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.

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 leading to viral clearance, and HIV persists in the organism as a latent provirus.

Current antiretroviral therapy efficiently suppresses viral replication but cannot eliminate latent HIV reservoirs. Moreover, the associated high costs, side effects, and drug resistance have stimulated a need for the development of alternative methods of HIV-1/AIDS treatment, such as peptide inhibitors or gene editing. Recently, we have developed Surface Oligopeptide knock-in for Rapid Target Selection (SORTS), a method for the rapid selection of CRISPR/Cas9 gene-edited cells via knock-in of the Flag and HA epitope tags embedded into the shortest GPI-protein, CD52. By targeting the capsid region of the HIV-1 genome, we demonstrate that SORTS can be applied in provirus eradication. However, the cells with inactivated provirus will be susceptible to HIV re-infection. We hypothesized that knocking in one of the peptides from the CHR-domain of gp41, which are known potent inhibitors of HIV-1 fusion, instead of the epitope tag, will provide “post-curable” HIV-1 resistance. While these peptides were extensively studied as soluble substances, their inhibitory effects on HIV after expression on cell surfaces via GPI-anchor are largely unknown. In this study, we established HEK293T/CD4/R5 and Raji/CD4/R5 HIV-1 permissive cell lines that stably expressed one of the gp41 peptides C34, MT-C34, MT-C34-R, and MT34-15D, or alfa-helix mimetics HP23L, p52, and MT-WQ-IDL. For cell surface delivery, the indicated peptides were embedded into the CD52 molecule, and upstream GFP was used to select transformed cells. Using a single-cycle replication assay with the inLuc reporter vector and different Envs, we demonstrated that C34-based GPI-anchored peptides inhibited both cell-free and cell-to-cell HIV-1 infection by at least two orders of magnitude. With the exception of HP23L, the alfa-helix mimetics were less potent inhibitors. Thus, peptides from gp41 associated with lipid rafts and exerted a strong inhibitory activity which can far exceed that determined for soluble peptides, but this should be tested further.

Purpose to study the tumor-forming activity of wild-type MC F-7 cells carrying a full set of porins (VDAC 1, VDAC 2, VDAC 3), as well as their genetically modified cells, from which one of the isoforms was removed (MC F-7 VDAC 1 KO, MC F-7 VDAC 2 KO, MC F -7 VDAC 3 KO).Material and Methods. The study was aimed at establishing of an animal model of orthotopic tumors in the mammary gland of immunodeficient BAL B/c nude mice by implanting a suspension of human breast cancer cells (MC F-7) and derivatives of these cells generated by targeted knockout of one of the selected mitochondrial porin isoforms (VDAC 1, VDAC 2 or VDAC 3). Suspensions of either wild-type MC F-7 cell lines containing all three porin isoforms (VDAC 1, VDAC 2 and VDAC 3) or their VDAC -deficient derivatives (MC F-7 VDAC 1 KO, MC F-7 VDAC 2 KO and MC F-7 VDAC 3 KO) were injected into mammary fat pads of BAL B/c nude mice at a dose of 4x106 cells per injection. A pathomorphological analysis of the place of implantation of tumor cells, the tumor itself, as well as the organs of the abdominal and thoracic cavity was carried out.Results. The study shows the feasibility of successful creation of orthotopic tumors in the adipose tissue of immunodeficient BAL B/c nude mice with MC F-7 human breast cancer epithelial cells containing a complete set of mitochondrial porin isoforms and their VDAC -deficient derivatives. The tumor-forming activity of the implanted cells was shown to correlate with their cytotoxic effect on the internal organs of animals. Pathological analysis showed that all implanted cell cultures, such as MC F-7 WT, MC F-7 VDAC 2 KO and MC F-7 VDAC 3 KO, except for MC F-7 VDAC 1 KO cells, which did not form tumors, caused pathological changes in the lungs, liver and spleen, as well as the presence of other tumor-like lesions.Conclusion. The data obtained will be used to optimize the injection volume and cell number, as well as to refine the dynamics of tumor growth, suitable for studying the effect of anticancer drugs on tumors formed by human breast cancer cells (MC F-7) and its genetically modified VDAC -deficient derivatives.

We have recently developed surface oligopeptide knock-in for rapid target selection (SORTS), a novel method to isolate mammalian cells with gene modifications using FACS-sorting. It relies on CRISPR/Cas9-mediated targeted knock-in of a very short promoterless expression construct (250 bp) comprising a Flag or HA epitope embedded into the smallest GPI-protein CD52, and a polyA signal from the beta-globin. CD52 efficiently delivers the epitope to the cell surface, where it serves as a marker for selection, while polyA terminates transcription and silences target gene expression. Primarily, SORTS was developed to knock out genes encoding intracellular or secreted proteins, which cannot be used as markers for selection of live cells. Using in-frame modification of SORTS, we demonstrated the possibility of HIV-1 provirus inactivation via sorting of GPI-tag positive cells. In order to make the “cured” cells resistant to a subsequent HIV-1 infection, the epitope tag in the CD52 molecule was substituted by one of the fusion inhibitor peptides from the CHR-domain of gp41. We selected a series of cell-surface-expressed, GPI-anchored, C34-based peptides that confer a strong cellular resistance to HIV-1 infection mediated by NL4-3, JRFL, or ZM153 Env. These findings together with a monoclonal antibody raised against the C34 peptide provide an opportunity to generate and select HIV-resistant lymphocytes for a therapeutic goal. SORTS was also adapted to engineer transgenic HIV-1 effector Т cells and to study cell-to-cell transmission. To facilitate transgenesis, we developed a knock-in strategy to express GPI-tag from the intronic region of the human PPP1R12C gene (AAVS1 locus) and delivered FRT sites of recombination into both alleles. In summary, SORTS is a novel instrument to isolate rare cells with precise genomic modifications with broad applications, including HIV biology. This work was supported by the Russian Science Foundation (grant 18-14-00333) and the Russian Foundation for Basic Research (grants 18-29-07052, 18-04-01016).

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 cloned a series of HIV-1 fusion inhibitory peptides and embedded them in CD52, the shortest 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. Unlike membrane-bound peptides, the shed variant of MT-C34 provided insufficient protection against HIV-1 due to its low concentrations in the culture medium. Using Cas9 plasmids or ribonucleoprotein electroporation and cell sorting with antibodies raised against gp41 peptides, we generated CEM/R5 cells with biallelic KI of MT-C34 (embedded in CD52 for expression in lipid rafts) and 2P23 (N-terminally fused to CXCR4). In combination, these peptides provided a higher level of protection than individual KI. By extending homology arms and substituting PCR donor DNA with a plasmid containing signals for nuclear localization, we achieved KI of MT-C34 into CXCR4 loci and HIV-1 proviral DNA at levels of up to 35% in CEM/R5 cells and increased KI occurrence from undetectable to 4-5% in CD4 lymphocytes. Comparative analysis of lentiviral and HDR-based delivery strategies showed that KI led to the higher MT-C34 expression and stronger protection of primary CD4 lymphocytes from HIV-1 than lentiviral transduction, albeit the efficiency of KI needs further improvements in order to meet clinical requirements. Thus, the developed CRISPR/Cas9 platform offers a new opportunity for antiviral peptide delivery with a concomitant precise genetic modification of targeted locus that can be employed to strengthen cell protection against HIV. AUTHOR SUMMARY: HIV is a human lentivirus that infects CD4-positive immune cells and, when left untreated, manifests in the fatal disease known as acquired immunodeficiency syndrome. Antiretroviral therapy (ART) is not leading 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.