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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-1 AD8 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-1 AD8 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 HIV AD8 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-1 AD8 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., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: The authors declare no conflict of interest.

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.

Monitoring of the level of the virus-neutralizing activity of serum immunoglobulins ensures that one can reliably assess the effectiveness of any protection against the SARS-CoV-2 infection. For SARS-CoV-2, the RBD-ACE2 neutralizing activity of sera is almost equivalent to the virus-neutralizing activity of their antibodies and can be used to assess the level of SARS-CoV-2 neutralizing antibodies. We are proposing an ELISA platform for performing a quantitative analysis of SARS-CoV-2 RBD-neutralizing antibodies, as an alternative to the monitoring of the virus-neutralizing activity using pseudovirus or "live" virus assays. The advantage of the developed platform is that it can be adapted to newly emerging virus variants in a very short time (1-2 weeks) and, thereby, provide quantitative data on the activity of SARS-CoV-2 RBD-neutralizing antibodies. The developed platform can be used to (1) study herd immunity to SARS-CoV-2, (2) monitor the effectiveness of the vaccination drive (revaccination) in a population, and (3) select potential donors of immune plasma. The protective properties of the humoral immune response in hospitalized patients and outpatients, as well as after prophylaxis with the two most popular SARS-CoV-2 vaccines in Russia, were studied in detail using this platform. The highest RBD-neutralizing activity was observed in the group of hospitalized patients. The protective effect in the group of individuals vaccinated with Gam-COVID-Vac vaccine was 25% higher than that in outpatients and almost four times higher than that in individuals vaccinated with the CoviVac vaccine., (Copyright ® 2022 National Research University Higher School of Economics.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Maslennikova and Mazurov.)

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.

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.

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.

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., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

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., (© 2020 The Author(s).)

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.

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.

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.

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest.

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-β1 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-β1 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

Recombination can assort polymorphic alleles to increase diversity in the HIV-1 population. To better understand the recombination potential of subtype A HIV-1, we generated viruses containing sequences from two variants circulating in Russia and analyzed the polymerase gene (pol) of the recombinants after one round of HIV-1 replication using single-genome sequencing. We observed that recombination occurred throughout pol and could easily assort alleles containing mutations that conferred resistance to currently approved antivirals. We measured the recombination rate in various regions of pol including a G-rich region that has been previously proposed to be a recombination hot spot. Our study does not support a recombination hot spot in this G-rich region. Importantly, of the 58 proviral sequences containing crossover event(s) in pol, we found that each sequence was a unique genotype indicating that recombination is a powerful genetic mechanism in assorting the genomes of subtype A HIV-1 variants., (Published by Elsevier Inc.)

Unlabelled: Cell-to-cell transmission is an efficient mechanism to disseminate human immunodeficiency virus type 1 (HIV-1) and human T cell lymphotropic virus type 1 (HTLV-1). However, it has been challenging to quantify the level of cell-to-cell transmission because the virus-producing cells cannot be easily distinguished from infected target cells. We have previously described replication-dependent vectors that can quantify infection events in cocultured cells. These vectors contain an antisense-oriented promoter and reporter gene interrupted by a sense-oriented intron from the human gamma-globin gene. This strategy prevents expression of the reporter gene in the transfected cells but permits its expression in target cells after infection. However, the gamma-globin intron is not efficiently removed by splicing in the aforementioned vectors, thereby reducing the level of reporter gene expression after transduction into target cells. Here, we used two approaches to improve the replication-dependent vectors. First, we improved the splicing events that remove the gamma-globin intron by optimizing the intron insertion site within the reporter gene. Second, we improved the packaging of the spliced RNA without the gamma-globin intron by targeting the intron-containing RNA via microRNA 30 (miR30)-based short hairpin RNAs. Using two optimized fluorescent reporter vectors and flow cytometry, we determined that multiply HIV-1-infected cells were generated at a higher frequency in coculture than in cell-free infection; furthermore, this increase was dependent upon viruses bearing HIV-1 Env. Compared with previously described vectors, these improved vectors can quantify the infection in lymphocytes and in primary cells with a higher sensitivity and allow the detection and quantitation of multiply infected cells, providing better tools to study retroviral cell-mediated infection., Importance: The human-pathogenic retroviruses HTLV-1 and HIV-1 can be transmitted more efficiently in vivo via direct contact of infected cells with healthy target cells than through cell-free virion-mediated infection. Despite its importance, cell-to-cell transmission has been difficult to quantify because the previously infected cells and the newly infected cells are mixed together in the same culture. In the current study, we generated vectors that are significantly improved over the previously described replication-dependent vectors. As a result, these improved vectors can efficiently detect and quantify cell-to-cell transmission or new infection events in cells in mixed culture. These luciferase- or fluorescence protein-based reporter vectors can be used to quantify and study HIV-1 or HTLV-1 cell-mediated infection in a simple one-step transfection/infection assay., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Tetraspanins interact with a wide variety of transmembrane and intracellular proteins called molecular partners, and modulate their function. In this article, we describe a new partner of tetraspanin web, membrane metalloprotease CD10, which is selectively associated with CD9. By constructing chimeras between tetraspanins CD9 and CD82 (the latter does not interact with CD10) or by using site-directed mutagenesis, we determined that a portion of the large extracellular loop from the CCG motif to transmembrane domain 4, as well as the C-terminal tail of CD9, are involved in the interaction with CD10. The stable expression of wild-type CD9 in K562 CD10-positive cells enhanced the level of CD10 released with exosomes five-fold. In contrast, the expression of chimeric CD9, which contained the cytoplasmic C-terminal domain from CD82, had little effect on CD10 release. Short hairpin RNA knockdown of CD9 expression in Nalm-6 pre-B cells resulted in a two-fold reduction in the amount of endogenous CD10 released with microvesicles. The peptidase activity of CD10 measured either on cells or on exosomes correlated with the level of CD10 expression, and was not significantly modulated by CD9 expression as such. Our data suggest that the interaction of CD10 with tetraspanin CD9 can play an important role in the redistribution of peptidase activity from the cell surface to outer microenvironments. In bone marrow, where CD10 presumably contributes to the maturation of pre-B cells and migration of B cells to the blood circulation, release of CD10 peptidase activity with exosomes may effectively regulate extracellular matrix microenvironments., (© 2013 The Authors Journal compilation © 2013 FEBS.)

We used replication-dependent retroviral vectors to identify cell surface antigens involved in the cell-to-cell transmission of human T cell leukemia virus type 1 (HTLV-1). We generated monoclonal antibodies (MAbs) against Jurkat T cells and selected several IgM MAbs that strongly inhibited HTLV-1 but not human immune deficiency virus type 1 (HIV-1) cell-to-cell infection. These MAbs recognized the so-called Tn antigen (GalNAcα1-O-Ser/Thr) that arises on Jurkat cells from a mutation in the T-synthase-specific chaperone Cosmc and the consequent loss of O-glycan elongation. Anti-Tn MAbs precipitated two major O-glycan carrier proteins, CD43 and CD45, and caused a strong aggregation of Jurkat cells. The restoration of O-glycosylation in Jurkat cells by stably transducing the wild-type Cosmc gene resulted in a 3- to 4-fold increase in the level of surface expression of CD43 and enhanced HTLV-1 transmission 10-fold in comparison to that of parental cells. The short hairpin RNA (shRNA) knockdown of CD43 or CD45 expression in Jurkat-Cosmc, HBP-ALL, and CEM T cells decreased HTLV-1 infection severalfold. The knockdown of CD45 in Jurkat cells severely reduced both HTLV-1 and HIV-1 infections, but Cosmc coexpression partially rescued infection. HTLV-1 proteins, which assembled in small patches on Jurkat cells, formed large clusters on the surface of Jurkat-Cosmc cells. These data indicate that large aggregates of HTLV-1 assemblies are more infectious than multiple clustered virions. We suggest that heavily O-glycosylated CD43 and CD45 molecules render cells less adhesive, prevent inappropriate cell-cell contacts, and favor the assembly of HTLV-1 particles into large, highly infectious structures on the surface of T cells.

Interfering RNA (RNAi) is a powerful tool to silence gene expression on the level of mRNA. To knock-down gene expression by using RNAi two major methods of mRNA silencing exist. First method utilizes siRNA (small interfering RNA), a readily processed dsRNA, that enters RISC complex and destroy target mRNA after transfection into the cells. The second method based on the construction of plasmid DNA that expresses shRNA (short harpin RNA) from U6 or CMV promoter. shRNA gets processed by Drosha and Dicer RNAses inside the cell before it translocates to the cell cytoplasm and affects the level of target RNA. In this study we modified lentiviral vector pGIPZ expressing tFP-IRES-Puro-shRNA(mir30) cassette by introducing BamH I restriction site downstream of this cassette. This modification makes possible to clone specific shRNA sequences in pGIPZ vector using XhoI/BamHI restriction sites instead of the original recombination. Three shRNAs against phosphoprotein P of respiratory sinthitial virus (RSV) and shRNA against human CD43 as a control were generated and cloned into modified so-called pCIPD vector. Monkey kidney cells MA-104 were stably transduced with four shRNA constructs. In conclusion, the generated lentiviral vector pCIPD can be successfully used for efficient gene silencing and virus replication in a broad variety of cells.

We have developed an efficient method to quantify cell-to-cell infection with single-cycle, replication dependent reporter vectors. This system was used to examine the mechanisms of infection with HTLV-1 and HIV-1 vectors in lymphocyte cell lines. Effector cells transfected with reporter vector, packaging vector, and Env expression plasmid produced virus-like particles that transduced reporter gene activity into cocultured target cells with zero background. Reporter gene expression was detected exclusively in target cells and required an Env-expression plasmid and a viral packaging vector, which provided essential structural and enzymatic proteins for virus replication. Cell-cell fusion did not contribute to infection, as reporter protein was rarely detected in syncytia. Coculture of transfected Jurkat T cells and target Raji/CD4 B cells enhanced HIV-1 infection two fold and HTLV-1 infection ten thousand fold in comparison with cell-free infection of Raji/CD4 cells. Agents that interfere with actin and tubulin polymerization strongly inhibited HTLV-1 and modestly decreased HIV-1 cell-to-cell infection, an indication that cytoskeletal remodeling was more important for HTLV-1 transmission. Time course studies showed that HTLV-1 transmission occurred very rapidly after cell mixing, whereas slower kinetics of HIV-1 coculture infection implies a different mechanism of infectious transmission. HTLV-1 Tax was demonstrated to play an important role in altering cell-cell interactions that enhance virus infection and replication. Interestingly, superantigen-induced synapses between Jurkat cells and Raji/CD4 cells did not enhance infection for either HTLV-1 or HIV-1. In general, the dependence on cell-to-cell infection was determined by the virus, the effector and target cell types, and by the nature of the cell-cell interaction.

The tetraspanin superfamily proteins play important roles in organizing membrane protein complexes, modulating integrin function, and controlling T cell adhesion. Tetraspanins such as CD82 contain two extracellular loops with its N terminus, C terminus, and inner loop exposed to the cytoplasm. The matrix (MA) domain of human T cell lymphotrophic virus, type 1 (HTLV-1), Gag interacts with the cytoplasmic face of the plasma membrane and is concentrated at tetraspanin-enriched microdomains. To understand the basis of this association, we generated site-directed mutations in the various domains of CD82 and used coimmunoprecipitation and colocalization approaches to examine interactions with HTLV-1 MA. The large extracellular loop of CD82, which is important for interactions with integrins, was not required for the association with HTLV-1 MA. The cytoplasmic N terminus and C terminus of CD82 were also dispensable for CD82-MA interactions. In contrast, mutations of conserved amino acids in the inner loop of CD82 or of palmitoylated cysteines that flank the inner loop diminished CD82 association with MA. HTLV-1 MA also interacted with the inner loop of CD81. Thus, association of HTLV-1 Gag with tetraspanin-enriched microdomains is mediated by the inner loops of CD81 and CD82.

We examined the association of HTLV-1 Gag with tetraspanin-enriched microdomains in the plasma membrane. Immunofluorescent staining and confocal image analysis showed that HTLV-1 Gag protein colocalized with CD82 and other tetraspanins at the plasma membrane of T cells. HTLV-1 Gag, which is associated with the inner surface of the plasma membrane, was concentrated to the patches formed by antibody-mediated cross-linking of CD82 on the cell surface. Also, CD82 and HTLV-1 Gag rapidly segregated to the immune synapse that is formed between Raji B cells and Jurkat T cells in the presence of bacterial superantigen. CD82, which was immunoprecipitated from cell extracts prepared in Brij97 detergent conditions, was associated with the matrix (MA) protein. Stable interaction of MA and CD82 in Brij97-disrupted cell extracts required Gag multimerization and proteolytic processing. The form of MA that coimmunoprecipitated with CD82 was a cysteine-linked homodimer. The viral envelope glycoprotein was not required for the association of Gag with CD82-enriched membrane regions. In contrast to HTLV-1, HIV-1 Gag did not colocalize, cosegregate, or coimmunoprecipitate with CD82. Our data suggest that once at the plasma membrane, HTLV-1 virion components associate with CD82-containing microdomains, which may facilitate the mobilization of nascent virions to sites of intercellular adhesion.

The direct action of synthetic peptide preparations, analogous to thymic hormones, on the functions of phagocytic cells was studied. The preparations Thymogen, Neogen and Thymodepressin in a dose of 10 mM produced a stimulating effect on the ingestive activity of the neutrophil, but not monocytic, population. All three preparations also enhanced the formation of oxygen metabolites registered in the luminol-dependent chemiluminescent analysis. The characteristics of spontaneous chemiluminescence (CL) reflecting the basal level of the synthesis of the active forms of oxygen and CL induced by opsomized zymosan significantly increased also in those cases when the preparations were used in a dose of 10 mM. The level of the synthesis of hydrogen peroxide in individual cells could be appraised by the intensity of the luminescence of dichlorofluorescein diacetate (DCF-DA), evaluated with the use of flow cytometry. All preparations produced a stimulating effect on the formation of hydrogen peroxide in monocytes. The reaction of neutrophils was even more active: Neogen (10 mM) produced the twofold change in the intensity of the luminescence of DCF-DA) in neutrophils, Thymogen and Thynodepressin increased the average intensity of the luminescence of DCF-DA by 80% and 60%, respectively.