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Pretreatment hepatitis C virus (HCV)-specific lymphoproliferative (LP) responses, neutralizing antibody (NA) responses, intrahepatic cytotoxic T lymphocyte (CTL) responses, and HCV quasi-species (QS) diversity and complexity were examined in patients with advanced hepatic fibrosis (Ishak fibrosis score of > or = 3) and prior nonresponse to interferon (IFN)- alpha therapy who were enrolled in the initial phase of the Hepatitis C Antiviral Long-Term Treatment against Cirrhosis Trial. Positive baseline HCV E1- and/or E2-specific NA responses (P = .01) and higher baseline HCV QS diversity (P = .01) were more commonly found in patients who did not become sustained virologic responders (SVRs) at week 72 (W72) than they were in those who did. No patients with positive results for both the LP and NA assays achieved a sustained virologic response. Multiple logistic regression analysis revealed that, when the presence of cirrhosis, prior ribavirin therapy, genotype 1 infection, log serum HCV RNA level, and receipt of >80% of the prescribed medication were controlled for, a sustained virologic response (W72) was negatively correlated with positive baseline LP assay results (P = .02) and with 1 or more positive assays (LP, NA, or CTL) (P = .02). No differences were noted in baseline intrahepatic CTL activity between SVRs and non-SVRs. Thus, in patients with advanced hepatic fibrosis due to HCV infection, pretreatment HCV-specific immune responses and increased QS variability appear to hinder viral clearance by pegylated IFN- alpha 2a and ribavirin combination therapy. [ABSTRACT FROM AUTHOR]

Molecular details of genome packaging are little understood for the majority of viruses. In enteroviruses (EVs), cleavage of the structural protein VP0 into VP4 and VP2 is initiated by the incorporation of RNA into the assembling virion and is essential for infectivity. We have applied a combination of bioinformatic, molecular and structural approaches to generate the first high-resolution structure of an intermediate in the assembly pathway, termed a provirion, which contains RNA and intact VP0. We have demonstrated an essential role of VP0 E096 in VP0 cleavage independent of RNA encapsidation and generated a new model of capsid maturation, supported by bioinformatic analysis. This provides a molecular basis for RNA-dependence, where RNA induces conformational changes required for VP0 maturation, but that RNA packaging itself is not sufficient to induce maturation. These data have implications for understanding production of infectious virions and potential relevance for future vaccine and antiviral drug design., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kingston et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In a pivotal trial (EPIC-HR), a 5-day course of oral ritonavir-boosted nirmatrelvir, given early during symptomatic SARS-CoV-2 infection (within three days of symptoms onset), decreased hospitalization and death by 89.1% and nasal viral load by 0.87 log relative to placebo in high-risk individuals. Yet, nirmatrelvir/ritonavir failed as post-exposure prophylaxis in a trial, and frequent viral rebound has been observed in subsequent cohorts. We develop a mathematical model capturing viral-immune dynamics and nirmatrelvir pharmacokinetics that recapitulates viral loads from this and another clinical trial (PLATCOV). Our results suggest that nirmatrelvir's in vivo potency is significantly lower than in vitro assays predict. According to our model, a maximally potent agent would reduce the viral load by approximately 3.5 logs relative to placebo at 5 days. The model identifies that earlier initiation and shorter treatment duration are key predictors of post-treatment rebound. Extension of treatment to 10 days for Omicron variant infection in vaccinated individuals, rather than increasing dose or dosing frequency, is predicted to lower the incidence of viral rebound significantly., (© 2024. The Author(s).)

Existing pharmacodynamic (PD) mathematical models for drug combinations discriminate antagonistic, additive, multiplicative, and synergistic effects, but fail to consider how concentration-dependent drug interaction effects may vary across an entire dose-response matrix. We developed a two-way pharmacodynamic (TWPD) model to capture the PD of two-drug combinations. TWPD captures interactions between upstream and downstream drugs that act on different stages of viral replication, by quantifying upstream drug efficacy and concentration-dependent effects on downstream drug pharmacodynamic parameters. We applied TWPD to previously published in vitro drug matrixes for repurposed potential anti-Ebola and anti-SARS-CoV-2 drug pairs. Depending on the drug pairing, the model recapitulated combined efficacies as or more accurately than existing models and can be used to infer efficacy at untested drug concentrations. TWPD fits the data slightly better in one direction for all drug pairs, meaning that we can tentatively infer the upstream drug. Based on its high accuracy, TWPD could be used in concert with PK models to estimate the therapeutic effects of drug pairs in vivo ., Competing Interests: The authors declare no conflict of interest.

Background: Persons with HIV (PWH) on antiretroviral therapy (ART) have persistent immune activation associated with increased risk for non-AIDS related diseases. Latent tuberculosis infection (LTBI), endemic in Africa, may contribute to this immune dysregulation. We evaluated the impact of HIV and TB co-infection on plasma pro- and anti-inflammatory cytokines among Kenyan adults., Methods: We compared data from 221 PWH on long-term ART and 177 HIV-negative adults examining biomarkers of pro-[sCD14, interleukin (IL)-2, IL-6, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), IL-12p70, IL-17A] and anti(IL-4, IL-5, IL-13) inflammatory cytokines, by HIV/LTBI status (HIV+LTBI+, HIV+LTBI-, HIV-LTBI+, HIV-LTBI-). LTBI was diagnosed based on a positive QuantiFERON TB Gold-Plus test in the absence of active TB symptoms. Linear regression was used to evaluate the associations of HIV, LTBI, and HIV/LTBI status with biomarkers adjusting for clinical factors including HIV-specific factors., Results: Half of the participants were women and 52% had LTBI. HIV was independently associated with higher sCD14, IL-15, IL-6, IL-4, IL-5. LTBI was independently associated with higher TNF-α, IL-12p70, IL-17A, IL-4, IL-13 in adjusted models ( P  < 0.05). LTBI status was associated with higher IL-4 and IL-12p70 only among PWH, but not HIV-negative participants ( P  < 0.05 for interactions). In multivariate analysis, only HIV+LTBI+ demonstrated elevated levels of TNF-α, IL-6, IL-12p70, IL-15, IL-17A, IL4, IL-5, IL-13 in comparison to the HIV-LTBI- ( P  < 0.05 for all). The effect of LTBI on cytokines among PWH was independent of CD4 + T-cell count and ART duration., Conclusions: Despite viral suppression, persons with HIV and LTBI exhibit abnormal cytokine production accompanied by high concentrations of pro- and anti-inflammatory cytokines., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

The ongoing coronavirus disease 2019 (COVID-19) pandemic has highlighted the need to better understand virus-host interactions. We developed a network-based method that expands the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-host protein interaction network and identifies host targets that modulate viral infection. To disrupt the SARS-CoV-2 interactome, we systematically probed for potent compounds that selectively target the identified host proteins with high expression in cells relevant to COVID-19. We experimentally tested seven chemical inhibitors of the identified host proteins for modulation of SARS-CoV-2 infection in human cells that express ACE2 and TMPRSS2. Inhibition of the epigenetic regulators bromodomain-containing protein 4 (BRD4) and histone deacetylase 2 (HDAC2), along with ubiquitin-specific peptidase (USP10), enhanced SARS-CoV-2 infection. Such proviral effect was observed upon treatment with compounds JQ1, vorinostat, romidepsin and spautin-1, when measured by cytopathic effect and validated by viral RNA assays, suggesting that the host proteins HDAC2, BRD4 and USP10 have antiviral functions. We observed marked differences in antiviral effects across cell lines, which may have consequences for identification of selective modulators of viral infection or potential antiviral therapeutics. While network-based approaches enable systematic identification of host targets and selective compounds that may modulate the SARS-CoV-2 interactome, further developments are warranted to increase their accuracy and cell-context specificity., (© The Author(s) 2022. Published by Oxford University Press.)

Three directly acting antivirals (DAAs) demonstrated substantial reduction in COVID-19 hospitalizations and deaths in clinical trials. However, these agents did not completely prevent severe illness and are associated with cases of rebound illness and viral shedding. Combination regimens can enhance antiviral potency, reduce the emergence of drug-resistant variants, and lower the dose of each component in the combination. Concurrently targeting virus entry and virus replication offers opportunities to discover synergistic drug combinations. While combination antiviral drug treatments are standard for chronic RNA virus infections, no antiviral combination therapy has been approved for SARS-CoV-2. Here, we demonstrate that combining host-targeting antivirals (HTAs) that target TMPRSS2 and hence SARS-CoV-2 entry, with the DAA molnupiravir, which targets SARS-CoV-2 replication, synergistically suppresses SARS-CoV-2 infection in Calu-3 lung epithelial cells. Strong synergy was observed when molnupiravir, an oral drug, was combined with three TMPRSS2 (HTA) oral or inhaled inhibitors: camostat, avoralstat, or nafamostat. The combination of camostat plus molnupiravir was also effective against the beta and delta variants of concern. The pyrimidine biosynthesis inhibitor brequinar combined with molnupiravir also conferred robust synergistic inhibition. These HTA+DAA combinations had similar potency to the synergistic all-DAA combination of molnupiravir plus nirmatrelvir, the protease inhibitor found in paxlovid. Pharmacodynamic modeling allowed estimates of antiviral potency at all possible concentrations of each agent within plausible therapeutic ranges, suggesting possible in vivo efficacy. The triple combination of camostat, brequinar, and molnupiravir further increased antiviral potency. These findings support the development of HTA+DAA combinations for pandemic response and preparedness. IMPORTANCE Imagine a future viral pandemic where if you test positive for the new virus, you can quickly take some medicines at home for a few days so that you do not get too sick. To date, only single drugs have been approved for outpatient use against SARS-CoV-2, and we are learning that these have some limitations and may succumb to drug resistance. Here, we show that combinations of two oral drugs are better than the single ones in blocking SARS-CoV-2, and we use mathematical modeling to show that these drug combinations are likely to work in people. We also show that a combination of three oral drugs works even better at eradicating the virus. Our findings therefore bode well for the development of oral drug cocktails for at home use at the first sign of an infection by a coronavirus or other emerging viral pathogens.

Broadly effective antiviral therapies must be developed to be ready for clinical trials, which should begin soon after the emergence of new life-threatening viruses. Here, we pave the way towards this goal by reviewing conserved druggable virus-host interactions, mechanisms of action, immunomodulatory properties of available broad-spectrum antivirals (BSAs), routes of BSA delivery, and interactions of BSAs with other antivirals. Based on the review, we concluded that the range of indications of BSAs can be expanded, and new pan- and cross-viral mono- and combinational therapies can be developed. We have also developed a new scoring algorithm that can help identify the most promising few of the thousands of potential BSAs and BSA-containing drug cocktails (BCCs) to prioritize their development during the critical period between the identification of a new virus and the development of virus-specific vaccines, drugs, and therapeutic antibodies., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Precision-cut human liver slice cultures (PCLS) have become an important alternative immunological platform in preclinical testing. To further evaluate the capacity of PCLS, we investigated the innate immune response to TLR3 agonist (poly-I:C) and TLR4 agonist (LPS) using normal and diseased liver tissue. Pathological liver tissue was obtained from patients with active chronic HCV infection, and patients with former chronic HCV infection cured by recent Direct-Acting Antiviral (DAA) drug therapy. We found that hepatic innate immunity in response to TLR3 and TLR4 agonists was not suppressed but enhanced in the HCV-infected tissue, compared with the healthy controls. Furthermore, despite recent HCV elimination, DAA-cured liver tissue manifested ongoing abnormalities in liver immunity: sustained abnormal immune gene expression in DAA-cured samples was identified in direct ex vivo measurements and in TLR3 and TLR4 stimulation assays. Genes that were up-regulated in chronic HCV-infected liver tissue were mostly characteristic of the non-parenchymal cell compartment. These results demonstrated the utility of PCLS in studying both liver pathology and innate immunity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wu, Roberto, Knupp, Greninger, Truong, Hollingshead, Kenerson, Tuefferd, Chen, Koelle, Horton, Jerome, Polyak, Yeung and Crispe.)

The world was unprepared for coronavirus disease 2019 (COVID-19) and remains ill-equipped for future pandemics. While unprecedented strides have been made developing vaccines and treatments for COVID-19, there remains a need for highly effective and widely available regimens for ambulatory use for novel coronaviruses and other viral pathogens. We posit that a priority is to develop pan-family drug cocktails to enhance potency, limit toxicity, and avoid drug resistance. We urge cocktail development for all viruses with pandemic potential both in the short term (<1 to 2 years) and longer term with pairs of drugs in advanced clinical testing or repurposed agents approved for other indications. While significant efforts were launched against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in vitro and in the clinic, many studies employed solo drugs and had disappointing results. Here, we review drug combination studies against SARS-CoV-2 and other viruses and introduce a model-driven approach to assess drug pairs with the highest likelihood of clinical efficacy. Where component agents lack sufficient potency, we advocate for synergistic combinations to achieve therapeutic levels. We also discuss issues that stymied therapeutic progress against COVID-19, including testing of agents with low likelihood of efficacy late in clinical disease and lack of focus on developing virologic surrogate endpoints. There is a need to expedite efficient clinical trials testing drug combinations that could be taken at home by recently infected individuals and exposed contacts as early as possible during the next pandemic, whether caused by a coronavirus or another viral pathogen. The approach herein represents a proactive plan for global viral pandemic preparedness.

Objectives: Heightened systemic inflammation is common in obese individuals and persons with HIV (PWH) and is independently associated with an increased risk of cardiovascular diseases (CVDs). We investigated the combined effect of central obesity, a surrogate measure of visceral fat and HIV on circulating levels of inflammatory cytokines among Kenyan adults., Design: A cross-sectional study., Methods: We analysed and compared data from 287 virally suppressed PWH and 277 noninfected Kenyan adults, including biomarkers of gut epithelial dysfunction (intestinal fatty acid binding protein), monocyte activation (soluble CD163 and CD14) and inflammation [interleukin (IL)-1β, IL-6, TNF-α and hsCRP] by HIV/central obesity status (HIV-positive/obese, HIV-negative/obese, HIV-positive/nonobese and HIV-negative/nonobese). Central obesity was defined as waist circumference more than 80 cm for women and more than 94 cm for men. We assessed the association of HIV/obesity status with elevated biomarkers (>75th percentile) using logistic regression., Results: Median age for participants was 44 years and 37% were centrally obese. Levels of all biomarkers were higher among the HIV-positive/obese compared with the HIV-negative/nonobese (P < 0.05 for all comparisons). The HIV-positive/obese group had the greatest odds of having elevated inflammatory biomarkers compared with other groups even after adjustment of age, BMI and other conventional CVD risk factors (P < 0.05 for all). Additional adjustment for sCD163 in the multivariate model substantially attenuated the association for HIV-positive/obesity with IL-1β, IL-6 and TNF-α but not hsCRP. The contribution of HIV-positive/obesity to inflammation was independent of the degree of immunosuppression., Conclusion: Central obesity is prevalent among virally suppressed African PWH and is associated with greater inflammation and monocyte activation independent of other comorbidities and HIV-specific factors., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

Neglected diseases caused by arenaviruses such as Lassa virus (LASV) and filoviruses like Ebola virus (EBOV) primarily afflict resource-limited countries, where antiviral drug development is often minimal. Previous studies have shown that many approved drugs developed for other clinical indications inhibit EBOV and LASV and that combinations of these drugs provide synergistic suppression of EBOV, often by blocking discrete steps in virus entry. We hypothesize that repurposing of combinations of orally administered approved drugs provides effective suppression of arenaviruses. In this report, we demonstrate that arbidol, an approved influenza antiviral previously shown to inhibit EBOV, LASV, and many other viruses, inhibits murine leukemia virus (MLV) reporter viruses pseudotyped with the fusion glycoproteins (GPs) of other arenaviruses (Junin virus [JUNV], lymphocytic choriomeningitis virus [LCMV], and Pichinde virus [PICV]). Arbidol and other approved drugs, including aripiprazole, amodiaquine, sertraline, and niclosamide, also inhibit infection of cells by infectious PICV, and arbidol, sertraline, and niclosamide inhibit infectious LASV. Combining arbidol with aripiprazole or sertraline results in the synergistic suppression of LASV and JUNV GP-bearing pseudoviruses. This proof-of-concept study shows that arenavirus infection in vitro can be synergistically inhibited by combinations of approved drugs. This approach may lead to a proactive strategy with which to prepare for and control known and new arenavirus outbreaks., (Copyright © 2021 Herring et al.)

Chronic hepatitis C (CHC) is a major cause of hepatocellular carcinoma (HCC) worldwide. While directly acting antiviral (DAA) drugs are now able to cure virtually all hepatitis C virus (HCV) infections, even in subjects with advanced liver disease, what happens to the liver and progression of the disease after DAA-induced cure of viremia is only beginning to emerge. Several large-scale clinical studies in different patient populations have shown that patients with advanced liver disease maintain a risk for developing HCC even when the original instigator, the virus, is eliminated by DAAs. Here we review emerging studies derived from multiple, complementary experimental systems involving patient liver tissues, human liver cell cultures, human liver slice cultures, and animal models, showing that HCV infection induces epigenetic, signaling, and gene expression changes in the liver associated with altered hepatic innate immunity and liver cancer risk. Of critical importance is the fact that these virus-induced abnormalities persist after DAA cure of HCV. These nascent findings portend the discovery of pathways involved in post-HCV immunopathogenesis, which may be clinically actionable targets for more comprehensive care of DAA-cured individuals.

Introduction: Markers of monocyte/macrophage activation and vascular inflammation are associated with HIV-related cardiovascular diseases (CVD) and mortality. We compared these markers among African people living with HIV (PLWH) and HIV-negative adults, and examined risk factors associated with elevated biomarkers (>75th percentile) in PLWH on antiretroviral therapy (ART)., Design: Cross-sectional study., Methods: We measured serum concentrations of a gut integrity biomarker (intestinal-fatty acid binding protein), monocyte/macrophage activation biomarkers (soluble CD14 and CD163), and vascular inflammation biomarkers [soluble intercellular adhesion molecule 1 (sICAM-1) and soluble vascular adhesion molecule 1 (sVCAM-1)]. We assessed the relationship of these inflammatory parameters with HIV, using logistic regression adjusting for traditional CVD risk factors., Results: Among the 541 participants, median age was 43 years and half were female. Among 275 PLWH, median CD4 T-cell count and duration of ART use was 509 cells/μl and 8 years, respectively. PLWH had significantly higher prevalence of elevated inflammatory biomarkers compared with HIV-negative individuals even after adjustment for traditional CVD risk factors. Compared with individuals without HIV, the prevalence of elevated biomarkers was highest among persons with detectable viral load and CD4 T-cell counts 200 cells/μl or less. In a subanalysis among PLWH, nadir CD4 T-cell count 200 cells/μl or less was associated with elevated soluble CD14 (sCD14); dyslipidemia with elevated sCD14, sICAM-1, and sVCAM-1; and overweight/obesity with reduced sCD14. Longer ART exposure (>4 years) was associated with reduced sVCAM-1 and sICAM-1., Conclusion: HIV and not traditional CVD risk factors is a primary contributor of monocyte/macrophage activation and inflammation despite ART. Anti-inflammatory therapies in addition to ART may be necessary to reduce these immune dysregulations and improve health outcomes of African PLWH.

Chronic hepatitis B virus (HBV) infection is a major public health problem. New treatment approaches are needed because current treatments do not target covalently closed circular DNA (cccDNA), the template for HBV replication, and rarely clear the virus. We harnessed adeno-associated virus (AAV) vectors and CRISPR- Staphylococcus aureus ( Sa )Cas9 to edit the HBV genome in liver-humanized FRG mice chronically infected with HBV and receiving entecavir. Gene editing was detected in livers of five of eight HBV-specific AAV- Sa Cas9-treated mice, but not control mice, and mice with detectable HBV gene editing showed higher levels of Sa Cas9 delivery to HBV + human hepatocytes than those without gene editing. HBV-specific AAV- Sa Cas9 therapy significantly improved survival of human hepatocytes, showed a trend toward decreasing total liver HBV DNA and cccDNA, and was well tolerated. This work provides evidence for the feasibility and safety of in vivo gene editing for chronic HBV infections, and it suggests that with further optimization, this approach may offer a plausible way to treat or even cure chronic HBV infections., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

Background: Residual monocyte activation may contribute to increased risk for endothelial dysfunction and subsequent atherosclerotic cardiovascular diseases (CVDs) among people with HIV (PWH) on antiretroviral therapy (ART). We examined the relationship between monocyte activation and endothelial activation in PWH in Kenya., Methods: Serum levels of markers of endothelial activation (soluble/circulating intercellular [sICAM-1] and vascular [sVCAM-1] cell adhesion molecule-1), intestinal barrier dysfunction (intestinal fatty acid binding protein [I-FABP]), and monocyte activation (soluble CD14 [sCD14]) were measured in 275 PWH on ART and 266 HIV-negative persons. Linear regression was used to evaluate associations, adjusting for demographic and traditional CVD risk factors., Results: Among 541 participants, the median age was 43 years, 50% were female, and most PWH were virally suppressed (97%). sICAM-1 and sVCAM-1 levels were significantly higher in PWH than in HIV-negative participants ( P  < .001 for both). After further adjustment for traditional CVD risk factors, HIV infection remained associated with 49% (95% CI, 33% to 67%) greater sICAM-1 and 30% (95% CI, 14% to 48%) greater sVCAM-1 relative to uninfected controls. Adjustment for sCD14 substantially attenuated the difference between PWH and HIV-negative individuals. In a stratified analysis of PWH, both sICAM-1 and sVCAM-1 were positively associated with sCD14 ( P  < .001)., Conclusions: Despite viral suppression, African PWH have evidence of enhanced endothelial activation associated with sCD14, suggesting that monocyte activation plays a role in atherosclerotic plaque development. Future studies are needed to determine mechanistic pathways leading to monocyte activation in this population., (© The Author(s) 2020. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

To determine the prevalence and correlates of metabolic syndrome (MetS) and compare 10-year cardiovascular disease (CVD) risk among Kenyan adults with and without HIV infection.We conducted a cross-sectional study among adults ≥30 years of age with and without HIV infection seeking care at Kisumu County Hospital. Participants completed a health questionnaire and vital signs, anthropomorphic measurements, and fasting blood were obtained. MetS was defined using 2009 Consensus Criteria and 10-year Atherosclerotic CVD (ASCVD) risk score was calculated. Chi-square, independent t tests, Wilcoxon ranksum test and multivariable logistic regression were used to determine differences and associations between HIV and MetS, CVD risk factors and ASCVD risk score.A total of 300 people living with HIV (PLWHIV) and 298 HIV-negative participants with median age 44 years enrolled, 50% of whom were female. The prevalence of MetS was 8.9% overall, but lower among PLWHIV than HIV-negative participants (6.3% vs 11.6%, respectively; P = .001). The most prevalent MetS components were elevated blood pressure, decreased high density lipoprotein, and abdominal obesity. Adjusting for covariates, PLWHIV were 66% less likely to have MetS compared to HIV-negative participants (adjusted odds ratio [aOR] 0.34; 95% confidence interval [95%CI] 0.18, 0.65; P = .005). Median ASCVD risk score was also lower among PLWHIV compared to HIV-negative participants (1.7% vs 3.0%, P = .002).MetS was more common among HIV-negative than HIV-positive adults, and HIV-negative adults were at greater risk for CVD compared to PLWHIV. These data support integration of routine CVD screening and management into health programs in resource-limited settings, regardless of HIV status.

Pharmacological HIV-1 reactivation to reverse latent infection has been extensively studied. However, HIV-1 reactivation also occurs naturally, as evidenced by occasional low-level viremia ("viral blips") during antiretroviral treatment (ART). Clarifying where blips originate from and how they happen could provide clues to stimulate latency reversal more effectively and safely or to prevent viral rebound following ART cessation. We studied HIV-1 reactivation in the female genital tract, a dynamic anatomical target for HIV-1 infection throughout all disease stages. We found that primary endocervical epithelial cells from several women reactivated HIV-1 from latently infected T cells. The endocervical cells' HIV-1 reactivation capacity further increased upon Toll-like receptor 3 stimulation with poly(I·C) double-stranded RNA or infection with herpes simplex virus 2 (HSV-2). Notably, acyclovir did not eliminate HSV-2-induced HIV-1 reactivation. While endocervical epithelial cells secreted large amounts of several cytokines and chemokines, especially tumor necrosis factor alpha (TNF-α), CCL3, CCL4, and CCL20, their HIV-1 reactivation capacity was almost completely blocked by TNF-α neutralization alone. Thus, immunosurveillance activities by columnar epithelial cells in the endocervix can cause endogenous HIV-1 reactivation, which may contribute to viral blips during ART or rebound following ART interruption. IMPORTANCE A reason that there is no universal cure for HIV-1 is that the virus can hide in the genome of infected cells in the form of latent proviral DNA. This hidden provirus is protected from antiviral drugs until it eventually reactivates to produce new virions. It is not well understood where in the body or how this reactivation occurs. We studied HIV-1 reactivation in the female genital tract, which is often the portal of HIV-1 entry and which remains a site of infection throughout the disease. We found that the columnar epithelial cells lining the endocervix, the lower part of the uterus, are particularly effective in reactivating HIV-1 from infected T cells. This activity was enhanced by certain microbial stimuli, including herpes simplex virus 2, and blocked by antibodies against the inflammatory cytokine TNF-α. Avoiding HIV-1 reactivation could be important for maintaining a functional HIV-1 cure when antiviral therapy is stopped., (Copyright © 2020 American Society for Microbiology.)

Arbidol (ARB, also known as umifenovir) is used clinically in several countries as an anti-influenza virus drug. ARB inhibits multiple enveloped viruses in vitro and the primary mode of action is inhibition of virus entry and/or fusion of viral membranes with intracellular endosomal membranes. ARB is also an effective inhibitor of non-enveloped poliovirus types 1 and 3. In the current report, we evaluate the antiviral potential of ARB against another picornavirus, foot-and-mouth disease virus (FMDV), a member of the genus Aphthovirus and an important veterinary pathogen. ARB inhibits the replication of FMDV RNA sub-genomic replicons. ARB inhibition of FMDV RNA replication is not a result of generalized inhibition of cellular uptake of cargo, such as transfected DNA, and ARB can be added to cells up to 3 h post-transfection of FMDV RNA replicons and still inhibit FMDV replication. ARB prevents the recovery of FMDV replication upon withdrawal of the replication inhibitor guanidine hydrochloride (GuHCl). Although restoration of FMDV replication is known to require de novo protein synthesis upon GuHCl removal, ARB does not suppress cellular translation or FMDV internal ribosome entry site (IRES)-driven translation. ARB also inhibits infection with the related Aphthovirus, equine rhinitis A virus (ERAV). Collectively, the data demonstrate that ARB can inhibit some non-enveloped picornaviruses. The data are consistent with inhibition of picornavirus genome replication, possibly via the disruption of intracellular membranes on which replication complexes are located.

Antiviral therapies that impede virus entry are attractive because they act on the first phase of the infectious cycle. Drugs that target pathways common to multiple viruses are particularly desirable when laboratory-based viral identification may be challenging, e.g., in an outbreak setting. We are interested in identifying drugs that block both Ebola virus (EBOV) and Lassa virus (LASV), two unrelated but highly pathogenic hemorrhagic fever viruses that have caused outbreaks in similar regions in Africa and share features of virus entry: use of cell surface attachment factors, macropinocytosis, endosomal receptors, and low pH to trigger fusion in late endosomes. Toward this goal, we directly compared the potency of eight drugs known to block EBOV entry with their potency as inhibitors of LASV entry. Five drugs (amodiaquine, apilimod, arbidol, niclosamide, and zoniporide) showed roughly equivalent degrees of inhibition of LASV and EBOV glycoprotein (GP)-bearing pseudoviruses; three (clomiphene, sertraline, and toremifene) were more potent against EBOV. We then focused on arbidol, which is licensed abroad as an anti-influenza drug and exhibits activity against a diverse array of clinically relevant viruses. We found that arbidol inhibits infection by authentic LASV, inhibits LASV GP-mediated cell-cell fusion and virus-cell fusion, and, reminiscent of its activity on influenza virus hemagglutinin, stabilizes LASV GP to low-pH exposure. Our findings suggest that arbidol inhibits LASV fusion, which may partly involve blocking conformational changes in LASV GP. We discuss our findings in terms of the potential to develop a drug cocktail that could inhibit both LASV and EBOV. IMPORTANCE Lassa and Ebola viruses continue to cause severe outbreaks in humans, yet there are only limited therapeutic options to treat the deadly hemorrhagic fever diseases they cause. Because of overlapping geographic occurrences and similarities in mode of entry into cells, we seek a practical drug or drug cocktail that could be used to treat infections by both viruses. Toward this goal, we directly compared eight drugs, approved or in clinical testing, for the ability to block entry mediated by the glycoproteins of both viruses. We identified five drugs with approximately equal potencies against both. Among these, we investigated the modes of action of arbidol, a drug licensed abroad to treat influenza infections. We found, as shown for influenza virus, that arbidol blocks fusion mediated by the Lassa virus glycoprotein. Our findings encourage the development of a combination of approved drugs to treat both Lassa and Ebola virus diseases., (Copyright © 2019 American Society for Microbiology.)

There are many emerging and re-emerging globally prevalent viruses for which there are no licensed vaccines or antiviral medicines. Arbidol (ARB, umifenovir), used clinically for decades in several countries as an anti-influenza virus drug, inhibits many other viruses. In the current study, we show that ARB inhibits six different isolates of Zika virus (ZIKV), including African and Asian lineage viruses in multiple cell lines and primary human vaginal and cervical epithelial cells. ARB protects against ZIKV-induced cytopathic effects. Time of addition studies indicate that ARB is most effective at suppressing ZIKV when added to cells prior to infection. Moreover, ARB inhibits pseudoviruses expressing the ZIKV Envelope glycoprotein. Thus, ARB, a broadly acting anti-viral agent with a well-established safety profile, inhibits ZIKV, likely by blocking viral entry.

Silymarin (SM), and its flavonolignan components, alter cellular metabolism and inhibit inflammatory status in human liver and T cell lines. In this study, we hypothesized that SM suppresses both acute and chronic immune activation (CIA), including in the context of HIV infection. SM treatment suppressed the expression of T cell activation and exhaustion markers on CD4+ and CD8+ T cells from chronically-infected, HIV-positive subjects. SM also showed a trend towards modifying CD4+ T cell memory subsets from HIV+ subjects. In the HIV-negative setting, SM treatment showed trends towards suppressing pro-inflammatory cytokines from non-activated and pathogen-associated molecular pattern (PAMP)-activated primary human monocytes, and non-activated and cytokine- and T cell receptor (TCR)-activated mucosal-associated invariant T (MAIT) cells. The data suggest that SM elicits broad anti-inflammatory and immunoregulatory activity in primary human immune cells. By using novel compounds to alter cellular inflammatory status, it may be possible to regulate inflammation in both non-disease and disease states., Competing Interests: The authors have declared that no competing interests exist.

Chronic viral infections like those caused by hepatitis C virus (HCV) and human immunodeficiency virus (HIV) cause disease that establishes an ongoing state of chronic inflammation. While there have been tremendous improvements towards curing HCV with directly acting antiviral agents (DAA) and keeping HIV viral loads below detection with antiretroviral therapy (ART), there is still a need to control inflammation in these diseases. Recent studies indicate that many natural products like curcumin, resveratrol and silymarin alter cellular metabolism and signal transduction pathways via enzymes such as adenosine monophosphate kinase (AMPK) and mechanistic target of rapamycin (mTOR), and these pathways directly influence cellular inflammatory status (such as NF-κB) and immune function. Natural products represent a vast toolkit to dissect and define how cellular metabolism controls cellular immune and inflammatory function.

Unlabelled: Cytokines are a group of small secreted proteins that mediate a diverse range of immune and nonimmune responses to inflammatory and microbial stimuli. Only a few of these cytokines mount an antiviral response, including type I, II, and III interferons (IFNs). During viral infections and under inflammatory conditions, a number of cytokines and chemokines are coproduced with IFN; however, no systematic study exists on the interactions of the cytokine repertoire with the IFN response. Here, we performed the largest cytokine and chemokine screen (the human cytokinome, with >240 members) to investigate their modulation of type I and type II IFN responses in a cell line model. We evaluated the cytokine activities in both IFN-stimulated response element (ISRE) and IFN-γ activation sequence (GAS) reporter systems. Several cytokine clusters that augment either or both ISRE- and GAS-mediated responses to IFNs were derived from the screen. We identified novel modulators of IFN response-betacellulin (BTC), interleukin 11 (IL-11), and IL-17F-that caused time-dependent induction of the IFN response. The ability to induce endogenous IFN-β and IFN-stimulated genes varies among these cytokines and was largely dependent on Stat1, as assessed by Stat1 mutant fibroblasts. Certain cytokines appear to augment the IFN-β response through the NF-κB pathway. The novel IFN-like cytokines augmented the antiviral activity of IFN-α against several RNA viruses, including encephalomyocarditis virus, vesicular stomatitis virus, and influenza virus, in susceptible cell lines. Overall, the study represents a large-scale analysis of cytokines for enhancing the IFN response and identified cytokines capable of enhancing Stat1, IFN-induced gene expression, and antiviral activities., Importance: Innate immunity to viruses is an early defense system to ward off viruses. One mediator is interferon (IFN), which activates a cascade of biochemical events that aim to control the virus life cycle. In our work, we examined more than 200 cytokines, soluble mediators produced within the body as a result of infection, for the ability to enhance IFN action. We identified enhanced interactions with specific IFNs and cytokines. We also revealed that betacellulin, IL-17, and IL-11 cytokines have the novel property of enhancing the antiviral action of IFN against several viruses. These results demonstrate that the human genome codes for previously unknown proteins with unrelated functions that can augment the innate immunity to viruses. Knowing these interactions not only helps our understanding of immunity to viruses and emerging diseases, but can also lead to devising possible new therapeutics by enhancing the mediator of antiviral action itself, IFN., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Plasmacytoid Dendritic Cells (pDCs) represent a key immune cell population in the defense against viruses. pDCs detect viral pathogen associated molecular patterns (PAMPs) through pattern recognition receptors (PRR). PRR/PAMP interactions trigger signaling events that induce interferon (IFN) production to initiate local and systemic responses. pDCs produce Type I and Type III (IFNL) IFNs in response to HCV RNA. Extracellular HCV core protein (Core) is found in the circulation in chronic infection. This study defined how Core modulates PRR signaling in pDCs. Type I and III IFN expression and production following exposure to recombinant Core or β-galactosiade was assessed in human GEN2.2 cells, a pDC cell line. Core suppressed type I and III IFN production in response to TLR agonists and the HCV PAMP agonist of RIG-I. Core suppression of IFN induction was linked with decreased IRF-7 protein levels and increased non-phosphorylated STAT1 protein. Circulating Core protein interferes with PRR signaling by pDCs to suppress IFN production. Strategies to define and target Core effects on pDCs may serve to enhance IFN production and antiviral actions against HCV.

Hepatitis C virus (HCV) infection of hepatocytes leads to transcriptional induction of the chemokine CXCL10, which is considered an interferon (IFN)-stimulated gene. However, we have recently shown that IFNs are not required for CXCL10 induction in hepatocytes during acute HCV infection. Since the CXCL10 promoter contains binding sites for several proinflammatory transcription factors, we investigated the contribution of these factors to CXCL10 transcriptional induction during HCV infection in vitro. Wild-type and mutant CXCL10 promoter-luciferase reporter constructs were used to identify critical sites of transcriptional regulation. The proximal IFN-stimulated response element (ISRE) and NF-κB binding sites positively regulated CXCL10 transcription during HCV infection as well as following exposure to poly(I·C) (a Toll-like receptor 3 [TLR3] stimulus) and 5' poly(U) HCV RNA (a retinoic acid-inducible gene I [RIG-I] stimulus) from two viral genotypes. Conversely, binding sites for AP-1 and CCAAT/enhancer-binding protein β (C/EBP-β) negatively regulated CXCL10 induction in response to TLR3 and RIG-I stimuli, while only C/EBP-β negatively regulated CXCL10 during HCV infection. We also demonstrated that interferon-regulatory factor 3 (IRF3) is transiently recruited to the proximal ISRE during HCV infection and localizes to the nucleus in HCV-infected primary human hepatocytes. Furthermore, IRF3 activated the CXCL10 promoter independently of type I or type III IFN signaling. The data indicate that sensing of HCV infection by RIG-I and TLR3 leads to direct recruitment of NF-κB and IRF3 to the CXCL10 promoter. Our study expands upon current knowledge regarding the mechanisms of CXCL10 induction in hepatocytes and lays the foundation for additional mechanistic studies that further elucidate the combinatorial and synergistic aspects of immune signaling pathways.

In this report, we further characterized the effects of silibinin (SbN), derived from milk thistle extract, and Legalon-SIL (SIL), a water-soluble derivative of SbN, on T cell metabolism and HIV infection. We assessed the effects of SbN and SIL on peripheral blood mononuclear cells (PBMC) and CEM-T4 cells in terms of cellular growth, ATP content, metabolism, and HIV infection. SIL and SbN caused a rapid and reversible (upon removal) decrease in cellular ATP levels, which was associated with suppression of mitochondrial respiration and glycolysis. SbN, but not SIL inhibited glucose uptake. Exposure of T cells to SIL (but not SbN or metabolic inhibitors) during virus adsorption blocked HIV infection. Thus, both SbN and SIL rapidly perturb T cell metabolism in vitro, which may account for its anti-inflammatory and anti-proliferative effects that arise with prolonged exposure of cells. However, the metabolic effects are not involved in SIL's unique ability to block HIV entry., (© 2013 Elsevier Inc. All rights reserved.)

Hepatitis C virus (HCV) is a global health concern infecting 170 million people worldwide. Previous studies indicate that the extract from milk thistle known as silymarin and its main component silibinin inhibit HCV infection. Here we investigated the mechanism of anti-HCV action of silymarin-derived compounds at the molecular level. By using live-cell confocal imaging, single particle tracking, transmission electron microscopy and biochemical approaches on HCV-infected human hepatoma cells and primary hepatocytes, we show that silibinin potently inhibits HCV infection and hinders HCV entry by slowing down trafficking through clathrin-coated pits and vesicles. Detailed analyses revealed that silibinin altered the formation of both clathrin-coated pits and vesicles in cells and caused abnormal uptake and trafficking of transferrin, a well-known cargo of the clathrin endocytic pathway. Silibinin also inhibited infection by other viruses that enter cells by clathrin-mediated endocytosis including reovirus, vesicular stomatitis and influenza viruses. Our study demonstrates that silibinin inhibits HCV early steps of infection by affecting endosomal trafficking of virions. It provides new insights into the molecular mechanisms of action of silibinin against HCV entry and also suggests that silibinin is a potential broad-spectrum antiviral therapy., (© 2013 John Wiley & Sons Ltd.)

An estimated 170 million people worldwide are chronically infected with the hepatitis C virus (HCV), which is characterized histologically by a persistent immune and inflammatory response that fails to clear HCV from hepatocytes. This response is recruited to the liver, in part, by the chemokine CXCL10, the serum and intrahepatic levels of which have been inversely linked to the outcome of interferon-based therapies for hepatitis C. Bystander tissue damage from this ineffective response is thought to lead to increased hepatocyte turnover and the development of fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). However, CXCL10 is traditionally viewed as an orchestrator of the angiostatic and antitumor immune response. In this review, we will explore this duality and the pathways by which CXCL10 is produced by hepatocytes during HCV infection, its effects on resident and infiltrating immune cells, and how deregulation of these cell populations within the liver may lead to chronic liver inflammation. We will also discuss potential host-directed therapies to slow or reverse HCV-induced inflammation that leads to fibrosis, cirrhosis, and HCCs.

Purified silymarin-derived natural products from the milk thistle plant (Silybum marianum) block hepatitis C virus (HCV) infection and inhibit T cell proliferation in vitro. An intravenous formulation of silibinin (SIL), a major component of silymarin, displays anti-HCV effects in humans and also inhibits T-cell proliferation in vitro. We show that SIL inhibited replication of HIV-1 in TZM-bl cells, PBMCs, and CEM cells in vitro. SIL suppression of HIV-1 coincided with dose-dependent reductions in actively proliferating CD19+, CD4+, and CD8+ cells, resulting in fewer CD4+ T cells expressing the HIV-1 co-receptors CXCR4 and CCR5. SIL inhibition of T-cell growth was not due to cytotoxicity measured by cell cycle arrest, apoptosis, or necrosis. SIL also blocked induction of the activation markers CD38, HLA-DR, Ki67, and CCR5 on CD4+ T cells. The data suggest that SIL attenuated cellular functions involved in T-cell activation, proliferation, and HIV-1 infection. Silymarin-derived compounds provide cytoprotection by suppressing virus infection, immune activation, and inflammation, and as such may be relevant for both HIV mono-infected and HIV/HCV co-infected subjects.

Silymarin prevents liver disease in many experimental rodent models, and is the most popular botanical medicine consumed by patients with hepatitis C. Silibinin is a major component of silymarin, consisting of the flavonolignans silybin A and silybin B, which are insoluble in aqueous solution. A chemically modified and soluble version of silibinin, SIL, has been shown to potently reduce hepatitis C virus (HCV) RNA levels in vivo when administered intravenously. Silymarin and silibinin inhibit HCV infection in cell culture by targeting multiple steps in the virus lifecycle. We tested the hepatoprotective profiles of SIL and silibinin in assays that measure antiviral and anti-inflammatory functions. Both mixtures inhibited fusion of HCV pseudoparticles (HCVpp) with fluorescent liposomes in a dose-dependent fashion. SIL inhibited 5 clinical genotype 1b isolates of NS5B RNA dependent RNA polymerase (RdRp) activity better than silibinin, with IC50 values of 40-85 µM. The enhanced activity of SIL may have been in part due to inhibition of NS5B binding to RNA templates. However, inhibition of the RdRps by both mixtures plateaued at 43-73%, suggesting that the products are poor overall inhibitors of RdRp. Silibinin did not inhibit HCV replication in subgenomic genotype 1b or 2a replicon cell lines, but it did inhibit JFH-1 infection. In contrast, SIL inhibited 1b but not 2a subgenomic replicons and also inhibited JFH-1 infection. Both mixtures inhibited production of progeny virus particles. Silibinin but not SIL inhibited NF-κB- and IFN-B-dependent transcription in Huh7 cells. However, both mixtures inhibited T cell proliferation to similar degrees. These data underscore the differences and similarities between the intravenous and oral formulations of silibinin, which could influence the clinical effects of this mixture on patients with chronic liver diseases.

Assessment of post-transcriptional control relies on use of transcriptional inhibitors and is masked by copious and cryptic transcriptional induction. We screened several cellular promoters that are constitutively active yet noninducible to external stimuli. The ribosomal protein RPS30 promoter was chosen; its TATA signal and sp1 site location were optimized. The modified promoter (RPS30M) is selective to post-transcriptional effects of AU-rich elements (ARE) in the 3'UTR, while it is not transcriptionally responsive to a wide variety of agents including pro-inflammatory cytokines and RNA-binding proteins. Specific cis-acting elements can be appended to RPS30M by a cloning-free approach to allow coupled transcriptional/post-transcriptional assessment, as demonstrated with NF-kappaB and beta-catenin/wnt signaling experiments. Moreover, efficient tetracycline-regulated RPS30M was created for quantitative assessment of the half-lives of mRNAs containing AREs. The described approach provides enhanced versatility and suitability for selective post-transcriptional assessment with or without transcriptional induction.

Silymarin, also known as milk thistle extract, inhibits hepatitis C virus (HCV) infection and also displays antioxidant, anti-inflammatory, and immunomodulatory actions that contribute to its hepatoprotective effects. In the current study, we evaluated the hepatoprotective actions of the seven major flavonolignans and one flavonoid that comprise silymarin. Activities tested included inhibition of: HCV cell culture infection, NS5B polymerase activity, TNF-alpha-induced NF-kappaB transcription, virus-induced oxidative stress, and T-cell proliferation. All compounds were well tolerated by Huh7 human hepatoma cells up to 80 muM, except for isosilybin B, which was toxic to cells above 10 muM. Select compounds had stronger hepatoprotective functions than silymarin in all assays tested except in T cell proliferation. Pure compounds inhibited JFH-1 NS5B polymerase but only at concentrations above 300 muM. Silymarin suppressed TNF-alpha activation of NF-kappaB dependent transcription, which involved partial inhibition of IkappaB and RelA/p65 serine phosphorylation, and p50 and p65 nuclear translocation, without affecting binding of p50 and p65 to DNA. All compounds blocked JFH-1 virus-induced oxidative stress, including compounds that lacked antiviral activity. The most potent compounds across multiple assays were taxifolin, isosilybin A, silybin A, silybin B, and silibinin, a mixture of silybin A and silybin B. The data suggest that silymarin- and silymarin-derived compounds may influence HCV disease course in some patients. Studies where standardized silymarin is dosed to identify specific clinical endpoints are urgently needed.

Background: The hepatitis C virus (HCV) core protein is implicated in diverse aspects of HCV-induced pathogenesis. There is a paucity of information on core in acute hepatitis C infection., Methods: We analyzed core gene sequences and protein functions from 13 patients acutely infected with HCV genotype 1., Results: Although core isolates differed slightly between patients, core quasispecies were relatively homogeneous within each patient. In 2 of 4 patients studied temporally, core quasispecies did not change over time. Comparison with more than 2700 published core isolates indicated that amino acid changes from a prototype reference strain found in acute core isolates were present in chronically infected persons at low frequency (6.4%; range, 0%-32%). Core isolates associated with lipid droplets to similar degrees in Huh7 cells. Core diffusion in cells was not affected by nonconservative changes F130L and G161S in the lipid targeting domain of core. Core isolates inhibited interferon-stimulated response element- and nuclear factor kappaB-dependent transcription and tumor necrosis factor alpha-induced nuclear translocation of nuclear factor kappaB and were also secreted from Huh7 cells., Conclusions: The data suggest that upon transmission, core quasispecies undergo genetic homogenization associated with amino acid changes that are rarely found in chronic infection and that, despite genetic variation, acute core isolates retain similar functions in vitro.

Approximately 200 million people throughout the world are infected with hepatitis C virus (HCV). One of the most striking features of HCV infection is its high propensity to establish persistence (approximately 70-80%) and progressive liver injury. Galectins are evolutionarily conserved glycan-binding proteins with diverse roles in innate and adaptive immune responses. Here, we demonstrate that galectin-9, the natural ligand for the T cell immunoglobulin domain and mucin domain protein 3 (Tim-3), circulates at very high levels in the serum and its hepatic expression (particularly on Kupffer cells) is significantly increased in patients with chronic HCV as compared to normal controls. Galectin-9 production from monocytes and macrophages is induced by IFN-gamma, which has been shown to be elevated in chronic HCV infection. In turn, galectin-9 induces pro-inflammatory cytokines in liver-derived and peripheral mononuclear cells; galectin-9 also induces anti-inflammatory cytokines from peripheral but not hepatic mononuclear cells. Galectin-9 results in expansion of CD4(+)CD25(+)FoxP3(+)CD127(low) regulatory T cells, contraction of CD4(+) effector T cells, and apoptosis of HCV-specific CTLs. In conclusion, galectin-9 production by Kupffer cells links the innate and adaptive immune response, providing a potential novel immunotherapeutic target in this common viral infection.

Background & Aims: Silymarin, an extract from the seeds of the milk thistle plant Silybum marianum, has been used for centuries for the treatment of chronic liver diseases. Despite common use by patients with hepatitis C in the United States, its clinical efficacy remains uncertain. The goal of this study was to determine whether silymarin has in vitro effects on immune function that might have implications for its potential effect on hepatitis C virus (HCV)-induced liver disease., Methods: Freshly isolated peripheral blood mononuclear cells (PBMC) and T cells from HCV-infected and uninfected subjects were tested in vitro for responses to nonspecific and antigenic stimulation in the presence and absence of a standardized preparation of silymarin (MK001)., Results: Minimal MK001 toxicity on PBMC was found at concentrations between 5 and 40 microg/mL. MK001 dose dependently inhibited the proliferation and secretion of tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and interleukin (IL)-2 by PBMC stimulated with anti-CD3. In addition, MK001 inhibited proliferation by CD4(+) T cells to HCV, Candida, and tetanus protein antigens and by HLA-A2/HCV 1406-1415-specific CD8(+) T cells to allogeneic stimulation. MK001 inhibited T-cell TNF-alpha and IFN-gamma cytokine secretion to tetanus and Candida protein antigens. Finally, MK001 inhibited nuclear factor-kappaB transcriptional activation after T-cell receptor-mediated stimulation of Jurkat T cells, consistent with its ability to inhibit Jurkat T-cell proliferation and secretion of IL-2., Conclusions: Silymarin's ability to inhibit the proliferation and proinflammatory cytokine secretion of T cells, combined with its previously described antiviral effect, suggests a possible mechanism of action that could lead to clinical benefit during HCV infection.

Background & Aims: Chronic hepatitis C is a serious global medical problem necessitating effective treatment. Because standard of care with pegylated interferon plus ribavirin therapy is costly, has significant side effects, and fails to cure about half of all infections, many patients seek complementary and alternative medicine to improve their health, such as Silymarin, derived from milk thistle (Silybum marianum). Milk thistle's clinical benefits for chronic hepatitis C are unsettled due to variability in standardization of the herbal product., Methods: In the current study, we focused on the anti-inflammatory and antiviral properties of a standardized Silymarin extract (MK-001)., Results: MK-001 inhibited expression of tumor necrosis factor-alpha in anti-CD3 stimulated human peripheral blood mononuclear cells and nuclear factor kappa B-dependent transcription in human hepatoma Huh7 cells. Moreover, MK-001 dose dependently inhibited infection of Huh7 and Huh7.5.1 cells by JFH-1 virus. MK-001 displayed both prophylactic and therapeutic effects against HCV infection, and when combined with interferon-alpha, inhibited HCV replication more than interferon-alpha alone. Commercial preparations of Silymarin also displayed antiviral activity, although the effects were not as potent as MK-001. Antiviral effects of the extract were attributable in part to induction of Stat1 phosphorylation, while interferon-independent mechanisms were suggested when the extract was biochemically fractionated by high-performance liquid chromatography. Silybin A, silybin B, and isosilybin A, isosilybin B elicited the strongest anti-NF-kappaB and anti-HCV actions. These effects were independent of MK-001-induced cytotoxicity., Conclusions: The data indicate that Silymarin exerts anti-inflammatory and antiviral effects, and suggest that complementary and alternative medicine-based approaches may assist in the management of patients with chronic hepatitis C.

Hepatitis C virus (HCV) infection induces the alpha-chemokine interleukin-8 (CXCL-8), which is regulated at the levels of transcription and mRNA stability. In the current study, CXCL-8 regulation by double-stranded (ds)RNA pathways was analyzed in the context of HCV infection. A constitutively active mutant of the retinoic acid-inducible gene I (RIG-I), RIG-N, activated CXCL-8 transcription. Promoter mutagenesis experiments indicated that NF-kappaB and interferon (IFN)-stimulated response element (ISRE) binding sites were required for the RIG-N induction of CXCL-8 transcription. IFN-beta promoter stimulator 1 (IPS-1) expression also activated CXCL-8 transcription, and mutations of the ISRE and NF-kappaB binding sites reduced and abrogated CXCL-8 transcription, respectively. In the presence of wild-type RIG-I, transfection of JFH-1 RNA or JFH-1 virus infection of Huh7.5.1 cells activated the CXCL-8 promoter. Expression of IFN regulatory factor 3 (IRF-3) stimulated transcription from both full-length and ISRE-driven CXCL-8 promoters. Chromatin immunoprecipitation assays demonstrated that IRF-3 and NF-kappaB bound directly to the CXCL-8 promoter in response to virus infection and dsRNA transfection. RIG-N stabilized CXCL-8 mRNA via the AU-rich element in the 3' untranslated region of CXCL-8 mRNA, leading to an increase in its half-life following tumor necrosis factor alpha induction. The data indicate that HCV infection triggers dsRNA signaling pathways that induce CXCL-8 via transcriptional activation and mRNA stabilization and define a regulatory link between innate antiviral and inflammatory cellular responses to virus infection.

The chemokine CXCL-8 (interleukin-8) is induced by many viruses, including hepatitis C virus (HCV). In the current study, we examined CXCL-8 levels in the context of acute and chronic HCV replication in vitro. Two different small interfering RNAs were used to silence CXCL-8 mRNA and protein expression in Huh7 and BB7 replicon cells. HCV RNA synthesis in BB7 cells was inhibited by CXCL-8 knockdown. Furthermore, antibody neutralization of endogenous CXCL-8 activity inhibited HCV replication, while addition of recombinant human CXCL-8 stimulated NS5A protein expression. Moreover, CXCL-8 protein levels correlated positively with HCV RNA levels in four independent subgenomic and genomic replicon lines (R = 0.41, P = 0.0013). However, CXCL-8 mRNA levels correlated inversely with CXCL-8 protein and HCV RNA levels in all replicon lines and in Huh7 cells. Transient replication assays with strongly permissive and weakly permissive Huh7 cells and three independent subgenomic replicons with various replicative capacities revealed that CXCL-8 protein levels were higher in weakly than in strongly permissive cells. The JFH-1 subgenomic replicon, which replicated to high levels in both strongly and weakly permissive Huh7 cells, induced CXCL-8 protein to high levels in both cell types. The data indicate that in the replicon system, CXCL-8 protein levels are positively associated with chronic HCV replication and that CXCL-8 removal inhibits HCV replication. During acute HCV replication, CXCL-8 production may be inhibitory to viruses with low replicative capacity. The data underscore the complex regulation of CXCL-8 mRNA and protein expression and further suggest that in addition to contributing to HCV pathology via proinflammatory actions, CXCL-8 may have opposing antiviral and proviral effects depending on the level of HCV replication, the cellular context, and whether the infection is acute or chronic.

Arbidol (ARB) is an antiviral compound that was originally proven effective for treatment of influenza and several other respiratory viral infections. The broad spectrum of ARB anti-viral activity led us to evaluate its effect on hepatitis C virus (HCV) infection and replication in cell culture. Long-term ARB treatment of Huh7 cells chronically replicating a genomic length genotype 1b replicon resulted in sustained reduction of viral RNA and protein expression, and eventually cured HCV infected cells. Pre-treatment of human hepatoma Huh7.5.1 cells with 15 microM ARB for 24 to 48 hours inhibited acute infection with JFH-1 virus by up to 1000-fold. The inhibitory effect of ARB on HCV was not due to generalized cytotoxicity, nor to augmentation of IFN antiviral signaling pathways, but involved impaired virus-mediated membrane fusion. ARB's affinity for membranes may inhibit several aspects of the HCV lifecycle that are membrane-dependent.

Background: Previous studies have shown that levels of CXCL-8 are elevated in patients with chronic hepatitis C virus (HCV) infection and are highest in nonresponders to interferon (IFN) therapy and that CXCL-8 inhibits IFN antiviral action. CXCL-8 expression involves AU-rich elements (AREs) in 3' untranslated regions that regulate mRNA stability. CXCL-8 mRNA stability was, therefore, investigated in the context of HCV replication in 4 replicon cell lines, Huh7 and Huh7.5 cells, and primary human fetal hepatocytes., Methods: The half-life of CXCL-8 mRNA was measured by use of real-time reverse-transcription polymerase chain reaction following tumor necrosis factor (TNF)- alpha induction in the presence and absence of inhibitors of transcription and translation. Cellular mRNAs containing AREs were assessed by custom microarray analyses., Results: The half-life of CXCL-8 mRNA increased in 3 of 4 HCV replicon cell lines, particularly after treatment with TNF- alpha , a potent inducer of CXCL-8. CXCL-8 mRNA was superinduced by TNF- alpha in the presence of the protein-synthesis inhibitor cycloheximide. Analysis of >1500 ARE-containing cellular mRNAs, by use of microarrays, revealed that CXCL-8 and other newly identified ARE genes were induced by TNF- alpha in Huh7 cells and were coordinately regulated., Conclusion: The data suggest that increased CXCL-8 gene expression in the context of HCV replication involves posttranscriptional events.

Alcohol abuse reduces response rates to IFN therapy in patients with chronic hepatitis C. To model the molecular mechanisms behind this phenotype, we characterized the effects of ethanol on Jak-Stat and MAPK pathways in Huh7 human hepatoma cells, in HCV replicon cell lines, and in primary human hepatocytes. High physiological concentrations of acute ethanol activated the Jak-Stat and p38 MAPK pathways and inhibited HCV replication in several independent replicon cell lines. Moreover, acute ethanol induced Stat1 serine phosphorylation, which was partially mediated by the p38 MAPK pathway. In contrast, when combined with exogenously applied IFN-alpha, ethanol inhibited the antiviral actions of IFN against HCV replication, involving inhibition of IFN-induced Stat1 tyrosine phosphorylation. These effects of alcohol occurred independently of i) alcohol metabolism via ADH and CYP2E1, and ii) cytotoxic or cytostatic effects of ethanol. In this model system, ethanol directly perturbs the Jak-Stat pathway, and HCV replication. Infection with Hepatitis C virus is a significant cause of morbidity and mortality throughout the world. With a propensity to progress to chronic infection, approximately 70% of patients with chronic viremia develop histological evidence of chronic liver diseases including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The situation is even more dire for patients who abuse ethanol, where the risk of developing end stage liver disease is significantly higher as compared to HCV patients who do not drink 12.Recombinant interferon alpha (IFN-alpha) therapy produces sustained responses (ie clearance of viremia) in 8-12% of patients with chronic hepatitis C 3. Significant improvements in response rates can be achieved with IFN plus ribavirin combination 456 and pegylated IFN plus ribavirin 78 therapies. However, over 50% of chronically infected patients still do not clear viremia. Moreover, HCV-infected patients who abuse alcohol have extremely low response rates to IFN therapy 9, but the mechanisms involved have not been clarified.MAPKs play essential roles in regulation of differentiation, cell growth, and responses to cytokines, chemokines and stress. The core element in MAPK signaling consists of a module of 3 kinases, named MKKK, MKK, and MAPK, which sequentially phosphorylate each other 10. Currently, four MAPK modules have been characterized in mammalian cells: Extracellular Regulated Kinases (ERK1 and 2), Stress activated/c-Jun N terminal kinase (SAPK/JNK), p38 MAP kinases, and ERK5 11. Interestingly, ethanol modulates MAPKs 12. However, information on how ethanol affects MAPKs in the context of innate antiviral pathways such as the Jak-Stat pathway in human cells is extremely limited. When IFN-alpha binds its receptor, two receptor associated tyrosine kinases, Tyk2 and Jak1 become activated by phosphorylation, and phosphorylate Stat1 and Stat2 on conserved tyrosine residues 13. Stat1 and Stat2 combine with the IRF-9 protein to form the transcription factor interferon stimulated gene factor 3 (ISGF-3), which binds to the interferon stimulated response element (ISRE), and induces transcription of IFN-alpha-induced genes (ISG). The ISGs mediate the antiviral effects of IFN. The transcriptional activities of Stats 1, 3, 4, 5a, and 5b are also regulated by serine phosphorylation 14. Phosphorylation of Stat1 on a conserved serine amino acid at position 727 (S727), results in maximal transcriptional activity of the ISGF-3 transcription factor complex 15. Although cross-talk between p38 MAPK and the Jak-Stat pathway is essential for IFN-induced ISRE transcription, p38 does not participate in IFN induction of Stat1 serine phosphorylation 1416171819. However, cellular stress responses induced by stimuli such as ultraviolet light do induce p38 MAPK mediated Stat1 S727 phosphorylation 18. In the current report, we postulated that alcohol and HCV proteins modulate MAPK and Jak-Stat pathways in human liver cells. To begin to address these issues, we characterized the interaction of acute ethanol on Jak-Stat and MAPK pathways in Huh7 cells, HCV replicon cells lines, and primary human hepatocytes.

Background: Hepatitis C virus (HCV) circulates as quasispecies (QS), whose evolution is associated with pathogenesis. Previous studies have suggested that the use of thermostable polymerases without proofreading function may contribute to inaccurate assessment of HCV QS. In this report, we compared non-proofreading (Taq) with proofreading (Advantage High Fidelity-2; HF-2) polymerases in the sensitivity, robustness, and HCV QS diversity and complexity in the second envelope glycoprotein gene hypervariable region 1 (E2-HVR1) on baseline specimens from 20 patients in the HALT-C trial and in a small cohort of 12 HCV/HIV co-infected patients. QS diversity and complexity were quantified using heteroduplex mobility assays (HMA)., Results: The sensitivities of both enzymes were comparable at 50 IU/ml, although HF-2 was more robust and slightly more sensitive than Taq. Both enzymes generated QS diversity and complexity scores that were correlated (r = 0.68; p < 0.0001, and r = 0.47; p < 0.01; Spearman's rank correlation). QS diversity was similar for both Taq and HF-2 enzymes, although there was a trend for higher diversity in samples amplified by Taq (p = 0.126). Taq amplified samples yielded complexity scores that were significantly higher than HF-2 samples (p = 0.033). HALT-C patients who were HCV positive or negative following 20 weeks of pegylated IFN plus ribavirin therapy had similar QS diversity scores for Taq and HF-2 samples, and there was a trend for higher complexity scores from Taq as compared with HF-2 samples. Among patients with HCV and HIV co-infection, HAART increased HCV QS diversity and complexity as compared with patients not receiving therapy, suggesting that immune reconstitution drives HCV QS evolution. However, diversity and complexity scores were similar for both HF-2 and Taq amplified specimens., Conclusion: The data suggest that while Taq may overestimate HCV QS complexity, its use does not significantly affect results in cohort-based studies of HCV QS analyzed by HMA. However, the use of proofreading enzymes such as HF-2 is recommended for more accurate characterization of HCV QS in vivo.

The assembly of hepatitis C virus (HCV) is poorly understood, largely due to the lack of mammalian cell culture systems that are easily manipulated and produce high titers of virus. This problem is highlighted by the inability of the recently established HCV replicon systems to support HCV capsid assembly despite high levels of structural protein synthesis. Here we demonstrate that up to 80% of HCV core protein synthesized de novo in cell-free systems containing rabbit reticulocyte lysate or wheat germ extracts assembles into HCV capsids. This contrasts with standard primate cell culture systems, in which almost no core assembles into capsids. Cell-free HCV capsids, which have a sedimentation value of approximately 100S, have a buoyant density (1.28 g/ml) on cesium chloride similar to that of HCV capsids from other systems. Capsids produced in cell-free systems are also indistinguishable from capsids isolated from HCV-infected patient serum when analyzed by transmission electron microscopy. Using these cell-free systems, we show that HCV capsid assembly is independent of signal sequence cleavage, is dependent on the N terminus but not the C terminus of HCV core, proceeds at very low nascent chain concentrations, is independent of intact membrane surfaces, and is partially inhibited by cultured liver cell lysates. By allowing reproducible and quantitative assessment of viral and cellular requirements for capsid formation, these cell-free systems make a mechanistic dissection of HCV capsid assembly possible.