Vaccine-Induced,Pseudorabies Virus-Specific,Extrathymic CD4+CD8+ Memory T-Helper Cells in Swine

Pseudorabies virus (PRV; suid herpesvirus 1) infection causes heavy economic losses in the pig industry. Therefore, vaccination with live attenuated viruses is practiced in many countries. This vaccination was demonstrated to induce extrathymic virus-specific memory CD4⁺CD8⁺ T lymphocytes. Due to their major histocompatibility complex (MHC) class II-restricted proliferation, it is generally believed that these T lymphocytes function as memory T-helper cells. To directly prove this hypothesis, 15-amino-acid, overlapping peptides of the viral glycoprotein gC were used for screening in proliferation assays with peripheral blood mononuclear cells of vaccinated d/d haplotype inbred pigs. In these experiments, two naturally processed T-cell epitopes (T1 and T2) which are MHC class II restricted were identified. It was shown that extrathymic CD4⁺CD8⁺ T cells are the T-lymphocyte subpopulation that responds to epitope T2. In addition, we were able to show that cytokine secretion can be induced in these T cells through recall with inactivated PRV and demonstrated that activated PRV-primed CD4⁺CD8⁺ T cells are able to induce PRV-specific immunoglobulin synthesis by PRV-primed, resting B cells. Taken together, these results demonstrate that the glycoprotein gC takes part in the priming of humoral anti-PRV memory responses. The experiments identified the first T-cell epitopes so far known to induce the generation of virus-specific CD4⁺CD8⁺ memory T lymphocytes and showed that CD4⁺CD8⁺ T cells are memory T-helper cells. Therefore, this study describes the generation of virus-specific CD4⁺CD8⁺ T cells, which is observed during vaccination, as a part of the potent humoral anti-PRV memory response induced by the vaccine.Pseudorabies virus (PRV), a member of the Alphaherpesvirinae, is the causative agent of Aujeszky’s disease. This disease is lethal to young pigs and causes important economic losses (52). Therefore, vaccination of pigs is practiced in many countries.Several humoral immune system effector mechanisms are involved in the protection of pigs from PRV infection. Virus-neutralizing antibodies, antibodies mediating antibody-dependent cell-mediated cytotoxicity, and antibodies mediating complement-mediated lysis of PRV-infected target cells have been demonstrated (22, 23, 53, 54). The main targets of this humoral immune response were shown to be the viral glycoproteins (3, 45), and passive immunization with monoclonal antibodies (MAbs) against gB, gC, and gD protects pigs from a lethal challenge (20, 49).The protection conferred through cell-mediated immunity is poorly understood. An increase in major histocompatibility complex (MHC)-unrestricted cell-mediated cytotoxicity against uninfected and PRV-infected cells has been detected after infection or vaccination of pigs with PRV (16, 53, 54), and specific cellular immune responses to PRV infections could be demonstrated by stimulation of proliferation and lymphokine secretion of porcine PRV-immune lymphocytes (10, 17, 42, 43, 51) as well as by the detection of PRV-specific cytotoxic lymphocytes (21, 56).There are some difficulties in defining more precisely the impact of cell-mediated immune effector mechanisms to protection from PRV-infection and their interplay with the observed humoral immune response. Considerably fewer porcine than human or mouse differentiation markers are available (34). In addition, the immune system of swine differs considerably from that of humans and mice. The pig has a substantial number of CD4⁻CD8⁻ T lymphocytes in the peripheral blood (4, 6, 12, 36, 39). In young animals, this subpopulation of T lymphocytes comprises up to 60% of the T lymphocytes and contains mainly γδ T lymphocytes. The pig is also the only species so far known to contain a substantial number of resting extrathymic CD4⁺CD8⁺ T lymphocytes (28, 36, 39). This T-lymphocyte population shows morphologically the phenotype of mature T lymphocytes (40) and increases with age to up to 60% of peripheral T lymphocytes (29, 35, 39, 55). Further, it was demonstrated that CD4⁺CD8⁺ T lymphocytes comprise memory T cells which proliferate upon stimulation with recall antigen (43, 55). Since the observed proliferative response was shown to be MHC class II-restricted, it was speculated that the porcine CD4⁺CD8⁺ T-cell subset contains memory T-helper lymphocytes (43). However, the ability of these T lymphocytes to secrete cytokines or to provide help to B cells has so far not been demonstrated.To gain a better understanding of immune effector mechanisms conferring protection from PRV infection, the function of these unusual extrathymic T-lymphocyte subsets has to be elucidated. In the present study, we identified two T-cell epitopes on glycoprotein gC which are primed during vaccination of d/d haplotype inbred pigs (41) against PRV and demonstrated that MHC class II-restricted, peripheral CD4⁺CD8⁺ memory T lymphocytes are the responding T lymphocytes. We were further able to show that PRV-specific, extrathymic CD4⁺CD8⁺ T lymphocytes are able to secrete cytokines and have the capacity to stimulate the secretion of PRV-specific immunoglobulins (Ig) by PRV-primed B cells. These results demonstrate that porcine CD4⁺CD8⁺ T lymphocytes can function as memory T-helper cells and can direct humoral anti-PRV memory responses.     

Protective CD4+ and CD8+ T Cells against Influenza Virus Induced by Vaccination with Nucleoprotein DNA

DNA vaccination is an effective means of eliciting both humoral and cellular immunity, including cytotoxic T lymphocytes (CTL). Using an influenza virus model, we previously demonstrated that injection of DNA encoding influenza virus nucleoprotein (NP) induced major histocompatibility complex class I-restricted CTL and cross-strain protection from lethal virus challenge in mice (J. B. Ulmer et al., Science 259:1745–1749, 1993). In the present study, we have characterized in more detail the cellular immune responses induced by NP DNA, which included robust lymphoproliferation and Th1-type cytokine secretion (high levels of gamma interferon and interleukin-2 [IL-2], with little IL-4 or IL-10) in response to antigen-specific restimulation of splenocytes in vitro. These responses were mediated by CD4⁺ T cells, as shown by in vitro depletion of T-cell subsets. Taken together, these results indicate that immunization with NP DNA primes both cytolytic CD8⁺ T cells and cytokine-secreting CD4⁺ T cells. Further, we demonstrate by adoptive transfer and in vivo depletion of T-cell subsets that both of these types of T cells act as effectors in protective immunity against influenza virus challenge conferred by NP DNA.Cellular immune responses play an important role in protection from disease caused by infectious pathogens, such as viruses and certain bacteria (e.g., Mycobacterium tuberculosis). The specific T cells involved in conferring immunity can include both CD4⁺ and CD8⁺ T cells, often through the action of secreted cytokines and cytolytic activity, respectively. Certain types of vaccines, such as subunit proteins and whole or partially purified preparations of inactivated organisms, in general induce CD4⁺ T-cell responses but not CD8⁺ cytotoxic T lymphocytes (CTL). In contrast, live attenuated organisms and subunit proteins formulated with certain experimental adjuvants can induce both types of responses. Recently, a different approach consisting of direct immunization with plasmid DNA expression vectors (i.e., DNA vaccines) has shown promise as a viable means of inducing broad-spectrum T-cell responses. The effectiveness of DNA vaccines in animal models is likely due, at least in part, to expression of antigens in situ (35), leading to the induction of CTL (29), antibodies (3, 4, 10, 21, 22, 32), and cytokine-secreting lymphocyte responses (12, 36). During the past 5 years, many reports have been published on the immunogenicity of DNA vaccines encoding various antigens in several animal models, thereby illustrating the applicability of the technology to many pathogens (for a review, see reference 6). However, in only a few instances has the nature of the effector cells responsible for protective immunity been described (7, 16). In the present study, we have analyzed in detail the cellular immune responses induced by influenza virus nucleoprotein (NP) DNA and have established that both CD4⁺ T cells secreting Th1-type cytokines and CD8⁺ cytotoxic T cells play important effector roles in heterosubtypic protective immunity against lethal influenza virus challenge in mice.     

Adsorption of Extracellular Chromosomal DNA and Its Effects on Natural Transformation of Azotobacter vinelandii

To better understand the influence of environmental conditions on the adsorption of extracellular chromosomal DNA and its availability for natural transformation, the amount and conformation of adsorbed DNA were monitored under different conditions in parallel with transformation assays using the soil bacterium Azotobacter vinelandii. DNA adsorption was monitored using the technique of quartz crystal microbalance with dissipation (QCM-D). Both silica and natural organic matter (NOM) surfaces were evaluated in solutions containing either 100 mM NaCl or 1 mM CaCl₂. The QCM-D data suggest that DNA adsorbed to silica surfaces has a more compact and rigid conformation in Ca²⁺ solution than in Na⁺ solution and that the reverse is true when DNA is adsorbed to NOM surfaces. While the amounts of DNA adsorbed on a silica surface were similar for Ca²⁺ and Na⁺ solutions, the amount of DNA adsorbed on an NOM-coated surface was higher in Ca²⁺ solution than in Na⁺ solution. Transformation frequencies for dissolved DNA and DNA adsorbed to silica and to NOM were 6 × 10⁻⁵, 5 × 10⁻⁵, and 2.5 × 10⁻⁴, respectively. For NOM-coated surfaces, transformation frequencies from individual experiments were 2- to 50-fold higher in the presence of Ca²⁺ than in the presence of Na⁺. The results suggest that groundwater hardness (i.e., Ca²⁺ concentration) will affect the amount of extracellular DNA adsorbed to the soil surface but that neither adsorption nor changes in the conformation of the adsorbed DNA will have a strong effect on the frequency of natural transformation of A. vinelandii.Horizontal gene transfer contributes to microbial evolution and provides mechanisms for the spread of both antimicrobial resistance genes and genetically engineered DNA. While most studies on horizontal gene transfer focus on conjugation, recent reviews on extracellular DNA (16, 27) document the need to consider also natural transformation. The amount of extracellular DNA in the soil is on the order of hundreds of ng/g of dry soil (27). Extracellular DNA adsorbs to many common soil constituents, including sand, clay, and natural organic matter (NOM) (16, 27), and once adsorbed may persist for days or years (16).As first reported by Lorenz et al. (18), adsorbed DNA is available for natural transformation and therefore represents a potential environmental reservoir facilitating horizontal gene transfer. DNA adsorbed on sand surfaces has been successfully transferred to both Gram-positive and Gram-negative soil bacteria, including Bacillus subtilis (18), Pseudomonas stutzeri (19), and Acinetobacter calcoaceticus (4). Other studies have focused on transformation with DNA adsorbed to other surfaces (for example, clay minerals [15], humic acids [6], and intact soils [25, 31]). In most previous studies, adsorbed DNA transformed at a lower frequency than dissolved DNA (see, for example, references 4 and 11). However, higher transformation frequencies for adsorbed DNA than for dissolved DNA have been reported in two studies (18, 19). In addition, Demaneche et al. were unable to detect Pseudomonas fluorescens transformants in a variety of liquid media despite successful transformations in sterile soil columns (8).Several studies have evaluated the influence of nutrients (24, 34) and soil (8, 11, 23, 31) on transformation efficiency. Less information is available on the influence of adsorbed DNA conformation on transformation efficiency. Pietramellara et al. speculated that the decrease in transformation rates they observed upon repeated wetting and drying cycles of adsorbed DNA was due to conformational changes (28). Cai et al. also speculated that differences in the conformation of adsorbed DNA could be responsible for lower transformation efficiencies for DNA bound to kaolinte and inorganic clays (2), based on their previous work characterizing adsorption to different surfaces (3). Detailed characterizations of the conformation of adsorbed DNA only recently became feasible, and the influence of the conformation of adsorbed DNA on transformation frequencies has not, to our knowledge, been systematically investigated.Characterization of the mass and conformation of DNA adsorbed on different surfaces can be accomplished using quartz crystal microbalance with dissipation (QCM-D) (7, 22). QCM-D measurements are based on the shift in frequency (ΔF) and the decay in vibrating energy (ΔD) that occur as molecules adsorb to piezoelectric sensors (29, 33). Viscosity, elastic shear modulus, and effective thickness of the adsorbed material can be determined by fitting the frequency and dissipation data to the viscoelastic Voigt model (7). Based on Nguyen and colleagues'' previous work with plasmid DNA adsorption on silica and NOM-coated surfaces, increasing electrolyte concentrations and the presence of divalent cations favor DNA adsorption (20-22). In addition, inner sphere complexation by Ca²⁺ with DNA phosphate backbone allows the formation of DNA-adsorbed layers that are more compact than the DNA-adsorbed layer formed by charge shielding in solution with a high Na⁺ concentration (20-22).The objective of this study was to investigate both the adsorption of chromosomal DNA to representative soil particle surfaces and the effect of such adsorption on natural transformation. We used QCM-D to characterize the conformation of adsorbed DNA on silica and NOM surfaces under two different solution chemistries (100 mM Na⁺ and 1 mM Ca²⁺). The influences of adsorption and of differences in the conformation of the adsorbed DNA on transformation frequencies were tested in a common soil bacterium, Azotobacter vinelandii. A. vinelandii is naturally competent (26) but had not been previously reported to be transformed with adsorbed DNA.     

Duration of Priming of Two Indirect Plant Defenses

When plants are infested by herbivores, they emit herbivore-induced plant volatiles (HIPVs) that attract carnivorous natural enemies of herbivores. Furthermore, there are increasing evidences that defenses of intact plants against herbivores are primed when exposed to HIPVs. We previously reported that lima bean leaf volatiles induced by the herbivorous mites Tetranychus urticae primed two T. urtiae-induced indirect defenses in neighboring conspecific plants: HIPV emission and extrafloral nectar (EFN) secretion. An intriguing unanswered question is whether the durations of these two defenses are the same. Here, we show that the durations of the two defenses were the same for up to two days after the initiation of T. urticae damage. The two induced primed defense would act as a battery of defense in exposed plants.Key Words: herbivore-induced plant volatiles, indirect, defense, induced response, plant-plant interaction, primingWhen infested by herbivores, plants defend themselves indirectly by emitting herbivore-induced plant volatiles (HIPVs). One of the ecological functions of HIPVs is to attract carnivorous natural enemies of the herbivores.^1,2 Recently, it was reported that the emission of HIPVs primed defenses against herbivores in neighboring intact plants.^3–7 Thus, HIPVs also mediate interactions between infested and intact plants.⁸ The enhanced defense in response to HIPVs in intact plants is called ‘priming’, which has been studied intensively in plant-pathogen interactions,⁹ but not so in plant-insect interactions.We previously reported that exposure to HIPVs emitted from lima bean leaves infested by Tetranychus urticae primed HIPV production in detached intact conspecific leaves.³ We also reported that exposure to HIPVs, produced in response to T. urticae damage,⁴ primed the induced production of extrafloral nectar (EFN; an alternative food source for predators^10,11 in lima bean plants. An intriguing question is whether the two primed defenses work as a battery against T. urticae. To answer this, we examined the duration of primed HIPV production by lima bean plants using the same experimental set-up as our previous study of EFN priming by conspecific plants.⁴For exposure of plants to HIPVs, we used a 60 × 60 × 60 cm cage with two 30 × 30 cm windows on opposite sides of the cage.¹² As odor sources, we used eight plants that had been infested with 60 adult T. urticae females per plant for 1 day. Eight uninfested plants were used as control odor sources. Two uninfested plants were placed in a cage with the odor source plants and exposed to either HIPVs or uninfested plant volatiles (UPVs) for 10 days in a climate-controlled room (25 ± 2°C, 60–70% RH, 16:8; L:D).A Y-tube olfactometer¹³ was used to examine the response of the predators to HIPVs. Adult female P. persimilis were randomly selected from a colony and individually positioned at the beginning of the iron wire. When test mites reached the end of one arm of the olfactometer, their choice was recorded. We tested the olfactory responses of the predator toward (1) plants infested by T. urticae for two days after exposure to UPVs vs. plants infested by T. urticae for two days after exposure to HIPVs, and (2) plants infested by T. urticae for four days after exposure to UPVs vs. plants infested by T. urticae for four days after exposure to HIPVs.HIPV-exposed plants attracted more predators than UPV-exposed plants in a Y-tube olfactometer when infested by T. urticae for two days (Fig. 1A). By contrast, the predators did not distinguish between HIPV- and UPV-exposed plants when infested by T. urticae for four days (Fig. 1B). Our previous study showed that HIPV-exposed plants secreted significantly larger amounts of EFN secretion than UPV-exposed plants infested by T. urticae for two days under the same experimental condition as in this study.⁴ However, the difference was not significant when they were infested for four days.⁴Open in a separate windowFigure 1The olfactory response of P. persimilis females to volatiles from the odor-exposed plants, as determined in a Y-tube olfactometer: (A) plants infested by T. urticae for two days after exposure to UPVs (UPV-exposed—T. urticae 2d) vs. plants infested by T. urticae for two days after exposure to HIPVs (HIPV-exposed—T. urticae 2d), and (B) plants infested by T. urticae for four days after exposure to UPVs (UPV-exposed—T. urticae 4d) vs. plants infested by T. urticae for four days after the exposure to HIPVs (HIPV-exposed—T. urticae 4d). Asterisks beside each bar indicate a significant difference between the first trifoliate leaves and the primary leaves. Asterisks beside a bar indicate a significant difference (binomial test: p < 0.001).Lima bean plants increase the amount of endogenous jasmonic acid after exposure to HIPVs.¹⁴ Jasmonic acid, an important plant hormone regulating a defense signaling pathway against herbivores and pathogens,^15,16 is reported to be involved in the induction of both volatile emission^17,18 and EFN secretion¹⁹ in response to T. urticae damage in lima bean plants. The increase of endogenous jasmonic acid in HIPV-exposed plants may partly explain the simultaneous priming of the two defenses.In this study, we showed that the durations of priming of two indirect defenses were roughly the same for up to two days. Priming of these two indirect defenses would thus be a battery of defense at the outset of T. urticae damage. Further study is necessarily to test whether the primed battery of induced defense increases the fitness of the exposed plants.     

Comparison of Immune Restoration in Early versus Late Alpha Interferon Therapy against Hepatitis C Virus

Early alpha interferon (IFN-α) therapy against hepatitis C virus (HCV) rescues polyfunctional, virus-specific memory CD8⁺ T cells, but whether immune restoration is possible during late therapy remains controversial. We compared immune restoration of HCV-specific memory T cells in patients who cleared HCV infection spontaneously and following early or late IFN therapy. Multifunctional CD4⁺ and CD8⁺ memory T cells were detected in spontaneous resolvers and in individuals treated early following an acute infection. In contrast, limited responses were detected in patients treated during chronic infection, and the phenotype of HCV-specific cells was influenced by autologous viral sequences. Our data suggest that irreversible damage to the HCV-specific memory T-cell response is associated with chronic HCV infection.The majority of acute hepatitis C virus (HCV) infections become chronic, with persistent viremia and serious liver complications (12). Alpha interferon (IFN-α)-based therapy is the only approved treatment for chronic HCV; its success rate ranges from 40 to 90% depending on the infecting genotype (9, 18). The success of therapy is characterized by a sustained virological response (SVR), defined as undetectable HCV RNA in plasma at 6 months after termination of therapy. SVR rates are greatly enhanced if therapy is started between 3 and 6 months following acute HCV infection, but the underlying mechanisms are not well understood (27, 28). We have demonstrated that early interferon therapy for HCV can rescue and select for long-lived polyfunctional CD8⁺ memory T cells (1). Treatment-induced memory T cells were similar in phenotype and function to natural memory T cells generated following spontaneously resolved infection. They expressed high levels of CD127 and Bcl-2 (CD127^hi, Bcl-2^hi) and low levels of PD1 (PD1^lo) and were polyfunctional in nature (1). However, restoration of HCV-specific memory CD4⁺ T cells has not been examined. Furthermore, whether immune restoration is possible following the late initiation of therapy during the chronic phase remains controversial. Kamal et al. demonstrated that SVR is associated with a recovery in HCV-specific CD4⁺ T-cell responses (13). In contrast, Barnes et al. and Rahman et al. demonstrated that the induction of HCV-specific immunity during therapy does not correlate with outcomes (2, 21).     

The Nematode Eukaryotic Translation Initiation Factor 4E/G Complex Works with a trans-Spliced Leader Stem-Loop To Enable Efficient Translation of Trimethylguanosine-Capped RNAs

Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5′ end through the eIF4F initiation complex binding to the 5′ m⁷G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5′ end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m⁷G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5′ untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs.Cap-dependent translation initiation in eukaryotes is a complex process involving many factors and serves as the primary mechanism for eukaryotic translation (37, 44). The first step in the initiation process, recruitment of the m⁷G (7-methylguanosine)-capped mRNA to the ribosome, is widely considered the rate-limiting step. It begins with recognition of and binding to the m⁷G cap at the 5′ end of the mRNA by the eukaryotic translation initiation factor 4F (eIF4F) complex, which contains three proteins: eIF4E (a cap-binding protein), eIF4G (a scaffold protein with RNA binding sites), and eIF4A (an RNA helicase). eIF4G''s interaction with eIF3, itself a multisubunit complex that interacts with the 40S ribosome, facilitates the actual recruitment of capped RNA to the ribosome. With the help of several other initiation factors, the small ribosomal subunit scans the mRNA from 5′ to 3′ until a translation initiation codon (AUG) in appropriate context is identified and an 80S ribosomal complex is formed, after which the first peptide bond is formed, thus ending the initiation process (37, 44). The AUG context can play an important role in the efficiency of translation initiation (23, 44). The length, structure, and presence of AUGs or open reading frames in the mRNA 5′ untranslated region (UTR) can negatively affect cap-dependent translation and ribosomal scanning. In general, long and highly structured 5′ UTRs, as well as upstream AUGs leading to short open reading frames, can impede ribosome scanning and lead to reduced translation (23, 44). In addition, 5′ UTRs less than 10 nucleotides (nt) in length are thought to be too short to enable preinitiation complex assembly and scanning (24). Thus, several attributes of the mRNA 5′ UTR are known to negatively affect translation initiation, whereas only the AUG context and the absence of negative elements are known to have a positive effect on translation initiation (44).Two of the important mRNA features associated with cap-dependent translation, the cap and the 5′ UTR, are significantly altered by an RNA processing event known as spliced leader (SL) trans splicing (3, 8, 17, 26, 36, 47). This takes place in members of a diverse group of eukaryotic organisms, including some protozoa, sponges, cnidarians, chaetognaths, flatworms, nematodes, rotifers, crustaceans, and tunicates (17, 28, 39, 55, 56). In SL trans splicing, a separately transcribed small exon (16 to 51 nucleotides [nt]) with its own cap gets added to the 5′ end of pre-mRNAs. This produces mature mRNAs with a unique cap and a conserved sequence in the 5′ UTR. In metazoa, the m⁷G cap is replaced with a trimethylguanosine (TMG) cap (m^2,2,7GpppN) (27, 30, 46, 49). In nematodes, ∼70% of all mRNAs are trans spliced and therefore have a TMG cap and an SL (2). In general, eukaryotic eIF4E proteins do not effectively recognize the TMG cap (35). This raises the issues of how the translation machinery in trans-splicing metazoa effectively recognizes TMG-capped trans-spliced mRNAs, what role the SL sequence plays in translation initiation, and how the conserved translation initiation machinery has adapted to effectively translate trans-spliced mRNAs.Previous work has shown that efficient translation of TMG-capped messages in nematodes requires the SL sequence (22 nt) immediately downstream of the cap (5, 25, 29). In the current studies, we sought to understand the manner in which the SL enhanced the translation of TMG-capped mRNAs. Using a cell-free nematode in vitro translation system, we carried out mutational analyses that define the specific sequences in the SL that are required and sufficient for efficient translation of TMG-capped mRNAs. These analyses led to the discovery of a small, discrete stem-loop immediately adjacent to the TMG cap in trans-spliced messages required for efficient translation. Notably, the sequences involved in the base pairing of the stem are highly conserved in alternative SL sequences found in nematodes. We further show that the nematode eIF4E/G complex plays a major role in facilitating the SL enhancement of TMG-capped mRNA that likely occurs after the initial cap-binding step. The results demonstrate the importance of specific enhancing elements in the 5′ UTR and adaptation in the eIF4F complex necessary for optimal cap-dependent translation.