Zinc is an essential micronutrient which regulates diverse physiological functions and has been shown to play a crucial role in viral infections. Zinc has a necessary role in the replication of many viruses, however, antiviral action of zinc has also been demonstrated in in vitro infection models most likely through induction of host antiviral responses. Therefore, depending on the host machinery that the virus employs at different stages of infection, zinc may either facilitate, or inhibit virus infection. In this study, we show that zinc plays divergent roles in rotavirus and dengue virus infections in epithelial cells. Dengue virus infection did not perturb the epithelial barrier functions despite the release of virus from the basolateral surface whereas rotavirus infection led to disruption of epithelial junctions. In rotavirus infection, zinc supplementation post-infection did not block barrier disruption suggesting that zinc does not affect rotavirus life-cycle or protects epithelial barriers post-infection suggesting the involvement of cellular pathways in the beneficial effect of zinc supplementation in enteric infections. Zinc depletion by N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) inhibited dengue virus and Japanese encephalitis virus (JEV) infection but had no effect on rotavirus. Time-of-addition experiments suggested that zinc chelation affected both early and late stages of dengue virus infectious cycle and zinc chelation abrogated dengue virus RNA replication. We show that transient zinc chelation induces ER stress and antiviral response by activating NF-kappaB leading to induction of interferon signaling. These results suggest that modulation of zinc homeostasis during virus infection could be a component of host antiviral response and altering zinc homeostasis may act as a potent antiviral strategy against flaviviruses.
Mycobacterium tuberculosis (Mtb) endures a combination of metal scarcity and toxicity throughout the human infection cycle, contributing to complex clinical manifestations. Pathogens counteract this paradoxical dysmetallostasis by producing specialized metal trafficking systems. Capture of extracellular metal by siderophores is a widely accepted mode of iron acquisition, and Mtb iron-chelating siderophores, mycobactin, have been known since 1965. Currently, it is not known whether Mtb produces zinc scavenging molecules. Here, we characterize low-molecular-weight zinc-binding compounds secreted and imported by Mtb for zinc acquisition. These molecules, termed kupyaphores, are produced by a 10.8 kbp biosynthetic cluster and consists of a dipeptide core of ornithine and phenylalaninol, where amino groups are acylated with isonitrile-containing fatty acyl chains. Kupyaphores are stringently regulated and support Mtb survival under both nutritional deprivation and intoxication conditions. A kupyaphore-deficient Mtb strain is unable to mobilize sufficient zinc and shows reduced fitness upon infection. We observed early induction of kupyaphores in Mtb-infected mice lungs after infection, and these metabolites disappeared after 2 wk. Furthermore, we identify an Mtb-encoded isonitrile hydratase, which can possibly mediate intracellular zinc release through covalent modification of the isonitrile group of kupyaphores. Mtb clinical strains also produce kupyaphores during early passages. Our study thus uncovers a previously unknown zinc acquisition strategy of Mtb that could modulate host–pathogen interactions and disease outcome.
We screened a siRNA library targeting human tyrosine kinases in Huh-7 cells and identified c-terminal Src kinase (Csk) as one of the kinases involved in dengue virus replication. Knock-down of Csk expression by siRNAs or inhibition of Csk by an inhibitor reduced dengue virus RNA levels but did not affect viral entry. Csk partially colocalized with viral replication compartments. Dengue infection was drastically reduced in cells lacking the three ubiquitous src family kinases, Src, Fyn and Yes. Csk knock-down in these cells failed to block dengue virus replication suggesting that the effect of Csk is via regulation of Src family kinases. Csk was found to be hyper-phosphorylated during dengue infection and inhibition of protein kinase A led to a block in Csk phosphorylation and dengue virus replication. Overexpression studies suggest an important role for the kinase and SH3 domains in this process. Our results identified a novel role for Csk as a host tyrosine kinase involved in dengue virus replication and provide further insights into the role of host factors in dengue replication.
We previously reported interferon gamma secretion by human CD4+ and CD8+ T cells in response to recombinant E. coli-expressed Rv1860 protein of Mycobacterium tuberculosis (MTB) as well as protection of guinea pigs against a challenge with virulent MTB following prime-boost immunization with DNA vaccine and poxvirus expressing Rv1860. In contrast, a Statens Serum Institute Mycobacterium bovis BCG (BCG-SSI) recombinant expressing MTB Rv1860 (BCG-TB1860) showed loss of protective ability compared to the parent BCG strain expressing the control GFP protein (BCG-GFP). Since Rv1860 is a secreted mannosylated protein of MTB and BCG, we investigated the effect of BCG-TB1860 on innate immunity. Relative to BCG-GFP, BCG-TB1860 effected a significant near total reduction both in secretion of cytokines IL-2, IL-12p40, IL-12p70, TNF-α, IL-6 and IL-10, and up regulation of co-stimulatory molecules MHC-II, CD40, CD54, CD80 and CD86 by infected bone marrow derived dendritic cells (BMDC), while leaving secreted levels of TGF-β unchanged. These effects were mimicked by BCG-TB1860His which carried a 6-Histidine tag at the C-terminus of Rv1860, killed sonicated preparations of BCG-TB1860 and purified H37Rv-derived Rv1860 glycoprotein added to BCG-GFP, but not by E. coli-expressed recombinant Rv1860. Most importantly, BMDC exposed to BCG-TB1860 failed to polarize allogeneic as well as syngeneic T cells to secrete IFN-γ and IL-17 relative to BCG-GFP. Splenocytes from mice infected with BCG-SSI showed significantly less proliferation and secretion of IL-2, IFN-γ and IL-17, but secreted higher levels of IL-10 in response to in vitro restimulation with BCG-TB1860 compared to BCG-GFP. Spleens from mice infected with BCG-TB1860 also harboured significantly fewer DC expressing MHC-II, IL-12, IL-2 and TNF-α compared to mice infected with BCG-GFP. Glycoproteins of MTB, through their deleterious effects on DC may thus contribute to suppress the generation of a TH1- and TH17-dominated adaptive immune response that is vital for protection against tuberculosis.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) initiates infection by attachment of the surface-exposed spike glycoprotein to the host cell receptors. The spike glycoprotein (S) is a promising target for inducing immune responses and providing protection; thus the ongoing efforts for the SARS-CoV-2 vaccine and therapeutic developments are mostly spiraling around S glycoprotein. The matured functional spike glycoprotein is presented on the virion surface as trimers, which contain two subunits, such as S1 (virus attachment) and S2 (virus fusion). The S1 subunit harbors the N-terminal domain (NTD) and the receptor-binding domain (RBD). The RBD is responsible for binding to host-cellular receptor angiotensin-converting enzyme 2 (ACE2). The NTD and RBD of S1, and the S2 of S glycoprotein are the major structural moieties to design and develop spike-based vaccine candidates and therapeutics. Here, we have identified three novel epitopes (20-amino acid peptides) in the regions NTD, RBD, and S2 domains, respectively, by structural and immunoinformatic analysis. We have shown as a proof of principle in the murine model, the potential role of these novel epitopes in-inducing humoral and cellular immune responses. Further analysis has shown that RBD and S2 directed epitopes were able to efficiently inhibit the replication of SARS-CoV-2 wild-type virus in vitro suggesting their role as virus entry inhibitors. Structural analysis revealed that S2-epitope is a part of the heptad repeat 2 (HR2) domain which might have plausible inhibitory effects on virus fusion. Taken together, this study discovered novel epitopes that might have important implications in the development of potential SARS-CoV-2 spike-based vaccine and therapeutics.
Over 90% of the COVID-19 patients manifest mild/moderate symptoms or are asymptomatic. Although comorbidities and dysregulation of immune response have been implicated in severe COVID-19, the host factors that associate with asymptomatic or mild infections have not been characterized. We have collected serial samples from 23 hospitalized COVID-19 patients with mild symptoms and measured the kinetics of SARS-CoV-2 viral load in respiratory samples and markers of inflammation in serum samples. We monitored seroconversion during the acute phase of illness and quantitated the amount of total IgG against the receptor-binding domain of SARS-CoV-2 and estimated the virus neutralization potential of these antibodies. Viral load decreased by day 8 in all the patients but the detection of viral RNA in saliva samples did not correlate well with viral RNA detection in nasopharyngeal/oropharyngeal swab samples. 25% of the virus-positive patients had no detectable neutralizing antibodies in the serum and in other cases, the efficiency of antibodies to neutralize SARS-CoV-2 B1.1.7 strain was lower as compared to the circulating virus isolate. Decrease in viral load coincided with increase in neutralizing antibodies and interferon levels in serum. Most patients showed no increase in inflammatory cytokines such as IL-1β or IL-6, however, elevated levels of IL-7 and other inflammatory mediators such as TNF-α and IL-8 was observed. These data suggest that most mild infections are associated with absence of inflammation coupled with an active innate immune response, T-cell activation and neutralizing antibodies.
Chandipura virus (CHPV), a cytoplasmic RNA virus, has been implicated in several outbreaks of acute encephalitis in India. Despite the relevance of CHPV to human health, how the virus interacts with the host signaling machinery remains obscure. In response to viral infections, mammalian cells activate RelA/NF-B heterodimers, which induce genes encoding interferon beta (IFN-) and other immune mediators. Therefore, RelA is generally considered to be an antiviral transcription factor. However, RelA activates a wide spectrum of genes in physiological settings, and there is a paucity of direct genetic evidence substantiating antiviral RelA functions. Using mouse embryonic fibroblasts, we genetically dissected the role of RelA in CHPV pathogenesis. We found that CHPV indeed activated RelA and that RelA deficiency abrogated the expression of IFN- in response to virus infections. Unexpectedly, infection of Rela Ϫ/Ϫ fibroblasts led to a decreased CHPV yield. Our investigation clarified that RelA-dependent synthesis of prosurvival factors restrained infection-inflicted cell death and that exacerbated cell death processes prevented multiplication of CHPV in RelAdeficient cells. Chikungunya virus, a cytopathic RNA virus associated also with epidemics, required RelA, and Japanese encephalitis virus, which produced relatively minor cytopathic effects in fibroblasts, circumvented the need of RelA for their propagation. In sum, we documented a proviral function of the pleiotropic factor RelA linked to its prosurvival properties. RelA promoted the growth of cytopathic RNA viruses by extending the life span of infected cells, which serve as the replicative niche of intracellular pathogens. We argue that our finding bears significance for understanding host-virus interactions and may have implications for antiviral therapeutic regimes. IMPORTANCE RelA/NF-B participates in a wide spectrum of physiological processes, including shaping immune responses against invading pathogens. In virusinfected cells, RelA typically induces the expression of IFN-, which restrains viral propagation in neighboring cells involving paracrine mechanisms. Our study suggested that RelA might also play a proviral role. A cell-autonomous RelA activity amplified the yield of Chandipura virus, a cytopathic RNA virus associated with human epidemics, by extending the life span of infected cells. Our finding necessitates a substantial revision of our understanding of host-virus interactions and indicates a dual role of NF-B signaling during the course of RNA virus infections. Our study also bears significance for therapeutic regimes which alter NF-B activities while alleviating the viral load.
Reactive oxygen species (ROS) are chemically active species which are involved in maintaining cellular and signalling processes at physiological concentrations. Therefore, cellular components that regulate redox balance are likely to play a crucial role in viral life-cycle either as promoters of viral replication or with antiviral functions. Zinc is an essential micronutrient associated with anti-oxidative systems and helps in maintaining a balanced cellular redox state. Here, we show that zinc chelation leads to induction of reactive oxygen species (ROS) in epithelial cells and addition of zinc restores ROS levels to basal state. Addition of ROS (H2O2) inhibited dengue virus (DENV) infection in a dose-dependent manner indicating that oxidative stress has adverse effects on DENV infection. ROS affects early stages of DENV replication as observed by quantitation of positive and negative strand viral RNA. We observed that addition of ROS specifically affected viral titres of positive strand RNA viruses. We further demonstrate that ROS specifically altered SEC31A expression at the ER suggesting a role for SEC31A-mediated pathways in the life-cycle of positive strand RNA viruses and provides an opportunity to identify drug targets regulating oxidative stress responses for antiviral development.
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