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.

[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.

[Antiviral Activity of CRISPR/Cas9 Ribonucleoprotein Complexes on a Hepatitis B Virus Model In Vivo].

Chronic hepatitis B (CHB) is caused by hepatitis B virus (HBV) infection. This disease is a key issue for global health. Modern methods of therapy do not completely eliminate HBV from infected cells and do not cure chronic infection. The CRISPR/Cas9 systems of site-specific nucleases can effectively cleave do not target DNA including viral genomes. The cleavage of the major form of the HBV genome, i.e., covalently closed circular DNA (cccDNA), leads to a robust reduction in viral replication and degradation or mutational inactivation of cccDNA. CRISPR/Cas9-based approaches are one of the most promising ways to achieve a 'sterilizing' cure of CHB, i.e., complete elimination of the virus from the body. Here, the HBV mouse model in vivo has been used to analyze the antiviral activity of the high-specific Cas9 protein and sgRNA targeting HBV genome. We have found that a single injection of short-lived ribonucleoprotein complexes of CRISPR/Cas9 results in a ~10-fold reduction in HBV DNA levels in the serum and liver of mice as early as 48 h after the start of the experiment. The remaining HBV DNAs have been found to harbor rare indel mutations. Developing new antivirals for treating CHB based on CRISPR/Cas9 ribonucleoprotein complexes could substantially reduce the duration of CHB therapy and, potentially, achieve complete elimination of viral infection.

[HBx Protein Potentiates Hepatitis B Virus Reactivation].

Hepatitis B virus (HBV) can cause chronic hepatitis B, one of the most prevalent infectious diseases in the world. Global estimates suggest that over 2 billion people are affected by HBV, with over 250 million people developing chronic infection. Upon treatment of comorbidities, patients with chronic infection may develop an abrupt increase of viral replication-HBV reactivation-leading to liver decompensation and, in some cases, death. HBV reactivation occurs mostly due to suppression of antiviral immune response and activation of intracellular pro-viral signaling. Defining the mechanisms of HBV reactivation is necessary for the rational use of drugs and reduction of mortality rates in patients with chronic infection. In this study, for the first time we analyzed the effects of HBx protein on HBV reactivation, described reactivation of HBV from the transcriptionally inactivated state at the methylated recombinant HBV genome model, and investigated HBV reactivation upon treatment with genotoxic agents (doxorubicin and hydrogen peroxide) and targeted drug therapies (sunitinib and bortezomib). We report that both wild-type HBx protein and, to a greater extent, the mutant form of HBx protein lacking the nuclear exportation signal, potentiate viral replication and promote HBV reactivation. For the first time, we demonstrate that HBV can reactivate from the transcriptionally inactive state. Doxorubicin and hydrogen peroxide induce HBV reactivation at models of both transcriptionally active and transcriptionally silenced viral genome. Sunitinib weakly reactivates HBV, while bortezomib does not affect HBV replication in vitro.

[Small Molecular Inhibitors of DNA Double Strand Break Repair Pathways Increase the ANTI-HBV Activity of CRISPR/Cas9].

The CRISPR/Cas9 nuclease system can effectively suppress the replication of the hepatitis B virus (HBV), while covalently closed circular DNA (cccDNA), a highly resistant form of the virus, persists in the nuclei of infected cells. The most common outcome of DNA double-strand breaks (DSBs) in cccDNA caused by CRISPR/Cas9 is double-strand break repair by nonhomologous end-joining, which results in insertion/deletion mutations. Modulation of the DNA double-strand break repair pathways by small molecules was shown to stimulate CRISPR/Cas9 activity and may potentially be utilized to enhance the elimination of HBV cccDNA. In this work, we used inhibitors of homologous (RI-1) and nonhomologous (NU7026) end-joining and their combination to stimulate antiviral activity of CRISPR/Cas9 on two cell models of HBV in vitro, i.e., the HepG2-1.1merHBV cells containing the HBV genome under the tet-on regulated cytomegalovirus promoter and the HepG2-1.5merHBV cells containing constitutive expression of HBV RNA under the wild-type promoter. The treatment of the cells with RI-1 or NU7026 after lentiviral transduction of CRISPR/Cas9 drops the levels of cccDNA compared to the DMSO-treated control. RI-1 and NU7026 resulted in 5.0-6.5 times more significant reduction in the HBV cccDNA level compared to the mock-control. In conclusion, the inhibition of both homologous and nonhomologous DNA double-strand break repair pathways increases the elimination of HBV cccDNA by CRISPR/Cas9 system in vitro, which may potentially be utilized as a therapeutic approach to treat chronic hepatitis B.

INCREASED FORMATION OF PHOSPHORYLATED H2AX FOCI IN NUCLEI OF CELLS INFECTED BY HEPATITIS B AND B+D VIRUSES.

Liver cirrhosis and hepatocellular carcinoma are the most common outcomes of chronic hepatitis B. Hepatitis B virus (HBV) induces transformation and cell death in chronic hepatitis B (CHB). DNA double strand breaks (DSBs) represent the most dangerous type of genome damage. It was shown previously that generation of phosphorylated histone H2AX foci is a reliable marker of DSBs. The aim of this study was to analyse generation of yH2AX foci in HBV and hepatitis D virus (HDV) infection in vitro and in liver biopsies of patients with CHB and CHB with delta-agent (CHD). Human hepatoma cell line HepG2-1.1merHBV with activated HBV life cycle was used to perform real-time PCR for analysis of pregenomic RNA, HBV DNA, HBV cccDNA and for immunocytochemical analysis of yH2AX. Liver biopsies from CHB and CHD patients were analyzed to confirm the results. HBV induces multiple discrete yH2AX foci in HepG2-1.1merHBV cells in vitro and in biopsies of CHB and CHB+D patients. The ratio of hepatocytes w/o yH2AX foci is significantly lower (49,9+/-12,3% vs. 85,5+/-0,9%, p.