A Physical Interaction Network of Dengue Virus and Human Proteins
2011; Elsevier BV; Volume: 10; Issue: 12 Linguagem: Inglês
10.1074/mcp.m111.012187
ISSN1535-9484
AutoresSudip Khadka, A.D. Vangeloff, Chaoying Zhang, Prasad Siddavatam, Nicholas S. Heaton, Ling Wang, Ranjan Sengupta, Sudhir Sahasrabudhe, Glenn Randall, Michael Gribskov, Richard Kühn, Rushika Perera, Douglas LaCount,
Tópico(s)Viral Infections and Vectors
ResumoDengue virus (DENV), an emerging mosquito-transmitted pathogen capable of causing severe disease in humans, interacts with host cell factors to create a more favorable environment for replication. However, few interactions between DENV and human proteins have been reported to date. To identify DENV-human protein interactions, we used high-throughput yeast two-hybrid assays to screen the 10 DENV proteins against a human liver activation domain library. From 45 DNA-binding domain clones containing either full-length viral genes or partially overlapping gene fragments, we identified 139 interactions between DENV and human proteins, the vast majority of which are novel. These interactions involved 105 human proteins, including six previously implicated in DENV infection and 45 linked to the replication of other viruses. Human proteins with functions related to the complement and coagulation cascade, the centrosome, and the cytoskeleton were enriched among the DENV interaction partners. To determine if the cellular proteins were required for DENV infection, we used small interfering RNAs to inhibit their expression. Six of 12 proteins targeted (CALR, DDX3X, ERC1, GOLGA2, TRIP11, and UBE2I) caused a significant decrease in the replication of a DENV replicon. We further showed that calreticulin colocalized with viral dsRNA and with the viral NS3 and NS5 proteins in DENV-infected cells, consistent with a direct role for calreticulin in DENV replication. Human proteins that interacted with DENV had significantly higher average degree and betweenness than expected by chance, which provides additional support for the hypothesis that viruses preferentially target cellular proteins that occupy central position in the human protein interaction network. This study provides a valuable starting point for additional investigations into the roles of human proteins in DENV infection. Dengue virus (DENV), an emerging mosquito-transmitted pathogen capable of causing severe disease in humans, interacts with host cell factors to create a more favorable environment for replication. However, few interactions between DENV and human proteins have been reported to date. To identify DENV-human protein interactions, we used high-throughput yeast two-hybrid assays to screen the 10 DENV proteins against a human liver activation domain library. From 45 DNA-binding domain clones containing either full-length viral genes or partially overlapping gene fragments, we identified 139 interactions between DENV and human proteins, the vast majority of which are novel. These interactions involved 105 human proteins, including six previously implicated in DENV infection and 45 linked to the replication of other viruses. Human proteins with functions related to the complement and coagulation cascade, the centrosome, and the cytoskeleton were enriched among the DENV interaction partners. To determine if the cellular proteins were required for DENV infection, we used small interfering RNAs to inhibit their expression. Six of 12 proteins targeted (CALR, DDX3X, ERC1, GOLGA2, TRIP11, and UBE2I) caused a significant decrease in the replication of a DENV replicon. We further showed that calreticulin colocalized with viral dsRNA and with the viral NS3 and NS5 proteins in DENV-infected cells, consistent with a direct role for calreticulin in DENV replication. Human proteins that interacted with DENV had significantly higher average degree and betweenness than expected by chance, which provides additional support for the hypothesis that viruses preferentially target cellular proteins that occupy central position in the human protein interaction network. 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The optimal concentration of 3-amino-1,2,4-triazole (3-AT) to suppress yeast growth in the absence of an interacting activation domain fusion (self-activation) was determined by growth on synthetic dropout (S.D.) medium lacking tryptophan, leucine, uracil, and histidine (S.D.-TLUH) and containing increasing amounts of 3-AT (1, 3, 5, 10, 20, and 50 mm). The lowest concentration of 3-AT that was able to suppress yeast growth - typically 1 or 3 mm - was used for the yeast two-hybrid assays. Library screens were performed by mating as described by Soellick and Uhrig (49Soellick T.R. Uhrig J.F. Development of an optimized interaction-mating protocol for large-scale yeast two-hybrid analyses.Genome Biol. 2001; 2 (RESEARCH0052)Crossref PubMed Google Scholar). All DNA-binding domain clones were screened at least twice against the human liver activation domain library. Colonies expressing interacting proteins were selected on S.D.-TLUH medium containing the concentration of 3-AT as determined above. The human gene inserts from the activation domain plasmids were PCR-amplified, sequenced from the 5′ end, and identified by querying the human RefSeq database (downloaded 3/4/08) using Cross_Match (129Green, P., Cross_Match program (part of Phrap package from Phil Green's documentation, www.phrap.org)Google Scholar). To retest the yeast two-hybrid interactions, PCR products of activation domain inserts from unique interactions were recloned into pOAD.103 by in vivo recombination in the yeast strain BK100. In addition, all unique interactions identified in a similar screen of HCV proteins against the same human liver library were recloned in the same manner (the full data set of HCV-human interactions will be presented elsewhere; manuscript in preparation). The resulting yeast strains were arrayed in quadruplicate in 384-spot format, mated with yeast expressing DNA binding domain fusions, and selected for growth on: (1) S.D.-TLUH containing the minimum concentration of 3-AT to suppress background growth; (2) S.D.-TLUH containing 3-AT at a concentration one step above that required to suppress background growth; and (3) S.D. medium lacking tryptophan, leucine, uracil, and adenine (S.D.-TLUA). Yeast growth was assessed on a scale of 0 to 4, with 0 being no growth above that observed for the negative control and 4 being robust growth. To standardize the results from different screens, each plate included a set of control yeast strains that displayed a range of growth rates on S.D.-TLUH+3-AT and S.D.-TLUA. Interactions were scored as positive if
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