Replenishment of hepatitis B virus cccDNA pool is restricted by baseline expression of host restriction factors in vitro

Funding Information: National Medical Research Center for Tuberculosis and Infectious Diseases, 127994 Moscow, Russia; [email protected] (A.K.); [email protected] (V.C.) Institute of Immunology, Federal Medical Biological Agency, 115522 Moscow, Russia; [email protected] NF Gamaleya Research Center of Epidemiology and Microbiology, 123098 Moscow, Russia; [email protected] (E.B.); [email protected] (I.G.); [email protected] (M.I.) Chumakov Federal Scientific Center for Research and Development of Immune and Biological Products of Russian Academy of Sciences, 108819 Moscow, Russia Sechenov First Moscow State Medical University, 119146 Moscow, Russia; [email protected] Riga Stradins University, LV-1007 Riga, Latvia Karolinska Institutet, SE-171 76 Stockholm, Sweden Izmerov Research Institute of Occupational Health, 105275 Moscow, Russia; [email protected] (I.G.); [email protected] (A.N.) Institute of Medical Virology, University of Giessen, 35392 Giessen, Germany; [email protected] 10 Central Research Institute of Epidemiology, 111123 Moscow, Russia Funding Information: Funding: This work was funded by the Russian Science Foundation grant No. 16-15-10426. D.K. received a scholarship from the President of Russia no. SP1439.2019.4. Publisher Copyright: © 2019 by the authors. Licensee MDPI, Basel, Switzerland.Background: Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the major cause of viral persistence in patients with chronic HBV infection. Understanding the mechanisms underlying stability and persistence of HBV cccDNA in hepatocytes is critical for developing novel therapeutics and managing chronic hepatitis B. In this study, we observed an unexpected increase in HBV cccDNA levels upon suppression of transcription by de novo DNA methyltransferase DNMT3A and uncovered additional mechanisms potentially involved in HBV cccDNA maintenance. Methods: HBV-expressing cell lines were transfected with a DNMT3A-expressing plasmid. Real-time PCR and HBsAg assays were used to assess the HBV replication rate. CeSavell cycling was analyzed by fluorescent cell sorting. CRISPR/Cas9 was utilized to abrogate expression of APOBEC3A and APOBEC3B. Alterations in the expression of target genes were measured by real-time PCR. Results: Similar to previous studies, HBV replication induced DNMT3A expression, which in turn, led to reduced HBV transcription but elevated HBV cccDNA levels (4-to 6-fold increase). Increased levels of HBV cccDNA were not related to cell cycling, as DNMT3A accelerated proliferation of infected cells and could not contribute to HBV cccDNA expansion by arresting cells in a quiescent state. At the same time, DNMT3A suppressed transcription of innate immunity factors including cytidine deaminases APOBEC3A and APOBEC3B. CRISPR/Cas9-mediated silencing of APOBEC3A and APOBEC3B transcription had minor effects on HBV transcription, but significantly increased HBV cccDNA levels, similar to DNMT3A. In an attempt to further analyze the detrimental effects of HBV and DNMT3A on infected cells, we visualized γ-H2AX foci and demonstrated that HBV inflicts and DNMT3A aggravates DNA damage, possibly by downregulating DNA damage response factors. Additionally, suppression of HBV replication by DNMT3A may be related to reduced ATM/ATR expression. Conclusion: Formation and maintenance of HBV cccDNA pools may be partially suppressed by the baseline expression of host inhibitory factors including APOBEC3A and APOBEC3B. HBV inflicts DNA damage both directly and by inducing DNMT3A expression.publishersversionPeer reviewe

Proteolytic Resistance Determines Albumin Nanoparticle Drug Delivery Properties and Increases Cathepsin B, D, and G Expression

Proteolytic activity is pivotal in maintaining cell homeostasis and function. In pathological conditions such as cancer, it covers a key role in tumor cell viability, spreading to distant organs, and response to the treatment. Endosomes represent one of the major sites of cellular proteolytic activity and very often represent the final destination of internalized nanoformulations. However, little information about nanoparticle impact on the biology of these organelles is available even though they represent the major location of drug release. In this work, we generated albumin nanoparticles with a different resistance to proteolysis by finely tuning the amount of cross-linker used to stabilize the carriers. After careful characterization of the particles and measurement of their degradation in proteolytic conditions, we determined a relationship between their sensitivity to proteases and their drug delivery properties. These phenomena were characterized by an overall increase in the expression of cathepsin proteases regardless of the different sensitivity of the particles to proteolytic degradation