Creation of new UniProtKB/SwissProt keywords

At the inception of this project, only two UniProtKB keywords were applicable to protein sequences involved in interaction between virus and host. These were the keyword “Host-virus interaction” – used for any type of interaction between viral and host proteins – and “viral immunoevasion”, used to group viral proteins counteracting the host immune system. In this work we created 63 new keywords to capture all host pathways commonly usurped by viruses (Table 1). To design these terms, we performed an exhaustive review of peer-reviewed literature concerning the interactions between host and viruses, with strong emphasis on viruses affecting humans (interactions between phages and bacteria were not considered in this work). Knowledge from textbooks and reviews was also considered. New keywords were only defined for host pathways affected by several different viruses or molecular mechanisms - keywords were not created for processes affected by a single viral protein. 54 of these new terms described specific interactions between virus and host such as “Inhibition of host RIG-I by virus”. 10 are parent terms, such as “Inhibition of host cell cycle by virus”, which has 5 child terms such as “G0/G1 host cell cycle checkpoint dysregulation by virus”. These concepts need to be refined constantly to keep pace with the latest biological knowledge, and new keywords will be added to the list once sufficient evidence of their importance is available in the literature.

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Table 1. List of the 65 host-virus interaction processes.

https://doi.org/10.1371/journal.pone.0108075.t001

Creation of new ViralZone keyword pages

Representation of pathways remains a challenging task in the field of curation, and the relationship between individual proteins which are part of a larger process is often not easy to describe in text form. To facilitate understanding of how viruses impact their hosts we therefore developed 57 ViralZone pages corresponding to the majority of the newly created keywords (Table 1). Each includes a manually created illustration of the host pathway described in the keyword, with points of interaction with viral proteins clearly marked. These ViralZone controlled vocabulary pages also provide a list of all annotated UniProtKB/SwissProt entries containing the keyword as well as all the PubMed references used to generate this annotation. They can be accessed either by host-virus interactions menu (Figure 2) or by fact sheets linking all related process (Figure 3).

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Figure 2. Host virus interactions in ViralZone A) Main entry page (http://viralzone.expasy.org/all_by_species/886.html).

All global processes are listed and some links allow browsing down in the controlled vocabulary hierarchy. B) Inhibition of host RLR pathway by virus ViralZone. An illustration describes the RLR antiviral signaling pathway. Known viral proteins inhibiting this pathway are indicated in red circles. A side menu allows reaching description pages for each part of the pathway. (C) “Inhibition of host RIG-I by virus” page describes the host process targeted by viruses, and displays viral proteins inhibiting this activity coded by Lyssavirus, Arenavirus, Influenzavirus A and Pneumovirus. The text describes this host-virus interaction at a molecular level, and a table displays known viruses and their method used to evade RIG-I antiviral defense. To the left, links to the corresponding UniProtKB keyword and GO term allow users to visit these resources and access to all annotated proteins related to the corresponding ontology term (Figure 4). The field “virus” contains the list of viruses attacking this process, a publication source, and a link to ViralZone virus fact sheet for each virus. Most pages give direct access to reviewed UniProtKB entries annotated with corresponding keyword within “protein by strain” and “protein by name” tabs. An illustration describes the RLR antiviral signaling pathway, and identified viral proteins interfering with RIG-I are indicated in red circles.

https://doi.org/10.1371/journal.pone.0108075.g002

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Figure 3. ViralZone fact sheet for Influenza virus A (http://viralzone.expasy.org/all_by_species/6.html) contains information about virion, molecular biology and cross references to different sequence databases.

A tab “Host-virus interactions” gives access to essential interactions occurring during Influenza A infection. In turn, these interactions are linked to the controlled vocabulary pages where more information concerning the particular interaction of interest is available. From there, users can visit UniProtKB or QuickGO pages related to the keyword or corresponding GO term.

https://doi.org/10.1371/journal.pone.0108075.g003

Mapping of UniProtKB/SwissProt keywords to GO ontology

The GO editorial team at EMBL-EBI collaborated with the ViralZone team to update the Gene Ontology (GO) with terms corresponding to the new host-virus interaction ontology. This effort led to the development of 57 GO terms (most newly created) exactly matching new UniProtKB keywords (Table 1). In some cases, one keyword was used to create several GO terms detailing individual aspects of specific functional interactions. The keyword “Inhibition of RIG-I by virus” gave rise to 3 GO terms, specifying different modes of RIG-I suppression - “suppression by virus of host RIG-I via RIG-I binding” and “suppression by virus of host RIG-I activity by viral RNA 5′ processing” – and the functional impact on RIG-I - “suppression by virus of host RIG-I K63-linked ubiquitination”. UniProtKB keywords are not designed to provide such a high level of functional detail.

Viral gene product curation with the new ontology

The goal of this work was to curate viral reference proteomes in order to capture host-virus interactions. This curation has been performed in different ways (Figure 1). Keywords were manually curated in UniProtKB/SwissProt records describing viral proteins after careful reading of the literature, using an editor available only to UniProtKB curators. GO terms were also curated manually using the Protein2GO editor, and also annotated automatically (Table 1) based on the mapping to curated keywords. Note that manual of KWs and GO terms is subject to a quality check to ensure the relevance of the information added. The manner in which UniProtKB and QuickGO provide access to information on host-virus interactions is shown in figure 4.

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Figure 4. Relationship between ViralZone, UniProtKB and QuickGO databases.

Because most host virus-interactions vocabulary or ontology share the same concept, users can browse in the three databases and have easy access to different formats about the same process. Links are indicated by large arrows.

https://doi.org/10.1371/journal.pone.0108075.g004

Inhibition of host IFN-mediated response initiation by virus

Upon entering a host cell, viruses must deal with host innate immunity. Pathogen recognition receptors (PRRs) [6], [7] are “foreign” sensors triggered by molecular patterns present in most types of viruses and/or bacteria. Upon activation they induce signaling events that ultimately lead to a cellular antiviral state mediated in vertebrates by the production of interferons and inflammatory cytokines. Many viruses directly block signaling components of these pathways in order to prevent the establishment of the antiviral state. RIGI is a PRR activated by cytoplasmic 5′-triphosphate-RNA [8], a type of RNA that appears in the cell cytoplasm of virus-infected cells. Upon recognition of this ligand, RIGI initiates a cascade resulting in the expression of antiviral genes and interferon beta. This induced antiviral state is potent at preventing virus replication and exit. To replicate in vertebrate cells, many RNA viruses including influenza virus, human metapneumovirus, arenavirus, or poliovirus inhibit RIGI through different strategies [9]. The RIGI downstream effector MAVS is targeted by the hepatitis viruses A [10], B [11] and C [12], suggesting a crucial role for MAVS in repressing these viruses in hepatocytes. Many other viruses counteract downstream key effectors of the pathway through direct interaction with IRF3, IRF7 or NF-kappaB transcription factors (figure 5).

Inhibition of host interferon signaling pathway by virus

Upon virus pattern recognition, most vertebrate cells release interferon (IFN) alpha or beta. IFNs signal infection to neighboring cells [13]. They bind to the cellular IFNalpha/beta receptors and trigger a signaling cascade that activates hundreds of antiviral genes [14]. Signaling from IFN receptors involves phosphorylation of proteins of the signal transducers and activators of transcription family (STAT). These migrate into the nucleus and activate interferon stimulated genes (ISG). This innate defense pathway is so efficient that almost all vertebrate viruses encode proteins to block it. They disrupt the pathway by degradation or inactivation of different cellular proteins involved: interferon receptors, JAK kinases, STAT1 and 2, or IRF9.

Modulation of host antiviral effectors

Both PRR and interferon signaling trigger an antiviral state in which more than 300 interferon stimulated genes (ISGs) are up regulated [15]. These gene products display a wide range of activities that together contribute to inhibition of virus replication: messenger and ribosomal RNA degradation, inhibition of cellular translation, viral genome hyper mutation, capsid sequestration, and inhibition of budding. Some ISGs target specific classes of viruses, like APOBEC3G which induces mutations in ssDNA [16] and is efficient against retroviruses. Others are specific for cellular compartments or components like PML proteins, which display an antiviral activity against nuclear replicating viruses [17].

The long history of viral and host coevolution has seen the development of a variety of evasive adaptations to circumvent or inactivate these antiviral effectors. One of the most early and effective means to do so is to prevent ISG transcription and the establishment of the antiviral state. This early attempt at evasion may fail though, and latent viruses will eventually have to face antiviral effectors. Therefore many viruses have evolved other ways to rescue their replication cycle by inactivating key effectors of cellular defense. For example the HIV-1 vif protein counteracts APOBEC3G activity [18], while Herpes simplex virus ICP0 protein prevents PML activity [19].

Inhibition of host adaptive immune response by virus

In innate immunity, pathogen-specific receptors are encoded by the genome, whereas in adaptive immunity these receptors are “acquired” during the lifetime of the organism. This immunity is said to be “adaptive” because it can memorize the pathogen offence and prepare the body to fight future challenges.

Adaptive immunity relies on the distinction between the bodys self-antigens and the foreign-antigens of unwanted invaders. Infected cells display viral antigens through major histocompatibility complexes (MHCs). These are recognized as non-self by T-lymphocytes, thereby triggering the adaptive response and inducing destruction of the infected cells and the synthesis of neutralizing antibodies. To escape this defense, many viruses inhibit MHC peptide presentation [20]. The herpes virus simplex 1 protein ICP47 binds to the peptide binding site of the transporter associated with antigen (TAP) and inhibits the first step of the translocation pathway [21]. Other strategies for preventing antigen presentation include inhibition of host tapasin, proteasome or class I and II MHC molecules. Viral superantigens are proteins secreted that bridge nonspecifically MHC to T-cell receptors, thereby interfering with the specificity of MHC antigen presentation.

Modulation of adaptive immunity: inhibition/activation of host NF-kappaB by virus, inhibition of host chemokines by virus

In response to viral infection, the NFkappaB transcription factor can be activated and induce the production of numerous cytokines and chemokines by different cell types including macrophages, dendritic cells or epithelial cells [22]. Some viruses produce their own interleukins, chemokine regulators, or IgG Fc receptor-like proteins [23] which either inhibit the immune response or attract lymphoid cells (in the case of lymphotrophic viruses). The cytomegalovirus gene UL146 encodes for a product similar to cellular alpha chemokine, which attracts neutrophils. These are in turn infected and carry the virus to new sites in the organism [24].

Suppressor of RNA silencing by plant and insect infecting viruses

In higher plants and insects, post transcription gene silencing (PTGS, also known as RNA interference, RNAi) operates as an adaptive antiviral defense mechanism [25]. Double-stranded RNA molecules from viruses are processed into small single-stranded molecules (short interfering RNA, or siRNA), that hybridize to viral RNAs and target them to the degradation pathway. To counteract this almost all plant viruses and many animal viruses encode (viral) suppressors of RNA silencing (VSRs) which inhibit key steps of PTGS. The enamovirus P0 [26] and cucumovirus suppressor 2b proteins [27] inhibit RNA silencing through inhibition of argonaute 1/AGO1, a component of the RNA silencing pathway. The insect flock house virus B2 protein (FHVB2) suppresses siRNA biogenesis by inhibiting host RISC [28].

Host gene expression shutoff by virus

After genome replication, and in the late phase of the infection, many viruses synthesize a large number of structural proteins and assemble a huge number of virions within the cell. The high demand for viral protein synthesis is often supported by shutoff of host gene expression, ensuring that all cellular resources are devoted to viral synthesis [29]. Host shutoff prevents also the activation of innate defenses. Host shutoff can be achieved by modulating host transcription, mRNA processing and translation.

Inhibition of host transcription can be mediated by preventing host RNA polymerase II initiation. The TATA-binding protein (TBP) is targeted by the adenovirus E1A protein, which disrupts the interaction between the TBP and the TATA box [30]. The thogoto virus ML protein targets the general transcription factor IIB [31]. RNA polymerase II itself may be directly targeted, for example by the alphavirus nsP2 protein which induces rapid degradation of Rpb1, a catalytic subunit of RNA polII [32].

Host messenger RNAs can be modulated at various stages including pre-RNA processing, nuclear export or modification of RNA decay. Whatever the means, viral messenger RNAs rely on alternative ways to be expressed. For example, viruses shutting off host nuclear gene transcription or mRNA export are often transcribed in the cytoplasm.

The main mechanism by which viruses inhibit host gene expression is by specifically targeting the translation of host mRNAs – which requires proteins not used for translation of viral proteins. Viruses inhibiting cap dependent translation use an internal ribosome entry site (IRES) or similar structures for translation initiation of their proteins [33]. The protease 3C from enteroviruses cleaves host eiF5B [34] while the protease from retroviruses cleaves eiF4G, completely abrogating cellular translation [35].

Modulation of host cell cycle by virus

Viral replication is limited by the ability of the host metabolic machinery to produce the resources – nucleic acids and proteins – necessary for the assembly of viral progeny [36]. These resources are most abundant during the S-phase of the cell cycle, and many DNA viruses modulate the G1/S transition to initiate DNA synthesis. Viruses such as Epstein-Barr virus, human cytomegalovirus, adenoviruses and SV40 modulate the activity of retinoblastoma (RB) protein family members to drive cells in S-phase [37]–[39]. Viruses infecting quiescent cells have also evolved mechanisms to force entry into the cell cycle. Myxomavirus M-T5 promotes phosphorylation, ubiquitination and degradation of the cyclin-dependent kinase inhibitor p27/KIP1 [40]. Viruses also retard the initiation of mitosis – the G2/M transition – to allow replication of their own genome before mitosis and sometimes to prevent clonal expansion of infected lymphocytes [41].

Inhibition or activation of host autophagy pathway by virus

Several pathogens interfere with or exploit the host autophagic pathway for their life-cycle or in order to evade or immune responses [42]. Autophagy is a fundamental eukaryotic cellular process for maintaining homeostasis by degrading cellular proteins, organelles and intracellular pathogens. This process is tightly associated with innate and adaptive immunity. The autophagic machinery may promote the production of type-I interferon (IFN) by delivering the cytosolic replication intermediates to the lysosomes and thereby activating endosomal toll-like receptors (TLRs) [43]. Moreover, lysosomal degradation of cytoplasmic compounds contributes to the pool of MHC class II displayed peptides. Herpes simplex virus ICP34.5 interacts with host Beclin-1 and interferes with autophagosome maturation and antigen presentation in dendritic cells [44]. A similar function has been attributed to protein TRS1 from Human cytomegalovirus that modulates Beclin-1. Alternatively, some viruses use autophagy to generate intracellular membranes useful for viral replication. For example autophagy may serve as a generator of intracellular membrane vesicles for picornavirus replication [45].

Modulation of host cell apoptosis by virus

In order to prevent viruses from spreading within the infected host organism, cells can commit apoptosis, a genetically controlled program of cell death. Many viruses have evolved strategies to inhibit apoptosis by blocking both intrinsic and extrinsic host-initiated cell death pathways. Several adenoviruses, herpesviruses and poxviruses encode proteins that are homologous to the cellular anti-apoptotic Bcl-2 protein. These viral proteins sequester pro-apoptotic Bcl-2 family members including Bak and Bax, thereby inhibiting apoptosis. Viral FLIPs (vFLIPs) block the interaction of the death receptor-adapter complex with the cellular effector FLICE (caspase-8) to prevent the initiation of the downstream caspase cascade.