A stable and persistent Hepatitis C virus (HCV) replication cell culture model was developed to examine clearance of viral replication during long-term treatment using interferon-α (IFN-α), IFN-λ, and ribavirin (RBV). Persistently HCV-infected cell culture exhibited an impaired antiviral response to IFN-α+RBV combination treatment, whereas IFN-λ treatment produced a strong and sustained antiviral response that cleared HCV replication. HCV replication in persistently infected cells induced chronic endoplasmic reticulum (ER) stress and an autophagy response that selectively down-regulated the functional IFN-α receptor-1 chain of type I, but not type II (IFN-γ) or type III (IFN-λ) IFN receptors. Down-regulation of IFN-α receptor-1 resulted in defective JAK-STAT signaling, impaired STAT phosphorylation, and impaired nuclear translocation of STAT. Furthermore, HCV replication impaired RBV uptake, because of reduced expression of the nucleoside transporters ENT1 and CNT1. Silencing ER stress and the autophagy response using chemical inhibitors or siRNA additively inhibited HCV replication and induced viral clearance by the IFN-α+RBV combination treatment. These results indicate that HCV induces ER stress and that the autophagy response selectively impairs type I (but not type III) IFN signaling, which explains why IFN-λ (but not IFN-α) produced a sustained antiviral response against HCV. The results also indicate that inhibition of ER stress and of the autophagy response overcomes IFN-α+RBV resistance mechanisms associated with HCV infection.
Decay of the HIV reservoir is slowed over time in part by expansion of the pool of HIV-infected cells. This expansion reflects homeostatic proliferation of infected cells by interleukin-7 (IL-7) or antigenic stimulation, as well as new rounds of infection of susceptible target cells. As novel therapies are being developed to accelerate the decay of the latent HIV reservoir, it will be important to identify interventions that prevent expansion and/or repopulation of the latent HIV reservoir. Our previous studies showed that HIV protease cleaves the host protein procaspase 8 to generate Casp8p41, which can bind and activate Bak to induce apoptosis of infected cells. In circumstances where expression of the anti-apoptotic protein BCL2 is high, Casp8p41 instead binds BCL2, and cell death does not occur. This effect can be overcome by treating cells with the clinically approved BCL2 antagonist venetoclax, which prevents Casp8p41 from binding BCL2, thereby allowing Casp8p41 to bind Bak and kill the infected cell. Here we assess whether the events that maintain the HIV reservoir are also antagonized by venetoclax. Using the J-Lat 10.6 model of persistent infection, we demonstrate that proliferation and HIV expression are countered by the use of venetoclax, which causes preferential killing of the HIV-expressing cells. Similarly, during new rounds of infection of primary CD4 T cells, venetoclax causes selective killing of HIV-infected cells, resulting in decreased numbers of HIV DNA-containing cells. Cure of HIV infection requires an intervention that reduces the HIV reservoir size. A variety of approaches are being tested for their ability to impact HIV reservoir size. Even if successful, however, these approaches will need to be combined with additional complementary approaches that prevent replenishment or repopulation of the HIV reservoir. Our previous studies have shown that the FDA-approved BCL2 antagonist venetoclax has a beneficial effect on the HIV reservoir size following HIV reactivation. Here we demonstrate that venetoclax also has a beneficial effect on HIV reservoir size in a model of homeostatic proliferation of HIV as well as in acute spreading infection of HIV in primary CD4 T cells. These results suggest that venetoclax, either alone or in combination with other approaches to reducing HIV reservoir size, is a compound worthy of further study for its effects on HIV reservoir size.
BackgroundHepatic steatosis is recognized as a major risk factor for liver disease progression and impaired response to interferon based therapy in chronic hepatitis C (CHC) patients. The mechanism of response to interferon-alpha (IFN-α) therapy under the condition of hepatic steatosis is unexplored. We investigated the effect of hepatocellular steatosis on hepatitis C virus (HCV) replication and IFN-α antiviral response in a cell culture model.MethodsSub-genomic replicon (S3-GFP) and HCV infected Huh-7.5 cells were cultured with a mixture of saturated (palmitate) and unsaturated (oleate) long-chain free fatty acids (FFA). Intracytoplasmic fat accumulation in these cells was visualized by Nile red staining and electron microscopy then quantified by microfluorometry. The effect of FFA treatment on HCV replication and IFN-α antiviral response was measured by flow cytometric analysis, Renilla luciferase activity, and real-time RT-PCR.ResultsFFA treatment induced dose dependent hepatocellular steatosis and lipid droplet accumulation in the HCV replicon cells was confirmed by Nile red staining, microfluorometry, and by electron microscopy. Intracellular fat accumulation supports replication more in the persistently HCV infected culture than in the sub-genomic replicon (S3-GFP) cell line. FFA treatment also partially blocked IFN-α response and viral clearance by reducing the phosphorylation of Stat1 and Stat2 dependent IFN-β promoter activation. We show that FFA treatment induces endoplasmic reticulum (ER) stress response and down regulates the IFNAR1 chain of the type I IFN receptor leading to defective Jak-Stat signaling and impaired antiviral response.ConclusionThese results suggest that intracellular fat accumulation in HCV cell culture induces ER stress, defective Jak-Stat signaling, and attenuates the antiviral response, thus providing an explanation to the clinical observation regarding how hepatocellular steatosis influences IFN-α response in CHC.
TNF-related apoptosis-inducing ligand (TRAIL) was initially described to induce apoptosis of tumor cells and/or virally infected cells, although sparing normal cells, and has been implicated in the pathogenesis of HIV disease. We previously identified TRAILshort, a TRAIL splice variant, in HIV-infected patients and characterized it as being a dominant negative ligand to subvert TRAIL-mediated killing. Herein, using single-cell genomics we demonstrate that TRAILshort is produced by HIV-infected cells, as well as by uninfected bystander cells, and that the dominant stimulus which induces TRAILshort production are type I IFNs and TLR7, TLR8, and TLR9 agonists. TRAILshort has a short by virtue of containing a PEST domain, which targets the protein toward the ubiquitin proteasome pathway for degradation. Further we show that TRAILshort binds preferentially to TRAIL receptors 1 and 2 with significantly reduced interaction with the decoy TRAIL receptors 3 and 4. Recombinant TRAILshort is sufficient to protect cells against TRAIL-induced killing, whereas immunodepletion of TRAILshort with a specific Ab restores TRAIL sensitivity. Importantly we show that TRAILshort is shed in microvesicles into the cellular microenvironment and therefore confers TRAIL resistance not only on the cell which produces it, but also upon neighboring bystander cells. These results establish a novel paradigm for understanding and overcoming TRAIL resistance, in particular how HIV-infected cells escape immune elimination by the TRAIL:TRAILshort receptor axis.
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