miRNA independent hepacivirus variants suggest a strong evolutionary pressure to maintain miR-122 dependence

Hepatitis C virus (HCV) requires the liver specific micro-RNA (miRNA), miR-122, to replicate. This was considered unique among RNA viruses until recent discoveries of HCV-related hepaciviruses prompting the question of a more general miR-122 dependence. Among hepaciviruses, the closest known HCV relative is the equine non-primate hepacivirus (NPHV). Here, we used Argonaute cross-linking immunoprecipitation (AGO-CLIP) to confirm AGO binding to the single predicted miR-122 site in the NPHV 5’UTR in vivo. To study miR-122 requirements in the absence of NPHV-permissive cell culture systems, we generated infectious NPHV/HCV chimeric viruses with the 5’ end of NPHV replacing orthologous HCV sequences. These chimeras were viable even in cells lacking miR-122, although miR-122 presence enhanced virus production. No other miRNAs bound this region. By random mutagenesis, we isolated HCV variants partially dependent on miR-122 as well as robustly replicating NPHV/HCV variants completely independent of any miRNAs. These miRNA independent variants even replicate and produce infectious particles in non-hepatic cells after exogenous delivery of apolipoprotein E (ApoE). Our findings suggest that miR-122 independent HCV and NPHV variants have arisen and been sampled during evolution, yet miR-122 dependence has prevailed. We propose that hepaciviruses may use this mechanism to guarantee liver tropism and exploit the tolerogenic liver environment to avoid clearance and promote chronicity

Replication and single-cycle delivery of SARS-CoV-2 replicons

Molecular virology tools are critical for basic studies of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and for developing new therapeutics. There remains a need for experimental systems that do not rely on viruses capable of spread that could potentially be used in lower containment settings. Here, we develop spike-deleted SARS-CoV-2 self-replicating RNAs using a yeast-based reverse genetics system. These non-infectious self-replicating RNAs, or replicons, can be trans-complemented with viral glycoproteins to generate Replicon Delivery Particles (RDPs) for single-cycle delivery into a range of cell types. This SARS-CoV-2 replicon system represents a convenient and versatile platform for antiviral drug screening, neutralization assays, host factor validation, and characterizing viral variants

Evidence for a Hepatitis B Virus Short RNA Fragment Directly Targeting the Cellular RRM2 Gene

The hepatitis B virus (HBV) is one of the smallest but most highly infectious human pathogens. With a DNA genome of only 3.2 kb and only four genes, HBV successfully completes its life cycle by using intricate processes to hijack the host machinery. HBV infects non-dividing liver cells in which dNTPs are limited. As a DNA virus, HBV requires dNTPs for its replication. HBV induces the ATR-mediated cellular DNA damage response pathway to overcome this constraint. This pathway upregulates R2 (RRM2) expression in generating an active RNR holoenzyme catalyzing de novo dNTP synthesis. Previously we reported that ERE, a small RNA fragment within the HBx ORF, is sufficient to induce R2 upregulation. Interestingly, there is high sequence similarity between ERE and a region within the R2 5′UTR that we named R2-box. Here, we established a mutant cell line in the R2-box region of the R2 gene using CRISPR-Cas9 technology to investigate the R2 regulation by ERE. This cell line expresses a much lower R2 level than the parental cell line. Interestingly, the HBV infection and life cycle were severely impaired. These cells became permissive to HBV infection upon ectopically R2 expression. These results validate the requirement of the R2 gene expression for HBV replication. Remarkably, the R2-box mutated cells became ERE refractory, suggesting that the homology region between ERE and R2 gene is critical for ERE-mediated R2 upregulation. Thus, along with the induction of the ATR pathway of the DNA damage response, ERE might also directly target the R2 gene via the R2-box

RNR-R2 Upregulation by a Short Non-Coding Viral Transcript

DNA viruses require dNTPs for replication and have developed different strategies to increase intracellular dNTP pools. Hepatitis B virus (HBV) infects non-dividing cells in which dNTPs are scarce and the question is how viral replication takes place. Previously we reported that the virus induces the DNA damage response (DDR) pathway culminating in RNR-R2 expression and the generation of an active RNR holoenzyme, the key regulator of dNTP levels, leading to an increase in dNTPs. How the virus induces DDR and RNR-R2 upregulation is not completely known. The viral HBx open reading frame (ORF) was believed to trigger this pathway. Unexpectedly, however, we report here that the production of HBx protein is dispensable. We found that a small conserved region of 125 bases within the HBx ORF is sufficient to upregulate RNR-R2 expression in growth-arrested HepG2 cells and primary human hepatocytes. The observed HBV mRNA embedded regulatory element is named ERE. ERE in isolation is sufficient to activate the ATR-Chk1-E2F1-RNR-R2 DDR pathway. These findings demonstrate a non-coding function of HBV transcripts to support its propagation in non-cycling cells

Growth kinetics of wild type HCV and NPHV/HCV chimeras in ΔmiR-122 Huh-7.5 cells.

<p><b>(A)</b> NS5A positive cells post transfection in Huh-7.5 (open bars) and ΔmiR-122 Huh-7.5 cells (hatched bars). Results represent mean±SEM from 3 independent transfections. <b>(B)</b> Infectious virus production of supernatants from transfected ΔmiR-122 Huh-7.5 and Huh-7.5 cells. Virus titers were quantified by limiting dilution assay on naïve Huh-7.5 cells (n = 3).</p

Evolutionary selected NPHV/HCV variants can replicate and produce infectious particles in non-hepatic cells.

<p><b>(A)</b> Quantification of NS5A positive cells on day 2 post-transfection of 293T cells with HCV and NPHV/HCV variants. <b>(B)</b> Quantification of NS5A positive 293T cells post-transfection as in (A) but compared to a daclatasvir (DCV) treated culture. <b>(C)</b> Infectious virus production from 293T cells with or without exogenous ApoE expression. Virus titers were quantified by limiting dilution assay on naïve Huh-7.5 cells. In (A)-(C), results represent mean±SEM from 3–6 independent experiments. Asterisks, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Student’s t test.</p

Identification of NPHV/HCV chimeras capable of replication and infectious virus production in Huh-7.5 cells.

<p><b>(A)</b> Schematic showing the secondary structures of 5’UTR regions of HCV/NPHV chimeras that contain the NPHV entire 5’UTR (NPHV-5’UTR), IRES (NPHV-IRES), Stem Loop 1 with microRNA Binding Region (miRBR) (NPHV-SL1/miRBR), or only SL1 (NPHV-SL1). All chimeras were constructed on the HCV J6/JFH Clone 2 backbone. <b>(B,E)</b> NS5A positive cells post transfection of Huh-7.5 cells. Results represent mean±SEM from 3 independent transfections. <b>(C,F)</b> Infectious virus production quantified by limiting dilution assay on naïve Huh-7.5 cells post transfection (n = 3). <b>(D)</b> Schematic of predicted miR-122 binding modes to NPHV and HCV. The adaptive mutation of NPHV-SL1/miRBR at C83A site is indicated. Asterisks, *p < 0.05, **p < 0.01, Student’s t test.</p

Expansion, in vivo–ex vivo cycling, and genetic manipulation of primary human hepatocytes

Primary human hepatocytes (PHHs) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture, and resistance to genetic manipulation have limited their use. Here, we show that the pyrrolizidine alkaloid retrorsine improves PHH repopulation of chimeric mice on average 10-fold and rescues the ability of even poorly plateable donor hepatocytes to provide cells for subsequent ex vivo cultures. These mouse-passaged (mp) PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum. mpPHH can be efficiently genetically modified in culture, mobilized, and then recultured as spheroids or retransplanted to create highly humanized mice that carry a genetically altered hepatocyte graft. Together, these advances provide flexible tools for the study of human liver disease and evaluation of hepatocyte-targeted gene therapy approaches

AGO-CLIP provides a miRNA binding map for NPHV <i>in vivo</i>.

<p><b>(A)</b> Schematic of standard AGO-CLIP and CLEAR-CLIP. After UV-induced cross-linking of RNA-protein complexes, precipitation of the AGO complex and RNA library preparation, standard AGO-CLIP provides a map of AGO/miRNA interactions across the transcriptome. In CLEAR-CLIP, chimeras of miRNAs with cellular or viral RNAs are induced. Analysis of these allows unambiguous detection of specific miRNA interactions. <b>(B)</b> <i>In vivo</i> AGO/miRNA binding across the NPHV genome from horse liver. Binding is observed across the four miR-122 seed sites conserved among all published isolates. Non-conserved sites present in the NZP1 isolate are indicated.</p