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. 2014 Jun 6;4(8):808-22.
doi: 10.7150/thno.8255. eCollection 2014.

Exploration of Panviral Proteome: High-Throughput Cloning and Functional Implications in Virus-Host Interactions

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Free PMC article

Exploration of Panviral Proteome: High-Throughput Cloning and Functional Implications in Virus-Host Interactions

Xiaobo Yu et al. Theranostics. .
Free PMC article

Abstract

Throughout the long history of virus-host co-evolution, viruses have developed delicate strategies to facilitate their invasion and replication of their genome, while silencing the host immune responses through various mechanisms. The systematic characterization of viral protein-host interactions would yield invaluable information in the understanding of viral invasion/evasion, diagnosis and therapeutic treatment of a viral infection, and mechanisms of host biology. With more than 2,000 viral genomes sequenced, only a small percent of them are well investigated. The access of these viral open reading frames (ORFs) in a flexible cloning format would greatly facilitate both in vitro and in vivo virus-host interaction studies. However, the overall progress of viral ORF cloning has been slow. To facilitate viral studies, we are releasing the initiation of our panviral proteome collection of 2,035 ORF clones from 830 viral genes in the Gateway® recombinational cloning system. Here, we demonstrate several uses of our viral collection including highly efficient production of viral proteins using human cell-free expression system in vitro, global identification of host targets for rubella virus using Nucleic Acid Programmable Protein Arrays (NAPPA) containing 10,000 unique human proteins, and detection of host serological responses using micro-fluidic multiplexed immunoassays. The studies presented here begin to elucidate host-viral protein interactions with our systemic utilization of viral ORFs, high-throughput cloning, and proteomic technologies. These valuable plasmid resources will be available to the research community to enable continued viral functional studies.

Keywords: Gateway® cloning; Gene synthesis; Nucleic Acid Programmable Protein Arrays (NAPPA).; ORFeome; Panviral proteome; Virus-host interactions.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
High-throughput viral gene cloning and repository construction. (A) The pipeline of high-throughput viral gene cloning, repository management and clone distribution. (B) The strategies of viral gene cloning and repository construction.
Figure 2
Figure 2
The representative images of viral gene cloning by 2-step PCR and de novo gene synthesis. (A) Examination of the 2nd PCR products from PCR-based cloning in E-gel. Here, 72 HSV1 ORFs amplified from their templates are shown as 9 columns × 8 rows. (B) Synthesis of HBV polymerase ORF using gene synthesis. The total 84 oligos (~60bp/oligo) were designed and automatically allocated into seven blocks by Gene Design 3.0. The oligos before (~60 bp/oligo) and after assembly (~400 bp/block) are shown at their expected sizes in an agarose gel. These assembled blocks were further annealed into a full-length HBV polymerase with the size of 2592bp, including attB sites.
Figure 3
Figure 3
Panviral proteome has the largest viral ORF clone collection to date. (A) Comparison of the number of viral ORF clones shared by panviral proteome and viralORFeome collections. (B) Examination of the expression of HCMV ORFeome clones from panviral proteome collection using western blot. The detection of GST-protein was performed using mouse anti-GST antibody and HRP labeled sheep anti-mouse IgG antibody. The band of expressed proteins at their expected sizes was visually inspected and the results is listed in Additional file 1: Table S1.
Figure 4
Figure 4
Rubella virus - host interactions. (A) The representative images show the host target identified for rubella virus using NAPPA arrays displaying 10,000 human proteins. The color of the microarray spot from blue to red corresponds to the fluorescent signal from weak to strong. (B) Rubella virus - host interaction network. The host proteins with red letters are the previous known targets and solid lines indicate independent validation. The annotation of biological process was performed by the PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification System. The interaction network was constructed with Cytoscape. (C) Selective validation of host targets using bead-based pull-down assays. PD: Pull-down, WB: Western blot. Halo protein is used as a query control in both microarray and pull-down assays.
Figure 5
Figure 5
Co-localization analysis of rubella virus capsid and its host targets in HeLa cells. The co-transfection of HeLa cells were executed for 20 hrs using the constructs of mChery tagged capsid (Red) and GFP tagged host targets (Green). The cells were fixed in 4% paraformaldehyde and imaged using confocal fluorescence microscopy. The nucleus was stained with Hoechst dye and shown as blue. Scale bar, 5µm.
Figure 6
Figure 6
Immunological profiling of human serological response to coxsackievirus antigens. (A) The work flow of micro-fluidic multiplexed immunoassays, (B) The fluorescent image of coxsackievirus protein expression and detection using anti-Halo antibody, (C) The fluorescent signal intensity of viral proteins coated in the antigen channels (left, y-axis) and the coefficient variations from channel to channel (right, y-axis), (D) and (E) are the fluorescent images and the heatmap of serological antibody profiling using micro-fluidic multiplexed immunoassays, respectively. 00 is buffer alone and 01-11 are the human sera samples. n.c. is HeLa cell lysate as negative control, and (F) is the validation of immune-dominant antigens (VP0 and VP1) using RAPID-ELISA.

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