Hepatitis C virus infection restricts human LINE-1 retrotransposition in hepatoma cells.
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ABSTRACT:
SUBMITTER: Schobel A
PROVIDER: S-EPMC8084336 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Adenosine deaminases acting on RNA (ADARs) are involved in RNA editing that converts adenosines to inosines in double-stranded RNAs. ADAR1 was demonstrated to be functional on different viruses exerting either antiviral or proviral effects. Concerning HIV-1, several studies showed that ADAR1 favors viral replication. The aim of this study was to investigate the composition of the ADAR1 ribonucleoprotein complex during HIV-1 expression. By using a dual-tag affinity purification procedure in cells expressing HIV-1 followed by mass spectrometry analysis, we identified 14 non-ribosomal ADAR1-interacting proteins, most of which are novel. A significant fraction of these proteins were previously demonstrated to be associated to the Long INterspersed Element 1 (LINE1 or L1) ribonucleoparticles and to regulate the life cycle of L1 retrotransposons that continuously re-enter host-genome.Hence, we investigated the function of ADAR1 in the regulation of L1 activity.By using different cell-culture based retrotransposition assays in HeLa cells, we demonstrated a novel function of ADAR1 as suppressor of L1 retrotransposition. Apparently, this inhibitory mechanism does not occur through ADAR1 editing activity. Furthermore, we showed that ADAR1 binds the basal L1 RNP complex. Overall, these data support the role of ADAR1 as regulator of L1 life cycle.
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Project description:Members of the APOBEC3 (A3) family of cytidine deaminase enzymes act as host defense mechanisms limiting both infections by exogenous retroviruses and mobilization of endogenous retrotransposons. Previous studies revealed that the overexpression of some A3 proteins could restrict engineered human Long INterspersed Element-1 (LINE-1 or L1) retrotransposition in HeLa cells. However, whether endogenous A3 proteins play a role in restricting L1 retrotransposition remains largely unexplored. Here, we show that HeLa cells express endogenous A3B and A3C, whereas human embryonic stem cells (hESCs) express A3B, A3C, A3DE, A3F, and A3G. To study the relative contribution of endogenous A3 proteins in restricting L1 retrotransposition, we first generated small hairpin RNAs (shRNAs) to suppress endogenous A3 mRNA expression, and then assessed L1 mobility using a cell-based L1 retrotransposition assay. We demonstrate that in both HeLa and hESCs, shRNA-based knockdown of A3B promotes a ?2-3.7-fold increase in the retrotransposition efficiency of an engineered human L1. Knockdown of the other A3s produced no significant increase in L1 activity. Thus, A3B appears to restrict engineered L1 retrotransposition in a broad range of cell types, including pluripotent cells.
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Project description:LINE-1 retrotransposition is tightly restricted by layers of regulatory control, with epigenetic pathways being the best characterized. Looking at post-transcriptional regulation, we now show that LINE-1 mRNA 3' ends are pervasively uridylated in various human cellular models and in mouse testes. TUT4 and TUT7 uridyltransferases catalyze the modification and function in cooperation with the helicase/RNPase MOV10 to counteract the RNA chaperone activity of the L1-ORF1p retrotransposon protein. Uridylation potently restricts LINE-1 retrotransposition by a multilayer mechanism depending on differential subcellular localization of the uridyltransferases. We propose that uridine residues added by TUT7 in the cytoplasm inhibit initiation of reverse transcription of LINE-1 mRNAs once they are reimported to the nucleus, whereas uridylation by TUT4, which is enriched in cytoplasmic foci, destabilizes mRNAs. These results provide a model for the post-transcriptional restriction of LINE-1, revealing a key physiological role for TUT4/7-mediated uridylation in maintaining genome stability.
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Project description:An estimated number of 71 million people are living with chronic hepatitis C virus (HCV) infection worldwide and 400,000 annual deaths are related to the infection. HCV entry into the hepatocytes is complex and involves several host factors. The tetraspanin human CD81 (hCD81) is one of the four essential entry factors and is composed of one large extracellular loop, one small extracellular loop, four transmembrane domains, one intracellular loop and two intracellular tails. The large extracellular loop interacts with the E2 glycoprotein of HCV. Regions outside the large extracellular loop (backbone) of hCD81 have a critical role in post-binding entry steps and determine susceptibility of hepatocytes to HCV. Here, we investigated the effect of five non-synonymous single-nucleotide variants in the backbone of hCD81 on HCV susceptibility. We generated cell lines that stably express the hCD81 variants and infected the cells using HCV pseudoparticles and cell culture-derived HCV. Our results show that all the tested hCD81 variants support HCV pseudoparticle entry with similar efficiency as wild-type hCD81. In contrast, variants A54V, V211M and M220I are less supportive to cell culture-derived HCV infection. This altered susceptibility is HCV genotype dependent and specifically affected the cell entry step. Our findings identify three hCD81 genetic variants that are impaired in their function as HCV host factors for specific viral genotypes. This study provides additional evidence that genetic host variation contributes to inter-individual differences in HCV infection and outcome.
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Project description:C-type lectin domain-containing proteins (CTLDcps) shape host responses to pathogens and infectious disease outcomes. Previously, we identified the murine CTLDcp Cd302 as restriction factor, limiting hepatitis C virus (HCV) infection of murine hepatocytes. In this study, we investigated in detail the human orthologue's ability to restrict HCV infection in human liver cells. CD302 overexpression in Huh-7.5 cells potently inhibited infection of diverse HCV chimeras representing seven genotypes. Transcriptional profiling revealed abundant CD302 mRNA expression in human hepatocytes, the natural cellular target of HCV. Knockdown of endogenously expressed CD302 modestly enhanced HCV infection of Huh-7.5 cells and primary human hepatocytes. Functional analysis of naturally occurring CD302 transcript variants and engineered CD302 mutants showed that the C-type lectin-like domain (CTLD) is essential for HCV restriction, whereas the cytoplasmic domain (CPD) is dispensable. Coding single nucleotide polymorphisms occurring in human populations and mapping to different domains of CD302 did not influence the capacity of CD302 to restrict HCV. Assessment of the anti-HCV phenotype at different life cycle stages indicated that CD302 preferentially targets the viral entry step. In contrast to the murine orthologue, overexpression of human CD302 did not modulate downstream expression of nuclear receptor-controlled genes. Ectopic CD302 expression restricted infection of liver tropic hepatitis E virus (HEV), while it did not affect infection rates of two respiratory viruses, including respiratory syncytial virus (RSV) and the alpha coronavirus HVCoV-229E. Together, these findings suggest that CD302 contributes to liver cell-intrinsic defense against HCV and might mediate broader antiviral defenses against additional hepatotropic viruses. IMPORTANCE The liver represents an immunoprivileged organ characterized by enhanced resistance to immune responses. However, the importance of liver cell-endogenous, noncytolytic innate immune responses in pathogen control is not well defined. Although the role of myeloid cell-expressed CTLDcps in host responses to viruses has been characterized in detail, we have little information about their potential functions in the liver and their relevance for immune responses in this organ. Human hepatocytes endogenously express the CTLDcp CD302. Here, we provide evidence that CD302 limits HCV infection of human liver cells, likely by inhibiting a viral cell entry step. We confirm that the dominant liver-expressed transcript variant, as well as naturally occurring coding variants of CD302, maintain the capacity to restrict HCV. We further show that the CTLD of the protein is critical for the anti-HCV activity and that overexpressed CD302 limits HEV infection. Thus, CD302 likely contributes to human liver-intrinsic antiviral defenses.
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Project description:Long interspersed element 1 (LINE-1 or L1) is the only active autonomous retrotransposon in the human genome that can serve as an endogenous upstream activator of cytoplasmic nucleic acid sensing pathways to elicit an antiviral immune response. In this study, we investigated the influence of enteroviral infection on L1 mobility. The results showed that infection with different enteroviruses, both EV-D68 and EV-A71, blocked L1 transposition. We screened diverse viral accessory proteins for L1 activity and identified EV-D68 2A, 3A, 3C, and EV-A71 ORF2p proteins as viral L1 inhibitors. EV-D68 2A suppressed L1 mobility by expression suppression of L1 proteins. Viral proteins 3A and 3C restricted ORF2p-mediated L1 reverse transcription in isolated L1 ribonucleoproteins. The newly identified enteroviral protein ORF2p inhibited the expression of L1 ORF1p. Altogether, our findings shed light on the strict modulation of L1 retrotransposons during enterovirus replication.
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Project description:Recent evidence indicates there is a role for small membrane vesicles, including exosomes, as vehicles for intercellular communication. Exosomes secreted by most cell types can mediate transfer of proteins, mRNAs, and microRNAs, but their role in the transmission of infectious agents is less established. Recent studies have shown that hepatocyte-derived exosomes containing hepatitis C virus (HCV) RNA can activate innate immune cells, but the role of exosomes in the transmission of HCV between hepatocytes remains unknown. In this study, we investigated whether exosomes transfer HCV in the presence of neutralizing antibodies. Purified exosomes isolated from HCV-infected human hepatoma Huh7.5.1 cells were shown to contain full-length viral RNA, viral protein, and particles, as determined by RT-PCR, mass spectrometry, and transmission electron microscopy. Exosomes from HCV-infected cells were capable of transmitting infection to naive human hepatoma Huh7.5.1 cells and establishing a productive infection. Even with subgenomic replicons, lacking structural viral proteins, exosome-mediated transmission of HCV RNA was observed. Treatment with patient-derived IgGs showed a variable degree of neutralization of exosome-mediated infection compared with free virus. In conclusion, this study showed that hepatic exosomes can transmit productive HCV infection in vitro and are partially resistant to antibody neutralization. This discovery sheds light on neutralizing antibodies resistant to HCV transmission by exosomes as a potential immune evasion mechanism.
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Project description:LINE-1 (L1) retroelements have retained their ability to mobilize. Mechanisms regulating L1 mobility include DNA methylation in somatic cells and the piRNA pathway in the germline. During pre-implantation stages of mouse embryonic development, however, both pathways are inactivated leading to a window necessitating alternate means of L1 regulation. We previously reported an increase in L1 levels in Dicer_KO mouse embryonic stem cells (mESCs), which was accompanied by only a marginal increase in retrotransposition, suggesting additional mechanisms suppressing L1 mobility. Here, we demonstrate that L1 Ribonucleoprotein complexes (L1 RNP) accumulate as aggregates in the cytoplasm of Dicer_KO mESCs along with the RNA helicase MOV10. The combined overexpression of L1 ORF1p and MOV10 is sufficient to create L1 RNP aggregates. In Dicer_KO mESCs, MOV10 is upregulated due to the loss of its direct regulation by miRNAs. The newly discovered post-transcriptional regulation of Mov10, and its role in preventing L1 retrotransposition by driving cytosolic aggregation provides routes to explore for therapy in disease conditions where L1s are upregulated.
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