Hepatitis C Virus Indirectly Disrupts DNA Damage-Induced p53 Responses by Activating Protein Kinase R

ABSTRACT Many DNA tumor viruses promote cellular transformation by inactivating the critically important tumor suppressor protein p53. In contrast, it is not known whether p53 function is disrupted by hepatitis C virus (HCV), a unique, oncogenic RNA virus that is the leading infectious cause of liver cancer in many regions of the world. Here we show that HCV-permissive, liver-derived HepG2 cells engineered to constitutively express microRNA-122 (HepG2/miR-122 cells) have normal p53-mediated responses to DNA damage and that HCV replication in these cells potently suppresses p53 responses to etoposide, an inducer of DNA damage, or nutlin-3, an inhibitor of p53 degradation pathways. Upregulation of p53-dependent targets is consequently repressed within HCV-infected cells, with potential consequences for cell survival. Despite this, p53 function is not disrupted by overexpression of the complete HCV polyprotein, suggesting that altered p53 function may result from the host response to viral RNA replication intermediates. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated ablation of double-stranded RNA (dsRNA)-activated protein kinase R (PKR) restored p53 responses while boosting HCV replication, showing that p53 inhibition results directly from viral activation of PKR. The hepatocellular abundance of phosphorylated PKR is elevated in HCV-infected chimpanzees, suggesting that PKR activation and consequent p53 inhibition accompany HCV infection in vivo . These findings reveal a feature of the host response to HCV infection that may contribute to hepatocellular carcinogenesis. IMPORTANCE Chronic infection with hepatitis C virus (HCV) is the leading cause of liver cancer in most developed nations. However, the mechanisms whereby HCV infection promotes carcinogenesis remain unclear. Here, we demonstrate that HCV infection inhibits the activation of p53 following DNA damage. Contrary to previous reports, HCV protein expression is insufficient to inhibit p53. Rather, p53 inhibition is mediated by cellular protein kinase R (PKR), which is activated by HCV RNA replication and subsequently suppresses global protein synthesis. These results redefine our understanding of how HCV infection influences p53 function. We speculate that persistent disruption of p53-mediated DNA damage responses may contribute to hepatocellular carcinogenesis in chronically infected individuals

Mouse model of SARS-CoV-2 reveals inflammatory role of type I interferon signaling

Severe acute respiratory syndrome–coronavirus 2 (SARS-Cov-2) has caused over 13,000,000 cases of coronavirus disease (COVID-19) with a significant fatality rate. Laboratory mice have been the stalwart of therapeutic and vaccine development; however, they do not support infection by SARS-CoV-2 due to the virus’s inability to use the mouse orthologue of its human entry receptor angiotensin-converting enzyme 2 (hACE2). While hACE2 transgenic mice support infection and pathogenesis, these mice are currently limited in availability and are restricted to a single genetic background. Here we report the development of a mouse model of SARS-CoV-2 based on adeno-associated virus (AAV)–mediated expression of hACE2. These mice support viral replication and exhibit pathological findings found in COVID-19 patients. Moreover, we show that type I interferons do not control SARS-CoV-2 replication in vivo but are significant drivers of pathological responses. Thus, the AAV-hACE2 mouse model enables rapid deployment for in-depth analysis following robust SARS-CoV-2 infection with authentic patient-derived virus in mice of diverse genetic backgrounds

Kynurenic acid may underlie sex-specific immune responses to COVID-19

Coronavirus disease 2019 (COVID-19) has poorer clinical outcomes in males than in females, and immune responses underlie these sex-related differences. Because immune responses are, in part, regulated by metabolites, we examined the serum metabolomes of COVID-19 patients. In male patients, kynurenic acid (KA) and a high KA–to–kynurenine (K) ratio (KA:K) positively correlated with age and with inflammatory cytokines and chemokines and negatively correlated with T cell responses. Males that clinically deteriorated had a higher KA:K than those that stabilized. KA inhibits glutamate release, and glutamate abundance was lower in patients that clinically deteriorated and correlated with immune responses. Analysis of data from the Genotype-Tissue Expression (GTEx) project revealed that the expression of the gene encoding the enzyme that produces KA, kynurenine aminotransferase, correlated with cytokine abundance and activation of immune responses in older males. This study reveals that KA has a sex-specific link to immune responses and clinical outcomes in COVID-19, suggesting a positive feedback between metabolites and immune responses in males

Utilizing miR-122 to examine hepatitis C virus-host interactions

The relationship between hepatitis C virus (HCV) and the liver specific microRNA miR- 122 is unusual. While most miRNAs inhibit translation when bound to their targets, miR- 122 increases HCV protein expression by binding to its RNA genome to enhance RNA stability and replication. Developing a cell system that supports the HCV lifecycle took almost 10 years of research. Huh-7 cells, which are one of the only cell lines found to support the entire HCV lifecycle, are also one of the only cell lines found to express miR-122. We hypothesized that insufficient miR-122 expression in most other hepatocyte derived cell lines was the reason they could not support HCV replication. Indeed, when we engineered HepG2 cells to express miR-122, HCV replication was greatly enhanced. We went on to show that HepG2 cells overexpressing both the missing entry factor CD81 in addition to miR-122 supported the entire HCV lifecycle. We found a striking difference in these cells' ability to sustain HCV infection and spread when compared to Huh-7 and Huh-7.5 cells, which was accompanied by robust antiviral and proinflammatory response reminiscent of HCV infections in vivo. Furthermore, blocking RIG-I like receptor and IFN-λ signaling pathways promoted HCV infection and spread in these cells. These studies further solidify the importance of IFN-λ in the hepatic response to HCV infection and revealed non-redundant roles of RIG-I and MDA5 in HCV recognition and antiviral response. Finally, we set out to better understand how miR-122 inhibition influences HCV replication over time and if HCV can develop resistance to miR-122 inhibition. We found a single nucleotide change at position 28 (G28A) of the HCV genome, which falls between the two miR-122 seed- binding sites, and enhanced HCV replication in cells with low miR-122 activity. Naturally occurring HCV isolates encoding an A at nucleotide 28 were similarly resistant to miR- 122 inhibition, indicating that subtle differences in viral sequence, even outside the seed-binding site, greatly influence HCV's requirement for miR-122. These studies provide insight into the interaction between miR-122 and HCV, and have important implications for the development of anti-miR-122-based HCV drugs. Taken together, these studies demonstrate how manipulation of miR-122 can reveal new and important information about HCV host-cell interactions

Species-Specific Regions of Occludin Required by Hepatitis C Virus for Cell Entry▿

Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. As HCV infects only human and chimpanzee cells, antiviral therapy and vaccine development have been hampered by the lack of a convenient small-animal model. In this study we further investigate how the species tropism of HCV is modulated at the level of cell entry. It has been previously determined that the tight junction protein occludin (OCLN) is essential for HCV host cell entry and that human OCLN is more efficient than the mouse ortholog at mediating HCV cell entry. To further investigate the relationship between OCLN sequence and HCV species tropism, we compared OCLN proteins from a range of species for their ability to mediate infection of naturally OCLN-deficient 786-O cells with lentiviral pseudoparticles bearing the HCV glycoproteins. While primate sequences function equivalently to human OCLN, canine, hamster, and rat OCLN had intermediate activities, and guinea pig OCLN was completely nonfunctional. Through analysis of chimeras between these OCLN proteins and alanine scanning mutagenesis of the extracellular domains of OCLN, we identified the second half of the second extracellular loop (EC2) and specific amino acids within this domain to be critical for modulating the HCV cell entry factor activity of this protein. Furthermore, this critical region of EC2 is flanked by two conserved cysteine residues that are essential for HCV cell entry, suggesting that a subdomain of EC2 may be defined by a disulfide bond

Temporal analysis of hepatitis C virus cell entry with occludin directed blocking antibodies.

Hepatitis C virus (HCV) is a major cause of liver disease worldwide. A better understanding of its life cycle, including the process of host cell entry, is important for the development of HCV therapies and model systems. Based on the requirement for numerous host factors, including the two tight junction proteins claudin-1 (CLDN1) and occludin (OCLN), HCV cell entry has been proposed to be a multi-step process. The lack of OCLN-specific inhibitors has prevented a comprehensive analysis of this process. To study the role of OCLN in HCV cell entry, we created OCLN mutants whose HCV cell entry activities could be inhibited by antibodies. These mutants were expressed in polarized HepG2 cells engineered to support the complete HCV life cycle by CD81 and miR-122 expression and synchronized infection assays were performed to define the kinetics of HCV cell entry. During these studies, OCLN utilization differences between HCV isolates were observed, supporting a model that HCV directly interacts with OCLN. In HepG2 cells, both HCV cell entry and tight junction formation were impaired by OCLN silencing and restored by expression of antibody regulatable OCLN mutant. Synchronized infection assays showed that glycosaminoglycans and SR-BI mediated host cell binding, while CD81, CLDN1 and OCLN all acted sequentially at a post-binding stage prior to endosomal acidification. These results fit a model where the tight junction region is the last to be encountered by the virion prior to internalization

Viral Determinants of miR-122-Independent Hepatitis C Virus Replication.

Hepatitis C virus (HCV) replication requires binding of the liver-specific microRNA (miRNA) miR-122 to two sites in the HCV 5' untranslated region (UTR). Although we and others have shown that viral genetics impact the amount of active miR-122 required for replication, it is unclear if HCV can replicate in the complete absence of this miRNA. To probe the absolute requirements for miR-122 and the genetic basis for those requirements, we used clustered regularly interspaced short palindromic repeat (CRISPR) technology to knock out miR-122 in Huh-7.5 cells and reconstituted these knockout (KO) cells with either wild-type miR-122 or a mutated version of this miRNA. We then characterized the replication of the wild-type virus, as well as a mutated HCV bearing 5' UTR substitutions to restore binding to the mutated miR-122, in miR-122 KO Huh-7.5 cells expressing no, wild-type, or mutated miR-122. We found that while replication was most efficient when wild-type or mutated HCV was provided with the matched miR-122, inefficient replication could be observed in cells expressing the mismatched miR-122 or no miR-122. We then selected viruses capable of replicating in cells expressing noncognate miR-122 RNAs. Unexpectedly, these viruses contained multiple mutations throughout their first 42 nucleotides that would not be predicted to enhance binding of the provided miR-122. These mutations increased HCV RNA replication in cells expressing either the mismatched miR-122 or no miR-122. These data provide new evidence that HCV replication can occur independently of miR-122 and provide unexpected insights into how HCV genetics influence miR-122 requirements. IMPORTANCE Hepatitis C virus (HCV) is the leading cause of liver cancer in the Western Hemisphere. HCV infection requires miR-122, which is expressed only in liver cells, and thus is one reason that replication of this virus occurs efficiently only in cells of hepatic origin. To understand how HCV genetics impact miR-122 usage, we knocked out miR-122 using clustered regularly interspaced short palindromic repeat (CRISPR) technology and adapted virus to replicate in the presence of noncognate miR-122 RNAs. In doing so, we identified viral mutations that allow replication in the complete absence of miR-122. This work provides new insights into how HCV genetics influence miR-122 requirements and proves that replication can occur without this miRNA, which has broad implications for how HCV tropism is maintained

Mutant OCLN cell entry factor activities differ between HCV isolates.

<p>(A–G) HepG2+miR-122+CD81 cells that express an shRNA to silence endogenous OCLN expression or (H–K) 786-O cells were transduced to express either shRNA-resistant wild type OCLN (WT), GFP alone, or shRNA-resistant OCLN with the indicated FLAG insertional mutations and challenged with the indicated viruses in the absence of antibody (dark), in the presence of the M2 FLAG antibody (light), or, as positive inhibition controls, either an E2 monoclonal antibody, for HCVpp, or the HCV polymerase inhibitor 2′CMA, for HCVcc (white). <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003244#s2" target="_blank">Results</a> are normalized to infection of cells expressing wild type OCLN for each virus in the absence of antibody. Gray boxes highlight mutants that exhibited different entry factor activities between isolates. *<i>P</i><0.05, **<i>P</i><0.01, *** <i>P</i><0.001 (Mann-Whitney test). Black asterisks represent statistically significant differences between the indicated value and cells expressing GFP alone. Light gray asterisks represent statistically significant differences between infections of the same cells without and with antibody.</p

The OCLN EC2-F5 mutant can complement HCV entry and tight junction defects in OCLN silenced HepG2 cells.

<p>(A) Immunoblots for either OCLN or β-actin of lysates from HepG2+miR-122+CD81 cell populations transduced to express either no target (NT) or OCLN specific shRNAs. Approximate molecular weight (kDa) marker positions are indicated to the left of each blot. (B) These cells were either mock transduced (Mock), or transduced to express either shRNA-resistant wild type (WT) or the EC2-F5 OCLN mutant along with either NT or OCLN specific shRNAs, and then challenged with either HCVpp (dark) or HCVcc (light). ‘Relative infectivity’ refers to values that are normalized to parallel VSVGpp infections, to control for variations in cell number, and set relative to infections of cells expressing wild type human OCLN. (C) To analyze tight junction formation, cells transduced as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003244#ppat-1003244-g004" target="_blank">Figure 4B</a> were grown for three days on collagen coated coverslips, and then stained for the bile canalicular marker MRP2 (red) or an isotype control and with Hoechst 33342 nuclear DNA stain (blue). A representative example of three independent experiments is shown. **<i>P</i><0.01, *** P<0.001 (Mann-Whitney test).</p

OCLN directed FLAG antibody inhibits HCVpp infection of 786-O cells.

<p>(A) Illustration of OCLN membrane topology. The region flanked by two conserved cysteine residues (C216 and C237) that was previously shown to be essential for HCV entry is marked by a dotted line and a star highlights the location of species-specific determinants of OCLN entry factor activity. F2–F5 indicate the location of the EC2 FLAG insertions that did not disrupt HCV cell entry factor activity. (B) 786-O cells expressing wild type OCLN, GFP alone, or the indicated FLAG insertion OCLN mutants were challenged with H77 genotype 1a HCVpp in the presence of 10 µg/ml of an irrelevant isotype control (dark gray) or the FLAG M2 monoclonal antibody (light gray). <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003244#s2" target="_blank">Results</a> were normalized to parallel VSVGpp infections and set relative to infection of cells expressing wild type OCLN. (C) To demonstrate the specificity and dose dependence of FLAG antibody inhibition, 786-O cells expressing either the OCLN EC2-5 mutant or wild type OCLN were challenged with HCVpp in the absence of antibody (white), or in the presence of the indicated concentration of either isotype control (dark gray) or FLAG antibody (light gray). *** P<0.001 (Mann-Whitney test).</p