key: cord-318749-k91oku7h
authors: Dong, Hui-Jun; Zhang, Rui; Kuang, Yu; Wang, Xiao-Jia
title: Selective regulation in ribosome biogenesis and protein production for efficient viral translation
date: 2020-10-29
journal: Arch Microbiol
DOI: 10.1007/s00203-020-02094-5
sha: 
doc_id: 318749
cord_uid: k91oku7h

As intracellular parasites, viruses depend heavily on host cell structures and their functions to complete their life cycle and produce new viral particles. Viruses utilize or modulate cellular translational machinery to achieve efficient replication; the role of ribosome biogenesis and protein synthesis in viral replication particularly highlights the importance of the ribosome quantity and/or quality in controlling viral protein synthesis. Recently reported studies have demonstrated that ribosome biogenesis factors (RBFs) and ribosomal proteins (RPs) act as multifaceted regulators in selective translation of viral transcripts. Here we summarize the recent literature on RBFs and RPs and their association with subcellular redistribution, post-translational modification, enzyme catalysis, and direct interaction with viral proteins. The advances described in this literature establish a rationale for targeting ribosome production and function in the design of the next generation of antiviral agents.

Initiation, as the first stage of translation, can be carried out by either cap-dependent or cap-independent mechanism in eukaryotic cells (Kapp and Lorsch 2004) . The majority of the eukaryotic genome is translated via cap-dependent translation initiation, mainly through the 43S preinitiation complex (PIC) binding to the mRNA 5′-end cap structure and scanning along the mRNA to the AUG initiation codon to initiate translation (Haimov et al. 2015) . In contrast to the cap-dependent mechanism, the cap-independent mechanism is translated by 43S PIC (or a single 40S unit in some specific mRNA templates) directly binding to the internal ribosome entry site (IRESs) in the genome intergenic region (Kapp and Lorsch 2004) . The number and types of translation initiation factors required for IRES-mediated translation initiation can vary significantly among specific mRNA species. For example, eIF4E is not required for IRES-mediated picornavirus genome translation (Avanzino et al. 2017) , and IRES elements present in the hepatitis C virus (HCV) genome require fewer translation initiation factors to initiate translation (Torrecilla et al. 2016; Kerr et al. 2016) .

Ribosomes are apparatuses that catalyze protein synthesis. The eukaryotic 80S ribosome is composed of two subunits: the 40S subunit contains the decoding center, which monitors the complementarity of transfer RNA (tRNA) and messenger RNAs (mRNA) and is composed of 33 ribosomal proteins (RPs), and the 60S subunit, which catalyzes the formation of peptide bonds and is composed of 47 RPs (Khatter et al. 2015; Barna 2013) . Ribosome biogenesis is a complex process, which takes place mainly within the nucleoli of eukaryotes, and requires more than 300 ribosomal biogenesis factors (RBFs) (Tafforeau et al. 2013) . RPs are coordinated with ribosomal RNA (rRNA) to synthesize, mature, assemble, and export into the cytoplasm, and they are crucial participants in the cellular process of translation from mRNA (Lafontaine and Tollervey 2001; Moore 2012) .

Ribosome-mediated translational regulation can be explained by two different models Simsek et al. 2017) . The ribosome-concentration hypothesis Communicated by Erko Stackebrandt. proposes that the changes in cellular ribosome abundance may be a major driver of changes in translation of mRNA pools. The specialized ribosomes hypothesis is based on the finding that some RPs differ in composition and specifically regulate a subset of specific mRNA (such as RPL38 for Hox mRNA) translation (Shi and Barna, 2015; Xue and Barna, 2012; Simsek et al. 2017; Briggs et al. 2017; Genuth and Barna, 2018) . RBFs control ribosome biogenesis to participate in the cellular process of mRNA translation. Several viruses have evolved sophisticated mechanisms that utilize cellular translational machinery to obtain efficient viral protein synthesis and viral replication (Hilton et al. 1986; Narayanan et al. 2008; Li et al. 2018a, b) .

The ribosome itself (primarily ribosomal proteins), and the ribosomal biogenic processes that are exploited by viruses to facilitate their own replication, should be considered as targets in the development of antiviral agents, as depicted in Table 1 and as summarized in this study.

Ribosome biogenesis is critical for cell proliferation and stress response. The synthesis of viral proteins depends on the host ribosome, and ribosome biogenesis has implications for viral infection through the effects of RBFs on viral transcription, proliferation, and antiviral immune responses, as well as the effects of viral proteins on ribosomal biosynthesis.

Ribosomal RNA transcription, catalyzed by RNA polymerase I (Pol I), plays a critical role in ribosome biogenesis. Transcriptional activation of rRNA is also closely associated with viral infection. However, restricting ribosome biogenesis by interfering with rRNA accumulation-triggered ribosomal stress stimulated the viral replication of human cytomegalovirus (HCMV); the process inhibited the expression of innate immune-related genes, such as the high-mobility group Box 2 (HMGB2). This reveals that rRNA accumulation and/or ribosome biogenesis regulate innate immune responses to restrict virus reproduction and regulate inflammation (Bianco and Mohr 2019) . Similarly, ribosomes are required for the protein synthesis of host cells and viruses, but the biogenesis factor RBFs can also impact the proliferation of virus and cell-intrinsic immune responses.

At this intersection of apparently competing processes, it is intriguing to find RBFs that are required specifically for the viral protein biosynthesis, but do not affect global translation. NOP53 (GLTSCR2/PICT-1), for example, shares homology with the yeast 60S ribosomal protein Nop53p, which acts as an essential ribosome biogenesis factor (Sydorskyy et al. 2005; Thomson et al. 2005) . A previous study by the present author showed that NOP53, expressed as a set of discrete globular structures within the nucleolus, is migrated to the cytoplasm upon infection by herpes simplex virus 1 (HSV-1) (Meng et al. 2018) . Furthermore, the cytoplasmic translocation of NOP53 is required for efficient viral replication, which occurs via depression of the activity of innate immune receptor RIG-I and decrease of the phosphorylation level of eIF2α to facilitate the production of viral proteins without affecting global protein synthesis (Meng et al. 2018 (Meng et al. , 2019 . Similarly, both nucleolar proteins of ribosomal RNA processing 1 homolog B (RRP1B) and nucleolin are involved in ribosomal biogenesis (Chamousset et al. 2010) . RRP1B associated with pre-60S ribosomal subunits was translocated to the nucleoplasm upon viral infection of influenza virus, where RRP1B improves viral RNA-dependent RNA polymerase (RdRp) activity, which is responsible for virus transcription and replication (Su et al. 2015) . Nucleolin, which is thought to control RNA metabolism and ribosome biogenesis (Cong, et al. 2012; Allain 2000) , is prevented from entering the nucleus in poliovirusinfected cells (Waggoner 1998) ; this allows stimulation of IRES-mediated translation of viral transcripts (Izumi 2001) . Upstream binding factor (UBF) plays a major role in the regulation of rRNA synthesis and associates with viral replication of adenovirus; the antibody against UBF reduces viral replication (Lawrence et al. 2006) .

In mammalian cells, there are two sets of ribosome particles, which reside in the cytoplasm and the mitochondria. Some viruses manipulate mitochondrial biogenesis to support replication. It has been reported that the protein levels involved in mitochondrial ribosome biogenesis are significantly up-regulated by infection with human cytomegalovirus (HCMV). The inhibition of mitochondrial translation with chloramphenicol or knockdown of ribosome biogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrial coding proteins and significantly impaired viral growth (Karniely et al., 2016) .

The main function of the nucleolus is in ribosome biogenesis, regulation of the cell cycle, and the response to cellular stress. Many viral proteins, such as the nucleocapsid protein of coronavirus (Chen et al. 2002; Cawood et al. 2007; Dove et al. 2006; Emmott et al. 2010; Hiscox et al. 2001; You et al. 2005) , the matrix protein of Newcastle disease virus (NDV) (Peeples et al. 1992) , and the non-structural protein 1 of influenza virus (Melén et al. 2002) , transport to the nucleolus to regulate ribosome biosynthesis, change the nucleolar morphology, or affect the function of the nucleolus. During nucleocytoplasmic shuttling, viral proteins can recruit nucleolar RBFs to modulate ribosomal biosynthesis for efficient viral replication. The multifunctional nucleolar phosphoprotein nucleophosmin (NPM) plays a lead role in ribosome biogenesis to stimulate RNA Pol I-dependent transcription (Li and Hann 2013) and regulates SARS-CoV, IBV, HIV-1, HCV Recruited by viral proteins to facilitate viral replication Chen et al. 2009; Zhou et al. 2013; Goh et al. 2004; Bortz et al. 2011 the export of ribosomal proteins and mature ribosomes to the cytoplasm (Yu et al. 2006) . The HBx oncoprotein of hepatitis B virus (HBV) directly interacted with the C-terminal domain of NPM to stimulate viral transcription (Ahuja et al. 2015) . Several RNA helicases have been demonstrated to facilitate ribosome biogenesis, involving the processing of ribosomal RNA (rRNA) as well as its assembly into functional ribonucleoprotein complexes (Bleichert et al. 2007 ). DDX5 promotes the synthesis and maturation of rRNA and ultimately increases ribosome output and proliferation (Jalal et al. 2007) . Many viral proteins, such as coronavirus nsp13 (Chen et al. 2009 ), Rev of human immunodeficiency virus 1 (HIV-1) (Zhou et al. 2013 ), NS5B of hepatitis C virus (HCV) (Goh et al. 2004) , and NP protein of influenza virus (Bortz et al. 2011 ) have evolved to hijack DDX5 in order to facilitate viral replication. Studies on the regulation or manipulation of viral protein in ribosome biosynthesis provides new insights into efficient viral replication.

The ribosome was once considered to be a large protein synthesis machine that merely translated mRNAs into proteins. In recent years, however, the role of ribosomes in the regulation of initiation and selection of mRNAs has been the subject of increased attention. Most RPs are essential for translation and viral replication, and regulate translation of viral mRNAs as constituents of the ribosome, although a few represent the defense signaling of host cells (Zhou et al. 2015) . (The role of RPs in activating an immune response is beyond the scope of the present paper.) Of particular interest are the RPs that are non-essential to the function of the ribosome, such as the large ribosomal subunit (RPL40, RPL38) and the small ribosomal subunit (RPS27, RPS25) (Jack et al. 2011; Karlas et al. 2010; Xue et al. 2015; Lee et al. 2013 ).

In the process of coevolution with hosts, viruses have evolved different strategies to inhibit host translation or induce host translation shutoff while protecting their own protein synthesis. Many viruses globally interfere with host translation by impairing cap-dependent ribosome recruitment to host mRNAs. For example, vesicular stomatitis virus (VSV) inhibits the translation of host mRNAs in infected cells, but allows the translation of its own capped mRNAs (Whitlow et al. 2008; Wertz and Youngner 1970) . RPL40 promotes efficient translation of VSV mRNAs in a cap-dependent manner, but is not required for global protein synthesis (Lee et al. 2013) . This may represent an endogenous specialized translation function for RPL40. Influenza A virus (IAV) viral mRNAs, on the other hand, are not preferentially translated compared to their host counterparts, and the extensive translation of viral proteins is the result of viral takeover of the mRNA pool in the cell (Bercovich-Kinori et al. 2016) . Ribosome footprints and mRNA read density analysis reveal that RPs are not strongly affected by IAV infection but are enriched for genes involved in pathways that the virus may depend on (Bercovich-Kinori et al. 2016) . Moreover, IAV encodes C4-type zinc finger peptide (ZFP) motif-containing viral protein (such as matrix protein M1) for replication and virus budding (Fernandez-Pol et al. 2001) ; RPS27 also contains a ZFP motif, and has recently been reported to facilitate translation and viral replication (Fernandez-Pol et al. 2001; Fernandez-Pol 2011) . When RPS27 is eliminated, the replication and infectivity of IAV is abolished, but it is dispensable for global protein synthesis (Karlas et al. 2010) . Research on the essential viral and cellular ZFP motif-containing proteins has implications for the prevention and therapy of viral diseases, and these proteins should be considered as targets for novel antiviral compounds.

In eukaryotes, certain RPs control selectivity during IRES-driven translation. For example, RPL38 and RPL35 control the selective translation of a specific mRNA by promoting translation primarily at the initiation stage (Kondrashov et al. 2011; Jiang et al. 2015; Xue et al. 2015) . Several viruses whose genomes lack a 5′-end cap structure use capindependent mechanisms for translation initiation (Daijogo and Semler 2011; Martinez-Salas et al. 2015) . These viruses have in fact evolved to hijack specific RPs to achieve optimal viral protein synthesis; they facilitate translation of viral transcripts of IRES-containing viruses. The RPs include RPS25 (Landry et al. 2009 ), RPS5 (Fukushi et al. 2001; Bhat et al. 2015) , RPS6 (Huang et al. 2012) , RPS9 (Fukushi et al. 2001) , and RACK1 (Majzoub et al. 2014) , as well as RPL10a , RPL22 (Wood et al. 2001) , and RPLp1/2 (Campos et al. 2017) . Of these, RACK1 (Majzoub et al. 2014 ) and RPS6 (Huang et al. 2012 ) are specifically required for IRES activity in various types of viral IRESs. RPL10a regulates translation from the HCV and cricket paralysis virus (CrPV) IRESs but not from the encephalomyocarditis virus (EMCV) IRES . In other words, RPL10a is an example of an RP that plays a specific role in promoting translation of particular classes of viral IRESs. RPS25 is essential for initiation from the HCV and CrPV IRES (Landry et al. 2009 ). RPS25 deletion in yeast or mammalian cells has minimal effects on cellular protein synthesis, which implies that this ribosomal protein may be selectively required for viral IRES-mediated translation (Jack et al. 2011; Hertz et al. 2013) . Similarly, RACK1 is not required for cellular IRESs or global synthesis (Majzoub et al. 2014) . Overall, RPs specifically required for protein synthesis in particular viruses but not for global synthesis may provide an effective target for methods to fight viral infection.

Some of the related functions of RPs are ribosome-dependent, as when RPs participate in viral protein biosynthesis, and other functions, typically involved in the regulation of infection in host cells, are independent of the ribosome. Alterations in RP characteristics for optional translation are implicated in interaction with viral proteins, post-translational modification, and redistribution, directly or with the assistance of RNA helicase.

Some viruses have developed to hijack specific RPs using viral proteins to achieve optimal viral translation. IBDV VP3 is a multifunctional protein playing a key role in virus assembly and pathogenesis. Ribosomal protein L4 (RPL4) ) and ribosomal protein L18 were identified as interacting partners of VP3 protein and as being involved in the regulation of IBDV replication. RPL18 interacts with nucleocapsid protein of rice stripe tenuivirus (RSV), and silencing RPL18 significantly reduces viral mRNA translation and replication of RSV (Li et al. 2018a, b) . RPL7 interacts with the VP51 to participate in viral infection of white spot syndrome virus (WSSV), and the addition of anti-RPL7 antibody inhibits WSSV infection in Litopenaeus vannamei . Among flaviviruses, the envelope protein of West-Nile virus (WNV), dengue virus (DENV), and yellow fever virus (YFV) bind with RPSA, which serves as the viral receptor (Zidane et al. 2012) . As regards alphaviruses, RPSA includes different binding sites for Venezualian equine encephalitis virus (VEEV) and Sindbis virus (SINV) (Jamieson et al. 2008; Malygin et al. 2009 ). HIV-1 Tat inhibits cell proliferation via an interaction with RPS3 and increases the level of RPS3 in the nucleus, thereby disrupting mitotic spindle formation during HIV-1 infection (Kim and Kim 2018) . FMDV VP1 inhibits the antiviral response of RPSA by interacting with RPSA to promote FMDV replication (Zhu et al. 2019) . Binding of viral proteins to specific RPs plays a crucial role in viral infection. Effective inhibition of the binding between RPs and viral proteins can be a potential target for antiviral design.

Post-translational modifications such as phosphorylation, ubiquitination, acetylation, methylation, or O-GlcNAcylation activate, deactivate, or modify RP's functions. Over 2,500 modifications of human RPs have been reported; these modifications impact RP activity and subsequently modify the global rate of translation by modulating initiation, elongation, and termination rates (Emmott et al. 2019) . One wellstudied modification on the ribosome is phosphorylation following activation or inhibition of intra and extracellular signaling cascades in response to stimuli occurring in physiological or pathological conditions (Emmott et al. 2019) . Phosphorylation of RPS6 is perhaps the most widely studied ribosomal protein phosphorylation among the RPs involved in virus infection; it is induced by various viral infections Decker 1981; Banham et al. 1993; Beaud et al. 1994 ). In addition, an early study revealed that phosphorylation of RPL30 was induced by herpes simplex virus-1 (HSV-1) (Simonin et al. 1995) . RPSa, RPS2 and RPS13 appear specifically phosphorylated in cells early after infection with vaccinia virus (Kaerlin and Horak 1978; Buendia et al. 1987) . Efficient phosphorylation of the ribosomal proteins correlates well with possible translational mechanisms, ensuring efficient expression of early and late genes of vaccinia virus. It has been shown that phosphorylation of the RACK1A protein on two residues-Ser-122 and Thr162-by an atypical serine/ threonine protein kinase WNK8 (With No Lysine8) negatively regulates RACK1A function in the glucose responsiveness pathway by influencing its protein stability (Urano et al. 2015) . RACK1A proteins are phosphorylated by tyrosine, depending on diverse environmental stresses (Sabila et al. 2016 ). Viral infection leads to activation of ribosomal protein phosphorylation, which may be closely related to effective viral translation. The change of phospho-RP level may affect viral translation. However, despite reports on the increasing phosphorylation of RPs induced by viral infection (Emmott et al. 2019; Diaz et al. 2002) , the details of the role of this modification in virus infection remain poorly understood.

Another extensively studied post-translational modification on the ribosome is ubiquitylation, or covalent bonding of ubiquitin to substrate proteins for disposal by the proteasome (degradative ubiquitylation) or alteration of the function of the protein (regulatory ubiquitylation) (Haas et al. 1982; Thrower et al. 2000) . Degradative ubiquitylation is important for ribosome-independent function. In fact, excess RPs that are not incorporated into the ribosome are degraded by the proteasome (Lam et al. 2007; Genuth and Barna, 2018) . Several RPs have been shown to be dynamically ubiquitylated in response to cellular conditions; RPS2, RPS3, and RPS20, for instance, undergo regulatory ubiquitylation during the unfolded protein response (UPR) in yeast, fruit fly, and human cell lines (Higgins et al. 2015) . Notably, the ubiquitination of RPS20 was found to be specifically required for viral replication and synthesis of poxvirus proteins (DiGiuseppe et al. 2018 ). In addition to ubiquitin, other related proteins have been found to modify the ribosome. RPL26, for instance, is the principal target of UFM1 conjugation, a ubiquitin-related modification (ufmylation), and UFMylated RPL26 is highly enriched on ER membranebound ribosomes and polysomes (Walczak et al. 2019; Wang et al. 2019) . UFM1 is also conjugated to RPS3, RPS20, and RPL10 (Simsek et al. 2017) . The overlap of ufmylation and ubiquitylation on RPS3 and RPS20 also occurs, and this suggests that certain RPs have combinatorial modifications. O-GlcNAcylation modification was also found to be activated in response to adverse stress to protect cellular proteins from damage Hart 2004, 2006; Slawson et al. 2006 ). In the stress response, O-GlcNAc-modified proteins are prominent components of stress granules, including small and large ribosomal subunit proteins (RPS3, 9, 11 and 24, and RPL6, 13a, 14, and the ribosomeassociated protein RACK1 (Ohn et al. 2008) . Although the effect of RP O-GlcNAcylation on viral infection has been as yet little studied, we can to some extent understand RP O-GlcNAcylation under viral infection through other forms of cellular stimulation, and start to develop an understanding of the possible role of post-translational modification of RPs in antiviral strategy.

Although RPs are all synthesized in the cytoplasm, they assemble into functional subunits in different subcellular compartments (Kressler et al. 2010; Li 2019) . Sometimes, ribosomal proteins change their localization under virus infection to exercise functions outside the ribosome. RPL22, for instance, is translocated from the nucleoli to the nucleoplasm and colocalized with ICP4 in infection by HSV-1. The location change of RPL22 plays a role in the regulatory functions expressed by ICP4 (Leopardi and Roizman, 1996) . In another example of similar function, after DENV infection, RPL18 is redistributed to the perinuclear region for regulation of viral replication (Cervantes-Salazar et al. 2015) . In eukaryotic cells, acidic ribosomal protein RPLp0 binds two P1/P2 protein heterodimers to form a pentameric P-stalk (Ballesta et al. 1996; Naganuma et al. 2007 ). Eukaryotic RPLp1 and RPLp2 proteins also exist in free form in the cytoplasm, and the exchange between the ribosome-bound RPLp1 and RPLp2 proteins and the cytoplasmic pools is thought to regulate the activity of the ribosome (Lee et al. 2010) . This property may lead to changes in the localization of RPLp1/2 in the ribosome and cytoplasmic pools under stress conditions. Viral infection is one of numerous possible stress stimuli, so viral infection may cause RPLp1/2 to produce this subcellular compartment exchange to affect virushost translation regulation. Under nucleolar stress induction, RPLp0 accumulates in the cytoplasm of mammalian cells as a free, ribosome-unbound protein (Deryło et al. 2018) . A ribosome-free pool of RPLp0 has been shown to be a population of proteins released from pre-existing ribosomes. The presence of RPLp0 on the ribosome seems to be affected in stressed cells, and it might be considered as a regulatory element responding to environmental fluctuations. In addition, the mitochondrial RPs (MRPs) are encoded by nuclear genes and then imported into mitochondria for assembly; they are responsible for the translation of 13 mitochondrial mRNAs (Christian and Spremulli 2012) . Mitochondrial RPL18 acts as a critical regulator of the stress response and generates a cytosolic isoform in a stress-dependent manner. The cytosolic RPL18 is incorporated into the 80S ribosome and facilitates ribosome engagement in heat-shock protein (HSP) mRNA translation to escape from the shutoff of global protein synthesis during stress (Zhang et al. 2015) .

Accumulating evidence has revealed the roles of RPs belonging to the large 60S subunit in regulating selective translation of specific mRNAs, although they are primarily involved in catalyzing peptide-bond formation (Wilson et al. 2012) . A previous study by the present author identified RPL13 as a critical regulator of IRES-driven translation during foot-and-mouth disease virus (FMDV) infection, but found that it is not essential for cellular global translation (Han et al. 2020) . Our results support a model whereby the viral IRESs recruit helicase DDX3 downstream of the RPL13 to facilitate IRES-driven translation and viral replication. Specifically, the depletion of DDX3 disrupts binding of RPL13 to the FMDV IRES, whereas the reduction in RPL13 expression impairs the ability of DDX3 to promote IRES-driven translation directly. This work was the first to identify a connection between DDX3 and RPs in modulating viral IRES-dependent translation (Han et al. 2020) . DDX3 is well known, however, to play roles in various key aspects of RNA metabolism, including transcriptional regulation, splicing, mRNA export, ribosome biogenesis, and translational regulation (Chuang et al. 1997; Rocak and Linder 2004; Guenther et al. 2018) . Improved knowledge about these RNA helicases and their relation to translation initiation could have important implications for the understanding of selective translation of viral mRNA, and thus for the development of effective antivirals.

The strategy of targeting a host factor, instead of the virus directly, could circumvent the danger of resistance in mutation-associated variations and the evolution of new viral variants after prolonged use of otherwise-promising antiviral drugs. After 60 years of research on ribosomes, the ribosome has emerged as a major player in translational regulation, and its place in the list of potential antiviral therapeutic targets has been reinforced. Targeting the ribosome, either biogenesis or function, may provide an efficient and focused approach for design of antiviral agents.

As our understanding of the function of RBFs and RPs in viral translation has evolved, the design of therapeutic strategies for RBFs and RPs has been explored. Recent structural analysis of prokaryotic and eukaryotic ribosomes has demonstrated that certain molecules, including some antibiotics and chemical inhibitors of translation, can bind to the ribosome (Svetlov et al. 2017; Tereshchenkov et al. 2018) . CX-5461, for example, is a ribosomal biogenesis inhibitor that disrupts RNA Pol I-mediated transcription (Drygin et al. 2011) , and inhibits viral DNA synthesis and virus production in the early and late stages of the HCMV infection (Westdorp and Terhune 2018) . Toyocamycin is a small molecule inhibitor of Rio1 kinase activity (Kiburu et al. 2012) ; Rio1 is an essential ribosome biogenesis factor required for maturation of 40 S ribosomal subunit (Angermayr et al. 2002) . Toyocamycin inhibits ribosome biogenesis to produce antiviral effects (Ríman et al. 1969) . Notably, translation of IRES-containing viruses (such as HCV, HTLV-1, CrPV, poliovirus, and adenovirus) is dependent on RPS25 (Landry et al. 2009; Hertz et al. 2013; Olivares et al. 2014) , and its activity is highly sensitive to the antibiotic Edeine (Olivares et al. 2014) . Picolinic acid (PA) and fusaric acid (FU) disrupt the ZFP motifs of RPS27 or crucial viral proteins to produce antiviral effects (Yu et al. 1995; Everett et al. 1993; Wakefield and Brownlee 1989; Love et al. 1996) . Ricin is an RPLp protein inhibitor (May et al. 2012) ; RPLp1/2 is an essential host factor for flavivirus replication (Campos et al.2017) . RACK1 is essential for the translation of many viruses, including HCV, Drosophila C (DCV), Cricket Paralysis (CrpV) (Majzoub et al. 2014) , and vaccinia viruses (Jha et al. 2017) . Thus, RACK1 inhibitors may be developed as novel antiviral therapeutics. The drug SD-29 and analogues show high efficacy in inhibition of virus proliferation (Ullah et al. 2019) . Exploring strategies for targeting RBFs and RPs is a natural path for further study in this field.

Once the function of RBFs and RPs in viral translation is understood, targeting inhibitors will need to be identified, designed, and screened. To accelerate the process of drug discovery for both novel targets and existing targets that suffer from drug resistance, basic platforms for drug design and screening are required. In a recent study, structure-based screening of two million commercially available compounds was used to screen for small molecules to target the RACK1 Y248 phosphorylation site. (As noted, host RACK1 protein is an attractive target for developing broad antiviral drugs.) Dozens of small compounds were identified that could potentially bind to the experimentally determined functional site of the RACK1A protein; the drugs show high efficacy in inhibition of the proliferation of viruses that require RACK1 for translation (Ullah et al. 2019) . This approach can be used to study the ribosomal proteins that are essential for the translation of many viruses, and eventually to design broad-spectrum antiviral drugs. Furthermore, depletion of regulatory RBFs and RPs is not essential for basic ribosomal function in experiments, making them potential targets for the design of small molecule antiviral drugs. RNA interference (RNAi) and strategies based on CRISPR/cas9 are also promising approaches in targeting of RBF and RP genes. Of course, projecting forward to the practical application of antiviral drugs, the adverse cell response caused by targeting of RBF and RP genes must be studied in detail.

Deciphering the functional details of translation of viral mRNA will require the in-depth scrutiny of RPs. A number of technological efforts have been made over the last decade to facilitate exploration of RP biogenesis and functions. Ribosome profiling is an emerging technique that allows the distribution of ribosome units on each transcript to be obtained, enabling systematic probing of translation and increased sensitivity and efficiency. This method, in conjunction with RNA-seq, has been used to probe the complex viral replication of several viruses Irigoyen et al. 2016 ). Using high-coverage tandem mass tag (TMT) mass spectrometry, the relative monosome and polysome fractions of each RP were compared. A thorough examination of each RP revealed that some RPs (such as RPS9 and RPL11) are more abundant in monosomes than in polysomes (Slavov et al. 2015) . TMT technology has been extended to the investigation of potential heterogeneity of RPs. Mass spectrometry has also been improved to address directly and more accurately the possibility of variability in RP stoichiometry. This novel technology, called selected reaction monitoring (SRM)-based proteomics, uses the spiking of samples with known amounts of labeled peptides derived from RPs as a standard for absolute quantification. Using this technology, absolute quantification of 15 RPs isolated from polysomes was assessed and 4 RPs, RPL10A, RPL38, RPS7, and RPS25, were identified as being substoichiometric in murine ESC ribosomes ). Based on this technology, the heterogeneity in ribosome composition within a single cell type and a single polysome profile fraction was revealed for the first time. These approaches provide a starting point in the attempt to identify and quantify each RP expression profile in cells challenged with viruses, and at different infectious stages. In a recent study, both the small and large ribosomal subunits in mouse ESCs were endogenously tagged, and affinity enrichment for each of the tagged ribosomal subunits was performed to define the intersection of the two separate ribosomal subunit datasets. This has led to the identification of ribosome-associated proteins (RAPs), which fall into unexpected functional categories, such as energy metabolism, cell cycle, and key protein and RNA modification enzymes (Simsek et al. 2017 ). This method may facilitate identification of a series of proteins (such as RAPs) that are specifically recruited to ribosomes with the assistance of RPs, and act as potential targets for antiviral therapeutic design. Technical limitations have thus far impeded the study of selective translation mediated by RPs in viral infection, and considerable research will be required before clinical application of viral therapeutics based on the targeting of RPs.

Historically, the ribosome has been viewed as a complex ribozyme, and ribosome activity has been considered to be highly regulated. Although the concepts of ribosome heterogeneity and specialization at the level of core RPs are still in infancy, solid data have been published in various areas, offering convincing evidence that the ribosome plays a constitutive role as well as a regulatory function in translation. Moreover, constitutive components of the ribosome may perform more specialized activities by virtue of their interactions with specific mRNA regulatory elements, such as IRESs. These findings add numerous layers of subtlety to the conventional interpretation of translational regulation.

Viruses depend on host cell structures and translation systems to complete their life cycle. In order to survive in the host cell and achieve rapid replication and proliferation, viruses have developed various mechanisms to allow the selective translation of viral mRNA and repress cellular mRNA translation. Inducing host translation shutoff is a strategy used by many viruses to optimize their replication and spread by fostering viral protein synthesis and crippling host antiviral responses. In this process, some RPs are activated to promote the translation of specific transcripts, and thus the virus can still synthesize its own proteins to optimize their replication and spread when host translation is shut off.

In this review, we focus on the important role of RP production and function in viral or host translation processes that support viral replication and infection. Understanding the research on the specialized RBFs and RPs utilized by a virus can deepen the understanding of selective translation, and expand the depth and breadth of the field of virus-host interactions. This research, data, and theoretical evidence will facilitate the identification of antiviral targets and the eventual design of antiviral drugs, and advance the development of therapeutic strategies to produce optimal antiviral agents for effective control of viral diseases.

The HBx oncoprotein of hepatitis B virus engages nucleophosmin to promote rDNA transcription and cellular proliferation

Molecular basis of sequence specific recognition of pre-ribosomal RNA by nucleolin

Yeast Rio1p is the founding member of a novel subfamily of protein serine kinases involved in the control of cell cycle progression

Cellular cap-binding protein, eIF4E, promotes picornavirus genome restructuring and translation

The large ribosomal subunit stalk as a regulatory element of the eukaryotic translational machinery

Phosphorylation of ribosomal proteins by the vaccinia virus B1R protein kinase

Ribosomes take control

Ribosomal protein S2/Sa kinase purified from HeLa cells infected with vaccinia virus corresponds to the B1R protein kinase and phosphorylates in vitro the viral ssDNA-binding protein

A systematic view on influenza induced host shutoff

Nucleolar trafficking of the mouse mammary tumor virus gag protein induced by interaction with ribosomal protein L9

The beta hairpin structure within ribosomal protein S5 mediates interplay between domains II and IV and regulates HCV IRES function

Ribosome biogenesis restricts innate immune responses to virus infection and DNA

The long unwinding road of RNA helicases

Host-and strain-specific regulation of influenza virus polymerase activity by interacting cellular proteins

Subtractional heterogeneity: a crucial step toward defining specialized ribosomes

Ribosomal protein phosphorylation in vivo and in vitro by vaccinia virus

RPLP1 and RPLP2 are essential flavivirus host factors that promote early viral protein accumulation

Cell cycle dependent nucleolar localization of the coronavirus nucleocapsid protein

Dengue virus NS1 protein interacts with the ribosomal protein RPL18: this interaction is required for viral translation and replication in Huh-7 cells

RRP1B targets PP1 to mammalian cell nucleoli and is associated with Pre-60S ribosomal subunits

Ribosomal protein S6 interacts with the latency-associated nuclear antigen of Kaposi's sarcomaassociated herpesvirus

Interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell

Interaction between SARS-CoV helicase and a multifunctional cellular protein (Ddx5) revealed by yeast and mammalian cell two-hybrid systems

Ribosomal protein L4 interacts with viral protein VP3 and regulates the replication of infectious bursal disease virus

Characterization of the interaction between hantavirus nucleocapsid protein (N) and ribosomal protein S19 (RPS19)

Mechanism of protein biosynthesis in mammalian mitochondria

Requirement of the DEAD-Box protein ded1p for messenger RNA translation

Interaction of nucleolin with ribosomal RNA genes and its role in RNA polymerase I transcription

Mechanistic intersections between picornavirus translation and RNA replication

Phosphorylation of ribosomal protein S6 in avian sarcoma virus-transformed chicken embryo fibroblasts

The uL10 protein, a component of the ribosomal P-stalk, is released from the ribosome in nucleolar stress

Alteration of ribosomal protein maps in herpes simplex virus type 1 infection

ZNF598 plays distinct roles in interferon-stimulated gene expression and poxvirus protein synthesis

Changes in nucleolar morphology and proteins during infection with the coronavirus infectious bronchitis virus

Targeting RNA polymerase I with an oral small molecule CX-5461 inhibits ribosomal RNA synthesis and solid tumor growth

Elucidation of the avian nucleolar proteome by quantitative proteomics using SILAC and changes in cells infected with the coronavirus infectious bronchitis virus

Ribosome stoichiometry: from form to function

A novel arrangement of zinc-binding residues and secondary structure in the C3HC4 motif of an alpha herpes virus protein family

Conservation of multifunctional ribosomal protein metallopanstimulin-1 (RPS27) through complex evolution demonstrates its key role in growth regulation in Archaea, eukaryotic cells, DNA repair, translation and viral replication

Essential viral and cellular zinc and iron containing metalloproteins as targets for novel antiviral and anticancer agents: Implications for prevention and therapy of viral diseases and cancer

Ribosomal protein S5 interacts with the internal ribosomal entry site of hepatitis C virus

Heterogeneity and specialized functions of translation machinery:from genes to organisms

Cellular RNA helicase p68 relocalization and interaction with the hepatitis C virus (HCV) NS5B protein and the potential role of p68 in HCV RNA replication

The helicase Ded1p controls use of near-cognate translation initiation codons in 5' UTRs

Ubiquitin-activating enzyme mechanism and role in protein-ubiquitin conjugation

Dikstein R (2015) Cap-dependent, scanning-free translation initiation mechanisms

Ribosomal protein L13 promotes IRES-driven translation of FMDV in helicase DDX3-dependent manner

Ribosomal protein S25 dependency reveals a common mechanism for diverse internal ribosome entry sites and ribosome shunting

The unfolded protein response triggers site-specific regulatory ubiquitylation of 40S ribosomal proteins

Translational control in murine hepatitis virus infection

The coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus

RPS27a enhances EBV-encoded LMP1-mediated proliferation and invasion by stabilizing of LMP1

Attenuation of 40S ribosomal subunit abundance differentially affects host and HCV translation and suppresses HCV replication

High-resolution analysis of coronavirus gene expression by RNA sequencing and ribosome profiling

Nucleolin stimulates viral internal ribosome entry site-mediated translation

rRNA pseudouridylation defects affect ribosomal ligand binding and translational fidelity from yeast to human cells

Redundant role of DEAD box proteins p68 (Ddx5) and p72/p82 (Ddx17) in ribosome biogenesis and cell proliferation

Crystal structure of the human laminin receptor precursor

Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase

60S ribosomal protein L35 regulates beta-casein translational elongation and secretion in bovine mammary epithelial cells

Identification and characterization of ribosomal proteins phosphorylated in vaccinia-virus-infected HeLa cells

The molecular mechanics of eukaryotic translation

Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication

Human cytomegalovirus infection upregulates the mitochondrial transcription and translation machineries

Increased phosphorylation of ribosomal protein S6 in hamster fibroblasts transformed by polyoma virus and simian virus 40

Phosphorylation of ribosomal proteins in hamster fibroblasts infected with pseudorabies virus or herpes simplex virus

Molecular analysis of the factorless internal ribosome entry site in Cricket Paralysis virus infection

Structure of the human 80S ribosome

Interaction of Rio1 kinase with toyocamycin reveals a conformational switch that controls oligomeric state and catalytic activity

Effect of HIV-1 Tat on the formation of the mitotic spindle by interaction with ribosomal protein S3

Ribosome-mediated specificity in Hox mRNA translation and vertebrate tissue patterning

Driving ribosome assembly

The function and synthesis of ribosomes

Analysis of nucleolar protein dynamics reveals the nuclear degradation of ribosomal proteins

RPS25 is essential for translation initiation by the Dicistroviridae and hepatitis C viral IRESs

Nucleolar protein upstream binding factor is sequestered into adenovirus DNA replication centres during infection without affecting RNA polymerase I location or ablating rRNA synthesis

Solution structure of the dimerization domain of ribosomal protein P2 provides insights for the structural organization of eukaryotic stalk

A ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mRNAs

Functional interaction and colocalization of the herpes simplex virus 1 major regulatory protein ICP4 with EAP, a nucleolar-ribosomal protein

Regulation of ribosomal proteins on viral infection

Nucleophosmin is essential for c-Myc nucleolar localization and c-Myc-mediated rDNA transcription

Ribosomal protein L18 is an essential factor that promote rice stripe virus accumulation in small brown planthopper

Porcine reproductive and respiratory syndrome virus infection induces both eIF2α phosphorylation-dependent and -independent host translation shutoff

White spot syndrome virus VP51 interact with ribosomal protein L7 of Litopenaeus vannamei

The crystal structure of hepatitis C virus NS3 proteinase reveals a trypsin-like fold and a structural zinc binding site

RACK1 controls IRES-mediated translation of viruses

C-terminal fragment of human laminin-binding protein contains a receptor domain for venezuelan equine encephalitis and tick-borne encephalitis viruses

Picornavirus IRES elements: RNA structure and host protein interactions

The P1/P2 proteins of the human ribosomal stalk are required for ribosome binding and depurination by ricin in human cells

Characterization of the interaction between the HIV-1 Gag structural polyprotein and the cellular ribosomal protein L7 and its implication in viral nucleic acid remodeling

Influenza A H3N2 subtype virus NS1 protein targets into the nucleus and binds primarily via its C-terminal NLS2/NoLS to nucleolin and fibrillarin

Multifunctional viral proteinγ345 manipulates nucleolar protein NOP53 for optimal viral replication of HSV-1

Targeting nuclear proteins for control of viral replication

How should we think about the ribosome?

The N-terminal regions of eukaryotic acidic phosphoproteins P1 and P2 are crucial for heterodimerization and assembly into the ribosomal GTPaseassociated center

Severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type I interferon, in infected cells

A functional RNAi screen links O-GlcNAc modification of ribosomal proteins to stress granule and processing body assembly

The 5 untranslated region of the human T-cell lymphotropic virus type 1 mRNA enables cap-independent translation initiation

Nuclear entry and nucleolar localization of the Newcastle disease virus (NDV) matrix protein occur early in infection and do not require other NDV proteins

Influence of toyocamycin on RNA synthesis in chick embryo cells noninfected and infected with strain MC29 avian leukosis virus

DEAD-box proteins: the driving forces behind RNA metabolism

Tyrosine phosphorylation based homo-dimerization of arabidopsis RACK1A proteins regulates oxidative stress signaling pathways in yeast

Ribosome protein L4 is essential for Epstein-Barr Virus nuclear antigen 1 function

Translating the genome in time and space: specialized ribosomes, RNA regulons, and RNA-binding proteins

Heterogeneous ribosomes preferentially translate distinct subpools of mRNAs genome-wide

Phosphorylation of ribosomal protein L30 after herpes simplex virus type 1 infection

The mammalian ribo-interactome reveals ribosome functional diversity and heterogeneity

Differential stoichiometry among core ribosomal proteins

O-GlcNAc cycling: how a single sugar post-translational modification is changing the way we think about signaling networks

A nucleolar protein, ribosomal RNA processing 1 homolog B (RRP1B), enhances the recruitment of cellular mRNA in influenza virus transcription

Kinetics of drugribosome interactions defines the cidality of macrolide antibiotics

Nop53p is a novel nucleolar 60S ribosomal subunit biogenesis protein

The complexity of human ribosome biogenesis revealed by systematic nucleolar screening of Pre-rRNA processing factors

Binding and action of amino acid analogs of chloramphenicol upon the bacterial ribosome

Nop53p is required for late 60S ribosome subunit maturation and nuclear export in yeast

Recognition of the polyubiquitin proteolytic signal

Silencing of hepatitis C virus replication by a non-viral vector based on solid lipid nanoparticles containing a shRNA targeted to the internal ribosome entry site (IRES)

Host targeted antiviral (HTA): functional inhibitor compounds of scaffold protein RACK1 inhibit herpes simplex virus proliferation

Arabidopsis receptor of activated C kinase1 phosphorylation by WITH NO LYSINE8 KINASE

Viral ribonucleoprotein complex formation and nucleolar-cytoplasmic relocalization of nucleolin in poliovirus-infected cells

RNA-binding properties of influenza A virus matrix protein M1

Ribosomal protein RPL26 is the principal target of UFMylation

Cloning of mouse genomic ribosomal protein L6 gene and analysis of its promoter

The association of ribosomal protein L18 (RPL18) with infectious bursal disease virus viral protein VP3 enhances viral replication

UFMylation of RPL26 links translocation-associated quality control to endoplasmic reticulum protein homeostasis

Interferon production and inhibition of host synthesis in cells infected with vesicular stomatitis virus

Impact of RNA polymerase I inhibitor CX-5461 on viral kinase-dependent and -independent cytomegalovirus replication

New mRNAs are preferentially translated during vesicular stomatitis virus infection

The structure and function of the eukaryotic ribosome

Hepatitis C virus 3'X region interacts with human ribosomal proteins

Specialized ribosomes: a new frontier in gene regulation and organismal biology

RNA regulons in Hox 5' UTRs confer ribosome specificity to gene regulation

Deciphering poxvirus gene expression by RNA sequencing and ribosome profiling

Subcellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein

Specific disulfide formation in the oxidation of HIV-1 zinc finger protein nucleocapsid p7

Nucleophosmin is essential for ribosomal protein L5 nuclear export

O-GlcNAc a sensor of cellular state: the role of nucleocytoplasmic glycosylation in modulating cellular function in response to nutrition and stress

Cell signaling, the essential role of O-GlcNAc

Translational control of the cytosolic stress response by mitochondrial ribosomal protein L18

DDX5 facilitates HIV-1 replication as a cellular co-factor of Rev

Ribosomal proteins: functions beyond the ribosome

Foot-and-mouth disease virus capsid protein VP1 interacts with host ribosomal protein SA to maintain the activation of MAPKs signal pathway and promote virus replication

The folded and disordered domains of human ribosomal protein SA have both idiosyncratic and shared functions as membrane receptors

Acknowledgments This work was supported by the National Natural Science Foundation of China (31772739) and a CAU-Grant for the Prevention and Control of Immunosuppressive Disease in Animals (CAU-G-PCIDA) of the China Agricultural University.

Conflict of interest The authors declare that they have no conflict of interest.