Exposure to low pH is not required for the penetration of mosquito cells by Sindbis virus 

Raquel Hernandez, Tianci Luo and Dennis T. Brown

Department of Biochemistry, North Carolina State University, Raleigh, NC 27695

It is widely held that the penetration of cells by alphaviruses is absolutely dependent upon exposure to the acid environment of an endosome. The alphaviruses Sindbis replicates in both vertebrate and invertebrate cell cultures. We have found that exposure to an acid environment may not be required for infection of cells of the insect host. In the research described below, we have investigated the effect of two  agents (NH₄Cl and chloroquine), which raise the pH of intracellular compartments (lysosomotropic weak bases) on the infection and replication of Sindbis virus in cells of the insect host Aedes albopictus.  The results show that both of  these agents do increase the pH of endosomes  as indicated by protection against diphtheria toxin intoxication.  NH₄Cl blocked the production of infectious virus and blocked virus RNA synthesis when added prior to infection.  Chloroquine in contrast to its effect on vertebrate cells had no inhibitory effect on infectious virus production in mosquito cells even when added prior to infection.  Treatment with NH₄CL blocked RNA synthesis and production of progeny virus.  NH₄Cl did not prevent the penetration of virus RNA into the cell cytoplasm or translation of the RNA to produce a precursor to virus nonstructural proteins.  These data suggest that while these two drugs raise the pH of endosomes, NH₄Cl has some other effect on the cell, which results in the inactivation of the virus/host machinery, which replicates virus RNA.  The mechanism of inactivation appears to be at the level of non-structural protein processing.  These data support previous experiments published from our laboratory suggesting that exposure to an acid environment within the cell may not be an obligatory step in the process of infection of cells by alphaviruses.