Cellular Initiation Factors

Another way in which picomaviruses could inhibit cellular protein synthesis would be to inactivate an initiation factor needed for cellular, but not viral, mENA translation. If this were the case one might expect to find a decreased capacity of extracts from infected cells to initiate translation of cellular mENAs compared to extracts from uninfected cells. Such studies have yielded a variety of results. In some laboratories no differences in activity were detected between extracts from xininfected cells and from EMC infected plasmacytoma cells or mengovirus infected Ehrlich ascites tumor cells (26, 44) In one laboratory the ability of extracts from infected cells to translate exogenously added encephalomyocarditis (EMC) virus ENA and total Krebs II ascites cell mENA was markedly diminished, but no evidence for the selective inhibition of translation of host cell mENA was obtained (52). 

Most recently Rose et al. (55) have identified an initiation factor that is inactivated after infection of HeLa cells with poliovirus. In their experiments Rose al. (55) took advantage of the finding that translation of YSV mRNA, like host mRNA translation, is inhibited in cells superinfected with poliovirus (52, 56). They prepared extracts from poliovirus-infected and uninfected HeLa cells, and after a preincubation period and RNase treatment to eliminate endogenous mRNA translation, tested the ability of the extracts to translate exogenous poliovirus and 7SV mRNA. Poliovirus mRNA was translated by both extracts, but YSV mRNA was translated only in the extracts from uninfected cells. 

Their results further indicated that extracts from infected cells contain an activity that causes the slow inactivation of eIF-4B. Apparently, the translation of poliovirus mRNA in cell-free systems requires little or no eIF-4B, or can use an altered form of 

Hackett and co-workers (57 5 ) have also observed that in a cell-free system from mengovirus infected Ehrlich ascites tumor cells, host mENA was translated less well than in extracts from uninfected cells. F-urthermore, at supraoptimal concentrations of Mg and K, the system from virus infected cells supported the translation of mengovirus ENA but not host mENA, thereby demonstrating a selective translation of viral over host mENA. A large part of the selectivity found in the translational system derived from infected cells was due to the initiation factor fraction. 

In summaxy, the available evidence suggests that an initiation factor is inactivated after infection of HeLa cells by poliovirus, possibly by a virus-coded factor. The variety of results seen in comparative studies using extracts from infected and uninfected cells in terms of translation of cellular and viral messages is not understood, but may be a property of the virus or cell system under study. 

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Also, we do not yet know how viruses such as YSY, which have capped messengers, inhibit cellular protein synthesis. Whatever the strategy that YSY uses to turn off the host, poliovirus is apparently resistent to this step, since poliovirus grows in YSY infected cells. If YSY does inactivate an initiation factor other than eIF-4B, then this would suggest that poliovirus mENA translation by-passes the requirement for several cellular initiation factors. This finding emphasizes our lack of knowledge of the exact initiation factor requirement for viral and cellular protein synthesis in cell-free systems. 

Exposure of cells to interferon normally results in the induction of a protein kinase termed eIF-2a protein kinase. The enzyme, which is synthesized in a catalytically inactive form, is activated by exposure to dsRNA. The activated kinase then phosplorylates its substrate, i.e. eIF-2a, which is the smallest subunit of initiation factor 2 (eIF2). This, in turn, blocks construction of the smaller ribosomal subunit, thereby preventing translation of all viral (and cellular) mRNA (Figure 8.6). 

The CSN3 subunit interacts with IKKy, a component of the IsrB-kinase complex controlling NF-kB activity [32]. Additionally, it is the binding site for the CSN-associated kinases CK2 and PKD [31]. The subunit of the translation-initiation factor 3 complex, Int6/eIF3e, and the ubiquitin-conjugating enzyme variant, COPIO, have been identified as other cellular interactors [33, 34]. Also the HIV-1 Tat protein interacts with CSN 3 (our unpublished data). 

Although the cellular and molecular mechanisms underlying fibrosis are not fully explained, it is assumed that an inflammatory reaction is the initiating factor in the early stage of fibrosis and that this inflammatory process continues during the fibrotic process [77, 105, 106], Kupffer and endothelial cells are considered to be the most important resident cells involved in the local production of inflammatory mediators [24, 28, 29, 76], Besides causing the activation of HSC, the inflammatory mediators induce the expression of adhesion molecules, such as ICAM-1 and VCAM on endothelial cells, that direct neutrophils and monocytes into the inflamed liver tissue [41-43], Expression of adhesion molecules is also shown for KC and stellate cells [42, 107], Furthermore, chemotactic compounds are released by endothelial and KC to attract immune competent cells... 

In this experiment, you will perform a number of in vitro translation reactions. The ribosomes used in this experiment were obtained from wheat germ, a eukaryotic organism. After the material is ground into a fine paste, the mixture is diluted with buffer to extract most of the proteins and other small molecules from the cells. This cellular extract is then subjected to centrifugation at 30,000 x -The insoluble material harvested following this step contains unlysed cells, cellular debris, and intact mitochondria. The supernatant, or S-30 fraction, contains all of the components needed to perform in vitro translation (ribosomes, tRNA, initiation factor, elongation factor, etc.). 

The eukaryotic protein-synthesizing machinery begins translation of most cellular mRNAs within about 100 nucleotides of the 5 capped end as just described. However, some cellular mRNAs contain an internal ribosome entry site (IRES) located far downstream of the 5 end. In addition, translation of some viral mRNAs, which lack a 5 cap, is initiated at IRESs by the host-cell machinery of infected eukaryotic cells. Some of the same translation initiation factors that assist in ribosome scanning from a 5 cap are required for locating an internal AUG start codon, but exactly how an IRES is recognized is less clear. Recent results indicate that some IRESs fold into an RNA structure that binds to a third site on the ribosome, the E site, thereby positioning a nearby internal AUG start codon in the P site. 

FIGURE 49-3 Interferon-mediated antiviral activity occurs via mub le mechanisms. The binding of IFN to specific cell surface receptor molecules signals die cell to produce a series of antiviral proteins. The stages of viral replication diat are inhibited by various IFN-induced antiviral proteins are shown. Most of these act to inhibit the translation of viral proteins (mechanism 2), but odier steps in viral replication also are affected (mechanisms 1,3, and 4). The roles of diese mechanisms in the other actions of IFNs are under study. Key IFN = interferon mRNA = messenger RNA Mx = specific cellular protein tRNA = transfer RNA RNase L = latent cellular endoribonuclease 2 5 A = 2 -5 -oligoadeny-lates eIF-2a = protein synthesis initiation factor. 

Mechanisms of analgesic nephropathy remain unclear. The renal lesion begins in the papillary tip as a result of accumulated toxic metabolites, decreased blood flow, and impaired cellular energy production. The metabolism of phenacetin to acetaminophen, which is then oxidized to toxic free radicals that are concentrated in the papilla, appears to be the initiating factor that causes toxicity by mechanisms analogous to acetaminophen hepatotoxicity. Toxicity is prevented by availability of reduced glutathione. However, salicylates deplete renal glutathione and thereby facilitate phenacetin and acetaminophen toxicity (Silva 2004 Braden et al. 2005). 

To determine which of the initiation factors was responsible for the resumption of globin ENA synthesis, the purified initiation factors from reticulocytes were added one by one (4 ) These studies suggested that when initiation factor eIP-4B(lF-M3)was present in limiting quantities, viral ENA outcompetes cellular mENA for translation. When the factor is present in excess, both messages are translated. 

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