Requirement of cholesterol in the viral envelope for dengue virus infection.

CARRO, A.C.; ACOSTA, E.G.; DAMONTE, E.B.

Cartagena

Congreso; Third Pan American Dengue Research Network Meeting; 2012

Infection of host cells by enveloped viruses relies on the fusion of the viral envelope with a cellular membrane and, consequently, the composition of both membranes is a key factor in virus infection. Especially, cholesterol was found to play an important role for entry into the cell and other steps of virus life cycle. For flaviviruses, controversial results have been reported. Here we have examined the role of chlolesterol in both the dengue virus (DENV) envelope and cellular target membrane for proper infection. Cholesterol depletion was achieved by using methyl-beta-cyclodextrin (MCD) or nystatin (N). First, cell viability was evaluated by trypan blue exclusion to determine noncytotoxic concentrations of MCD and N. Pretreatment of different mammalian cells such as U937, K562, Raji DC-SIGN, Vero and A549 cells with these compounds produced cholesterol depletion, measured with Amplex Red Cholesterol Assay kit, but did not affect DENV-2 multiplication, determined by plaque formation. Herpes simplex virus and vesicular stomatitis virus were simultaneously assayed as controls of host membrane cholesterol dependent or independent infection, respectively. By contrast, extraction of cholesterol from DENV-2 by pretreatment of virions strongly reduced virus infectivity in a dose-dependent manner. The virucidal action of MCD was exerted against all DENV serotypes. On the other hand, soluble cholesterol also proved to have a great virucidal activity against DENV-2.  Then, it can be concluded that there is a delicate balance in the requirement of cholesterol in viral envelope to generate a productive dengue infection. For this reason DENV-2 infectivity could not be fully restored in MCD-treated virions by cholesterol replenishment at non-virucidal concentrations. The amount of bound and internalized virions, determined by real time-PCR, was not diminished in cells infected with MCD-inactivated DENV-2. These results indicate that the presence of cholesterol within the viral envelope is not necessary for viral uptake, but it is required for a later step in the viral life cycle.