The Arabidopsis eukaryotic initiation factor (iso)4E is dispensable for plant growth but required for susceptibility to potyviruses

An Arabidopsis thaliana line bearing a transposon insertion in the gene coding for the isozyme form of the plant-specific cap-binding protein, eukaryotic initiation factor (iso) 4E (eIF (iso) 4E), has been isolated. This mutant line completely lacks both eIF(iso)4E mRNA and protein, but was found to have a phenotype and fertility indistinguishable from wild-type plants under standard laboratory conditions. In contrast, the amount of the related eIF4E protein was found to increase in seedling extracts. Furthermore, polysome analysis shows that the mRNA encoding eIF4E was being translated at increased levels. Given the known interaction between cap-binding proteins and potyviral genome-linked proteins (VPg), this plant line was challenged with two potyviruses, Turnip mosaic virus (TuMV) and Lettuce mosaic virus (LMV) and was found resistant to both, but not to the Nepovirus, Tomato black ring virus (TBRV) and the Cucumovirus, Cucumber mosaic virus (CMV). Together with previous data showing that the VPg-eIF4E interaction is necessary for virus infectivity and upregulates genome amplification, this shows that the eIF4E proteins are specifically recruited for the replication cycle of potyviruses.     

Potyvirus terminal protein VPg, effector of host eukaryotic initiation factor eIF4E

Potyvirus RNA contains at the 5' end a covalently linked virus-encoded protein VPg, which is required for virus infectivity. This role has been attributed to VPg interaction with the eukaryotic translation initiation factor eIF4E, a cap-binding protein. We characterized the dissociation constants for the interaction of the potato virus Y VPg with different plant eIF4Es and its isoforms and mapped the eIF(iso)4E attachment region on VPg. VPg/eIF4E interaction results in the inhibition of cell-free protein synthesis, and we show that it stems from the liberation of the cap moiety from the complex with eIF4E. Since VPg does not attach the cap, it appears that VPg induces changes in the eIF4E structure, diminishing its affinity to the cap. We show here that the initiation complex scaffold protein eIF(iso)4G increases VPg interaction with eIF(iso)4E. These data together suggest similar cap and VPg interactions with eIF4E and characterize VPg as a novel eIF4E-binding protein, which inhibits host protein synthesis at a very early stage of the initiation complex formation through the inhibition of cap attachment to the initiation factor eIF4E.     

Turnip mosaic virus VPg interacts with Arabidopsis thaliana eIF(iso)4E and inhibits in vitro translation

The interaction between turnip mosaic virus (TuMV) viral protein linked to the genome (VPg) and Arabidopsis thaliana eukaryotic initiation factor (iso)4E (eIF(iso)4E) was investigated to address the influence of potyviral VPg on host cellular translational initiation. Affinity chromatographic analysis showed that the region comprising amino acids 62-70 of VPg is important for the interaction with eIF(iso)4E. In vitro translation analysis showed that the addition of VPg significantly inhibited translation of capped RNA in eIF(iso)4E-reconstituted wheat germ extract. This result indicates that VPg inhibits cap-dependent translational initiation via binding to eIF(iso)4E. The inhibition by VPg of in vitro translation of RNA with wheat germ extract did not depend on RNase activity. Our present results may indicate that excess VPg produced at the encapsidation stage shuts off cap-dependent translational initiation in host cells by inhibiting complex formation between eIF(iso)4E and cellular mRNAs.     

When a knockout is an Achilles’ heel: Resistance to one potyvirus species triggers hypersusceptibility to another one in Arabidopsis thaliana

The translation initiation factors 4E are a small family of major susceptibility factors to potyviruses. It has been suggested that knocking out these genes could provide genetic resistance in crops when natural resistance alleles, which encode functional eIF4E proteins, are not available. Here, using the well-characterized Arabidopsis thaliana–potyvirus pathosystem, we evaluate the resistance spectrum of plants knocked out for eIF4E1, the susceptibility factor to clover yellow vein virus (ClYVV). We show that besides resistance to ClYVV, the eIF4E1 loss of function is associated with hypersusceptibility to turnip mosaic virus (TuMV), a potyvirus known to rely on the paralog host factor eIFiso4E. On TuMV infection, plants knocked out for eIF4E1 display striking developmental defects such as early senescence and primordia development stoppage. This phenotype is coupled with a strong TuMV overaccumulation throughout the plant, while remarkably the levels of the viral target eIFiso4E remain uninfluenced. Our data suggest that this hypersusceptibility cannot be explained by virus evolution leading to a gain of TuMV aggressiveness. Furthermore, we report that a functional eIF4E1 resistance allele engineered by CRISPR/Cas9 base-editing technology successfully circumvents the increase of TuMV susceptibility conditioned by eIF4E1 disruption. These findings in Arabidopsis add to several previous findings in crops suggesting that resistance based on knocking out eIF4E factors should be avoided in plant breeding, as it could also expose the plant to the severe threat of potyviruses able to recruit alternative eIF4E copies. At the same time, it provides a simple model that can help understanding of the homeostasis among eIF4E proteins in the plant cell and what makes them available to potyviruses.     

The potyvirus recessive resistance gene, sbm1, identifies a novel role for translation initiation factor eIF4E in cell-to-cell trafficking

From the characterization of the recessive resistance gene, sbm1, in pea we have identified the eukaryotic translation initiation factor, eIF4E, as a susceptibility factor required for infection with the Potyvirus, Pea seed-borne mosaic virus. A functional analysis of the mode of action of the product of the dominant allele revealed a novel function for eIF4E in its support for virus movement from cell-to-cell, in addition to its probable support for viral RNA translation, and hence replication. Different resistance specificities in two independent pea lines were explained by different mutations in eIF4E. On the modelled structure of eIF4E the coding changes were in both cases lying in and around the structural pocket involved in binding the 5'-m7G cap of eukaryotic mRNAs. Protein expression and cap-binding analysis showed that eIF4E encoded by a resistant plant could not bind to m7G-Sepharose, a result which may point to functional redundancy between eIF4E and the paralogous eIF(iso)4E in resistant peas. These observations, together with related findings for other potyvirus recessive resistances, provide a more complete picture of the potyvirus life cycle.     

Coordinated and selective recruitment of eIF4E and eIF4G factors for potyvirus infection in Arabidopsis thaliana

The translation initiation factors eIF4E and eIF(iso)4E play a key role during virus infection in plants. During mRNA translation, eIF4E provides the cap-binding function and is associated with the protein eIF4G to form the eIF4F complex. Susceptibility analyses of Arabidopsis mutants knocked-out for At-eIF4G genes showed that eIF4G factors are indispensable for potyvirus infection. The colonization pattern by a viral recombinant carrying GFP indicated that eIF4G is involved at a very early infection step. Like eIF4E, eIF4G isoforms are selectively recruited for infection. Moreover, the eIF4G selective involvement parallels eIF4E recruitment. This is the first report of a coordinated and selective recruitment of eIF4E and eIF4G factors, suggesting the whole eIF4F recruitment.     

Helper component proteinase of the genus Potyvirus is an interaction partner of translation initiation factors eIF(iso)4E and eIF4E and contains a 4E binding motif

The multifunctional helper component proteinase (HCpro) of potyviruses (genus Potyvirus; Potyviridae) shows self-interaction and interacts with other potyviral and host plant proteins. Host proteins that are pivotal to potyvirus infection include the eukaryotic translation initiation factor eIF4E and the isoform eIF(iso)4E, which interact with viral genome-linked protein (VPg). Here we show that HCpro of Potato virus A (PVA) interacts with both eIF4E and eIF(iso)4E, with interactions with eIF(iso)4E being stronger, as judged by the data of a yeast two-hybrid system assay. A bimolecular fluorescence complementation assay on leaves of Nicotiana benthamiana showed that HCpro from three potyviruses (PVA, Potato virus Y, and Tobacco etch virus) interacted with the eIF(iso)4E and eIF4E of tobacco (Nicotiana tabacum); interactions with eIF(iso)4E and eIF4E of potato (Solanum tuberosum) were weaker. In PVA-infected cells, interactions between HCpro and tobacco eIF(iso)4E were confined to round structures that colocalized with 6K2-induced vesicles. Point mutations introduced to a 4E binding motif identified in the C-terminal region of HCpro debilitated interactions of HCpro with translation initiation factors and were detrimental to the virulence of PVA in plants. The 4E binding motif conserved in HCpro of potyviruses and HCpro-initiation factor interactions suggest new roles for HCpro and/or translation factors in the potyvirus infection cycle.     

Regulation of Eukaryotic Initiation Factor 4E and Its Isoform： Implications for Antiviral Strategy in Plants

In recent years, biotechnology has permitted regulation of the expression of endogenous plant genes to improve agronomlcally important traits. Genetic modification of crops has benefited from emerging knowledge of new genes, especially genes that exhibit novel functions, one of which is eukaryotlc initiation factor 4E （eIF4E）. eIF4E Is one of the most important translation initiation factors Involved in eukaryotic initiation. Recent research has demonstrated that virus resistance mediated by eIF4E and Its isoform elf （Iso）4E occurs in several plant-virus interactions, thus indicating a potential new role for eIF4E/elF（Iso）4E In resistance strategies against plant viruses. In this review, we briefly describe eIF4E activity In plant translation, its potential role, and functions of the eIF4E subfamily In plant-virus interactions. Other initiation factors such as elF4G could also play a role In plant resistance against viruses. Finally, the potential for developing eIF4E-mediated resistance to plant viruses in the future Is discussed. Future research should focus on elucidation of the resistance mechanism and spectrum mediated by eIF4E. Knowledge of a particu- lar plant-virus interaction will help to deepen our understanding of eIF4E and other eukaryotic Initiation factors, and their involvement in virus disease control.     

Mimicking natural polymorphism in eIF4E by CRISPR‐Cas9 base editing is associated with resistance to potyviruses

In many crop species, natural variation in eIF4E proteins confers resistance to potyviruses. Gene editing offers new opportunities to transfer genetic resistance to crops that seem to lack natural eIF4E alleles. However, because eIF4E are physiologically important proteins, any introduced modification for virus resistance must not bring adverse phenotype effects. In this study, we assessed the role of amino acid substitutions encoded by a Pisum sativum eIF4E virus‐resistance allele (W69L, T80D S81D, S84A, G114R and N176K) by introducing them independently into the Arabidopsis thaliana eIF4E1 gene, a susceptibility factor to the Clover yellow vein virus (ClYVV). Results show that most mutations were sufficient to prevent ClYVV accumulation in plants without affecting plant growth. In addition, two of these engineered resistance alleles can be combined with a loss‐of‐function eIFiso4E to expand the resistance spectrum to other potyviruses. Finally, we use CRISPR‐nCas9‐cytidine deaminase technology to convert the Arabidopsis eIF4E1 susceptibility allele into a resistance allele by introducing the N176K mutation with a single‐point mutation through C‐to‐G base editing to generate resistant plants. This study shows how combining knowledge on pathogen susceptibility factors with precise genome‐editing technologies offers a feasible solution for engineering transgene‐free genetic resistance in plants, even across species barriers.     

Transgenic Brassica rapa plants over‐expressing eIF(iso)4E variants show broad‐spectrum Turnip mosaic virus (TuMV) resistance

The protein–protein interaction between VPg (viral protein genome‐linked) of potyviruses and eIF4E (eukaryotic initiation factor 4E) or eIF(iso)4E of their host plants is a critical step in determining viral virulence. In this study, we evaluated the approach of engineering broad‐spectrum resistance in Chinese cabbage (Brassica rapa) to Turnip mosaic virus (TuMV), which is one of the most important potyviruses, by a systematic knowledge‐based approach to interrupt the interaction between TuMV VPg and B. rapa eIF(iso)4E. The seven amino acids in the cap‐binding pocket of eIF(iso)4E were selected on the basis of other previous results and comparison of protein models of cap‐binding pockets, and mutated. Yeast two‐hybrid assay and co‐immunoprecipitation analysis demonstrated that W95L, K150L and W95L/K150E amino acid mutations of B. rapa eIF(iso)4E interrupted its interaction with TuMV VPg. All eIF(iso)4E mutants were able to complement an eIF4E‐knockout yeast strain, indicating that the mutated eIF(iso)4E proteins retained their function as a translational initiation factor. To determine whether these mutations could confer resistance, eIF(iso)4E W95L, W95L/K150E and eIF(iso)4E wild‐type were over‐expressed in a susceptible Chinese cabbage cultivar. Evaluation of the TuMV resistance of T₁ and T₂ transformants demonstrated that the over‐expression of the eIF(iso)4E mutant forms can confer resistance to multiple TuMV strains. These data demonstrate the utility of knowledge‐based approaches for the engineering of broad‐spectrum resistance in Chinese cabbage.     

Molecular Cloning and Characterization of a Gene Encoding Eukaryotic Initiation Factor iso4E in Tomato (Solanum lycopersicum)

The eukaryotic translation initiation factor 4E (eIF4E) and its isoform, eIF(iso)4E, play important roles in protein translation and recently reported to be involved in plant–virus interactions. A cDNA encoding the tomato eIF(iso)4E was cloned based on a tentative consensus (TC170275) in TIGR (), and was designated as SleIF(iso)4E, with an open reading frame of 603 nucleotides encoding a protein of 200 amino acids. The calculated molecular weight of the SleIF(iso)4E protein was 22.85 kD, and the theoretical isoelectric point was 5.76. The amino acid sequence of SleIF(iso)4E showed 66–91% identity with eIF(iso)4Es in pepper, tobacco, pea and maize, and 44–51% identity with eIF4Es from other plants. The phylogenetic relationship and tertiary structure comparisons indicate that SleIF(iso)4E share high homology and strict conserved regions with other members of the eIF4E family, a characteristic of all members of this family. Semi-quantitative RT-PCR showed varying expression levels of SleIF(iso)4E in different tissues. By comparing eIF(iso)4E coding sequences between resistant and susceptible tomato genotypes, correlation between sequence variations and virus resistance was identified. These findings provide good grounds for future research on the role of SleIF(iso)4E in translation initiation and plant–virus interactions. Sequence data of SleIF(iso)4E from this article have been deposited at GenBank under accession number EU119958.     

Activation of cap-independent translation by variant eukaryotic initiation factor 4G in vivo

Protein synthesis is tightly controlled by assembly of an intricate ribonucleoprotein complex at the m⁷GTP-cap on eukaryotic mRNAs. Ensuing linear scanning of the 5′ untranslated region (UTR) is believed to transfer the preinitiation complex to the initiation codon. Eukaryotic mRNAs are characterized by significant 5′ UTR heterogeneity, raising the possibility of differential control of translation initiation rate at individual mRNAs. Curiously, many mRNAs with unconventional, highly structured 5′ UTRs encode proteins with central biological roles in growth control, metabolism, or stress response. The 5′ UTRs of such mRNAs may influence protein synthesis rate in multiple ways, but most significantly they have been implicated in mediating alternative means of translation initiation. Cap-independent initiation bypasses strict control over the formation of initiation intermediates at the m⁷GTP cap. However, the molecular mechanisms that favor alternative means of ribosome recruitment are not understood. Here we provide evidence that eukaryotic initiation factor (eIF) 4G controls cap-independent translation initiation at the c-myc and vascular endothelial growth factor (VEGF) 5′ UTRs in vivo. Cap-independent translation was investigated in tetracycline-inducible cell lines expressing either full-length eIF4G or a C-terminal fragment (Ct) lacking interaction with eIF4E and poly(A) binding protein. Expression of Ct, but not intact eIF4G, potently stimulated cap-independent initiation at the c-myc/VEGF 5′ UTRs. In vitro RNA-binding assays suggest that stimulation of cap-independent translation initiation by Ct is due to direct association with the c-myc/VEGF 5′ UTR, enabling 43S preinitiation complex recruitment. Our work demonstrates that variant translation initiation factors enable unconventional translation initiation at mRNA subsets with distinct structural features.     

High overexpression of CERES,a plant regulator of translation,induces different phenotypical defence responses during TuMV infection

Mutations in the eukaryotic translation initiation factors eIF4E and eIF(iso)4E confer potyvirus resistance in a range of plant hosts. This supports the notion that, in addition to their role in translation of cellular mRNAs, eIF4E isoforms are also essential for the potyvirus cycle. CERES is a plant eIF4E- and eIF(iso)4E-binding protein that, through its binding to the eIF4Es, modulates translation initiation; however, its possible role in potyvirus resistance is unknown. In this article, we analyse if the ectopic expression of AtCERES is able to interfere with turnip mosaic virus replication in plants. Our results demonstrate that, during infection, the ectopic expression of CERES in Nicotiana benthamiana promotes the development of a mosaic phenotype when it is accumulated to moderate levels, but induces veinal necrosis when it is accumulated to higher levels. This necrotic process resembles a hypersensitive response (HR)-like response that occurs with different HR hallmarks. Remarkably, Arabidopsis plants inoculated with a virus clone that promotes high expression of CERES do not show signs of infection. These final phenotypical outcomes are independent of the capacity of CERES to bind to eIF4E. All these data suggest that CERES, most likely due to its leucine-rich repeat nature, could act as a resistance protein, able to promote a range of different defence responses when it is highly overexpressed from viral constructs.     

An exceptionally high nucleotide and haplotype diversity and a signature of positive selection for the eIF4E resistance gene in barley are revealed by allele mining and phylogenetic analyses of natural populations

In barley, the eukaryotic translation initiation factor 4E (eIF4E) gene situated on chromosome 3H is recognized as an important source of resistance to the bymoviruses Barley yellow mosaic virus and Barley mild mosaic virus. In modern barley cultivars, two recessive eIF4E alleles, rym4 and rym5, confer different isolate-specific resistances. In this study, the sequence of eIF4E was analysed in 1090 barley landraces and noncurrent cultivars originating from 84 countries. An exceptionally high nucleotide diversity was evident in the coding sequence of eIF4E but not in either the adjacent MCT-1 gene or the sequence-related eIF(iso)4E gene situated on chromosome 1H. Surprisingly, all nucleotide polymorphisms detected in the coding sequence of eIF4E resulted in amino acid changes. A total of 47 eIF4E haplotypes were identified, and phylogenetic analysis using maximum likelihood provided evidence of strong positive selection acting on this barley gene. The majority of eIF4E haplotypes were found to be specific to distinct geographic regions. Furthermore, the eI4FE haplotype diversity (uh) was found to be considerably higher in East Asia, whereas SNP genotyping identified a comparatively low degree of genome-wide genetic diversity in 16 of 17 tested accessions (each carrying a different eIF4E haplotype) from this same region. In addition, selection statistic calculations using coalescent simulations showed evidence of non-neutral variation for eIF4E in several geographic regions, including East Asia, the region with a long history of the bymovirus-induced yellow mosaic disease. Together these findings suggest that eIF4E may play a role in barley adaptation to local habitats.     

The eukaryotic translation initiation factor 4E controls lettuce susceptibility to the Potyvirus Lettuce mosaic virus

The eIF4E and eIF(iso)4E cDNAs from several genotypes of lettuce (Lactuca sativa) that are susceptible, tolerant, or resistant to infection by Lettuce mosaic virus (LMV; genus Potyvirus) were cloned and sequenced. Although Ls-eIF(iso)4E was monomorphic in sequence, three types of Ls-eIF4E differed by point sequence variations, and a short in-frame deletion in one of them. The amino acid variations specific to Ls-eIF4E(1) and Ls-eIF4E(2) were predicted to be located near the cap recognition pocket in a homology-based tridimensional protein model. In 19 lettuce genotypes, including two near-isogenic pairs, there was a strict correlation between these three allelic types and the presence or absence of the recessive LMV resistance genes mo1(1) and mo1(2). Ls-eIF4E(1) and mo1(1) cosegregated in the progeny of two separate crosses between susceptible genotypes and an mo1(1) genotype. Finally, transient ectopic expression of Ls-eIF4E restored systemic accumulation of a green fluorescent protein-tagged LMV in LMV-resistant mo1(2) plants and a recombinant LMV expressing Ls-eIF4E degrees from its genome, but not Ls-eIF4E(1) or Ls-eIF(iso)4E, accumulated and produced symptoms in mo1(1) or mo1(2) genotypes. Therefore, sequence correlation, tight genetic linkage, and functional complementation strongly suggest that eIF4E plays a role in the LMV cycle in lettuce and that mo1(1) and mo1(2) are alleles coding for forms of eIF4E unable or less effective to fulfill this role. More generally, the isoforms of eIF4E appear to be host factors involved in the cycle of potyviruses in plants, probably through a general mechanism yet to be clarified.     

Soybean RNA interference lines silenced for eIF4E show broad potyvirus resistance

Soybean mosaic virus (SMV), a potyvirus, is the most prevalent and destructive viral pathogen in soybean-planting regions of China. Moreover, other potyviruses, including bean common mosaic virus (BCMV) and watermelon mosaic virus (WMV), also threaten soybean farming. The eukaryotic translation initiation factor 4E (eIF4E) plays a critical role in controlling resistance/susceptibility to potyviruses in plants. In the present study, much higher SMV-induced eIF4E1 expression levels were detected in a susceptible soybean cultivar when compared with a resistant cultivar, suggesting the involvement of eIF4E1 in the response to SMV by the susceptible cultivar. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that soybean eIF4E1 interacted with SMV VPg in the nucleus and with SMV NIa-Pro/NIb in the cytoplasm, revealing the involvement of VPg, NIa-Pro, and NIb in SMV infection and multiplication. Furthermore, transgenic soybeans silenced for eIF4E were produced using an RNA interference approach. Through monitoring for viral symptoms and viral titers, robust and broad-spectrum resistance was confirmed against five SMV strains (SC3/7/15/18 and SMV-R), BCMV, and WMV in the transgenic plants. Our findings represent fresh insights for investigating the mechanism underlying eIF4E-mediated resistance in soybean and also suggest an effective alternative for breeding soybean with broad-spectrum viral resistance.     

Simultaneous CRISPR/Cas9‐mediated editing of cassava eIF4E isoforms nCBP‐1 and nCBP‐2 reduces cassava brown streak disease symptom severity and incidence

Cassava brown streak disease (CBSD) is a major constraint on cassava yields in East and Central Africa and threatens production in West Africa. CBSD is caused by two species of positive‐sense RNA viruses belonging to the family Potyviridae, genus Ipomovirus: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Diseases caused by the family Potyviridae require the interaction of viral genome‐linked protein (VPg) and host eukaryotic translation initiation factor 4E (eIF4E) isoforms. Cassava encodes five eIF4E proteins: eIF4E, eIF(iso)4E‐1, eIF(iso)4E‐2, novel cap‐binding protein‐1 (nCBP‐1), and nCBP‐2. Protein–protein interaction experiments consistently found that VPg proteins associate with cassava nCBPs. CRISPR/Cas9‐mediated genome editing was employed to generate ncbp‐1, ncbp‐2, and ncbp‐1/ncbp‐2 mutants in cassava cultivar 60444. Challenge with CBSV showed that ncbp‐1/ncbp‐2 mutants displayed delayed and attenuated CBSD aerial symptoms, as well as reduced severity and incidence of storage root necrosis. Suppressed disease symptoms were correlated with reduced virus titre in storage roots relative to wild‐type controls. Our results demonstrate the ability to modify multiple genes simultaneously in cassava to achieve tolerance to CBSD. Future studies will investigate the contribution of remaining eIF4E isoforms on CBSD and translate this knowledge into an optimized strategy for protecting cassava from disease.     

Complex formation between potyvirus VPg and translation eukaryotic initiation factor 4E correlates with virus infectivity

The interaction between the viral protein linked to the genome (VPg) of turnip mosaic potyvirus (TuMV) and the translation eukaryotic initiation factor eIF(iso)4E of Arabidopsis thaliana has previously been reported. eIF(iso)4E binds the cap structure (m(7)GpppN, where N is any nucleotide) of mRNAs and has an important role in the regulation in the initiation of translation. In the present study, it was shown that not only did VPg bind eIF(iso)4E but it also interacted with the eIF4E isomer of A. thaliana as well as with eIF(iso)4E of Triticum aestivum (wheat). The interaction domain on VPg was mapped to a stretch of 35 amino acids, and substitution of an aspartic acid residue found within this region completely abolished the interaction. The cap analogue m(7)GTP, but not GTP, inhibited VPg-eIF(iso)4E complex formation, suggesting that VPg and cellular mRNAs compete for eIF(iso)4E binding. The biological significance of this interaction was investigated. Brassica perviridis plants were infected with a TuMV infectious cDNA (p35Tunos) and p35TuD77N, a mutant which contained the aspartic acid substitution in the VPg domain that abolished the interaction with eIF(iso)4E. After 20 days, plants bombarded with p35Tunos showed viral symptoms, while plants bombarded with p35TuD77N remained symptomless. These results suggest that VPg-eIF(iso)4E interaction is a critical element for virus production.