Biology subjects

Seifert, L. L.

Publications and source records attributed to Seifert, L. L..

5 recordsLinked to original sources

bioRxiv2026

Genetic accessibility, not superior fitness, determines which anti-HBs antibody escape variants are selected in patients

Background and Aims: Genetic diversity within the hepatitis B virus (HBV) surface antigen (HBsAg) drives the emergence of immune escape variants, but studying this diversity in vivo has been limited by the difficulty of establishing infection from patient isolates. We aimed to perform unbiased in vivo screening of variants in the HBs antigenic loop for antibody escape and fitness, using synthetic DNA launch of HBV in human hepatocyte chimeric mice. Approach and Results: Direct intrahepatic injection (IHI) of barcoded, recombinant cccDNA established infection from up to 32,000 independent launch events of a genotype A strain and successfully launched clinical consensus sequences across the major genotypes. Using IHI, we performed deep mutational scanning of 4,608 codon variants across the HBs antigenic loop. Under control antibody treatment, diverse variants expanded, revealing differences in variant fitness in the absence of adaptive immune pressure. Anti-HBs monoclonal antibody treatment suppressed wild-type viremia while permitting breakthrough of resistant variants from the library. Sequencing identified strong enrichment for substitutions at G145 (G145Q, G145H, G145R). Notably, G145Q, rather than the clinically dominant G145R, showed the greatest fitness advantage, indicating that G145R's clinical prevalence does not reflect superior intrinsic fitness. Conclusions: Unbiased in vivo screening reveals that clinically observed HBV escape mutations reflect which substitutions are genetically accessible by single nucleotide change and compatible with the overlapping polymerase reading frame, rather than which confer optimal fitness. This approach establishes a platform for high-resolution mapping of viral variant fitness under antiviral selection pressure.

microbiology↗

bioRxiv2026

The HBV basal core promoter mutation confers a replicative advantage and transcriptionally reprograms hepatocytes toward HCC subtypes

Background: Hepatitis B virus (HBV) basal core promoter (BCP) and precore (PC) mutations occur during chronic HBV infection with the BCP-mutant being associated with increased hepatocellular carcinoma (HCC) risk. Objectives: The effects of these mutants on viral replication, hepatocyte biology, and carcinogenesis remain poorly defined. To address this, we characterized BCP- and PC-mutants using human hepatocyte chimeric mice and asked whether the resulting infection-induced transcriptomes correspond to subsets of human HBV-related hepatocellular carcinomas (HBV-HCCs). Design: Isogenic wild-type (WT), BCP- and PC-mutants of HBV genotypes A, C, and D (HBV-A, -C, -D) were generated from recombinant covalently-closed circular DNA to infect chimeric mice. HBV-HCC transcriptomic datasets from The Cancer Genome Atlas (TCGA) were used to stratify association of HBV variants with HCC subtypes. Results: WT, BCP-mutant, and PC-mutant HBV sequences remained genetically stable. The BCP mutation, but not the PC mutation, accelerated the rise in serum viremia in HBV-D- and HBV-A-infected chimeras; in HBV-C, acceleration required both (PC+BCP) mutations. Further comparisons of HBV-D variants revealed that the BCP-mutant increased intrahepatic viral DNA, viral protein expression, and upregulated cancer-related pathways, including transcripts associated with a subset of HBV-HCCs. Analysis of HBV-HCC samples from the TCGA revealed that tumors often harbor a mixture of WT and mutant transcripts, and that WT- and BCP-mutant-associated HCCs exhibit distinct transcriptional profiles. Conclusions: The HBV BCP-mutant directly perturbs hepatocyte homeostasis via virus-intrinsic mechanisms, selectively activating cancer-related pathways and defining a molecularly distinct subset of HBV-HCC. These findings suggest that HBV variants form distinct subcategories of HBV-HCCs.

molecular biology↗

bioRxiv2026

Orthogonal CRISPR screens and human liver chimeric mice identify hepatitis B virus host factors

Hepatitis B virus (HBV) chronically infects approximately 250 million people worldwide, and reliable curative therapies are lacking. A broader understanding of viral-host interactions could accelerate efforts to find new host-centric therapeutic targets. However, inefficient cell culture systems and limited replication markers compatible with pooled screening have precluded the widespread use of genetic perturbation screens. Here, we performed the first pooled, genome-wide CRISPR-Cas9 knockout (KO) screen with authentic HBV infection and integrated these results with two orthogonal pooled screens to identify host factors. We selected 72 genes for a multi-step assessment that included arrayed validation assays using both HBV infection and pgRNA transfection. We then independently tested thirteen genes using high-efficiency bulk KO experiments to guide further investigations of both antiviral and proviral factors. In both KO and siRNA-mediated knockdown experiments, depletion of the top antiviral factor, EXOC1, enhanced multiple HBV replication markers, and transcriptomic analysis revealed activation of hypoxia- and HIF-1 gene signatures. Three proviral factors, IRF2, WDR48, and ZCCHC14, were investigated in vivo using a human liver chimeric mouse model, which demonstrated that ZCCHC14 KO greatly reduced HBV replication and spread. Together, these complementary in vitro and in vivo platforms expand the catalog of HBV host factors and provide a scalable framework for host target discovery.

microbiology↗

bioRxiv2025

An immunocompetent murine model of virus-elicited liver fibrosis and hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third deadliest cancer worldwide. Over 75% of HCC cases are associated with chronic viral infections. Mechanistic studies and preclinical therapeutic development for virus-associated HCC have been limited by a paucity of small animal models of chronic hepatotropic virus infection that faithfully recapitulate human disease. Here we demonstrate the induction of chronic hepatitis, progressive liver fibrosis, and HCC in immunocompetent laboratory mice upon chronic viral infection with Norway rat hepacivirus (NrHV) - a virus closely related to hepatitis C virus (HCV). NrHV-elicited tumors resemble HCV-associated tumors and liver transcriptome analyses reveal numerous similarities between chronic NrHV and HCV. These findings establish an experimentally tractable, physiologically relevant, and immunocompetent mouse model of virus-elicited progressive liver fibrosis and oncogenesis.

pathology↗

bioRxiv2019

The regulatory factor ELF1 triggers a critical wave of transcription in the antiviral response to type I interferon

The transcription of interferon-stimulated genes (ISGs) is classically triggered via activation of the JAK-STAT pathway, and together, ISGs raise a multifaceted antiviral barrier. An increasing body of evidence reports the existence of additional, non-canonical pathways and transcription factors that coordinate ISG expression. Detailed knowledge of how heterogenous mechanisms regulate ISG expression is crucial for the rational design of drugs targeting the type I interferon response. Here, we characterize the first ETS transcription factor family member as a regulator of non-canonical ISG expression: E74-like ETS transcription factor 1 (ELF1). Using high-content microscopy to quantify viral infection over time, we found that ELF1, itself an ISG, inhibits eight diverse RNA and DNA viruses uniquely at multi-cycle replication. ELF1 did not regulate expression of type I or II interferons, and ELF1s antiviral effect was not abolished by the absence of STAT1 or by inhibition of JAK phosphorylation. Accordingly, comparative expression analyses by RNAseq revealed that the ELF1 transcriptional program is distinct from, and delayed with respect to, the immediate interferon response. Finally, knockdown experiments demonstrated that ELF1 is a critical component of the antiviral interferon response in vitro and in vivo. Our findings reveal a previously overlooked mechanism of non-canonical ISG regulation that both amplifies and prolongs the initial interferon response by expressing broadly antiviral restriction factors. AUTHOR SUMMARYOver 60 years after their discovery, we still struggle to understand exactly how interferons inhibit viruses. Our gap in knowledge stems, on one hand, from the sheer number of interferon-stimulated effector genes, of which only few have been characterized in mechanistic detail. On the other hand, our knowledge of interferon-regulated gene transcription is constantly evolving. We know that different regulatory mechanisms greatly influence the quality, magnitude, and timing of interferon-stimulated gene expression, all of which may contribute to the antiviral mechanism of interferons. Deciphering these regulatory mechanisms is indispensable for understanding this critical first line of host defense, and for harnessing the power of interferons in novel antiviral therapies. Here, we report a novel mechanism of interferon-induced gene regulation by an interferon-stimulated gene, which, paradoxically, inhibits viruses in the absence of additional interferon signaling: E74-like ETS transcription factor 1 (ELF1) raises an unusually delayed antiviral program that potently restricts propagation of all viruses tested in our study. Reduced levels of ELF1 significantly diminished interferon-mediated host defenses against influenza A virus in vitro and in vivo, suggesting a critical but previously overlooked role in the type I interferon response. The transcriptional program raised by ELF1 is vast and comprises over 400 potentially antiviral genes, which are almost entirely distinct from those known to be induced by interferon. Taken together, our data provide evidence for a critical secondary wave of antiviral protection that adds both "quality" and "time" to the type I interferon response.

immunology↗