Browsing by Subject "actin"
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Item Characterization of the entry mechanisms utilized by the alphavirus venezuelan equine encephalitis virus to infect mosquito cells(2007-08-13) Tonya Michelle Colpitts; Robert Davey, PhD; Scott Weaver, PhD; Peter Mason, PhD; Lisa Elferink, PhD; Christopher Broder, PhDVenezuelan 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.Item Examination of eukaryotic chaperonin-mediated nascent chain folding in the cytosol: a photocrosslinking approach(Texas A&M University, 2004-11-15) Etchells, Stephanie AnneTRiC (TCP-1 ring complex), a type II chaperonin, facilitates protein folding, and we previously showed that TRiC crosslinks to ribosome-bound actin and luciferase nascent chains. Here, it was found that actin and luciferase nascent chains were adjacent to more than one TRiC subunit at different stages of translation. Six and seven out of the eight TRiC subunits were photocrosslinked to the luciferase and actin nascent chains, respectively. Actin nascent chains with widely-spaced, site-specific probe locations were adjacent to the same three TRiC subunits (a, b and e) at different stages of translation. The exposure of other TRiC subunits to nascent chains varied with the length and identity of the nascent chain. In addition, the presence or absence of ATP influences the photocrosslinking yields. This suggests that ATP alters the conformation of the subunits and/or their affinity for the nascent chain. Photocrosslinking also revealed that TRiC is in close proximity to the exit site of the ribosomal tunnel, presumably to create a protected folding environment for the nascent chain. Immunoprecipitations under native conditions revealed that prefoldin photocrosslinks to the actin nascent chain and that these prefoldin-containing photoadducts are coimmunoprecipitated with antibodies specific for the TRiC a subunit. This result suggests that prefoldin and TRiC bind simultaneously to the same actin nascent chain. Photocrosslinking studies with probes at position 68 in the actin nascent chain revealed that prefoldin binds to the nascent chain subsequently to TRiC binding. An unknown protein with an apparent molecular mass of 105 kDa was shown to photocrosslink to the luciferase nascent chain in a length-dependent manner at specific probe locations close to the N-terminus of the nascent chain. Thus, the nascent chain sees a variety of proteins in its immediate environment as it emerges from the ribosomal tunnel and undergoes its chaperonin-assisted folding.