Characterization of the entry mechanisms utilized by the alphavirus venezuelan equine encephalitis virus to infect mosquito cells

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Title: Characterization of the entry mechanisms utilized by the alphavirus venezuelan equine encephalitis virus to infect mosquito cells
Author: Tonya Michelle Colpitts
Abstract: 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 .
URI: http : / /hdl .handle .net /2152 .3 /249
Date: 2007-08-13

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Characterization of the entry mechanisms utilized by the alphavirus venezuelan equine encephalitis virus to infect mosquito cells. Doctoral dissertation, The University of Texas Medical Branch. Available electronically from http : / /hdl .handle .net /2152 .3 /249 .

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