Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Dec;10(12):M111.012187.
doi: 10.1074/mcp.M111.012187. Epub 2011 Sep 12.

A physical interaction network of dengue virus and human proteins

Sudip Khadka  1 , Abbey D Vangeloff, Chaoying Zhang, Prasad Siddavatam, Nicholas S Heaton, Ling Wang, Ranjan Sengupta, Sudhir Sahasrabudhe, Glenn Randall, Michael Gribskov, Richard J Kuhn, Rushika Perera, Douglas J LaCount
Affiliations

A physical interaction network of dengue virus and human proteins

Sudip Khadka et al. Mol Cell Proteomics. 2011 Dec.

Abstract

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. This study provides a valuable starting point for additional investigations into the roles of human proteins in DENV infection.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
DENV-human protein interaction network. A, the complete set of DENV-human protein interactions identified in this study are shown. Diamonds indicate viral proteins; ovals, human proteins; black lines, DENV-human protein interactions identified in this study; blue lines, previously identified interactions between human proteins from HPIN-1. Shading identifies proteins implicated in the replication of other viruses. Red, human proteins previously implicated in DENV replication; blue, human proteins implicated in HCV replication; green, human proteins implicated in HIV replication; gray, human proteins implicated in INFV replication; yellow, proteins implicated in the replication of other viruses; and white, proteins not previously implicated in virus replication. Additional details can be found in supplemental Table S5. B, DENV-human protein interactions by fragment. Human proteins identified in the DENV-human yeast two-hybrid screens are shown above or below the smallest viral protein fragment they interacted with in the yeast two-hybrid retests. Brackets indicate fragments composed of two or more smaller fragments (for example, NS1 fragment CD). Complete results from the yeast two-hybrid screens listing all viral fragments that interacted with a particular human protein can be found in supplemental Table S4.
Fig. 2.
Fig. 2.
Validation of DENV-human protein interactions in the split-luciferase assay. DENV and human proteins were expressed in wheat germ extracts as fusions to the N- and C-terminal fragments of firefly luciferase, respectively. In vitro translated DENV and human proteins were mixed in PBS supplemented with 1% BSA and protease inhibitors (Roche) and incubated overnight at 4 °C. As negative controls, each viral protein was tested against the empty C-terminal luciferase vector and each human protein was tested against the empty N-terminal luciferase vector. Binding reactions were set up in duplicate and luciferase activity was measured twice for each reaction. Graph shows the average of the four luciferase readings normalized to the luciferase activity of the DENV protein with the empty C-terminal luciferase negative control, which was set at 100 relative light units (RLU). Error bars indicate the S.E. Asterisks indicate pairs of interacting proteins that yielded luciferase activity that was significantly greater than both negative controls (student's unpaired t test, p value <0.02).
Fig. 3.
Fig. 3.
Enriched annotation terms and pathways among the human proteins that interacted with DENV. A, Enriched terms identified by the DAVID Bioinformatics Database in the complete set of human proteins that interacted with DENV proteins in this study (68, 69). Graph shows the –log10-transformed Benjamini-corrected p values for each term. Terms were grouped according to their functional similarity. Enriched features of human proteins that interacted with individual viral proteins are listed in supplemental Table S6. B, Subnetwork of human plasma proteins that interacted with DENV. Diamonds indicate DENV proteins. Ovals represent human proteins. Shading indicates human proteins annotated with the term “Complement and coagulation cascade.”
Fig. 4.
Fig. 4.
Inhibition of DENV replication in cells with reduced expression of human genes. A, Luciferase production from a DENV replicon in siRNA treated cells. siRNAs targeting the indicated human genes, DENV NS5, or an irrelevant sequence were electroporated into Huh-7.5 cells along with DENV luciferase replicon RNA. Luciferase levels were measured in cell lysates prepared at 6 and 48 h after electroporation. DENV replication is represented as the ratio of the luciferase values at 48 and 6 h and normalized to the irrelevant siRNA control, which was defined as 100%. Values are averages of at least three replicates ± S.E. * p ≤ 0.05, ** p ≤ 0.001. B, Cell viability following siRNA treatment. siRNAs targeting the indicated genes or an irrelevant sequence (IRR) were electroporated into Huh-7.5 cells with DENV luciferase replicon RNAs. Cellular ATP levels were measured at 48 h using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega). Values are averages of at least three replicates ± S.E. C, DENV production in DDX3X siRNA-treated cells. Huh7 cells were transfected with the indicated siRNAs and infected with DENV at an MOI of 3. Supernatant was harvested at 24 h post infection and titered by plaque assay. Titers were normalized to nonspecific siRNA control (NS). D, Western blot analysis of DDX3X levels in siRNA-treated cells from part C. Bottom panel shows actin levels as a loading control.
Fig. 5.
Fig. 5.
Colocalization of CALR with DENV proteins and dsRNA in infected cells. Huh7 cells were infected with DENV at an MOI of 1, fixed 36 h post infection, and probed with antibodies against CALR and DENV NS5 (top row), DENV NS3 (middle row), or dsRNA (bottom row). Pseudo-colored merged images are shown in column 3, with CALR in red and NS5, NS3, and dsRNA in green. Inset shows a higher magnification of the region in the white box of the merged images. The white line indicates the region sampled for quantification of the fluorescent intensity of CALR (red lines) and NS3 or NS5 (green lines), as shown in the graphs on the right. Nuclear-localized NS5, which accounts for ∼95% of the total NS5 present in the infected cell, is overexposed to enable visualization of cytoplasmic NS5. Images represent a single optical slice.
Fig. 6.
Fig. 6.
Elevated degree and betweenness in cellular proteins implicated in virus infection. A, average degree (k) of human proteins that bound to viral proteins from DENV (this study), HCV, HIV, and INFV. B, Average betweenness centrality (b) of human proteins that bound to viral proteins from DENV (this study), HCV, HIV, and INFV. C, average degree (k) of human proteins that were identified in siRNA screens for cellular cofactors of DENV, HCV, HIV, INFV, and Mycobacterium tuberculosis. D, average betweenness centrality (b) of human proteins that were identified in siRNA screens for cellular cofactors of DENV, HCV, HIV, INFV, and Mycobacterium tuberculosis. Average degree and betweenness centrality were calculated for each data set and compared with the same values from a randomly chosen set of human proteins of the same size. Asterisks indicate statistical significance. * p ≤ 0.05, ** p ≤ 0.001, *** p ≤ 1 × 106. E, degree distribution of human proteins in the DENV-human protein interaction network (open circles) and the human-human protein interaction network (closed circles). Proteins were binned according to the number of binding partners as indicated.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Llano M., Saenz D. T., Meehan A., Wongthida P., Peretz M., Walker W. H., Teo W., Poeschla E. M. (2006) An essential role for LEDGF/p75 in HIV integration. Science 314, 461–464 - PubMed
    1. Maertens G., Cherepanov P., Pluymers W., Busschots K., De Clercq E., Debyser Z., Engelborghs Y. (2003) LEDGF/p75 is essential for nuclear and chromosomal targeting of HIV-1 integrase in human cells. J. Biol. Chem. 278, 33528–33539 - PubMed
    1. Galluzzi L., Kepp O., Morselli E., Vitale I., Senovilla L., Pinti M., Zitvogel L., Kroemer G. (2010) Viral strategies for the evasion of immunogenic cell death. J. Intern Med. 267, 526–542 - PubMed
    1. Chaurushiya M. S., Weitzman M. D. (2009) Viral manipulation of DNA repair and cell cycle checkpoints. DNA Repair 8, 1166–1176 - PMC - PubMed
    1. Skaug B., Chen Z. J. (2010) Emerging role of ISG15 in antiviral immunity. Cell 143, 187–190 - PMC - PubMed

Publication types

MeSH terms

Substances