Viviana Castilla

Functional entry of dengue virus into Aedes albopictus mosquito cells is dependent on clathrin-mediated endocytosis

Entry of dengue virus 2 (DENV-2) into Aedes albopictus mosquito C6/36 cells was analysed using biochemical and molecular inhibitors, together with confocal and electron microscopy observations. Treatment with monodansylcadaverine, chlorpromazine, sucrose and ammonium chloride inhibited DENV-2 virus yield and protein expression, whereas nystatin, a blocker of caveolae-mediated endocytosis, did not have any effect. Using confocal microscopy, co-localization of DENV-2 E glycoprotein and the marker protein transferrin was observed at the periphery of the cytoplasm. To support the requirement of clathrin function for DENV-2 entry, overexpression of a dominant-negative mutant of Eps15 in C6/36 cells was shown to impair virus entry. The disruption of actin microfilaments by cytochalasin D also significantly affected DENV-2 replication. In contrast, microtubule disruption by colchicine treatment did not impair DENV-2 infectivity, suggesting that DENV-2 does not require transport from early to late endosomes for successful infection of mosquito cells. Furthermore, using transmission electron microscopy, DENV-2 particles of approximately 44-52 nm were found attached within electron-dense invaginations of the plasma membrane and in coated vesicles that resembled those of clathrin-coated pits and vesicles, respectively. Together, these results demonstrate for the first time that DENV-2 enters insect cells by receptor-mediated, clathrin-dependent endocytosis, requiring traffic through an acidic pH compartment for subsequent uncoating and completion of a productive infection.

Alternative infectious entry pathways for dengue virus serotypes into mammalian cells

The entry of two dengue virus (DENV) serotypes into Vero cells was analysed using biochemical inhibitors, dominant negative mutants of cellular proteins involved in endocytic pathways, fluorescence microscopy and infectivity determinations. By treatment with dansylcadaverine and chlorpromazine and overexpression of a dominant negative form of the Eps15 protein, a clathrin‐mediated endocytosis for productive DENV‐1 internalization into Vero cells was demonstrated whereas the infectious entry of DENV‐2 in the same cell system was independent of clathrin. Treatment with the inhibitors nystatin and methyl‐β‐cyclodextrin, as well as transfection of Vero cells with dominant negative caveolin‐1, had no effect on DENV‐2 virus infection. It was also shown, by using the K44A mutant and the inhibitor dynasore, that dynamin was required for DENV‐2 entry. Consequently, the infectious entry of DENV‐2 into Vero cells occurs by a non‐classical endocytic pathway independent of clathrin, caveolae and lipid rafts, but dependent on dynamin. By contrast, DENV‐2 entry into A549 cells was clathrin‐dependent, as previously reported in HeLa, C6/36 and BS‐C‐1 cells. Our results conclusively show, for the first time, a differential mode of infective entry for DENV‐1 and DENV‐2 into a common host cell, Vero cells, as well as alternative entry pathways for a given serotype, DENV‐2, into different types of cells.

Enterocin CRL35 inhibits late stages of HSV-1 and HSV-2 replication in vitro

The replication of herpes simplex virus (HSV) type 1 and 2 in Vero cells is inhibited in the presence of enterocin CRL35 (ECRL), a bacteriocin produced by Enterococcus faecium CRL35. Attempts to resolve the mode of action of ECRL indicate that virus adsorption and penetration are not affected. Instead, a late step of virus multiplication is hindered since the addition of 100 microg/ml of ECRL at 8h post infection still causes a 90% inhibition of virus release. The effect of ECRL on HSV antigen expression was studied by immunofluorescence using a polyclonal serum and a monoclonal antibody against glycoprotein D (gamma protein). These studies indicated that ECRL impeded the second round of infection, apparently as a consequence of the inhibition of glycoprotein D expression. The replication of syncytial mutants of HSV-1 was significantly inhibited at a ECRL concentration of 25 microg/ml. Both the percentage of fused cells and the polykaryocyte size were affected. Studies on the effect of ECRL on viral protein synthesis showed that in the presence of ECRL, HSV late gamma proteins were not synthesized. From these findings, it is concluded that inhibition of HSV spreading by ECRL is due to the prevention of mainly late glycoprotein synthesis.

The entry of Junin virus into Vero cells

SummaryThe entry mechanism of Junin virus (JV) into Vero cells was studied analyzing the effect of lysosomotropic compounds and acid pH on JV infection. Ammonium chloride, amantadine, chlorpheniramine and procaine inhibited JV production. The action of ammonium chloride was exerted at early times of infection. Virus internalization was inhibited and viral protein expression was not detected. When the extracellular medium was buffered at low pH, the ammonium chloride induced block on JV infection was overcome. Furthermore, JV was able to induce fusion of infected cells at pH 5.5 leading to polykaryoctye formation. Taken together, these results demonstrate that JV entry occurs through an endocytic mechanism requiring a low pH dependent membrane fusion.

Low-pH-induced fusion of Vero cells infected with Junin virus.

SummaryJunin virus (JV) infected Vero cells were used to investigate virus capacity to induce cell-cell fusion. Polykaryocyte formation due to JV was found to be pH and temperature-dependent. A reduced fusion activity was detected on BHK-21 cells. Different JV-strains exhibited a similar extent and pH dependence of their fusion activity. Neutralizing antibodies against the main viral glycoprotein (GP38) inhibited syncytium production and GP38 conformational changes in response to acid treatment were detected by an immunoprecipitation assay.

Differential Requirements in Endocytic Trafficking for Penetration of Dengue Virus

The entry of DENV into the host cell appears to be a very complex process which has been started to be studied in detail. In this report, the route of functional intracellular trafficking after endocytic uptake of dengue virus serotype 1 (DENV-1) strain HW, DENV-2 strain NGC and DENV-2 strain 16681 into Vero cells was studied by using a susceptibility to ammonium chloride assay, dominant negative mutants of several members of the family of cellular Rab GTPases that participate in regulation of transport through endosome vesicles and immunofluorescence colocalization. Together, the results presented demonstrate that in spite of the different internalization route among viral serotypes in Vero cells and regardless of the viral strain, DENV particles are first transported to early endosomes in a Rab5-dependent manner. Then a Rab7-dependent pathway guides DENV-2 16681 to late endosomes, whereas a yet unknown sorting event controls the transport of DENV-2 NGC, and most probably DENV-1 HW, to the perinuclear recycling compartments where fusion membrane would take place releasing nucleocapsid into the cytoplasm. Besides the demonstration of a different intracellular trafficking for two DENV-2 strains that shared the initial clathrin-independent internalization route, these studies proved for the first time the involvement of the slow recycling pathway for DENV-2 productive infection.

Infectious dengue-1 virus entry into mosquito C6/36 cells.

The entry of dengue virus-1 (DENV-1) strain Hawaii into mosquito C6/36 cells was analyzed using a variety of biochemical inhibitors together with electron microscopy. The treatment with ammonium chloride, chlorpromazine, dansylcadaverine and dynasore inhibited virus yields, determined by infectivity titrations, whereas nystatin and methyl-β-cyclodextrin did not have any effect. The effect of the clathrin and dynamin inhibitors on DENV-1 entry was corroborated by detection of internalized virions using immunofluorescence staining. Furthermore, electron micrographs showed the incoming virions attached to electron-dense invaginations of the plasma membrane and within coated vesicles that resembled clathrin-coated pits and vesicles, respectively. The susceptibility to clathrin and dynamin inhibitors of clinical isolates from recent outbreaks was comparable to that shown by the cell culture-adapted reference strain. Similarly, DENV-3 strain H87 and DENV-4 strain 8124 were also inhibited in the presence of ammonium chloride, chlorpromazine and dynasore, allowing conclude that the infectious entry of DENV serotypes to mosquito cells occurs by low pH-dependent clathrin-mediated endocytosis.

Inhibition of foot and mouth disease virus (FMDV) uncoating by a plant-derived peptide isolated from Melia azedarach L leaves.

SummaryMeliacine (MA), a peptide isolated from leaves of the high plant Melia azedarach L inhibited the multiplication of foot and mouth disease virus (FMDV) in BHK-21 cells. In this report, we establish that the MA-inhibitable process takes place within the first hour of the viral reproductive cycle. MA had no virucidal effect and did not affect adsorption and penetration of the virus in cells. In experiments with neutral red-labeled virus, it was found that MA significantly suppressed the development of photoresistance of the virus in infected cells. In untreated cultures nearly all virus which adsorbed to cells was uncoated within 1 h at 37 °C, whereas in treated cultures, even after 3 h only 3% of the virus was uncoated. Labeling of BHK-21 cells with acridine orange showed that MA affects the pH of intracellular acidic vesicles. Therefore, it is concluded that MA prevents the process of uncoating of FMDV in BHK-21 cells by inhibiting vacuolar acidification.

In vitro anti-junin virus activity of a peptide isolated from Melia azedarach L. leaves

Meliacine, a peptide isolated from leaves of Melia azedarach L. inhibited the multiplication of Junin virus in Vero cells treated with the compound before infection (pre-treatment) or immediately after virus adsorption. Analysis of early events following infection demonstrated that meliacine blocks virus penetration by preventing the uncoating step. The addition of meliacine at different times after infection indicated that meliacine also interferes with the release of infectious particles to the extracellular medium and inhibits the low-pH-induced fusion of infected cells. Intracellular transport of viral glycoproteins to the cell membrane was not affected by meliacine, as revealed by immunofluorescence staining. Taken together, these results suggest that meliacine affects two events of the virus replicative cycle that require membrane fusion: uncoating and budding.

In vitro antiviral activity of dehydroepiandrosterone, 17 synthetic analogs and ERK modulators against herpes simplex virus type 1.

In the present study the in vitro antiviral activity of dehydroepiandrosterone (DHEA) and 17 synthetic derivatives against herpes simplex type 1 (HSV-1) was determined. DHEA, epiandrosterone (EA), two synthetic DHEA analogs and three synthetic EA analogs showed a selective inhibitory effect on HSV in vitro multiplication. DHEA and E2, a synthetic derivative of EA, were not found to be virucidal to cell-free HSV-1 and did not impair virus adsorption or penetration. We determined that treatment with both compounds decreased viral protein synthesis. Moreover, inhibitory effect of DHEA and E2 on extracellular viral titer was stronger than the inhibition found on total viral infectivity, suggesting that the antiherpetic activity of these compounds may also be in part due to an inhibition in virus formation and release. Since DHEA is a known Raf/MEK/ERK signaling pathway activator, we studied the role of this pathway on HSV-1 infection. ERK1/2 phosphorylation was stimulated in HSV-1 infected cultures. UO126, a Raf/MEK/ERK signaling pathway inhibitor, impaired viral multiplication, while anisomycin, an activator of this pathway, enhanced it. Treatment with DHEA 6 h before infection enhanced HSV-1 multiplication. On the contrary, pre-treatment with E2, which does not modulate Raf/MEK/ERK signaling pathway, did not produce an increase of viral replication. Taking together these results, the antiviral activity of DHEA seems to occur via a mechanism independent of its ability to modulate ERK phosphorylation.