Truncated initiation factor eIF4G lacking an eIF4E binding site can support capped mRNA translation.
(49/642)
Picornavirus proteases cleave translation initiation factor eIF4G into a C-terminal two-thirds fragment (hereafter named p100) and an N-terminal one-third fragment, which interacts with the cap-binding factor eIF4E. As the timing of this cleavage correlates broadly with the shut-off of host cell protein synthesis in infected cells, a very widespread presumption has been that p100 cannot support capped mRNA translation. Through the use of an eIF4G-depleted reticulocyte lysate system, we show that this presumption is incorrect. Moreover, recombinant p100 can also reverse the inhibition of capped mRNA translation caused either by m7GpppG cap analogue, by 4E-BP1, which sequesters eIF4E and thus blocks its association with eIF4G, or by cleavage of endogenous eIF4G by picornavirus proteases. The concentration of p100 required for maximum translation of capped mRNAs is approximately 4-fold higher than the endogenous eIF4G concentration in reticulocyte lysates. Our results imply that picornavirus-induced shut-off is not due to an intrinsic inability of p100 to support capped mRNA translation, but to the viral RNA outcompeting host cell mRNA for the limiting concentration of p100. (+info)
Composition and arrangement of genes define the strength of IRES-driven translation in bicistronic mRNAs.
(50/642)
In addition to the cap-dependent mechanism, eukaryotic initiation of translation can occur by a cap-independent mechanism which directs ribosomes to defined start codons enabled by internal ribosome entry site (IRES) elements. IRES elements from poliovirus and encephalomyocarditis virus are often used to construct bi- or oligocistronic expression vectors to co-express various genes from one mRNA. We found that while cap-dependent translation initiation from bicistronic mRNAs remains comparable to monocistronic expression, internal initiation mediated by these viral IRESs is often very inefficient. Expression of bicistronic expression vectors containing the hepatitis B virus core antigen (HBcAg) together with various cytokines in the second cistron of bicistronic mRNAs gave rise to very low levels of the tested cytokines. On the other hand, the HBcAg was well expressed when positioned in the second cistron. This suggests that the arrangement of cistrons in a bicistronic setting is crucial for IRES-dependent translation of the second cistron. A systematic examination of expression of reporter cistrons from bicistronic mRNAs with respect to position was carried out. Using the dual luciferase assay system we show that the composition of reading frames on a bicistronic mRNA and the order in which they are arranged define the strength of IRES-dependent translation. Although the cellular environment and the nature of the IRES element influence translation strength the dominant determinant is the nature and the arrangement of cistrons on the mRNA. (+info)
The novel picornavirus Equine rhinitis B virus contains a strong type II internal ribosomal entry site which functions similarly to that of Encephalomyocarditis virus.
(51/642)
Equine rhinitis B virus (ERBV) has recently been classified as an Erbovirus, a new genus in the Picornaviridae family. ERBV is distantly related to members of the Cardiovirus and Aphthovirus genera which utilize a type II internal ribosome entry sequence (IRES) to initiate translation. We show that ERBV also possesses the core stem-loop structures (H-L) of a type II IRES. The function of the ERBV IRES was characterized using bicistronic plasmids that were analysed both by transfection into BHK-21 cells and by in vitro transcription and translation in rabbit reticulocyte lysates. In both systems, a region encompassed by nucleotides (nt) 189-920 downstream of the poly(C) tract was required for maximal translation. This sequence includes stem-loops H-L as well as four additional upstream stem-loops. Nt 904 corresponds to the second of three in-frame AUG codons located immediately downstream of the polypyrimidine tract (nucleotides 869-880). Site-directed mutagenesis demonstrated that AUG2 is the major initiation codon despite the appropriate positioning of AUG1 16 nt downstream of the polypyrimidine tract. In direct IRES competition experiments, the ERBV IRES was able to compete strongly for translation factors with the IRES of Encephalomyocarditis virus (EMCV). This was true when the assays were performed in vitro (with the IRESs competing either in cis or trans) and in vivo (with the IRESs competing in cis). A comparative analysis of the strength of several IRESs revealed that the ERBV IRES, like that of the EMCV, is a powerful inducer of translation and may have similar potential for use in mammalian expression systems. (+info)
Kinetic analysis of the conjugation of ubiquitin to picornavirus 3C proteases catalyzed by the mammalian ubiquitin-protein ligase E3alpha.
(52/642)
The 3C proteases of the encephalomyocarditis virus and the hepatitis A virus are both type III substrates for the mammalian ubiquitin-protein ligase E3alpha. The conjugation of ubiquitin to these proteins requires internal ten-amino acid-long protein destruction signal sequences. To evaluate how these destruction signals modulate interactions that must occur between E3alpha and the 3C proteases, we have kinetically analyzed the formation of ubiquitin-3C protease conjugates in a reconstituted system of purified E1, HsUbc2b/E2(14Kb), and human E3alpha. Our measurements show that the encephalomyocarditis virus 3C protease is ubiquitinated in this system with K(m) = 42 +/- 11 microm and V(max) = 0.051 +/- 0.01 pmol/min whereas the parameters for the ubiquitination of the hepatitis A virus 3C protease are K(m) = 20 +/- 5 microm and V(max) = 0.018 +/- 0.003 pmol/min. Mutations in the destruction signal sequences resulted in changes in the rate at which E3alpha conjugates ubiquitin to the altered 3C protease proteins. The K(m) and V(max) values for these reactions change proportionally in the same direction. These results suggest differences in rates of conjugation of ubiquitin to 3C proteases are primarily a k(cat) effect. Replacing specific encephalomyocarditis virus 3C protease lysine residues with arginine residues was found to increase, rather than decrease, the rate of ubiquitin conjugation, and the K(m) and V(max) values for these reactions are both higher than for the wild type protein. The ability of E3alpha to catalyze the conjugation of ubiquitin to both 3C proteases was found to be inhibited by lysylalanine and phenylalanylalanine, demonstrating that the same sites on E3alpha that bind destabilizing N-terminal amino acids in type I and II substrates also interact with the 3C proteases. (+info)
Phosphatidylinositol 3-kinase confers resistance to encephalomyocarditis and herpes simplex virus-induced cell death through the activation of distinct downstream effectors.
(53/642)
The Janus kinase/STAT pathway has emerged as the paradigm of IFN-induced protection from viral infections. However, the possible participation of other signaling proteins in this protection is not clearly understood. In this report, we demonstrate that activation of phosphatidylinositol 3-kinase (PI3K) by either serum factors or IFNs blocks cell death induced by encephalomyocarditis virus (EMCV) and HSV. This increased resistance to virus-induced cell death does not involve the activation of the STAT pathway and occurs in the presence of normal viral replication. Interestingly, the cell uses two different PI3K regulated pathways to block EMCV- and HSV-induced cell death. The increased sensitivity of p85alpha(-/-) embryonic fibroblasts to EMCV-induced cell death is specifically corrected by overexpression of an activated allele of Akt/protein kinase B, but not activated mitogen-activated protein kinase extracellular kinase. Conversely, the augmented sensitivity of p85alpha(-/-) cells to HSV-induced cell death was compensated for by expression of an activated form of mitogen-activated protein kinase extracellular kinase, but not by activated Akt/protein kinase B. We conclude from these data that PI3K-activated pathways function in parallel with the Janus kinase/STAT pathway to protect cells from the lethal effects of viruses. (+info)
Multigene lentiviral vectors based on differential splicing and translational control.
(54/642)
Lentiviral vectors, so far, have been optimized for the expression of a single open reading frame. Certain practical applications of gene therapy will, however, require expression of multiple genes. The goal of this study was to explore the feasibility of directing expression of two marker genes from a lentiviral vector. We designed two types of multigene lentiviral vectors. First, we used a strategy based on the natural splicing signals of HIV-1, by which multiple mRNAs are generated from a single transcriptional unit. A second strategy was construction of a polycistronic mRNA using a translational cis-acting element, the encephalomyocarditis virus internal ribosome entry site (IRES). Our studies show that the inclusion of multiple genes in lentiviral vectors does not result in reduction in virus titers or in the loss of ability to infect nondividing cells. We introduced mutations in tat and/or rev to test whether splicing modulates the relative levels of expression of reporter genes. We also developed a truncated version of tat, which is devoid of the apoptosis-associated domain. Inclusion of this tat mutant in a lentiviral vector resulted in the generation of virus with titers similar to those of lentivirus vectors expressing wild-type tat. (+info)
Immunoglobulin treatment prevents congestive heart failure in murine encephalomyocarditis viral myocarditis associated with reduction of inflammatory cytokines.
(55/642)
We have previously shown that immunoglobulin therapy suppressed murine coxsackievirus B3 myocarditis. In the present study, we examined the effects of immunoglobulin upon murine myocarditis induced by encephalomyocarditis virus, which is not pathogenic to humans. Antiviral activity of immunoglobulin (Venilon) against encephalomyocarditis virus could not be detected in vitro. The production of cytokines was decreased in virus-infected macrophages by the treatment of immunoglobulin in vitro. Immunoglobulin (1 g/kg/day) was administered intraperitoneally to the virus-infected C3H/He mice daily for 2 weeks, beginning simultaneously with virus inoculation in experiment I and on day 14 after virus inoculation in experiment II. In experiment I, survival rate did not differ significantly between immunoglobulin-treated and untreated groups. In experiment II, survival rate was higher in immunoglobulin compared with control groups. Immunoglobulin administration suppressed the development of myocardial necrosis with T-lymphocyte infiltrates in mice not only in the acute viremic but in the chronic aviremic stages concomitantly associated with the reduction of inflammatory cytokines, i.e., tumor necrosis factor-alpha, interferon-gamma, macrophage inflammatory protein-2, and interleukin-6. Taken together, immunoglobulin therapy could have the potential to prevent congestive heart failure. (+info)
Poly(A)-binding protein interaction with elF4G stimulates picornavirus IRES-dependent translation.
(56/642)
The eukaryotic mRNA 3' poly(A) tail and the 5' cap cooperate to synergistically enhance translation. This interaction is mediated, at least in part, by elF4G, which bridges the mRNA termini by simultaneous binding the poly(A)-binding protein (PABP) and the cap-binding protein, elF4E. The poly(A) tail also stimulates translation from the internal ribosome binding sites (IRES) of a number of picornaviruses. elF4G is likely to mediate this translational stimulation through its direct interaction with the IRES. Here, we support this hypothesis by cleaving elF4G to separate the PABP-binding site from the portion that promotes internal initiation. elF4G cleavage abrogates the stimulatory effect of poly(A) tail on translation. In addition, translation in extracts in which elF4G is cleaved is resistant to inhibition by the PABP-binding protein 2 (Paip2). The elF4G cleavage-induced loss of the stimulatory effect of poly(A) on translation was mimicked by the addition of the C-terminal portion of elF4G. Thus, PABP stimulates picornavirus translation through its interaction with elF4G. (+info)