Enzymatic activity of HIV-1 protease defines migration of tumor cells in vitro and enhances their metastatic activity in vivo.

Overexpression of aspartic proteases, as cathepsin D, is an independent marker of poor prognosis in breast cancer, correlated with the incidence of clinical metastasis. We aimed to find if HIV-1 aspartic protease (PR) can play a similar role. Murine adenocarcinoma 4T1luc2 cells were transduced with lentivirus encoding inactivated drug-resistant PR, generating subclones PR20.1 and PR20.2. Subclones were assessed for production of reactive oxygen species (ROS), expression of epithelial-mesenchymal transition (EMT) factors, and in vitro migratory activity in the presence or absence of antioxidant N-acetyl cysteine and protease inhibitors. Tumorigenic activity was evaluated by implanting cells into BALB/c mice and following tumor growth by calipering and bioluminescence imaging in vivo, and metastases, by organ imaging ex vivo. Both subclones expressed PR mRNA, and PR20.2, also the protein detected by Western blotting. PR did not induce production of ROS, and had no direct effect on cell migration rate, however, treatment with inhibitors of drug-resistant PR suppressed the migratory activity of both subclones. Furthermore, expression of N-cadherin and Vimentin in PR20.2 cells and their migration were enhanced by antioxidant treatment. Sensitivity of in vitro migration to protease inhibitors and to antioxidant, known to restore PR activity, related the effects to the enzymatic activity of PR. In vivo, PR20.2 cells demonstrated higher tumorigenic and metastatic activity than PR20.1 or parental cells. Thus, HIV-1 protease expressed in breast cancer cells determines their migration in vitro and metastatic activity in vivo. This effect may aggravate clinical course of cancers in people living with HIV-1.

Experimental Use of Common Marmosets (Callithrix jacchus) in Preclinical Trials of Antiviral Vaccines.

Common marmoset (Callithrix jacchus, CM) is a New World primate species that is of interest for preclinical trials of immunobiological products. In this study, we describe the approaches to long-term laboratory breeding and maintenance of CMs. We also establish the reference values of the main complete blood count and serum chemistry parameters evaluated during preclinical trials of immunobiological products and describe the histological characteristics of CM lymphoid organs during the development of post-vaccination immune response. We show that CMs bred in laboratory conditions excluding background infectious pathology are a relevant model that allows for a high degree of reliability in characterizing the safety and immunogenicity profile of antiviral vaccines during preclinical trials.

[Stochastic Packaging of Cas Proteins into Exosomes].

CRISPR/Cas systems are perspective molecular tools for targeted manipulation with genetic materials, such as gene editing, regulation of gene transcription, modification of epigenome etc. While CRISPR/Cas systems proved to be highly effective for correcting genetic disorders and treating infectious diseases and cancers in experimental settings, clinical translation of these results is hampered by the lack of efficient CRISPR/Cas delivery vehicles. Modern synthetic nanovehicles based on organic and inorganic polymers have many disadvantages, including toxicity issues, the lack of targeted delivery, and complex and expensive production pipelines. In turn, exosomes are secreted biological nanoparticles that exhibit high biocompatibility, physico-chemical stability, and the ability to cross biological barriers. Early clinical trials found no toxicity associated with exosome injections. In the recent years, exosomes have been considered as perspective delivery vehicles for CRISPR/Cas systems in vivo. The aim of this study was to analyze the efficacy of CRISPR/Cas stochastic packaging into exosomes for several human cell lines. Here, we show that Cas9 protein is effectively localized into the compartment of intracellular exosome biogenesis, but stochastic packaging of Cas9 into exosomes turns to be very low (~1%). As such, stochastic packaging of Cas9 protein is very ineffective and cannot be used for gene editing purposes. Developing novel tools and technologies for loading CRISPR/Cas systems into exosomes is needed.

Murine Models of Chronic Viral Infections and Associated Cancers.

Viruses are now recognized as bona fide etiologic factors of human cancer. Carcinogenic viruses include Epstein- Barr virus (EBV), high-risk human papillomaviruses (HPVs), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell leukemia virus type 1 (HTLV-1), human immunodeficiency virus type 1 (HIV-1, indirectly), and several candidate human cancer viruses. It is estimated that 15% of all human tumors worldwide are caused by viruses. Tumor viruses establish long-term persistent infections in humans, and cancer is an accidental side effect of viral replication strategies. Viruses are usually not complete carcinogens, supporting the concept that cancer results from the accumulation of multiple cooperating events, in which human cancer viruses display different, often opposing roles. The laboratory mouse Mus musculus is one of the best in vivo experimental systems for modeling human pathology, including viral infections and cancer. However, mice are unsusceptible to infection with the known carcinogenic viruses. Many murine models were developed to overcome this limitation and to address various aspects of virus-associated carcinogenesis, from tumors resulting from xenografts of human tissues and cells, including cancerous and virus infected, to genetically engineered mice susceptible to viral infections and associated cancer. The review considers the main existing models, analyzes their advantages and drawbacks, describes their applications, outlines the prospects of their further development.

[Murine Models of Chronic Viral Infections and Associated Cancers].

Viruses are now recognized as bona fide etiologic factors of human cancer. Carcinogenic viruses include Epstein-Barr virus (EBV), high-risk human papillomaviruses (HPVs), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell leukemia virus type 1 (HTLV-1), human immunodeficiency virus type 1 (HIV-1, indirectly), and several candidate human cancer viruses. It is estimated that 15% of all human tumors worldwide are caused by viruses. Tumor viruses establish long-term persistent infections in humans, and cancer is an accidental side effect of viral replication strategies. Viruses are usually not complete carcinogens, supporting the concept that cancer results from the accumulation of multiple cooperating events, in which human cancer viruses display different, often opposing roles. The laboratory mouse Mus musculus is one of the best in vivo experimental systems for modeling human pathology, including viral infections and cancer. However, mice are unsusceptible to infection with the known carcinogenic viruses. Many murine models were developed to overcome this limitation and to address various aspects of virus-associated carcinogenesis, from tumors resulting from xenografts of human tissues and cells, including cancerous and virus infected, to genetically engineered mice susceptible to viral infections and associated cancer. The review considers the main existing models, analyzes their advantages and drawbacks, describes their applications, outlines the prospects of their further development.

[Expression of HIV-1 Reverse Transcriptase in Murine Cancer Cells Increases Mitochondrial Respiration].

Changes in metabolic pathways are often associated with the development of a wide range of pathologies. Increased glycolysis under conditions of sufficient tissue oxygen supply and its dissociation from the Krebs cycle, known as aerobic glycolysis or the Warburg effect, is a hallmark of many malignant neoplasms. Identification of specific metabolic shifts can characterize the metabolic programming of individual types of tumor cells, the stage of their transformation, and predict their metastatic potential. Viral infection can also alter the metabolism of cells to support the process of viral replication. Infection with human immunodeficiency virus type 1 (HIV-1) is associated with an increased incidence of various cancers, and for some viral proteins a direct oncogenic effect was demonstrated. In particular, we showed that the expression of HIV-1 reverse transcriptase (RT) in 4T1 breast adenocarcinoma cells increases the tumorigenic and metastatic potential of cells in vitro and in vivo by a mechanism associated with the ability of RT to induce reactive oxygen species in cells (ROS). The aim of this work was to study the molecular mechanism of this process, namely the effect of HIV-1 RT on the key metabolic pathways associated with tumor progression: glycolysis and mitochondrial respiration. Expression of HIV-1 RT had no effect on the glycolysis process. At the same time, it led to an increase in mitochondrial respiration and the level of ATP synthesis in the cell, while not affecting the availability of the substrates, carbon donors for the Krebs cycle, which excludes the effect of RT on the metabolic enzymes of cells. Increased mitochondrial respiration was associated with restoration of the mitochondrial network despite the RT-induced reduction in mitochondrial mass. Increased mitochondrial respiration may increase cell motility, which explains their increased tumorigenicity and metastatic potential. These data are important for understanding the pathogenesis of HIV-1 infection, including the stimulation of the formation and spread of HIV-1 associated malignancies.