Interaction of CSFV E2 Protein with Swine Host Factors as Detected by Yeast Two-Hybrid System

Abstract

E2 is one of the envelope glycoproteins of pestiviruses, including classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV). E2 is involved in several critical functions, including virus entry into target cells, induction of a protective immune response and virulence in swine. However, there is no information regarding any host binding partners for the E2 proteins. Here, we utilized the yeast two-hybrid system and identified fifty-seven host proteins as positive binding partners which bound E2 from both CSFV and BVDV with the exception of two proteins that were found to be positive for binding only to CSFV E2. Alanine scanning of CSFV E2 demonstrated that the binding sites for these cellular proteins on E2 are likely non-linear binding sites. The possible roles of the identified host proteins are discussed as the results presented here will be important for future studies to elucidate mechanisms of host protein-virus interactions during pestivirus infection. However, due to the limitations of the yeast two hybrid system, the proteins identified is not exhaustive and each interaction identified needs to be confirmed by independent experimental approaches in the context of virus-infected cells before any definitive conclusion can be drawn on relevance for the virus life cycle.

About the Authors:

Douglas P. Gladue

Affiliations Plum Island Animal Disease Center, Agriculture Research Service, United States Department of Agriculture, Greenport, New York, United States of America, Department of Pathobiology and Veterinary Science, University of Connecticut, Storrs, Connecticut, United States of America

Ryan Baker-Bransetter

Affiliation: Plum Island Animal Disease Center, Agriculture Research Service, United States Department of Agriculture, Greenport, New York, United States of America

Lauren G. Holinka

Affiliation: Plum Island Animal Disease Center, Agriculture Research Service, United States Department of Agriculture, Greenport, New York, United States of America

Ignacio J. Fernandez-Sainz

Affiliation: Plum Island Animal Disease Center, Agriculture Research Service, United States Department of Agriculture, Greenport, New York, United States of America

Vivian O’Donnell

Affiliations Plum Island Animal Disease Center, Agriculture Research Service, United States Department of Agriculture, Greenport, New York, United States of America, Department of Pathobiology and Veterinary Science, University of Connecticut, Storrs, Connecticut, United States of America

Paige Fletcher

Affiliation: Plum Island Animal Disease Center, Agriculture Research Service, United States Department of Agriculture, Greenport, New York, United States of America

Zhiqiang Lu

Affiliation: Plum Island Animal Disease Center, Department of Homeland Security, Greenport, New York, United States of America

Manuel V. Borca

* E-mail: [email protected]

Affiliation: Plum Island Animal Disease Center, Agriculture Research Service, United States Department of Agriculture, Greenport, New York, United States of America

Introduction

Classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV) are highly contagious diseases of swine and bovine, respectively. Both are small, enveloped viruses with a positive-sense, single-strand RNA genome and are classified as members of the pestivirus genus within the Flaviviridae family [1]. The approximately 12.5-kb pestivirus genome contains a single open reading frame that encodes a polyprotein composed of 3,898 amino acids that ultimately yields 11 to 12 final cleavage products (NH2-Npro-C-Erns-E1-E2-p7-NS2-NS3-NS4ANS4B-NS5A-NS5B-COOH) through co- and post-translational processing of the polyprotein by cellular and viral proteases [2]. Structural components of the virions include the capsid (C) protein and glycoproteins: Erns, E1 and E2. E1 and E2 are anchored to the envelope at their carboxyl termini, and Erns loosely associates with the viral envelope [3]–[5]. E1 and E2 are type I transmembrane proteins with an N-terminal ectodomain and a C-terminal hydrophobic anchor [5]. E2 is considered essential for CSFV replication, as virus mutants containing partial or complete deletions of the E2 gene are nonviable [6]. E2 is the most immunogenic of the CSFV and BVDV glycoproteins [3], [7], [8], inducing neutralizing antibodies and protection against lethal CSFV or BVDV challenge. E2 has been implicated, along with Erns [9] and E1 [10], in viral adsorption to host cells; indeed, chimeric pestiviruses exhibit infectivity and cell tropism phenotypes consistent with those of the E2 gene donor [7], [11]. Modifications introduced into this glycoprotein appear to have an important effect on CSFV virulence [12]–[16]. It is evident that pestivirus E2 plays many critical roles. Recently the E2 protein of BVDV has been crystallized, revealing a three domain structure. Domains I and II are similar to Ig-like domains and domain III is a series of three small β-sheet modules; this structure is believed to be similar to CSFV E2 by prediction analysis [17], [18]. Although it is obvious that E2 plays a critical role during virus infection, there is no direct evidence of any host binding partners to either CSFV or BVDV E2. To advance the current understanding of the functions of the pestivirus E2 protein, we attempted to identify host proteins that directly interact with the E2 protein of CSFV or BVDV by means of the yeast two-hybrid system using custom swine and bovine cDNA libraries. Results indicate that both CSFV and BVDV E2 interact with fifty-seven different host proteins, while two additional proteins interact solely with CSFV E2. Attempts to map any of the host protein binding sites within the CSFV E2 protein by using a poly-alanine scanning mutagenesis approach suggests that the host proteins bind a structurally non-linear portion of E2. The possible roles of the identified host proteins are discussed. Identification of host proteins directly interacting with pestivirus E2 may significantly improve the understanding of the role of E2 during infection and virulence. However, each interaction identified here needs to be confirmed by an independent experimental approach in the context of virus-infected cells before any definitive conclusion can be drawn on relevance for the virus life cycle.

Materials and Methods

Development of the cDNA Libraries

A porcine primary macrophage cDNA expression library was constructed (Clontech, Mountain View, CA) using monocytes/macrophages obtained from healthy CSFV-free swine exactly as previously described [19]. Macrophage cultures were prepared from defibrinated swine blood. Total RNA was extracted from adherent cells using an RNeasy Mini kit (Qiagen, Valencia, CA). Contaminant genomic DNA was removed by DNase treatment using TURBO DNA-free (Ambion, Austin, TX). After DNase treatment, genomic DNA contamination of RNA stocks was assessed by real-time PCR amplification targeting the porcine β-actin gene. RNA quality was assessed using RNA Nano Chips on an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). Cellular proteins were expressed as GAL4-AD fusion proteins while CSFV E2 was expressed as a GAL4-BD fusion protein. Following a similar procedure, a bovine cDNA expression library was constructed (Clontech, Mountain View, CA) using different tissues from healthy non-infected bovine [20].

Library Screening

The GAL4-based yeast two-hybrid system was used for this study [21], [22]. The ‘bait’ protein, CSFV Brescia E2 protein (amino acid residues 1–342) or BVDV NADL E2 protein (amino acid residues 1–342), was expressed with an N-terminus fusion to the GAL4 Binding Domain (BD). As ‘prey’, the previously described swine macrophage cDNA library and bovine cDNA library containing proteins fused to the GAL4 Activation Domain (AD) was used. Screening was done as previously described [19]. Sequencing of the identified library clones was performed by the dideoxynucleotide chain-termination method [23]. Sequencing reactions were prepared with the Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). Reaction products were sequenced using an ABI PRISM 3730xl automated DNA sequencer (Applied Biosystems, Foster City, CA). The identified sequence was checked to be in-frame with the GAL4-AD and the NCBI BLAST algorithm was used to identify the host protein.

PANTHER Classification of Proteins

Positive interacting host proteins that bound CSFV and BVDV E2 proteins were entered into the PANTHER classification program. The resulting classification was used to group proteins by biological process. The PANTHER program [24] can be found at http://www.pantherdb.org/.

Construction of an Alanine Scanning Mutagenesis Library for CSFV E2

Full-length CSFV E2 from Brescia strain was used as a template in which native amino acids were substituted with alanine, introduced by site-directed mutagenesis using the Quick Change XL Site-Directed Mutagenesis kit (Stratagene, Cedar Creek, TX), performed per manufacturer's instructions. Briefly, the full-length plasmid was amplified by PCR, digested with Dpn1 to leave only the newly amplified plasmid, transformed into XL10-Gold ultra competent cells, and grown on Terrific Broth plates containing ampicillin. Positive colonies were grown for plasmid purification using the Qiagen Maxiprep kit. The full-length E2 was sequenced to verify that only the desired mutation was present in the plasmid. Primers were designed using the Stratagene Primer Mutagenesis program. This program limits a maximum number of amino acid changes, based on primer length and the number of nucleotides that had to be changed for every mutant, setting the basis for deciding on the regions to be mutated. Primers were designed using the manufacturer’s primer design program: https://www.genomics.agilent.com/CollectionSubpage.aspx?PageType= Tool&SubPageType = TolQCPD&PageID = 15.

Results and Discussion

Conservation in E2 Amino Acid Sequence among Pestiviruses

A sequence alignment between CSFV E2 from the Brescia isolate (GenBank Accession #AF091661) or the BVDV E2 from the NADL isolate (GenBank Accession #AJ133738.1) was performed, and a comparison between the amino acid sequence of these two proteins revealed a 65% identity and a 74% similarity depending on which isolate was being compared [17] (Fig. 1). Exchanging the E2 protein from one pestivirus with another pestivirus E2 results in changes to cell tropism in vitro [11], [25], suggesting that although there are differences in sequence between the different E2 proteins, they share enough common features that they can be exchanged and allow for virus replication. However, it has been reported that a chimeric BVDV virus with the E2 protein of CSFV was unable to cause disease in swine although it was shown to efficiently grow in swine cells, suggesting that the E2 proteins of CSFV and BVDV have enough similarity to be exchanged for growth in cell culture, but additional factors are needed to cause disease in the non-native host [25].

[Figure omitted. See PDF.]

Figure 1. Multiple sequence alignment using Bioedit software was performed using CSFV E2 from the Brescia isolate (GenBank Accession #AF091661) and the BVDV E2 from the NADL isolate (GenBank Accession #AJ133738.1).

https://doi.org/10.1371/journal.pone.0085324.g001

Specificity of E2 Proteins in the Yeast Two-hybrid

The E2 protein from either CSFV or BVDV was cloned into the yeast two-hybrid vector with the N-terminus of E2 fused to the binding domain (BD) of gal4. Both the CSFV and BVDV E2 proteins tested lacked the C-terminal transmembrane domain to allow the protein to enter the nucleus, a requirement for the yeast two-hybrid model. The level of background activity for the E2 proteins was assessed by co-expressing E2-BD with the T-antigen coupled to the AD (TAg-AD), a common negative control protein used in the yeast two-hybrid. Both pestivirus E2s were determined to have a low degree of background when grown on media lacking adenine and histidine, two nutritional markers that are driven by the gal4 promoter only in the presence of binding between the two proteins being tested. P53-BD and TAg-AD were included as a positive control for the yeast two-hybrid (data not shown). Cells were grown on media lacking only plasmid selection nutritional markers (tryptophan and leucine), allowing growth in the absence of protein binding, as a control. Our results show that there was no background growth on media lacking adenine and histidine, and that both CSFV E2 and BVDV E2 are suitable for screening a host protein library.

Yeast Two-hybrid Screening Results

To identify host cellular proteins that interact with the CSFV or BVDV E2 proteins, both proteins were screened against both a custom bovine and a custom swine cDNA library. Approximately 1×107 independent yeast colonies were tested, representing approximately 3-fold library saturation, as both libraries were determined to contain approximately 3×106 independent clones. Positive colonies were selected for growth on selection media (-Leu/-Trp/-His/-Ade). Plasmids were recovered in E.coli and sequenced. In-frame proteins were retested for specificity to CSFV E2, BVDV E2, BD only, and Lam-BD, a negative control commonly used in the yeast two-hybrid. Fifty-seven proteins were identified as positive binding partners to both the CSFV E2 and the BVDV E2 protein (Table 1).

[Figure omitted. See PDF.]

Table 1. Proteins that interact with CSFV+BVDV E2.

https://doi.org/10.1371/journal.pone.0085324.t001

Differential Binding of Host Proteins to Pestivirus E2 Proteins

Despite the number of proteins identified that bound both BVDV E2 and CSFV E2, there were some differential binding partners for these viral proteins. Two of the proteins identified were positive binding partners for CSFV E2 and did not bind BVDV E2 (Table 2). One protein is a member of the heterogeneous nuclear ribonucleoprotein (HNRP) family (HNRPF). HNRPs have been shown to be binding partners for a wide range of viruses, and disruption of this binding or knockdown of HNRPs causes a decrease in viral replication or viral release. For example, HNRNPD has been shown to bind Epstein-Barr Virus-encoded RNA 1 (EBER1) [26] and HNRNPK binds Epstein-Barr virus nuclear antigen 2 (EBNA2) which enhances viral protein LMP2A expression by an unknown mechanism [27]. In Japanese encephalitis virus (JEV) infections, HNRNP A2 has been shown to be important for viral replication, binding both the core protein and the viral RNA [28]. HNRNP H1 also binds the core protein of Hepatitis C virus [29]. During an infection with herpes simplex virus-1, depletion of HNRNP K inhibits viral egress [30]. Although the exact mechanisms are not known for the requirement for HNRP’s that bind viral proteins or viral RNA, it can be hypothesized that viral binding of HNRNPs can regulate both host and viral RNA, and may provide some way for viruses to enhance their own RNA replication and translation, or possibly is a way to prevent host RNA translation during viral infection.

[Figure omitted. See PDF.]

Table 2. Host proteins exclusively binding CSFV E2.

https://doi.org/10.1371/journal.pone.0085324.t002

The other protein that was positive for CSFV E2, polypyrimidine tract-binding protein 2 (PTBP2), is highly homologous to polypyrimidine tract binding protein (PTB) but thought to be located primarily in brain and neural cells. PTBP2 has ribonucleoprotein domains, has been shown to enhance HNRNPH function, and could be involved with other HNRNPs, suggesting both proteins that were positive for CSFV E2, PTBP2 and HNRNPF, could be involved in a similar pathway. Interestingly, these are the only two proteins that were determined to be a positive binding partner for CSFV E2 and did not bind BVDV E2.

Classification of E2-binding Host Proteins by Biological Process

To gain some insight into the potential biological processes that are regulated by pestivirus E2 proteins, we classified all the identified host proteins using the PANTHER (Protein ANalysis THrough Evolutionary Relationships) classification system [24] (Fig. 2). For the proteins that bind both E2 proteins, the PANTHER analysis identified 15 different biological processes defined in Table 3, as the function of a particular protein in the context of a larger network of proteins that interact to accomplish a process at the level of the cell or organism. A brief analysis of these proteins in the context of their biological processes and their hypothetical significance in CSFV replication and virulence are discussed.

[Figure omitted. See PDF.]

Figure 2. A graphic view of the distribution of E2 positive interacting proteins, as separated into Biological processes, as defined by the PANTHER classification system.

https://doi.org/10.1371/journal.pone.0085324.g002

[Figure omitted. See PDF.]

Table 3. Biological process classification of E2 interacting proteins by PANTHER analysis.

https://doi.org/10.1371/journal.pone.0085324.t003

Twenty-one proteins were classified as being involved in PANTHER Metabolic Processes within the cell (Table 3). We, along with others, have previously used microarray analysis to identify subsets of genes in cellular metabolic pathways involved in DNA replication that were down-regulated during CSFV infection [31]–[33]. The host proteins identified in the yeast two-hybrid screening as being part of these metabolic processes are (Table 3). Several of these host proteins have been shown to be involved in the pathogenesis of other viruses. For example, Chaperonin containing TCP1, subunit 7 (CCT7) is a member of the TCP1 Ring complex (TRiC). This complex is involved in correctly folding various proteins, and has been shown to be involved in the replicative process of influenza A virus [34], type D retrovirus [35] and Hepatitis C virus (HCV) [36]. Another protein, DCTN6 (Dynactin 6), is part of the Dynactin complex, which binds cargo (organelles, virus, vesicle, etc) to dynein or kinesin for intracellular transport. This transport system has been implicated as critical for several viruses for cellular entry or cellular escape, including pseudorabies virus (PrV) [37], adenovirus [38] and the human immunodeficiency virus type 1 (HIV-1) [39]. During an Epstein-Barr virus (EBV) infection, the protein UXT (ubiquitously-expressed, prefoldin-like chaperone) is phosphorylated, causing the downregulation of NF-κB transactivation, and may possibly play a critical role in the lytic cycle of EBV. This class of proteins is important to the process of replication and pathogenesis for other viruses, suggesting they could play also an important role in pestivirus virulence.

Thirteen of the host proteins identified by the yeast two-hybrid screening are involved in the PANTHER biological pathway of cellular processes (Table 3). In addition, the PANTHER biological pathway of developmental processes, is represented by 10 of the host proteins identified (Table 3). There is a large amount of overlap in the developmental process and cellular process groups, likely because proteins that are involved in cellular processes can ultimately play a role in organism development; because of this overlap we will discuss the two process groups together. Some of these proteins have been described as playing an important role in viral processes. For example, LGALS3 (lectin galactoside-binding soluble protein 3), is known to be involved in the process of virus entry during viral infection with parvovirus [40] and HSV-1 [41]. In addition, LGALS3 is up-regulated during Junín virus (JUNV) infection although its role during infection is still unknown [42]. Other galectin proteins have been implicated as being important for pathogenesis during viral infection for both HIV [43] and influenza virus [44]. This suggests the possibility that pestiviruses could utilize LGALS3 as part of the mechanism for viral entry; however, further studies would be required to determine the specific role for E2 binding to LGALS3.

Viral subversion of the immune system during the replication of CSFV has been repeatedly shown [45]–[49]; thus, it is no surprise that several of the proteins that were identified in our screen are classified as being part of PANTHER immune system processes (Table 3). CFP, or complement factor properdin, is a regulator of the alternative complement activation pathway and it is a protein target for immune regulation by other viruses. In dengue virus infection, CFP is up-regulated [50], while in HSV-1 the CFP binding site in C3 is blocked by HSV-1 glycoprotein gC [51] and in HIV-1, glycoprotein gp12 has binding affinity to CFP. Many viruses have evolved ways to prevent activation of the complement pathway, and perhaps CSFV and BVDV block this pathway though the interaction between E2 and CFP.

Further analysis of identified host proteins using PANTHER classification schemes revealed host proteins that belong to ten other biological processes, cell communication, cell adhesion, system processes, response to stimulus, cellular component organization, cell cycle; transport, generation of precursor metabolites and energy, apoptosis, localization, and reproduction (Table 3). Some of these proteins overlap in several biological functions as cellular proteins often play multiple roles in multiple pathways. Further studies of the potential effect of these proteins binding to pestivirus E2 proteins will have to be done to gain a better understanding of which cellular pathways E2 may be involved in.

In addition, several proteins were unable to be classified by the PANTHER classification system as belonging to any predefined biological process. Those are host proteins CDC115, CEP57, CTSB, EMID1, FANCF, FANCL, GCA, MRPL45, NUP43, PELI1, POFUT2, POMP, RMD5, SERTAD1, SERTAD3, SIVA1, SPG11, TBL1XR1, TOR1AIP2, TRAPPC2, UPF0712 and UPF1 (Table 1). However, some of these proteins are known to be important during other viral infections. For example, host protein Cathepsin B (CTSB) promotes Heptatis B infection [52], while inhibition of CTSB decreases HIV particle release from the infected cell [53], [54]. Additionally, host protein Fanconi anemia complementation group L (FANCL), was determined to interact with CSFV protein NS2 [55], suggesting the possibility that FANCL could play multiple roles during CSFV infection by binding to different CSFV proteins. Interestingly, now we are reporting an additional member of this family, FANCF (group F), as an additional binding partner for E2. Host protein Nup43, or nuclear pore protein 43 kDA, is a component of the nuclear pore. In polio virus [56], vaccinia virus [57] and HIV-1 [58] infections, the viral proteins have been shown to interact with other nuclear pore proteins to either allow the viral genome to enter the nucleus or to prevent host mRNA from exiting the nucleus, thus preventing host transcription. Additionally, host protein PELI1, or Pellino-1, has been implicated in regulating the epithelial cellular response to rhinoviruses [59], while SIVA1, or apoptosis-inducing factor, has been shown to be required for influenza A virus replication [60] and is important for apoptosis regulation during viral infection by HIV-1 [61], Human papillomavirus-16 [62] and coxsackievirus B3 [63]. A regulator of nonsense transcripts homolog, UPF1, has been implicated in human T-lymphotropic virus type 1 to interact with tax protein, inhibiting nonsense-mediated mRNA decay [64]. During a Rous sarcoma virus (RSV) infection, UPF1 degrades viral RNA in the absence of the RSV stability element RSE, suggesting a role for RSE in avoiding UPF1 degradation. During HIV-1 infection, UPF1 is a component of the HIV-1 ribonucleoprotein (RNP), and expression of UPF1 directly influences HIV-1 RNA expression [65].

It is clear that many of the proteins identified by the yeast two-hybrid system as host proteins that interact with the E2 proteins of CSFV and BVDV are involved in different aspects of viral pathogenesis in other viral infections, strongly suggesting that these proteins could play a role in the pathogenesis of pestiviruses.

Construction of Poly-alanine Mutant Library

In an attempt to map the binding site(s) for the host proteins that interact with CSFV E2, an alanine scanning mutagenesis approach was used. This particular approach has been used in our laboratory to facilitate elucidation of specific areas within FMDV viral proteins recognized by host proteins [20], [66]. We used site-directed mutagenesis to construct a set of 76 CSFV E2 mutant proteins containing sequential stretches of amino acids where the native amino acid residues were substituted by alanine residues (Fig. 3). The complete set of mutated E2 proteins the yeast two-hybrid system, were assessed for their ability to bind all of the host proteins identified that interacted with native CSFV E2. After testing all of the potential mutants with all of the positive cellular proteins, we were unable to map any of the binding sites, as all of the host proteins were capable of binding all of the E2 alanine mutants. These results suggest that disruption of a single linear amino acid stretch is not sufficient to alter the binding of any of the positive cellular proteins. This was initially an unexpected result; however, the recent determination of the crystal structure of BVDV E2 has demonstrated that the structure of BVDV E2 is a rather complex spatial structure. Therefore, it is possible that most of the host proteins would bind multiple non-linearized arranged residues that would not be disrupted by changing a single linear residue stretch to alanine. To map the residues responsible for host protein binding would require a more complex mutagenesis approach to determine the areas of protein interaction within E2.

[Figure omitted. See PDF.]

Figure 3. Scheme showing CSFV E2 alanine mutants used in the present study.

All indicated residues were mutated to an alanine.

https://doi.org/10.1371/journal.pone.0085324.g003

Conclusion

We identified fifty-five host proteins that bind structural glycoprotein E2 of both CSFV and BVDV (Table 1). Two additional host proteins recognized CSFV E2 but did not interact with BVDV E2 (Table 2). Many of the host proteins identified have also been identified by other means to interact with other viruses, and in many cases determined to be important in the process of viral replication and/or pathogenesis. We cannot rule out the limitations of the yeast two-hybrid methodology in regards to the identification of host proteins that may not actually interact with E2 in the infected cells, as our studies determined proteins that were capable of interacting in the yeast two-hybrid system, and not in a virus infected cell. It is also possible, that some host proteins were not identified either due to the host protein not being present in either library or due to the inability of the protein to interact due to the limitations in the yeast two-hybrid system. For example, unidentified protein interactions that may only occur exist within the environment of the infected cells or in the presence of other viral proteins such as proteins that interact with the E2–E1 heterodimer. CD46 the cellular receptor for BVDV E2 [67] was also not detected, most likely due to CD46 being a membrane protein, making it unlikely that it would enter the nucleus in the yeast two-hybrid system. Nevertheless, the identification of cellular factors potentially interacting with structural glycoprotein E2 during virus infection is critical to explaining phenomena known to involve E2 as well as to discover novel roles for E2.

Further work is needed in order to truly evaluate the importance of the interactions described here in regards to their possible roles in virus replication or virus virulence, as our study was limited to identification of proteins in the yeast two-hybrid, and there is the possibility that some of these protein interactions due not occur in cells that are infected with the virus. However, the massive identification of the host proteins potentially interacting with E2, a viral protein that has been shown to be one of the most important proteins for virus virulence is very valuable information and can help us to understand the potential mechanisms of CSFV replication and pathogenesis. In order to determine the significance of each of the host proteins identified here, additional studies are required to explore the role(s) of each of these proteins during pestivirus infection.

Acknowledgments

We would like to thank Melanie Prarat for editing the manuscript.

Author Contributions

Conceived and designed the experiments: DPG MVB. Performed the experiments: DPG RBB ZL. Analyzed the data: DPG MVB. Contributed reagents/materials/analysis tools: DPG RBB LGH IFS PF VO. Wrote the paper: DPG MVB.

Citation: Gladue DP, Baker-Bransetter R, Holinka LG, Fernandez-Sainz IJ, O’Donnell V, Fletcher P, et al. (2014) Interaction of CSFV E2 Protein with Swine Host Factors as Detected by Yeast Two-Hybrid System. PLoS ONE 9(1): e85324. https://doi.org/10.1371/journal.pone.0085324

References

1. Becher P, Avalos Ramirez R, Orlich M, Cedillo Rosales S, Konig M, et al. (2003) Genetic and antigenic characterization of novel pestivirus genotypes: implications for classification. Virology 311: 96–104.

2. Lindenbach BD, Thiel JH, Rice CM (2007) Flaviviridae: The Viruses and Their Replication. In: Fields Virology 5th edition. Raven, Philadelphia: Lippincott. 931–959.

3. Weiland E, Stark R, Haas B, Rumenapf T, Meyers G, et al. (1990) Pestivirus glycoprotein which induces neutralizing antibodies forms part of a disulfide-linked heterodimer. J Virol 64: 3563–3569.

4. Weiland F, Weiland E, Unger G, Saalmuller A, Thiel HJ (1999) Localization of pestiviral envelope proteins E(rns) and E2 at the cell surface and on isolated particles. J Gen Virol 80 (Pt 5): 1157–1165.

5. Thiel HJ, Stark R, Weiland E, Rumenapf T, Meyers G (1991) Hog cholera virus: molecular composition of virions from a pestivirus. J Virol 65: 4705–4712.

6. van Gennip HG, Bouma A, van Rijn PA, Widjojoatmodjo MN, Moormann RJ (2002) Experimental non-transmissible marker vaccines for classical swine fever (CSF) by trans-complementation of E(rns) or E2 of CSFV. Vaccine 20: 1544–1556.

7. van Gennip HG, van Rijn PA, Widjojoatmodjo MN, de Smit AJ, Moormann RJ (2000) Chimeric classical swine fever viruses containing envelope protein E(RNS) or E2 of bovine viral diarrhoea virus protect pigs against challenge with CSFV and induce a distinguishable antibody response. Vaccine 19: 447–459.

8. Konig M, Lengsfeld T, Pauly T, Stark R, Thiel HJ (1995) Classical swine fever virus: independent induction of protective immunity by two structural glycoproteins. J Virol 69: 6479–6486.

9. Hulst MM, Moormann RJ (1997) Inhibition of pestivirus infection in cell culture by envelope proteins E(rns) and E2 of classical swine fever virus: E(rns) and E2 interact with different receptors. J Gen Virol 78 (Pt 11): 2779–2787.

10. Wang Z, Nie Y, Wang P, Ding M, Deng H (2004) Characterization of classical swine fever virus entry by using pseudotyped viruses: E1 and E2 are sufficient to mediate viral entry. Virology 330: 332–341.

11. Liang D, Sainz IF, Ansari IH, Gil LH, Vassilev V, et al. (2003) The envelope glycoprotein E2 is a determinant of cell culture tropism in ruminant pestiviruses. J Gen Virol 84: 1269–1274.

12. Risatti GR, Borca MV, Kutish GF, Lu Z, Holinka LG, et al. (2005) The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine. J Virol 79: 3787–3796.

13. Risatti GR, Holinka LG, Carrillo C, Kutish GF, Lu Z, et al. (2006) Identification of a novel virulence determinant within the E2 structural glycoprotein of classical swine fever virus. Virology 355: 94–101.

14. Risatti GR, Holinka LG, Fernandez Sainz I, Carrillo C, Kutish GF, et al. (2007) Mutations in the carboxyl terminal region of E2 glycoprotein of classical swine fever virus are responsible for viral attenuation in swine. Virology 364: 371–382.

15. Risatti GR, Holinka LG, Fernandez Sainz I, Carrillo C, Lu Z, et al. (2007) N-linked glycosylation status of classical swine fever virus strain Brescia E2 glycoprotein influences virulence in swine. J Virol 81: 924–933.

16. Van Gennip HG, Vlot AC, Hulst MM, De Smit AJ, Moormann RJ (2004) Determinants of virulence of classical swine fever virus strain Brescia. J Virol 78: 8812–8823.

17. Li Y, Wang J, Kanai R, Modis Y (2013) Crystal structure of glycoprotein E2 from bovine viral diarrhea virus. Proc Natl Acad Sci U S A 110: 6805–6810.

18. Iourin O, Harlos K, El Omari K, Lu W, Kadlec J, et al. (2013) Expression, purification and crystallization of the ectodomain of the envelope glycoprotein E2 from Bovine viral diarrhoea virus. Acta Crystallogr Sect F Struct Biol Cryst Commun 69: 35–38.

19. Gladue DP, Holinka LG, Fernandez-Sainz IJ, Prarat MV, O'Donell V, et al. (2010) Effects of the interactions of classical swine fever virus Core protein with proteins of the SUMOylation pathway on virulence in swine. Virology 407: 129–136.

20. Gladue DP, O'Donnell V, Baker-Branstetter R, Holinka LG, Pacheco JM, et al. (2012) Foot-and-mouth disease virus nonstructural protein 2C interacts with Beclin1, modulating virus replication. J Virol 86: 12080–12090.

21. Chien CT, Bartel PL, Sternglanz R, Fields S (1991) The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci U S A 88: 9578–9582.

22. Fields S, Song O (1989) A novel genetic system to detect protein-protein interactions. Nature 340: 245–246.

23. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74: 5463–5467.

24. Mi H, Muruganujan A, Thomas PD (2013) PANTHER in 2013: modeling the evolution of gene function, and other gene attributes, in the context of phylogenetic trees. Nucleic Acids Res 41: D377–386.

25. Reimann I, Depner K, Trapp S, Beer M (2004) An avirulent chimeric Pestivirus with altered cell tropism protects pigs against lethal infection with classical swine fever virus. Virology 322: 143–157.

26. Lee N, Pimienta G, Steitz JA (2012) AUF1/hnRNP D is a novel protein partner of the EBER1 noncoding RNA of Epstein-Barr virus. RNA 18: 2073–2082.

27. Gross H, Hennard C, Masouris I, Cassel C, Barth S, et al. (2012) Binding of the heterogeneous ribonucleoprotein K (hnRNP K) to the Epstein-Barr virus nuclear antigen 2 (EBNA2) enhances viral LMP2A expression. PLoS One 7: e42106.

28. Katoh H, Mori Y, Kambara H, Abe T, Fukuhara T, et al. (2011) Heterogeneous nuclear ribonucleoprotein A2 participates in the replication of Japanese encephalitis virus through an interaction with viral proteins and RNA. J Virol 85: 10976–10988.

29. Lee JW, Liao PC, Young KC, Chang CL, Chen SS, et al. (2011) Identification of hnRNPH1, NF45, and C14orf166 as novel host interacting partners of the mature hepatitis C virus core protein. J Proteome Res 10: 4522–4534.

30. Schmidt T, Striebinger H, Haas J, Bailer SM (2010) The heterogeneous nuclear ribonucleoprotein K is important for Herpes simplex virus-1 propagation. FEBS Lett 584: 4361–4365.

31. Gladue DP, Zhu J, Holinka LG, Fernandez-Sainz I, Carrillo C, et al. (2010) Patterns of gene expression in swine macrophages infected with classical swine fever virus detected by microarray. Virus Res 151: 10–18.

32. Hulst M, Loeffen W, Weesendorp E (2013) Pathway analysis in blood cells of pigs infected with classical swine fever virus: comparison of pigs that develop a chronic form of infection or recover. Arch Virol 158: 325–339.

33. Li J, Yu YJ, Feng L, Cai XB, Tang HB, et al. (2010) Global transcriptional profiles in peripheral blood mononuclear cell during classical swine fever virus infection. Virus Res 148: 60–70.

34. Fislova T, Thomas B, Graef KM, Fodor E (2010) Association of the influenza virus RNA polymerase subunit PB2 with the host chaperonin CCT. J Virol 84: 8691–8699.

35. Hong S, Choi G, Park S, Chung AS, Hunter E, et al. (2001) Type D retrovirus Gag polyprotein interacts with the cytosolic chaperonin TRiC. J Virol 75: 2526–2534.

36. Inoue Y, Aizaki H, Hara H, Matsuda M, Ando T, et al. (2011) Chaperonin TRiC/CCT participates in replication of hepatitis C virus genome via interaction with the viral NS5B protein. Virology 410: 38–47.

37. Zaichick SV, Bohannon KP, Hughes A, Sollars PJ, Pickard GE, et al. (2013) The herpesvirus VP1/2 protein is an effector of dynein-mediated capsid transport and neuroinvasion. Cell Host Microbe 13: 193–203.

38. Bremner KH, Scherer J, Yi J, Vershinin M, Gross SP, et al. (2009) Adenovirus transport via direct interaction of cytoplasmic dynein with the viral capsid hexon subunit. Cell Host Microbe 6: 523–535.

39. McDonald D, Vodicka MA, Lucero G, Svitkina TM, Borisy GG, et al. (2002) Visualization of the intracellular behavior of HIV in living cells. J Cell Biol 159: 441–452.

40. Garcin P, Cohen S, Terpstra S, Kelly I, Foster LJ, et al. (2013) Proteomic analysis identifies a novel function for galectin-3 in the cell entry of parvovirus. J Proteomics 79: 123–132.

41. Woodward AM, Mauris J, Argueso P (2013) Binding of transmembrane mucins to galectin-3 limits herpesvirus 1 infection of human corneal keratinocytes. J Virol 87: 5841–5847.

42. Jaquenod De Giusti C, Alberdi L, Frik J, Ferrer MF, Scharrig E, et al. (2011) Galectin-3 is upregulated in activated glia during Junin virus-induced murine encephalitis. Neurosci Lett 501: 163–166.

43. Sato S, Ouellet M, St-Pierre C, Tremblay MJ (2012) Glycans, galectins, and HIV-1 infection. Ann N Y Acad Sci 1253: 133–148.

44. Chernyy ES, Rapoport EM, Andre S, Kaltner H, Gabius HJ, et al. (2011) Galectins promote the interaction of influenza virus with its target cell. Biochemistry (Mosc) 76: 958–967.

45. Feng L, Li XQ, Li XN, Li J, Meng XM, et al. (2012) In vitro infection with classical swine fever virus inhibits the transcription of immune response genes. Virol J 9: 175.

46. Husser L, Ruggli N, Summerfield A (2012) N(pro) of classical swine fever virus prevents type I interferon-mediated priming of conventional dendritic cells for enhanced interferon-alpha response. J Interferon Cytokine Res 32: 221–229.

47. Chen LJ, Dong XY, Shen HY, Zhao MQ, Ju CM, et al. (2012) Classical swine fever virus suppresses maturation and modulates functions of monocyte-derived dendritic cells without activating nuclear factor kappa B. Res Vet Sci. 93: 529–537.

48. Fiebach AR, Guzylack-Piriou L, Python S, Summerfield A, Ruggli N (2011) Classical swine fever virus N(pro) limits type I interferon induction in plasmacytoid dendritic cells by interacting with interferon regulatory factor 7. J Virol 85: 8002–8011.

49. Fernandez-Sainz I, Gladue DP, Holinka LG, O'Donnell V, Gudmundsdottir I, et al. (2010) Mutations in classical swine fever virus NS4B affect virulence in swine. J Virol 84: 1536–1549.

50. Dalrymple NA, Mackow ER (2012) Endothelial cells elicit immune-enhancing responses to dengue virus infection. J Virol 86: 6408–6415.

51. Lubinski J, Wang L, Mastellos D, Sahu A, Lambris JD, et al. (1999) In vivo role of complement-interacting domains of herpes simplex virus type 1 glycoprotein gC. J Exp Med 190: 1637–1646.

52. Chen WN, Chen JY, Jiao BY, Lin WS, Wu YL, et al. (2012) Interaction of the hepatitis B spliced protein with cathepsin B promotes hepatoma cell migration and invasion. J Virol 86: 13533–13541.

53. Ha SD, Park S, Hattlmann CJ, Barr SD, Kim SO (2012) Inhibition or deficiency of cathepsin B leads defects in HIV-1 Gag pseudoparticle release in macrophages and HEK293T cells. Antiviral Res 93: 175–184.

54. Yoshii H, Kamiyama H, Goto K, Oishi K, Katunuma N, et al. (2011) CD4-independent human immunodeficiency virus infection involves participation of endocytosis and cathepsin B. PLoS One. 6: e19352.

55. Kang K, Guo K, Tang Q, Zhang Y, Wu J, et al. (2012) Interactive cellular proteins related to classical swine fever virus non-structure protein 2 by yeast two-hybrid analysis. Mol Biol Rep 39: 10515–10524.

56. Park N, Skern T, Gustin KE (2010) Specific cleavage of the nuclear pore complex protein Nup62 by a viral protease. J Biol Chem 285: 28796–28805.

57. Sivan G, Martin SE, Myers TG, Buehler E, Szymczyk KH, et al. (2013) Human genome-wide RNAi screen reveals a role for nuclear pore proteins in poxvirus morphogenesis. Proc Natl Acad Sci U S A 110: 3519–3524.

58. Di Nunzio F, Danckaert A, Fricke T, Perez P, Fernandez J, et al. (2012) Human nucleoporins promote HIV-1 docking at the nuclear pore, nuclear import and integration. PLoS One 7: e46037.

59. Bennett JA, Prince LR, Parker LC, Stokes CA, de Bruin HG, et al. (2012) Pellino-1 selectively regulates epithelial cell responses to rhinovirus. J Virol 86: 6595–6604.

60. Shiozaki T, Iwai A, Kawaoka Y, Takada A, Kida H, et al. (2011) Requirement for Siva-1 for replication of influenza A virus through apoptosis induction. J Gen Virol 92: 315–325.

61. Py B, Bouchet J, Jacquot G, Sol-Foulon N, Basmaciogullari S, et al. (2007) The Siva protein is a novel intracellular ligand of the CD4 receptor that promotes HIV-1 envelope-induced apoptosis in T-lymphoid cells. Apoptosis 12: 1879–1892.

62. Severino A, Abbruzzese C, Manente L, Valderas AA, Mattarocci S, et al. (2007) Human papillomavirus-16 E7 interacts with Siva-1 and modulates apoptosis in HaCaT human immortalized keratinocytes. J Cell Physiol 212: 118–125.

63. Henke A, Launhardt H, Klement K, Stelzner A, Zell R, et al. (2000) Apoptosis in coxsackievirus B3-caused diseases: interaction between the capsid protein VP2 and the proapoptotic protein siva. J Virol 74: 4284–4290.

64. Mocquet V, Neusiedler J, Rende F, Cluet D, Robin JP, et al. (2012) The human T-lymphotropic virus type 1 tax protein inhibits nonsense-mediated mRNA decay by interacting with INT6/EIF3E and UPF1. J Virol 86: 7530–7543.

65. Ajamian L, Abrahamyan L, Milev M, Ivanov PV, Kulozik AE, et al. (2008) Unexpected roles for UPF1 in HIV-1 RNA metabolism and translation. RNA 14: 914–927.

66. Gladue DP, O'Donnell V, Baker-Branstetter R, Holinka LG, Pacheco JM, et al. (2013) Foot-and-mouth disease virus modulates cellular vimentin for virus survival. J Virol.

67. Maurer K, Krey T, Moennig V, Thiel HJ, Rumenapf T (2004) CD46 is a cellular receptor for bovine viral diarrhea virus. J Virol 78: 1792–1799.