Our analyses pointed out that viral evasion mechanism observed in one virus could also be relevant for other viruses. To test this, we obtained known drug-gene interactions from DGIdb We added 12 agents as controls Table S1 and Figure S6.
Seven different concentrations of the compounds were added to HMPV or mock-infected cells. HMPV-induced GFP expression and cell viability was measured and after 48 h to determine compound efficiency and toxicity. We repeated the experiment with these compounds. In summary, our meta-analysis approach of the hvPPI could provide novel and faster approaches for the re-purposing of existing drugs as antiviral agents. Table 1.
Using integrative analysis of orthogonal datasets our study provides a comprehensive view of viral evasion mechanisms. In particular, our analysis of the hvPPI network revealed that all the viruses have evolved to target proteins that are central and have strong control over the human interactome. Host proteins targeted by viruses contain a high proportion of intrinsically disordered regions. We identified the core cellular processes and associated proteins that are targeted by all viruses.
Detailed comparative analysis of the subcellular localization of the host proteins showed commonality and specificity both between viral proteins from different strains of the same virus; and between viruses. This unique dataset can be used for further detailed interrogation of the mechanisms behind viral evasion. This could serve as a starting point for identifying novel host targets and generating hypothesis in the context of viral evasion and development of pan-viral therapeutic intervention strategies.
The methods described here also provide unique ways of dissecting the orthogonal datasets. Various analyses from this study have highlighted the existence on pan-viral evasion points that could be utilized for the development of host-directed antiviral therapies. It is also intriguing to see that there is commonality and specificity at the level of sub cellular localization of the viral targets.
Further detailed analysis in this context along with protein sequence features, such as Short Linear Motifs [SLiMs; 79 ] would provide novel insights as well as deeper understanding of how small sequence features are involved in the hijacking of the host machinery.
Integration of such data with known drug-gene interactions provides a clear estimate of the druggable proportion in the hvPPI. Our meta-analysis approach of the hvPPI could provide novel avenues of re-purposing existing drugs for antiviral targeting strategies. These safe-in-man drugs have already been used as investigational agents or experimental drugs in different virus infections Table S2.
We demonstrated novel antiviral effects of azacytidine, itraconazole, lopinavir, nitazoxanide, and oritavancin against HMPV, as well as cidofovir, dibucaine, azithromycin, gefitinib, minocycline, oritavancin, and pirlindole against HCV. Azithromycin, is an FDA-approved antibiotic of the macrolide family.
Dibucaine is an FDA-approved amide local anesthetic. Gefitinib is an FDA approved anticancer drug. Oritavancin is a semisynthetic glycopeptide antibiotic used for the treatment of Gram-positive bacterial skin infections. Azacitidine is a chemical analog of cytidine, which is used in the treatment of myelodysplastic syndrome. Itraconazole is an antifungal medication. Lopinavir is an FDA-approved antiretroviral of the protease inhibitor class. In addition to inhibition of viral proteases Table S2 , Lopinavir was reported to induce host RNaseL production in infected and non-infected cells RNaseL is endoribonuclease that is a part of interferon IFN antiviral response, which is the most critical node of virus-host interactions.
Although, the antiviral mechanisms of action of other compounds are still unknown, these agents could inhibit steps of viral infections, which precede reporter protein expression from viral RNA. In summary, our results indicate that existing BSAs could be re-purposed to other viral infections.
To further expand a spectrum of their activities, these BSAs could be tested against other viruses. Re-purposing these and other safe-in-man antiviral therapeutics could save resources and time needed for development of novel drugs to quickly address unmet medical needs, because safety profiles of these agents in humans are available.
Effective treatment with broad-spectrum-antivirals may shortly become available, pending the results of further pre-clinical studies and clinical trials. This, in turn, means that some broad-spectrum-antivirals could be used for rapid management of new or emerging drug-resistant strains, as well as for first-line treatment or for prophylaxis of acute virus infections or for viral co-infections. The most effective and tolerable compounds could expand the available therapeutics for the treatment of viral diseases, improving preparedness and the protection of the general population from viral epidemics and pandemics.
All datasets used for this study are accessible as stated in the Materials and Methods section 2. RK and KB performed all the bioinformatics and network analysis. DK supervised the drug re-purposing screen.
GS-F and BS provided data. RK conceived and supervised the study. All authors contributed, read, and approved the manuscript. 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.
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Please check for further notifications by email. View Metrics. Email alerts Article activity alert. Advance article alerts. New issue alert. Subject alert. Receive exclusive offers and updates from Oxford Academic. Related articles in Web of Science Google Scholar. These data indicate that the persistent phenotype of Cl13 was dominant over the acute phenotype of Arm.
To determine if Arm was able to trigger an immune response capable of blocking the establishment of Cl13 persistence, we primed mice first with Arm before Cl13 infection.
At 5, 10 and 19 days post-infection serum virus titers were determined by plaque assay on Vero cells. Data are representative of two independent experiments.
Serum virus titers were determined as in A at the indicated time points. Data in C—E are representative of two independent experiments. To understand how Arm triggers a primed immune response that facilitated control and clearance of a subsequent infection with Cl13, we used a non-propagating Arm for priming prior to Cl13 infection.
One mouse of five cleared the infection when primed four hours before Cl13 infection Fig. When mice were primed at 12 hours before Cl13 infection, viral titers at day 5 were lower than those of mock primed mice Fig. Adult immunocompetent mice infected with Cl13 never clear virus from their kidneys despite undetectable viral titers from all other organs and serum four months post-infection [61]. Mice primed with a non-propagating Cl13 virus also cleared the infection suggesting that neither tropism nor differences in Cl13 viral gene expression were responsible for a primed-mediated clearance of Cl13 Fig.
Altogether, these data demonstrate that the priming agent must be able to either express viral genes or replicate, or both, to trigger an immune response capable of clearing a Cl13 infection.
Cl13 infection prevented weight loss and mortality. T cells from adult immunocompetent mice infected with Cl13 have a decreased function T cell exhaustion that leads to their inability to clear the infection [57]. We therefore tested whether a cytotoxic T cell CTL response was responsible for clearance in primed mice.
Only T cells isolated seven days post Cl13 infection and stimulated with GP33 did not show significant differences, however significant differences were seen between these groups at 11 days post Cl13 infection. Blood samples were collected at 7 top panels or 11 bottom panels days post-infection and after erythrocyte depletion incubated with H-2b restricted peptides GP 33—41 and NP — , IL-2 and BrefeldinA.
Mice received intraperiotneal injections of 0. B Virus titers in serum were determined as in Fig. Two mice in the CD8 depleted group died at day Mice treated with non-relevant isotype control antibodies cleared a primed Cl13 infection while primed Cl13 infected mice depleted of CTLs were viremic at days 7 and 11 Fig.
Under this scenario, we would expect a similar, but earlier induction of the innate immune response in primed mice compared to mock primed mice in the first 24 hours after Cl13 infection. To examine this possibility we measured expression levels of a panel of cytokines in serum samples collected at several times after priming and infection.
We found that cytokine levels in the serum of Clinfected mice varied substantially between primed and mock primed mice. These differences fall roughly into two groups. The first group Fig. Data for cytokines that were not detected by the multiplex ELISA and those for which there was no discernible difference were not graphed.
The second group Fig. Importantly, results shown in Fig. Conversely, a dysregulated innate immune response early after Cl13 infection is associated with the establishment of viral persistence.
However, by three days post-infection, titers between both groups were similar Fig. Virus titers in clarified homogenates were determined as in Fig. Statistical analysis was done using t-tests of log transformed values. Graphs are representative of three independent experiments. Together with data from Fig. Splenic microarchitecture was assessed by visualization of mellalophilic macrophages using anti mouse MOMA-1 antibodies red. Data are from one of two independent experiments.
Highly significant differences were seen in plasmacytoid DCs pDCs mock primed: These cell types have been associated with persistent viral infection and essential for a proper anti-viral immune response [41] , [43] , [45] , [46] , [63] , [64]. Additionally, we observed significant differences in T mock primed: 1.
Higher numbers and percentages though low overall of infected T and B cells may be due to the increased presence of virus in splenic white pulp Fig. Cells were identified by flow cytometry through staining and gating with the following fluorescently conjugated anti-mouse antibodies: DCS: lin- CD Representative data from one of two independent experiments are shown with SEM calculated and displayed using GraphPad Prism software.
B—D Spleens treated as in Fig. Yellow arrows indicate areas of costaining. The two examples from each staining are from different mice. All micrographs are representative examples of micrographs from two independent experiments. Notably, some but not all of the reticular fibroblasts lining the red pulp sinus contained viral antigen as well as some FRCs present in the white pulp in the spleens of mock primed Cl13 infected mice three days post-infection Fig.
Together, our data show that viral propagation into red and white pulp was coincident with a wider viral cellular tropism. At the indicated times virus titers in serum were determined as in Fig. H Ly5. All data shown are representative of at least 2 independent experiments. The phenotype in feeble mice, is restricted to pDCs and has no effect on conventional DCs [20] , [63].
Feeble mice cleared the primed infection similarly to its wild-type counterparts Fig. Cytoplasmic RNA sensors are found in both hematopoetic and non-hematopoetic cells. Consistent with previous findings [18] , in the absence of IFN-I, viral titers were significantly higher than those measured in WT controls Fig.
In this paper we report how early events affect the establishment of a persistant infection by comparing the outcome of infection of mice with the Cl13 strain of LCMV under two different immune environments. In contrast, the LCMV Cl13 isolate overcomes this response through rapid and robust multiplication that leads to immune dysregulation through the upregulation of cytokines and chemokines, dissemination from marginal zones, infection of important immune cells, and hyporesponsive T cell response.
Notably, some mice primed with the non-persistent Arm strain of LCMV as early as 4—6 hours prior infection with Cl13 were able to control and clear Cl13 while all mice primed after 8 hours prior to infection were able to clear a Cl13 infection. Hence, a narrow temporal window exists between the host's ability to mount an appropriate anti-viral immune response through triggering of cytoplasmic PPR and a dysregulated immune response caused by rapid viral propagation.
When inoculated concurrently, Cl13 overcame the innate immune response triggered by cytoplasmic PRRs induced by the priming agent, a response also presumably generated by Cl13 itself.
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