Biology subjects

Lim, S. G.

Publications and source records attributed to Lim, S. G..

3 recordsLinked to original sources

bioRxiv2024

Identification of Cyclin L1 as a host factor regulating Hepatitis B Virus replication

Background & AimsUnderstanding the regulatory interactions between Hepatitis B virus (HBV) and human host factors is key to the development of next-generation host-directed antiviral therapies and achieving a functional HBV cure. In this study, we aimed to investigate HBV-induced alterations in host gene expression in primary human hepatocytes (PHH) to identify host-specific factors that are regulated and exploited by the virus for replication and survival. MethodsWe performed whole transcriptome sequencing (WTS) of HBV-infected PHH to identify host pathways that could potentially influence the HBV life cycle. RNA-interference-based validation of putative targets to evaluate the function of dysregulated candidate genes resulted in the identification of Cyclin L1 (CCNL1) as a key host factor. ResultsRNAi-knockdown of CCNL1 revealed that it is essential for HBV gene expression, including HBV-surface antigen (HBsAg). Mechanistically, we found that CCNL1 can phosphorylate the C-terminal domain (CTD) of RNA Polymerase II (RNAPII) at serine 2 (S2), likely to regulate HBV transcription. Furthermore, the knockdown of CCNL1 inhibited the binding of total and phospho- (Ser2-Ser5) RNAPII, pan-acetylated H3ac, and H3K27ac to HBV cccDNA, implicating its function in the regulation of cccDNA-dependent viral transcription. Finally, enhanced CCNL1 expression in chronic hepatitis B patients, as compared to those with resolved infection, underscores a functional link between this host factor and CHB. ConclusionOur data demonstrates that CCNL1 regulates HBV RNA transcription and replication by modulating RNAPII phosphorylation and activity, making it a potential host susceptibility factor for HBV. LAY SUMMARYHepatitis B requires human host cell factors and biological processes to establish an efficient infection. Identifying host factors that support and/or restrict HBV infection is essential for understanding the molecular basis of chronic HBV infection and for developing host-targeting anti-HBV drugs. Here, we report that CCNL1 can serve as a potential host susceptibility factor for HBV, as reduced CCNL1 function results in reduced viral replication and gene expression. Graphical SummaryGraphical summary, highlighting the approach and validation experiments. From whole transcriptomics analysis, we identified known HBV-host factors such as SRPK1, CDK1, NXF1 among others as well as new factors such as CCNL1. Employing various RNAi approaches and different cell models including primary human hepatocytes, we validated the role of CCNL1 during HBV infection cycle. Pull-down experiments followed by ChIP-PCR further showed a reduction in cccDNA-based transcription upon knockdown of CCNL1. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/619969v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1ee9cd9org.highwire.dtl.DTLVardef@7bac2dorg.highwire.dtl.DTLVardef@164c3bborg.highwire.dtl.DTLVardef@6e497a_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2020

LAG3 is a Central Regulator of NK Cell Cytokine Production

Natural killer (NK) cells are innate effectors, which play a crucial role in controlling viral infections. Administration of IFN- has shown promising results as a therapeutic, controlling HIV, and chronic viral hepatitis. However the downstream mechanisms by which IFN- mediates its anti-viral effects is largely unknown. In this investigation, we evaluated the impact of IFN- on peripheral blood NK cells from healthy donors. High dimensional flow cytometry analysis of NK cell surface receptors following exposure to IFN- showed an increased expression of the check point inhibitor LAG3. Further characterization revealed that LAG3 was expressed in a subset of NK cells with high expression of activation and maturation markers. Assessment of metabolic pathways showed that LAG3+ NK cells had enhanced rates of glycolysis and glycolytic capacity, suggesting that it is a primed effector subset with enhanced glucose metabolism. Inhibition of LAG3 on NK cells using antibody in vitro resulted in a profound increase in secretion of cytokines IFN-{gamma}, TNF-, MIP-1 and MIP-1{beta}, without affecting the cytotoxic activity. Taken together, these results showed that LAG3 is a negative regulator of cytokine production by mature NK cells.

immunology↗

bioRxiv2017

Single-Virion Sequencing Of Lamivudine Treated HBV Populations Reveal Population Evolution Dynamics And Demographic History

Viral populations are complex, dynamic, and fast evolving. The evolution of groups of closely related viruses in a competitive environment is termed quasispecies. To fully understand the role that quasispecies play in viral evolution, characterizing the trajectories of viral genotypes in an evolving population is the key. In particular, long-range haplotype information for thousands of individual viruses is critical; yet generating this information is non-trivial. Popular deep sequencing methods generate relatively short reads that do not preserve linkage information, while third generation sequencing methods have higher error rates that make detection of low frequency mutations a bioinformatics challenge. Here we applied BAsE-Seq, an Illumina-based single-virion sequencing technology, to eight samples from four chronic hepatitis B (CHB) patients - once before antiviral treatment and once after viral rebound due to resistance. We obtained 248-8,796 single-virion sequences per sample, which allowed us to find evidence for both hard and soft selective sweeps. We were also able to reconstruct population demographic history that was independently verified by clinically collected data. We further verified four of the samples independently on PacBio and Illumina sequencers. Overall, we showed that single-virion sequencing yields insight into viral evolution and population dynamics in an efficient and high throughput manner. We believe that single-virion sequencing is widely applicable to the study of viral evolution in the context of drug resistance, differentiating between soft or hard selective sweeps, and the reconstruction of intra-host viral population demographic history.

evolutionary biology↗