Mechanisms of vesicular stomatitis virus inactivation by protoporphyrin ix, zinc- protoporphyrin ix, and mesoporphyrin ix

© 2017 American Society for Microbiology. All Rights Reserved.Virus resistance to antiviral therapies is an increasing concern that makes the development of broad-spectrum antiviral drugs urgent. Targeting of the viral envelope, a component shared by a large number of viruses, emerges as a promising strategy to overcome this problem. Natural and synthetic porphyrins are good candidates for antiviral development due to their relative hydrophobicity and pro-oxidant character. In the present work, we characterized the antiviral activities of protoprophyrin IX (PPIX), Zn-protoporphyrin IX (ZnPPIX), and mesoporphyrin IX (MPIX) against vesicular stomatitis virus (VSV) and evaluated the mechanisms involved in this activity. Treatment of VSV with PPIX, ZnPPIX, and MPIX promoted dose-dependent virus inactivation, which was potentiated by porphyrin photoactivation. All three porphyrins inserted into lipid vesicles and disturbed the viral membrane organization. In addition, the porphyrins also affected viral proteins, inducing VSV glycoprotein cross-linking, which was enhanced by porphyrin photoactivation. Virus incubation with sodium azide and α-tocopherol partially protected VSV from inactivation by porphyrins, suggesting that singlet oxygen (1O2) was the main reactive oxygen species produced by photoactivation of these molecules. Furthermore, 1O2 was detected by 9,10-dimethylanthracene oxidation in photoactivated porphyrin samples, reinforcing this hypothesis. These results reveal the potential therapeutic application of PPIX, ZnPPIX, and MPIX as good models for broad antiviral drug design.Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ; Brazil; grant number E-26/201.167/2014), the Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq; Brazil; grant number 306669/2013-7), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; Brazil; grant number CsF 171/2012), the Fundacao para a Ciencia e Tecnologia-Ministério da Educação e Ciência (FCT-MEC; Portugal; project HIVERA/0002/2013), and Marie Skłodowska-Curie Actions (MSCA; European Commission project INPACT 644167). C.C.-O. acknowledges a Science without Borders postdoctoral fellowship from CAPES (171/2012), and J.M.F. acknowledges an FCT-MEC Ph.D. fellowship (SFRH/BD/70423/2010)info:eu-repo/semantics/publishedVersio

MIF Participates in Toxoplasma gondii-Induced Pathology Following Oral Infection

BACKGROUND: Macrophage migration inhibitory factor (MIF) is essential for controlling parasite burden and survival in a model of systemic Toxoplasma gondii infection. Peroral T. gondii infection induces small intestine necrosis and death in susceptible hosts, and in many aspects resembles inflammatory bowel disease (IBD). Considering the critical role of MIF in the pathogenesis of IBD, we hypothesized that MIF participates in the inflammatory response induced by oral infection with T. gondii. METHODOLOGY/PRINCIPAL FINDINGS: Mif deficient (Mif(-/-)) and wild-type mice in the C57Bl/6 background were orally infected with T. gondii strain ME49. Mif(-/-) mice had reduced lethality, ileal inflammation and tissue damage despite of an increased intestinal parasite load compared to wt mice. Lack of MIF caused a reduction of TNF-α, IL-12, IFN-γ and IL-23 and an increased expression of IL-22 in ileal mucosa. Moreover, suppressed pro-inflammatory responses at the ileal mucosa observed in Mif(-/-) mice was not due to upregulation of IL-4, IL-10 or TGF-β. MIF also affected the expression of matrix metalloproteinase-9 (MMP-9) but not MMP-2 in the intestine of infected mice. Signs of systemic inflammation including the increased concentrations of inflammatory cytokines in the plasma and liver damage were less pronounced in Mif(-/-) mice compared to wild-type mice. CONCLUSION/SIGNIFICANCE: In conclusion, our data suggested that in susceptible hosts MIF controls T. gondii infection with the cost of increasing local and systemic inflammation, tissue damage and death

Dengue Virus Capsid Protein Usurps Lipid Droplets for Viral Particle Formation

Dengue virus is responsible for the highest rates of disease and mortality among the members of the Flavivirus genus. Dengue epidemics are still occurring around the world, indicating an urgent need of prophylactic vaccines and antivirals. In recent years, a great deal has been learned about the mechanisms of dengue virus genome amplification. However, little is known about the process by which the capsid protein recruits the viral genome during encapsidation. Here, we found that the mature capsid protein in the cytoplasm of dengue virus infected cells accumulates on the surface of ER-derived organelles named lipid droplets. Mutagenesis analysis using infectious dengue virus clones has identified specific hydrophobic amino acids, located in the center of the capsid protein, as key elements for lipid droplet association. Substitutions of amino acid L50 or L54 in the capsid protein disrupted lipid droplet targeting and impaired viral particle formation. We also report that dengue virus infection increases the number of lipid droplets per cell, suggesting a link between lipid droplet metabolism and viral replication. In this regard, we found that pharmacological manipulation of the amount of lipid droplets in the cell can be a means to control dengue virus replication. In addition, we developed a novel genetic system to dissociate cis-acting RNA replication elements from the capsid coding sequence. Using this system, we found that mislocalization of a mutated capsid protein decreased viral RNA amplification. We propose that lipid droplets play multiple roles during the viral life cycle; they could sequester the viral capsid protein early during infection and provide a scaffold for genome encapsidation

Macrophages as target cells for Mayaro virus infection: involvement of reactive oxygen species in the inflammatory response during virus replication

ABSTRACT Alphaviruses among the viruses that cause arthritis, consisting in a public health problem worldwide by causing localized outbreaks, as well as large epidemics in humans. Interestingly, while the Old World alphaviruses are arthritogenic, the New World alphaviruses cause encephalitis. One exception is Mayaro virus (MAYV), which circulates exclusively in South America but causes arthralgia and is phylogenetically related to the Old World alphaviruses. Although MAYV-induced arthritis in humans is well documented, the molecular and cellular factors that contribute to its pathogenesis are completely unknown. In this study, we demonstrated for the first time that macrophages, key players in arthritis development, are target cells for MAYV infection, which leads to cell death through apoptosis. We showed that MAYV replication in macrophage induced the expression of TNF, a cytokine that would contribute to pathogenesis of MAYV fever, since TNF promotes an inflammatory profile characteristic of arthritis. We also found a significant increase in the production of reactive oxygen species (ROS) at early times of infection, which coincides with the peak of virus replication and precedes TNF secretion. Treatment of the cells with antioxidant agents just after infection completely abolished TNF secretion, indicating an involvement of ROS in inflammation induced during MAYV infection

MIF upregulates MMP-9 but not MMP-2 in the terminal ileum of <i>T. gondii</i> infected mice.

<p>Quantitative real-time PCR of (A) MMP-2 and (B) MMP-9 mRNAs in ileal biopsies of control and infected (9 dpi) wt and <i>Mif</i>⁻/⁻ mice. Results are expressed as fold changes to HPRT mRNA expression. One representative experiment out of two experiments is shown. Data of three to five mice per group are given as means ± SEM and p values were determined by Mann-Whitney (**p≤0.01). Cont, control not infected.</p

<i>T. gondii</i>-induced sepsis-like response is partially dependent of MIF.

<p>(A) Leukocyte counts were determined in peripheral blood at indicated times. (B) TNF-α and (C) IFN-γ concetrations in the plasma of wt and <i>Mif</i>⁻/⁻ mice (n = 3–5) 9 dpi were measured by ELISA. (D) Histopathologycal analysis of the liver at 9 dpi from wt and <i>Mif</i>⁻/⁻ mice. (E) Quantitative analysis of liver sections (3 slides/animal) of infected wt and <i>Mif</i>⁻/⁻ mice mice was performed. Liver damage was also determined by quantifying serum concentrations of biochemical marker as (F) alanine aminotransferase (ALT), and (G) aspartate aminotransferase (AST). (H) Toxo DNA concentration was determined in the liver of wt and <i>Mif</i>⁻/⁻ mice. One representative experiment out of six experiments is shown. Data of three to five mice/group are given as as means ± SEM and p values were determined by t-test (* p≤0.05, ** p≤0.01). Cont, control not infected.</p

<i>T. gondii</i>-induced ileitis is mediated by MIF that induces severe tissue damage despite efficient parasite control.

<p>(A) Shortening of small intestine of wt and <i>Mif</i>⁻/⁻ mice (n = 6) at 9 days post-infection (dpi). (B) Histopathologycal analysis by hematoxilin and eosin (HE) of terminal ileum was performed at 9 dpi in wt and <i>Mif</i>⁻/⁻. Magnification are ×200 (B1, 2, 5 and 6) and ×400 (B3 and 4). In (C) Histologycal scores of ileal biopsies of wt and <i>Mif</i>⁻/⁻ mice at 9 dpi. The horizontal line limits absence of inflammatory response (0 to 3) and necrosis (above 3). (D) Toxo DNA concentration was determined in ileal biopsies of wt and <i>Mif</i>⁻/⁻ mice. One representative experiment out of three independent experiments. Data from 3 to 6 mice per group is represented by means ± SEM and p values determined by t-test (* p≤0.05, ** p≤0.01). Length of intestinal segments of control not infected wt and <i>Mif</i>⁻/⁻ mice are represented by a dotted line in A. Cont, control not infected.</p

Reduced intestinal expression of IL-10 and TGF-β in <i>Mif^−/−</i> mice.

<p>Quantitative RT-PCR of of (A) IL-4, (B) IL-10 and (C) TGF-β mRNA expression in ileal explants of control and infected wt and <i>Mif</i>⁻/⁻ mice. Results are expressed as fold changes relative to HPRT mRNA expression. Data from 3 to 6 mice per group is represented by means ± SEM and p values determined by t-test (*p≤0.05). Cont, control not infected.</p