Characterization of the entry mechanisms utilized by the alphavirus venezuelan equine encephalitis virus to infect mosquito cells
Venezuelan equine encephalitis eirus (VEEV) is a New World alphavirus that can cause fatal encephalitis in humans. VEEV is an enveloped, positive-strand RNA virus that is transmitted by a mosquito vector. Most research on alphavirus entry was done with the Old World alphavirus Semliki Forest virus (SFV) in mammalian cells. Not much is known about the entry of New World alphaviruses, especially in cells of the viral vector, the mosquito. Work with SFV has shown that Old World alphaviruses enter mammalian cells via receptor-mediated, clathrin-mediated endocytosis. This endocytic pathway utilizes several proteins in the mammalian cell, including the clathrin protein, the small GTPases known as Rab proteins and the large GTPase dynamin. These proteins have been shown to play a role in the entry of several viruses and are thought to be involved in alphavirus entry in mammalian cells. Here mosquito homologs of these proteins are identified, isolated and characterized in the mosquito cell. Rab5, Rab7 and dynamin are shown to be involved and necessary for VEEV entry and infection in mosquito cells. A novel entry assay is used to confirm that VEEV requires a low pH to enter mosquito cells. This work represents the first characterization of the involvement of mosquito endocytic pathways for infection of a New World alphavirus and sheds light on an important aspect of virus infection in an insect vector. The role of actin in VEEV internalization was also examined. Actin is known to be involved in the mammalian endocytic pathway and to act together with dynamin to coordinate endocytosis. Here mosquito actin is identified and shown to colocalize with mosquito dynamin. Both proteins also colocalize with internalized VEEV. Inhibiting actin polymerization prevents entry of the virus both by microscopic examination as well as utilizing the luciferase entry assay. This work shows that VEEV enters the mosquito cell via a pH-dependent endocytic pathway that requires functional endocytic proteins including Rab5, Rab7 and dynamin. It is also shown that F-actin must be present for VEEV to enter mosquito cells and that actin and dynamin act together during virus internalization.