Mosquito cells persistently infected with dengue virus produce viral particles with host-dependent replication.

Dengue viruses (DENV) are important arboviruses that can establish a persistent infection in its mosquito vector Aedes. Mosquitoes have a short lifetime in nature which makes trying to study the processes that take place during persistent viral infections in vivo. Therefore, C6/36 cells have been used to study this type of infection. C6/36 cells persistently infected with DENV 2 produce virions that cannot infect BHK -21 cells. We hypothesized that the following passages in mosquito cells have a deleterious impact on DENV fitness in vertebrate cells. Here, we demonstrated that the viral particles released from persistently infected cells were infectious to mosquito but not to vertebrate cells. This host restriction occurs at the replication level and is associated with several mutations in the DENV genome. In summary, our findings provide new information about viral replication fitness in a host-dependent manner.

The calmodulin antagonist W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride) inhibits DENV infection in Huh-7 cells.

Dengue virus (DENV) replicative cycle occurs in the endoplasmic reticulum where calcium ions play an important role in cell signaling. Calmodulin (CaM) is the primary sensor of intracellular Ca2+ levels in eukaryotic cells. In this paper, the effect of the calmodulin antagonist W-7 in DENV infection in Huh-7 cells was evaluated. W7 inhibited viral yield, NS1 secretion and viral RNA and protein synthesis. Moreover, luciferase activity, encoded by a DENV replicon, was also reduced. A decrease in the replicative complexes formation was clearly observed in W7 treated cells. Docking simulations suggest 2 possible mechanisms of action for W7: the direct inhibition of NS2B-NS3 activity and/or inhibition of the interaction between NS2A with Ca2+-CaM complex. This last possibility was supported by the in vitro interaction observed between recombinant NS2A and CaM. These results indicate that Ca2+-CaM plays an important role in DENV replication.

Dengue virus NS1 protein interacts with the ribosomal protein RPL18: this interaction is required for viral translation and replication in Huh-7 cells.

Given dengue virus (DENV) genome austerity, it uses cellular molecules and structures for virion entry, translation and replication of the genome. NS1 is a multifunctional protein key to viral replication and pathogenesis. Identification of cellular proteins that interact with NS1 may help in further understanding the functions of NS1. In this paper we isolated a total of 64 proteins from DENV infected human hepatic cells (Huh-7) that interact with NS1 by affinity chromatography and immunoprecipitation assays. The subcellular location and expression levels during infection of the ribosomal proteins RPS3a, RPL7, RPL18, RPL18a plus GAPDH were determined. None of these proteins changed their expression levels during infection; however, RPL-18 was redistributed to the perinuclear region after 48hpi. Silencing of the RPL-18 does not affect cell translation efficiency or viability, but it reduces significantly viral translation, replication and viral yield, suggesting that the RPL-18 is required during DENV replicative cycle.

An embryonic heart cell line is susceptible to dengue virus infection.

Dengue virus (DENV) is the causative agent of dengue fever. In recent years, patients with more severe form of the disease with acute heart failure or progression to cardiogenic shock and death have been reported. However, the pathogenesis of myocardial lesions and susceptibility of cardiomyocytes to DENV infection have not been evaluated. Under this perspective, the susceptibility of the myoblast cell line H9c2, obtained from embryonic rat heart, to DENV infection was analyzed. Our findings indicate that H9c2 cells are susceptible to the infection with the four DENV serotypes. Moreover, virus translation/replication and viral production in this cell line is as efficient as in other susceptible cell lines, supporting the idea that DENV may target heart cells as evidenced by infection of H9c2 cells. This cell line may thus represent an excellent model for the study and characterization of cardiac physiopathology in DENV infection.

Nordihydroguaiaretic acid (NDGA) inhibits replication and viral morphogenesis of dengue virus.

Dengue is the most common mosquito borne viral disease in humans. The infection with any of the 4 dengue virus serotypes (DENV) can either be asymptomatic or manifest in two clinical forms, the mild dengue fever or the more severe dengue hemorrhagic fever that may progress into dengue shock syndrome. A DENV replicative cycle relies on host lipid metabolism; specifically, DENV infection modulates cholesterol and fatty acid synthesis, generating a lipid-enriched cellular environment necessary for viral replication. Thus, the aim of this work was to evaluate the anti-DENV effect of the Nordihydroguaiaretic acid (NDGA), a hypolipidemic agent with antioxidant and anti-inflammatory properties. A dose-dependent inhibition in viral yield and NS1 secretion was observed in supernatants of infected cells treated for 24 and 48 h with different concentrations of NDGA. To evaluate the effect of NDGA in DENV replication, a DENV4 replicon transfected Vero cells were treated with different concentrations of NDGA. NDGA treatment significantly reduced DENV replication, reiterating the importance of lipids in viral replication. NDGA treatment also led to reduction in number of lipid droplets (LDs), the neutral lipid storage organelles involved in DENV morphogenesis that are known to increase in number during DENV infection. Furthermore, NDGA treatment resulted in dissociation of the C protein from LDs. Overall our results suggest that NDGA inhibits DENV infection by targeting genome replication and viral assembly.

The increase in cholesterol levels at early stages after dengue virus infection correlates with an augment in LDL particle uptake and HMG-CoA reductase activity.

Several cellular molecules and components, specifically, cholesterol and lipid rafts have been described as necessary elements for dengue virus entry and signaling in several human cells. Thus, changes in lipid rafts formation and cholesterol levels were evaluated. Here we report that the amount of total cholesterol and lipid rafts formation increase early after infection of Huh-7 cells. This augment correlates with an increase in the amount of low density lipoprotein receptor (LDLr) on the surface of infected cells and also with a lower phosphorylation level of the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). None of the changes were observed in Huh 7 cells infected with VSV used as a control. These results suggest that dengue virus infection increases intracellular cholesterol levels at early times post infection by triggering the modulation of LDL particles uptake and the increase in the enzymatic activity of HMG-CoA reductase.