CRISPR/Cas9 genome-wide loss-of-function screening identifies druggable cellular factors involved in sunitinib resistance in renal cell carcinoma.

BACKGROUND: Multi-targeted tyrosine kinase inhibitors (TKIs) are the standard of care for patients with advanced clear cell renal cell carcinoma (ccRCC). However, a significant number of ccRCC patients are primarily refractory to targeted therapeutics, showing neither disease stabilisation nor clinical benefits. METHODS: We used CRISPR/Cas9-based high-throughput loss of function (LOF) screening to identify cellular factors involved in the resistance to sunitinib. Next, we validated druggable molecular factors that are synthetically lethal with sunitinib treatment using cell and animal models of ccRCC. RESULTS: Our screening identified farnesyltransferase among the top hits contributing to sunitinib resistance in ccRCC. Combined treatment with farnesyltransferase inhibitor lonafarnib potently augmented the anti-tumour efficacy of sunitinib both in vitro and in vivo. CONCLUSION: CRISPR/Cas9 LOF screening presents a promising approach to identify and target cellular factors involved in the resistance to anti-cancer therapeutics.

Distribution of Hepatitis B Virus Nuclear DNA.

Chronic hepatitis B affects over 300 million people who are at risk of developing liver cancer. The basis for the persistence of hepatitis B virus (HBV) in hepatocytes, even in the presence of available antiviral therapies, lies in the accumulation of covalently closed circular DNA (cccDNA) in nuclei of infected cells. While methods for cccDNA quantification from liver biopsy specimens and cell lines expressing the virus are known, information about cccDNA formation, stability, and turnover is lacking. In particular, little is known about the fate of cccDNA during cell division. To fill the gaps in knowledge concerning cccDNA biology, we have developed a fluorescence imaging in situ hybridization (FISH)-based assay for the detection of duck hepatitis B virus (DHBV) cccDNA and HBV nuclear DNA in established cell lines. Using FISH, we determined the distribution of cccDNA under conditions mimicking chronic infections with and without antiviral therapy, which prevents de novo viral replication. Our results showed that the copy numbers of viral nuclear DNA can vary by as much as 1.8 orders of magnitude among individual cells and that antiviral therapy leads to a reduction in nuclear DNA in a manner consistent with symmetrical distribution of viral DNA to daughter cells.IMPORTANCE A mechanistic understanding of the stability of HBV cccDNA in the presence of antiviral therapy and during cell division induced by immune-mediated lysis of infected hepatocytes will be critical for the future design of curative antiviral therapies against chronic hepatitis B. Current knowledge about cccDNA stability was largely derived from quantitative analyses of cccDNA levels present in liver samples, and little was known about the fate of cccDNA in individual cells. The development of a FISH-based assay for cccDNA tracking provided the first insights into the fate of DHBV cccDNA and nuclear HBV DNA under conditions mimicking antiviral therapy.

Complete Spectrum of CRISPR/Cas9-induced Mutations on HBV cccDNA.

Hepatitis B virus (HBV) causes chronic infections that cannot yet be cured. The virus persists in infected hepatocytes, because covalently closed circular DNA (cccDNA), the template for the transcription of viral RNAs, is stable in nondividing cells. Antiviral therapies with nucleoside analogues inhibit HBV DNA synthesis in capsids in the cytoplasm of infected hepatocytes, but do not destroy nuclear cccDNA. Because over 200 million people are still infected, a cure for chronic hepatitis B (CHB) has become one of the major challenges in antiviral therapy. As a first step toward the development of curative therapies, we previously demonstrated that the CRISPR/Cas9 system can be used to functionally inactivate cccDNA derived from infectious HBV. Moreover, some evidence suggests that certain cytokines might induce an APOBEC-mediated cascade leading to the destruction of cccDNA. In this report we investigated whether a combination of the two mechanisms could act synergistically to inactivate cccDNA. Using next generation sequencing (NGS), we determined the complete spectrum of mutations in cccDNA following Cas9 cleavage and repair by nonhomologous end joining (NHEJ). We found that over 90% of HBV DNA was cleaved by Cas9. In addition our results showed that editing of HBV DNA after Cas9 cleavage is at least 15,000 times more efficient that APOBEC-mediated cytosine deamination following treatment of infected cells with interferon alpha (IFNα). We also found that a previously used method to detect cytosine deaminated DNA, termed 3D-PCR, overestimates the amount and frequency of edited HBV DNA. Taken together, our results demonstrated that the CRISPR/Cas9 system is so far the best method to functionally inactivate HBV cccDNA and provide a cure for CHB.

Does Tyrosyl DNA Phosphodiesterase-2 Play a Role in Hepatitis B Virus Genome Repair?

Hepatitis B virus (HBV) replication and persistence are sustained by a nuclear episome, the covalently closed circular (CCC) DNA, which serves as the transcriptional template for all viral RNAs. CCC DNA is converted from a relaxed circular (RC) DNA in the virion early during infection as well as from RC DNA in intracellular progeny nucleocapsids via an intracellular amplification pathway. Current antiviral therapies suppress viral replication but cannot eliminate CCC DNA. Thus, persistence of CCC DNA remains an obstacle toward curing chronic HBV infection. Unfortunately, very little is known about how CCC DNA is formed. CCC DNA formation requires removal of the virally encoded reverse transcriptase (RT) protein from the 5' end of the minus strand of RC DNA. Tyrosyl DNA phosphodiesterase-2 (Tdp2) was recently identified as the enzyme responsible for cleavage of tyrosyl-5' DNA linkages formed between topoisomerase II and cellular DNA. Because the RT-DNA linkage is also a 5' DNA-phosphotyrosyl bond, it has been hypothesized that Tdp2 might be one of several elusive host factors required for CCC DNA formation. Therefore, we examined the role of Tdp2 in RC DNA deproteination and CCC DNA formation. We demonstrated Tdp2 can cleave the tyrosyl-minus strand DNA linkage using authentic HBV RC DNA isolated from nucleocapsids and using RT covalently linked to short minus strand DNA produced in vitro. On the other hand, our results showed that Tdp2 gene knockout did not block CCC DNA formation during HBV infection of permissive human hepatoma cells and did not prevent intracellular amplification of duck hepatitis B virus CCC DNA. These results indicate that although Tdp2 can remove the RT covalently linked to the 5' end of the HBV minus strand DNA in vitro, this protein might not be required for CCC DNA formation in vivo.

Targeting Hepatitis B Virus With CRISPR/Cas9.

Hepatitis B virus persistence in infected hepatocytes is due to the presence of covalently closed circular DNA (cccDNA), the template for the transcription of viral RNAs. Antiviral therapies with nucleoside analogues inhibit replication of HBV DNA in capsids present in the cytoplasm of infected cells, but do not reduce or destroy nuclear cccDNA. To investigate whether cccDNA derived from infectious HBV could be directly targeted for destruction, we used the CRISPR/Cas9 system in HepG2 cells expressing the HBV receptor sodium taurocholate cotransporting polypeptide (NTCP). We tested different HBV-specific guide RNAs and demonstrated that they could inhibit HBV infections up to eightfold. Inhibition was due to mutations and deletions in cccDNA similar to those observed with chromosomal DNA cleaved by Cas9 and repaired by nonhomologous end joining (NHEJ). Interferon alpha (IFN-α) did not have a measurable effect on the antiviral activity of the CRISPR/Cas9 system, suggesting that Cas9 and NHEJ activities are not affected by induction of an innate immune response with the cytokine. Taken together, our results demonstrated that Cas9 can be recruited to cccDNA, opening the possibility for the development of future antiviral strategies aimed at targeting cccDNA for endonucleolytic cleavage with small molecules.

West Nile virus infection induces depletion of IFNAR1 protein levels.

Productive virus infection requires evasion, inhibition, or subversion of innate immune responses. West Nile virus (WNV), a human pathogen that can cause symptomatic infections associated with meningitis and encephalitis, inhibits the interferon (IFN) signal transduction pathway by preventing phosphorylation of Janus kinases and STAT transcription factors. Inhibition of the IFN signal cascade abrogates activation of IFN-induced genes, thus attenuating an antiviral response. We investigated the mechanism responsible for this inhibition and found that WNV infection prevents accumulation of the IFN-α receptor subunit 1 (IFNAR1). The WNV-induced depletion of IFNAR1 was conserved across multiple cell types. Our results indicated that expression of WNV nonstructural proteins resulted in activated lysosomal and proteasomal protein degradation pathways independent of the unfolded protein response (UPR). Furthermore, WNV infection did not induce serine phosphorylation, a modification on IFNAR1 that precedes its natural turnover. These data demonstrate that WNV infection results in a reduction of IFNAR1 protein through a non-canonical protein degradation pathway, and may participate in the inhibition of the IFN response.

Mechanism for CCC DNA synthesis in hepadnaviruses.

Hepadnavirus replication requires the synthesis of a covalently closed circular (CCC) DNA from the relaxed circular (RC) viral genome by an unknown mechanism. CCC DNA formation could require enzymatic activities of the viral reverse transcriptase (RT), or cellular DNA repair enzymes, or both. Physical mapping of the 5' and 3' ends of RC DNA and sequence analysis of CCC DNA revealed that CCC DNA synthesis requires the removal of the RT and an RNA oligomer from the 5' ends of minus and plus strand DNA, respectively, removal of sequences from the terminally redundant minus strand, completion of the less than full-length plus strand, and ligation of the ends. Two models have been proposed that could explain CCC DNA formation. The first (model 1) invokes a role for the RT to catalyze a cleavage-ligation reaction leading to the formation of a unit length minus strand in CCC DNA and a DNA repair reaction for the completion and ligation of plus strand DNA; the second (model 2) predicts that CCC DNA formation depends entirely on cellular DNA repair enzymes. To determine which mechanism is utilized, we developed cell lines expressing duck hepatitis B virus genomes carrying mutations permitting us to follow the fate of viral DNA sequences during their conversion from RC to CCC DNA. Our results demonstrated that the oligomer at the 5' end of minus strand DNA is completely or at least partially removed prior to CCC DNA synthesis. The results indicated that both RC DNA strands undergo DNA repair reactions carried out by the cellular DNA repair machinery as predicted by model 2. Thus, our study provided the basis for the identification of the cellular components required for CCC DNA formation.

Focal distribution of hepatitis C virus RNA in infected livers.

BACKGROUND: Hepatitis C virus (HCV) is a plus-strand RNA virus that replicates by amplification of genomic RNA from minus strands leading to accumulation of almost one thousand copies per cell under in vitro cell culture conditions. In contrast, HCV RNA copy numbers in livers of infected patients appear to be much lower, estimated at a few copies per cell. METHODOLOGY/PRINCIPAL FINDINGS: To gain insights into mechanisms that control HCV replication in vivo, we analyzed HCV RNA levels as well as expression of interferon beta (IFNbeta) and several interferon stimulated genes (ISGs) from whole liver sections and micro-dissected subpopulations of hepatocytes in biopsy samples from 21 HCV-infected patients. The results showed that intrahepatic HCV RNA levels range form less than one copy per hepatocyte to a maximum of about eight. A correlation existed between viral RNA levels and IFNbeta expression, but not between viral RNA and ISG levels. Also, IFNbeta expression did not correlate with ISGs levels. Replication of HCV RNA occurred in focal areas in the liver in the presence of a general induction of ISGs. CONCLUSION/SIGNIFICANCE: The low average levels of HCV RNA in biopsy samples can be explained by focal distribution of infected hepatocytes. HCV replication directly induces IFNbeta, which then activates ISGs. The apparent lack of a correlation between levels of IFNbeta and ISG expression indicates that control of the innate immune response during HCV infections depends on multiple factors.

Mechanism of the interferon alpha response against hepatitis C virus replicons.

Interferon alpha (IFN-alpha) inhibits hepatitis C virus (HCV) replication in vivo and in cell cultures by one or several mechanisms that are not yet understood. We sought to identify the viral targets of the IFN-alpha-induced cellular antiviral program in Huh7 cells expressing HCV subgenomic replicons. Our results revealed a tight linkage between translation, assembly of replication complexes and viral RNA synthesis, and indicated that the stability of amplified plus strand RNA was reduced in the presence of the cytokine. Moreover, HCV internal ribosomal entry site (IRES)-directed translation was inhibited approximately 2-fold in IFN-treated cells. In contrast, the synthesis of viral RNA did not seem to be directly affected by the antiviral program induced by the cytokine. Our results were consistent with a model predicting that the IFN-alpha-induced antiviral program could inhibit multiple steps of the HCV replication cycle, leading to a reduction in viral protein synthesis and eventually inhibition of viral RNA amplification.