Tick-flavivirus interactions: Discovery of host proteins that are affected in virus infection
Tick-borne flavivirus (TBF) infections cause morbidity and mortality worldwide. A primary goal of vector-borne disease research is to identify ways to prevent pathogen and parasite transmission. The black-legged tick, Ixodes scapularis, is the only tick species with a sequenced and annotated genome and is a vector of TBFs. This project utilizes Langat virus (LGTV) to model TBF infection in I. scapularis ISE6 cells. The aims were to (1) characterize the LGTV-ISE6 system, (2) discover differentially-expressed proteins associated with LGTV infection, and (3) identify key genes of interest that were involved in LGTV infection. Two proteomic analyses by LC-MS/MS identified 486 and 579 ISE6 proteins (total of 764 ISE6 proteins combined). First and second proteomic analyses identified 68 and 246 ISE6 proteins with increased expression and 198 and 188 ISE6 proteins with decreased expression, respectively. Greater than 265 ISE6 proteins were mapped to cellular function/pathways using Kyoto Encyclopedia of Genes and Genomes (KEGG). Greater than 221 ISE6 proteins were identified with no KEGG function/pathway. After filtering based on strength of protein identification, cellular function/pathway involvement, and an increased expression following LGTV infection, ten proteins were selected. Ten selected proteins/genes of interest included eight with known function (Fumar, ERP29, DPCD, CNHydro, MDH2, PARP, CUKinase, and ACAT1) and two with unknown function (Hypo195 and Hypo576). RNA interference knockdown assays were established in ISE6 cells to determine effect on LGTV infection. Inhibition of genes of interest involved in cellular amino acid, carbohydrate/lipid, and cofactor/vitamin metabolism as well as unknown proteins were shown to affect LGTV genome replication and replication. In addition, LGTV replication was reduced by perturbing ISE6 metabolic function using trichostatin A and oligomycin A and glutamate metabolic function using hexachlorophene and epicatechin gallate small molecules in compound assays. This research suggested that carbohydrate and amino acid metabolic enzymes were involved in LGTV infection in the tick vector and possibly for transmission.
Richard, Purdue University.
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