Manipulation of the host translation initiation complex eIF4F by DNA viruses

In the absence of their own translational machinery, all viruses must gain access to host cell ribosomes to synthesize viral proteins and replicate. Ribosome recruitment and scanning of capped host mRNAs is facilitated by the multisubunit eIF (eukaryotic initiation factor) 4F, which consists of a cap-binding protein, eIF4E and an RNA helicase, eIF4A, assembled on a large scaffolding protein, eIF4G. Although inactivated by many viruses to inhibit host translation, a growing number of DNA viruses are being found to employ diverse strategies to stimulate eIF4F activity in infected cells and maximize viral protein synthesis. These strategies include stimulation of cellular mTOR (mammalian target of rapamycin) signalling to inactivate 4E-BPs (eIF4E-binding proteins), a family of translational repressors that limit eIF4E availability and eIF4F complex formation, together with modulating the activity of the eIF4E kinase Mnk (mitogen-activated protein kinase signal-integrating kinase) in a variety of manners to regulate both host and viral mRNA translation. In some cases, specific viral proteins that mediate these signalling events have been identified, whereas others have been shown to interact with host translation initiation factors or complexes and modify their activity and/or subcellular localization. The present review outlines current understanding of the role of eIF4F in the life cycle of various DNA viruses and discusses its potential as a therapeutic target to suppress viral infection.     

Regulation of translation is required for dendritic cell function and survival during activation

In response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation (maturation) that exhibits specific mechanisms to control antigen processing and presentation. Here, we show that in response to lipopolysaccharides, protein synthesis is rapidly enhanced in DCs. This enhancement occurs via a PI3K-dependent signaling pathway and is key for DC activation. In addition, we show that later on, in a manner similar to viral or apoptotic stress, DC activation leads to the phosphorylation and proteolysis of important translation initiation factors, thus inhibiting cap-dependent translation. This inhibition correlates with major changes in the origin of the peptides presented by MHC class I and the ability of mature DCs to prevent cell death. Our observations have important implications in linking translation regulation with DC function and survival during the immune response.     

Hijacking the translation apparatus by RNA viruses

As invading viruses do not harbor functional ribosomes in their virions, successful amplification of the viral genomes requires that viral mRNAs compete with cellular mRNAs for the host cell translation apparatus. Several RNA viruses have evolved remarkable strategies to recruit the host translation initiation factors required for the first steps in translation initiation by host cell mRNAs. This review describes the ways that three families of RNA viruses effectively usurp limiting translation initiation factors from the host.     

Concanavalin A agglutinability of some enveloped RNA viruses modified by host cell transformation

The dependence on concanavalin A (Con A) concentration of agglutinability of some enveloped RNA viruses grown in transformed cells was compared with that of those grown in nontransformed cells. The avian oncoviruses were purified by centrifuging to equilibrium in a combination equilibrium: viscosity gradient of potassium tartrate and glycerol after conventional isopycnic sucrose density gradient centrifugation. Avian oncoviruses were more agglutinable with Con A when grown in transformed cells than when grown in nontransformed cells. Vesicular stomatitis virus grown in transformed cells was also more agglutinable than the virus grown in nontransformed cells. These results agree with the concept that the envelopes are modified by host cell transformation and that, therefore, viruses grown in transformed cells are expected to be more agglutinable with Con A than those grown in nontransformed cell.     

Cleavage of poly(A)-binding protein by poliovirus 3C protease inhibits host cell translation: a novel mechanism for host translation shutoff

Cleavage of eukaryotic translation initiation factor 4GI (eIF4GI) by viral 2A protease (2Apro) has been proposed to cause severe translation inhibition in poliovirus-infected cells. However, infections containing 1 mM guanidine-HCl result in eIF4GI cleavage but only partial translation shutoff, indicating eIF4GI cleavage is insufficient for drastic translation inhibition. Viral 3C protease (3Cpro) cleaves poly(A)-binding protein (PABP) and removes the C-terminal domain (CTD) that interacts with several translation factors. In HeLa cell translation extracts that exhibit cap-poly(A) synergy, partial cleavage of PABP by 3Cpro inhibited translation of endogenous mRNAs and reporter RNA as effectively as complete cleavage of eIF4GI and eIF4GII by 2Apro. 3Cpro-mediated translation inhibition was poly(A) dependent, and addition of PABP to extracts restored translation. Expression of 3Cpro in HeLa cells resulted in partial PABP cleavage and similar inhibition of translation. PABP cleavage did not affect eIF4GI-PABP interactions, and the results of kinetics experiments suggest that 3Cpro might inhibit late steps in translation or ribosome recycling. The data illustrate the importance of the CTD of PABP in poly(A)-dependent translation in mammalian cells. We propose that enteroviruses use a dual strategy for host translation shutoff, requiring cleavage of PABP by 3Cpro and of eIF4G by 2Apro.     

Regulation of host cell cyclin D1 by Trypanosoma cruzi in myoblasts

Infection with the parasite Trypanosoma cruzi causes Chagas disease. In this study we demonstrated that there was an increase in cyclin D1 expression in T. cruzi (Tulahuen strain)-infected myoblasts. To examine a possible mechanism for the increased cyclin D1 expression we transfected L6E9 myoblasts with cyclin D1 luciferase reporter constructs and infected with T. cruzi. There was no evidence of an increase in promoter activity. Additionally, quantitative PCR did not demonstrate any change in cyclin D1 message during infection. Moreover, we demonstrated that the cyclin D1 protein was significantly stabilized after infection. Collectively, these data indicate that infection with T. cruzi increases cyclin D1 protein abundance post-translationally.     

Regulation of dendritic cell differentiation and function by estrogen receptor ligands

Estrogen receptor (ER) ligands can modulate innate and adaptive immunity and hematopoiesis, which may explain the clear sex differences in immune responses during autoimmunity, infection or trauma. Dendritic cells (DC) are antigen presenting cells important for initiation of innate and adaptive immunity, as well as immune tolerance. DC progenitors and terminally differentiated DC express ER, indicating the ER ligands may regulate DC at multiple developmental and functional stages. Although there are profound differences in innate immunity between males and females or upon systemic imposition of sex hormones, studies are just beginning to link these differences to DC. Our and others studies demonstrate that estradiol and other ER ligands regulate the homeostasis of bone marrow myeloid and lymphoid progenitors of DC, as well as DC differentiation mediated by GM-CSF and Flt3 Ligand. Since DC have a brief lifespan, these data suggest that relatively short exposures to ER ligands in vivo will alter DC numbers and intrinsic functional capacity related to their developmental state. Studies in diverse experimental models also show that agonist and antagonist ER ligands modulate DC activation and production of inflammatory mediators. These findings have implications for human health and disease since they suggest that both DC development and functional capacity will be responsive to the physiological, pharmacological and environmental ER ligands to which an individual is exposed in vivo.     

Regulation of cell membrane function and secretion by extracellular fluid viscosity

Plasma viscosity is elevated in various pathological states, due to increased levels of protein and other macromolecules. The possibility that elevation of extracellular fluid viscosity (EFV) affects cellular and biochemical functions was examined in cultured liver cells and in red blood cells. The viscosity was modified by the addition of various macromolecules, which differ in their capacity to increase viscosity and in their chemical nature. It was found that secretion of lipoproteins and lysosomal enzymes by liver cells is inhibited as a function of the medium viscosity. Correspondingly, elevation of plasma viscosity of hyperlipidemic rats reduced lipoprotein levels. In search for the mechanism of this phenomenon we examined the effects of EFV on two cell membrane components which are involved in transmembrane processes: Gangliosides (GMs), and phospholipase A2 (PLA2). It was found that the rate of GMs degradation is decreased with increasing EFV. Of special interest was the finding that the activity of cell membrane PLA2, a key enzyme in secretory processes, is inhibited by increasing EFV. This phenomena was not confined to cell membrane PLA2, as we further found that erythrocyte hemolysis, induced by soluble snake venom PLA2, is inhibited as the EFV is increased. It is proposed that the extracellular fluid viscosity may play an important role in regulation of cellular and biochemical processes in general.     

Propagation of viruses on micropatterned host cells

We have developed a technique to characterize the in vitro propagation of viruses. Microcontact printing was used to generate linear arrays of alkanethiols on gold surfaces, which served as substrates for the patterned culture of baby hamster kidney (BHK-21) cells. Vesicular stomatitis virus (VSV) was added to unpatterned cell reservoirs adjacent to the patterned cells and incubated, setting in motion a continuously advancing viral infection into the patterned cells. At different incubation times, multiple arrays were chemically fixed to stop the viral propagation. Viral propagation distances into the patterned cells were determined by indirect immunofluorescent labeling and visualization of the VSV surface glycoprotein (G). The infection spread at approximately 50 microm/h in the 140-microm lines. Moreover, different temporal stages of the infection process were simultaneously visualized along individual lines. These stages included initiation of infection, based on G protein expression; cell-cell fusion, based on virus-induced clustering of cell nuclei; and cytoskeletal degradation, based on localized release of cells from the surface. This work sets a foundation for parallel, high-throughput characterization of viral and cellular processes.     

Regulation of in vitro translation by double-stranded RNA in mammalian cell mRNA preparations.

Polyadenylated mRNA has been purified from a variety of human and mouse cell sources. These preparations are actively translated in the wheat germ cell-free system but have only poor ability to stimulate the nuclease-treated reticulocyte lysate. The translation of endogenous and exogenous globin mRNA is strongly inhibited by the poly(A)+ RNA preparations in reticulocyte lysates. Both polysomal and non-polysomal RNA have similar effects but poly(A)+ RNA is almost 2000-fold more inhibitory than poly(A)-RNA on a weight basis. The inhibition is abolished in the presence a high concentration of poly(I).poly(C). Analysis of endogenous eIF-2 in the lysate reveals that the subunit becomes extensively phosphorylated in the presence of the inhibitory poly(A)+ RNA. Prolonged incubation of lysate with poly(A)+ RNA also causes some nucleolytic degradation of polysomal globin mRNA. These characteristics suggest that some eukaryotic cell mRNAs contain regions of double-stranded structure which are sufficiently extensive to activate translational control mechanisms in the reticulocyte lysate.     

Regulation of translation and cancer

One of the effects of oncogenic signaling appears to be differentiated recruitment of mRNA's to ribosomes. The mRNA's so affected frequently encode proteins involved in growth regulation cell-cell interaction. These functions are critical for both cancer and development, potentially suggesting that the normal role of differential translation may be during development. It is not known whether this effect is sufficient to induce cancer from cells with an initial non-neoplastic gene expression profile.