engineered HIV-based pseudovirus with mutant IN defective in Ku70 binding and generated heterozygous Ku70, Ku80 and DNA-PKcs human knockout (KO) cells using CRISPR/Cas9. KO of either of these proteins or inhibition of DNA-PKcs catalytic activity substantially decreased the infectivity of HIV-1 with native IN but not with the mutant one. We used a recently developed qPCR assay for the measurement of gap repair efficiency to show that HIV-1 with mutant IN was defective in DNA post-integrational repair, whereas the wild type virus displayed such a defect only when NHEJ system was disrupted in any way. This effect was present in CRISPR/Cas9 modified 293T cells, in Jurkat and CEM lymphoid lines and in primary human PBMCs. CONCLUSIONS: Our data provide evidence that IN recruits DNA-PK to the site of HIV-1 post-integrational repair due to Ku70 binding-a novel finding that explains the involvement of DNA-PK despite the absence of free double stranded DNA breaks. In addition, our data clearly indicate the importance of interactions between HIV-1 IN and Ku70 in HIV-1 replication at the post-integrational repair step.

Distinct Requirements for HIV-1 Accessory Proteins during Cell Coculture and Cell-Free Infection.

The role of accessory proteins during cell-to-cell transmission of HIV-1 has not been explicitly defined. In part, this is related to difficulties in measuring virus replication in cell cocultures with high accuracy, as cells coexist at different stages of infection and separation of effector cells from target cells is complicated. In this study, we used replication-dependent reporter vectors to determine requirements for Vif, Vpu, Vpr, or Nef during one cycle of HIV-1 cell coculture and cell-free infection in lymphoid and nonlymphoid cells. Comparative analysis of HIV-1 replication in two cell systems showed that, irrespective of transmission way, accessory proteins were generally less required for virus replication in 293T/CD4/X4 cells than in Jurkat-to-Raji/CD4 cell cocultures. This is consistent with a well-established fact that lymphoid cells express a broad spectrum of restriction factors, while nonlymphoid cells are rather limited in this regard. Remarkably, Vpu deletion reduced the level of cell-free infection, but enhanced the level of cell coculture infection and increased the fraction of multiply infected cells. Nef deficiency did not influence or moderately reduced HIV-1 infection in nonlymphoid and lymphoid cell cocultures, respectively, but strongly affected cell-free infection. Knockout of BST2-a Vpu antagonizing restriction factor-in Jurkat producer cells abolished the enhanced replication of HIV-1 ΔVpu in cell coculture and prevented the formation of viral clusters on cell surface. Thus, BST2-tethered viral particles mediated cell coculture infection more efficiently and at a higher level of multiplicity than diffusely distributed virions. In conclusion, our results demonstrate that the mode of transmission may determine the degree of accessory protein requirements during HIV-1 infection.

Isolation of gene-edited cells via knock-in of short glycophosphatidylinositol-anchored epitope tags.

We describe Surface Oligopeptide knock-in for Rapid Target Selection (SORTS), a novel method to select mammalian cells with precise genome modifications that does not rely on cell cloning. SORTS is designed to disrupt the target gene with an expression cassette encoding an epitope tag embedded into human glycophosphatidylinositol (GPI)-anchored protein CD52. The cassette is very short, usually less than 250 nucleotides, which simplifies donor DNA construction and facilitates transgene integration into the target locus. The chimeric protein is then expressed from the target promoter, processed and exposed on the plasma membrane where it serves as a marker for FACS sorting with tag-specific antibodies. Simultaneous use of two different epitope tags enables rapid isolation of cells with biallelic knock-ins. SORTS can be easily and reliably applied to a number of genome-editing problems such as knocking out genes encoding intracellular or secreted proteins, protein tagging and inactivation of HIV-1 provirus.

Gene Editing in Human Lymphoid Cells: Role for Donor DNA, Type of Genomic Nuclease and Cell Selection Method.

Programmable endonucleases introduce DNA breaks at specific sites, which are repaired by non-homologous end joining (NHEJ) or homology recombination (HDR). Genome editing in human lymphoid cells is challenging as these difficult-to-transfect cells may also inefficiently repair DNA by HDR. Here, we estimated efficiencies and dynamics of knockout (KO) and knockin (KI) generation in human T and B cell lines depending on repair template, target loci and types of genomic endonucleases. Using zinc finger nuclease (ZFN), we have engineered Jurkat and CEM cells with the 8.2 kb human immunodeficiency virus type 1 (HIV-1) ∆Env genome integrated at the adeno-associated virus integration site 1 (AAVS1) locus that stably produce virus particles and mediate infection upon transfection with helper vectors. Knockouts generated by ZFN or clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) double nicking techniques were comparably efficient in lymphoid cells. However, unlike polyclonal sorted cells, gene-edited cells selected by cloning exerted tremendous deviations in functionality as estimated by replication of HIV-1 and human T cell leukemia virus type 1 (HTLV-1) in these cells. Notably, the recently reported high-fidelity eCas9 1.1 when combined to the nickase mutation displayed gene-dependent decrease in on-target activity. Thus, the balance between off-target effects and on-target efficiency of nucleases, as well as choice of the optimal method of edited cell selection should be taken into account for proper gene function validation in lymphoid cells.

p63 and p73 repress CXCR5 chemokine receptor gene expression in p53-deficient MCF-7 breast cancer cells during genotoxic stress.

Many types of chemotherapeutic agents induce of DNA-damage that is accompanied by activation of p53 tumor suppressor, a key regulator of tumor development and progression. In our previous study we demonstrated that p53 could repress CXCR5 chemokine receptor gene in MCF-7 breast cancer cells via attenuation of NFkB activity. In this work we aimed to determine individual roles of p53 family members in the regulation of CXCR5 gene expression under genotoxic stress. DNA-alkylating agent methyl methanesulfonate caused a reduction in CXCR5 expression not only in parental MCF-7 cells but also in MCF-7-p53off cells with CRISPR/Cas9-mediated inactivation of the p53 gene. Since p53 knockout was associated with elevated expression of its p63 and p73 homologues, we knocked out p63 using CRISPR/Cas9 system and knocked down p73 using specific siRNA. The CXCR5 promoter activity, CXCR5 expression and CXCL13-directed migration in MCF-7 cells with inactivation of all three p53 family genes were completely insensitive to genotoxic stress, while pairwise p53+p63 or p53+p73 inactivation resulted in partial effects. Using deletion analysis and site-directed mutagenesis, we demonstrated that effects of NFkB on the CXCR5 promoter inversely correlated with p63 and p73 levels. Thus, all three p53 family members mediate the effects of genotoxic stress on the CXCR5 promoter using the same mechanism associated with attenuation of NFkB activity. Understanding of this mechanism could facilitate prognosis of tumor responses to chemotherapy.

Constitutive and activation-dependent phosphorylation of lymphocyte phosphatase-associated phosphoprotein (LPAP).

Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a small transmembrane protein expressed exclusively in lymphocytes. LPAP is a component of a supramolecular complex composed of the phosphatase CD45, the co-receptor CD4, and the kinase Lck. In contrast to its immunologically important partners, the function of LPAP is unknown. We hypothesized that the biological role of LPAP may be determined by analyzing LPAP phosphorylation. In the present study, we identified LPAP phosphorylation sites by site-directed mutagenesis, phospho-specific antibodies, and protein immunoprecipitation coupled to mass spectrometry analysis. Our results confirmed previous reports that Ser-99, Ser-153, and Ser-163 are phosphorylated, as well as provided evidence for the phosphorylation of Ser-172. Using various SDS-PAGE techniques, we detected and quantified a set of LPAP phosphoforms that were assigned to a combination of particular phosphorylation events. The phosphorylation of LPAP appears to be a tightly regulated process. Our results support the model: following phorbol 12-myristate 13-acetate (PMA) or TCR/CD3 activation of T cells, LPAP is rapidly dephosphorylated at Ser-99 and Ser-172 and slowly phosphorylated at Ser-163. Ser-153 exhibited a high basal level of phosphorylation in both resting and activated cells. Therefore, we suggest that LPAP may function as a co-regulator of T-cell signaling.

Synthetic peptide TEKKRRETVEREKE derived from ezrin induces differentiation of NIH/3T3 fibroblasts.

Synthetic 14 AA peptide (Gepon) derived from the hinge region of ezrin, a protein that links cell surface molecules to intracellular actin filaments, accelerates and facilitates wound and ulcer healing in clinical applications. However, the molecular mechanisms underlying this phenomenon and involved in enhanced healing of wounds with Gepon are not yet understood. The purpose of current study was to investigate intracellular signaling pathways involved in the effect of this peptide on wild type and genetically modified (CD44 KO) NIH/3T3 embryonic mouse fibroblasts. Gepon treatment of NIH/3T3 cells resulted in morphological and biochemical changes, characteristic of differentiated fibroblasts. While treatment of NIH/3T3 cells with TGF-ß1 triggered the activation of both canonical and non-canonical signaling pathways, exposure of fibroblasts to Gepon activated only the ERK1/2 dependent pathway without modulating SMAD dependent signaling pathway. Knocking out hyaluronic acid CD44 receptor did not change Gepon or TGF-ß1 dependent activation of intracellular signaling pathways and assembling of α-SMA-positive filaments. Gepon dependent differentiation of NIH/3T3 fibroblasts is based on activation of ERK1/2 kinase, non-canonical intracellular signaling pathway. Our data suggest that the treatment of fibroblasts with Gepon triggers activation of the non-canonical (SMAD independent) intracellular signaling pathway that involves ERK1/2kinase phosphorylation. Activation of the MAPK signaling pathway and the increase in formation of α-SMA containing stress filaments induced by Gepon were independent on presence of CD44 receptor in NIH/3T3 fibroblasts. Thus, our observation designates the significance and sufficiency of MAPK pathway mediated activation of fibroblasts with Gepon for healing of erosion, ulcers and wounds.

Determining antigen specificity of a monoclonal antibody using genome-scale CRISPR-Cas9 knockout library.

An essential step in monoclonal antibody (mAb) development is the characterization and final identification of the specific target antigen and its epitope. Antibody validation is rather straightforward when immunization is carried out with peptide or purified protein, but is more difficult when whole cells or other complex antigens are used for the immunization. Determining antigen specificity of a mAb is further complicated, when reactivity of an antibody is not detected in Western blotting and/or immunoprecipitation assay. In addition to protein-based methods used for antibody characterization, a number of gene-based techniques, such as cDNA expression or short-interfering RNA (siRNA) knockdown have been applied for validation of antibodies with restricted reactivities. Earlier we have generated, characterized, but not identified the BF4 mAb that specifically stains viral biofilms on the surface of the Human T-lymphotropic Virus Type I (HTLV-1) infected T cells. In this study, using the recently developed genome-scale CRISPR-Cas9 knockout (GeCKO) library vectors, we have established the CEM T- and the Raji B cell lines with pooled libraries. After immunofluorescent staining of these cells, negative cell sorting, and guide-RNA (gRNA) sequencing, we have identified BF4 as an anti-CD82 mAb. A deep sequence analysis of GeCKO library transferred to the cells shows that the chance to succeed in the selection of antibody-negative cells and, therefore, to identify a mAb depends on the quality of cell library preparation. We believe that the described method is applicable for identification of many other hybridomas and represents a good alternative to the current protein- and gene-based methods used for mAb validation.

The Dual NOD1/NOD2 Agonism of Muropeptides Containing a Meso-Diaminopimelic Acid Residue.

Muropeptides are fragments of peptidoglycan that trigger innate immune responses by activating nucleotide-binding oligomerization domain (NOD) 1 and NOD2. Muropeptides from Gram-negative bacteria contain a meso-diaminopimelic acid (meso-DAP) residue in either a terminal or a non-terminal position. While the former ones are known to be recognized by NOD1, much less is known about recognition of muropeptides with non-terminal meso-DAP, which are most abundant moieties of Gram-negative peptidoglycans. Here, we developed a novel system to assess biological activity of muropeptides, based on CRISPR/Cas9-mediated knockout (KO) of NOD1 and NOD2 genes in modified HEK293T cells. Using NOD1/NOD2 knockout and overexpression systems, as well as human monocytes and macrophages, we refine the current view of muropeptide recognition. We show that NOD2 can recognize different natural muropeptides containing a meso-DAP residue (preferably in a non-terminal position), provided they are present at micromolar concentrations. NOD2 accepts muropeptides with long and branched peptide chains and requires an intact N-acetylmuramyl residue. Muropeptides with non-terminal meso-DAP can activate NOD1 as well, but, in this case, probably require peptidase pre-processing to expose the meso-DAP residue. Depending on NOD1/NOD2 ratio in specific cell types, meso-DAP-containing muropeptides can be recognized either primarily via NOD2 (in monocytes) or via NOD1 (in monocyte-derived macrophages and HEK293T-derived cells). The dual NOD1/NOD2 agonism of meso-DAP-containing muropeptides should be taken into account when assessing cellular responses to muropeptides and designing muropeptide immunostimulants and vaccine adjuvants.

Lymphocyte phosphatase-associated phosphoprotein proteoforms analyzed using monoclonal antibodies.

Phosphatase CD45 regulates the activation of lymphocytes by controlling the level of receptor and signal molecule phosphorylation. However, it remains unknown which molecules mediate the phosphatase activity of CD45. A candidate for such a molecule is a small transmembrane adapter protein called lymphocyte phosphatase-associated phosphoprotein (LPAP). LPAP forms a supramolecular complex that consists of not only CD45 molecule but also CD4 and Lck kinase. The function of LPAP has not been defined clearly. In our study, we determined the pattern of LPAP expression in various cell types and characterized its proteoforms using new monoclonal antibodies generated against the intracellular portion of the protein. We show that LPAP is a pan-lymphocyte marker, and its expression in cells correlates with the expression of CD45. The majority of T, B and NK cells express high levels of LPAP, whereas monocytes, granulocytes, monocyte-derived dendritic cells, platelets and red blood cells are negative for LPAP. Using one- and two-dimensional protein gel electrophoresis, we demonstrate that LPAP has at least four sites of phosphorylation. The resting cells express at least six different LPAP phosphoforms representing mono-, di- and tri-phosphorylated LPAP. T and B cells differ in the distribution of the protein between phosphoforms. The activation of lymphocytes with PMA reduces the diversity of phosphorylated forms. Our experiments on Lck-deficient Jurkat cells show that Lck kinase is not involved in LPAP phosphorylation. Thus, LPAP is a dynamically phosphorylated protein, the function of which can be understood, when all phosphosites and kinases involved in its phosphorylation will be identified.