Effect of Antiviral Treatment on Hepatitis B Virus Integration and Hepatocyte Clonal Expansion.

BACKGROUND: This study investigated the effect of nucleos(t)ide analogue (NUC) treatment on hepatitis B virus (HBV) DNA integration and hepatocyte clonal expansion, both of which are implicated in hepatocellular carcinoma (HCC) in chronic hepatitis B. METHODS: Twenty-eight patients receiving NUCs (11 lamivudine, 7 telbivudine, 10 entecavir) were included. All had liver biopsies at baseline and year 1, and 7 had a third biopsy at year 10. HBV DNA integration and hepatocyte clone size were assessed by inverse polymerase chain reaction. RESULTS: All patients had detectable HBV integration at baseline, with a median integration frequency of 1.01 × 109 per liver and hepatocyte clone size of 2.41 × 105. Neither integration frequency nor hepatocyte clone size correlated with age and HBV virologic parameters. After 1 year of treatment, HBV integration was still detectable in all patients, with a median of 5.74 × 108 integration per liver (0.22 log reduction; P = .008) and hepatocyte clone size of 1.22 × 105 (0.40 log reduction; P = .002). HBV integration remained detectable at year 10 of treatment, with a median integration frequency of 4.84 × 107 integration per liver (0.93 log reduction from baseline) and hepatocyte clone size of 2.55 × 104 (1.02 log reduction from baseline). From baseline through year 1 to year 10, there was a decreasing trend in both integration frequency and hepatocyte clone size (P = .066 and.018, respectively). CONCLUSIONS: NUCs reduced both HBV DNA integration and hepatocyte clonal expansion, suggesting another alternative pathway besides direct viral suppression to reduce HCC risk. Our findings supported the notion for a long-term NUC treatment to prevent HCC.

Cellular and molecular mechanisms of HIV-1 integration targeting.

Integration is central to HIV-1 replication and helps mold the reservoir of cells that persists in AIDS patients. HIV-1 interacts with specific cellular factors to target integration to interior regions of transcriptionally active genes within gene-dense regions of chromatin. The viral capsid interacts with several proteins that are additionally implicated in virus nuclear import, including cleavage and polyadenylation specificity factor 6, to suppress integration into heterochromatin. The viral integrase protein interacts with transcriptional co-activator lens epithelium-derived growth factor p75 to principally position integration within gene bodies. The integrase additionally senses target DNA distortion and nucleotide sequence to help fine-tune the specific phosphodiester bonds that are cleaved at integration sites. Research into virus-host interactions that underlie HIV-1 integration targeting has aided the development of a novel class of integrase inhibitors and may help to improve the safety of viral-based gene therapy vectors.

Liver damage favors the eliminations of HBV integration and clonal hepatocytes in chronic hepatitis B.

BACKGROUND: HBV integration is suspected to be an obstinate risk factor for hepatocellular carcinoma (HCC) in the era of antiviral therapy. Integration events start to occur in the immunotolerance phase, but their fates in the immune clearance phase have not yet been clarified. Here, we report the influences of liver damage on HBV integration and clonal hepatocyte expansion in patients with chronic hepatitis B (CHB). METHODS: HBV integration breakpoints in liver biopsy samples from 54 CHB patients were detected using a modified next-generation sequencing assay. RESULTS: A total of 3729 (69 per sample) integration breakpoints were found in the human genome, including some hotspot genes and KEGG pathways, especially in patients with abnormal transaminases. The number of breakpoint types, an integration risk parameter, was negatively correlated with HBV DNA load and transaminase levels. The average, maximum and total frequencies of given breakpoint types, parameters of clonal hepatocyte expansion, were negatively correlated with HBV DNA load, transaminase levels and liver inflammation activity grade score. The HBV DNA load and inflammation activity grade score were further found to be positively correlated with transaminase levels. Moreover, nucleos(t)ide analog (NUC) treatment that normalized transaminases nonsignificantly reduced the types, but significantly increased the average frequency and negated the enrichments of integration breakpoints. CONCLUSION: Liver damage mainly removed the inventories of viral integration and clonal hepatocytes in CHB. NUC treatment may have reduced HBV integration but clearly increased clonal hepatocyte expansion, which may explain why HCC risk cannot be ruled out by NUC treatment.

Causes and Consequences of HPV Integration in Head and Neck Squamous Cell Carcinomas: State of the Art.

A constantly increasing incidence in high-risk Human Papillomaviruses (HPV)s driven head and neck squamous cell carcinomas (HNSCC)s, especially of oropharyngeal origin, is being observed. During persistent infections, viral DNA integration into the host genome may occur. Studies are examining if the physical status of the virus (episomal vs. integration) affects carcinogenesis and eventually has further-reaching consequences on disease progression and outcome. Here, we review the literature of the most recent five years focusing on the impact of HPV integration in HNSCCs, covering aspects of detection techniques used (from PCR up to NGS approaches), integration loci identified, and associations with genomic and clinical data. The consequences of HPV integration in the human genome, including the methylation status and deregulation of genes involved in cell signaling pathways, immune evasion, and response to therapy, are also summarized.

Quantification of HIV DNA Using Droplet Digital PCR Techniques.

HIV persists, despite effective antiretroviral therapy, in long-lived cells, posing a major barrier toward a cure. A key step in the HIV replication cycle and a hallmark of the Retroviridae family is the integration of the viral DNA into the host genome. Once integrated, HIV expression is regulated by host machinery and the provirus persists until the cell dies. A reservoir of cells harboring replication-competent proviruses can survive for years, and mechanisms that maintain that reservoir are under investigation. The majority of integrated proviruses, however, are defective or have large deletions, and the composition of the proviral landscape during therapy remains unknown. Methods to quantify HIV proviruses are useful in investigating HIV persistence. Presented in this unit is a method for total HIV DNA quantification of various HIV genome targets that utilizes the next-generation PCR platform, digital PCR. The abundance of various HIV gene targets reflects the overall proviral composition. In this protocol, total genomic DNA is isolated from patient-derived cells and then used as a template for droplet digital PCR, in which the PCR reaction is partitioned into approximately 20,000 individual droplets, PCR amplified to an end point, and subjected to absolute quantification by counting the number of positive and negative droplets. Copy number is directly calculated using straightforward Poisson correction. Additionally, this methodological approach can be used to obtain absolute quantification of other DNA targets. © 2018 by John Wiley & Sons, Inc.