Ribosome from infected cells

Soon after T4 phage has infected E. coli, host protein synthesis is inhibited and the bacterium cannot be superinfected with RNA phage. This is reflected by the inability of ribosomes from T4 infected E. coli to translate MS2, f2, or R17 RNA in vitro or to bind to the initiation sites at the beginning of each cistron (Dube and Rudland, 1970 Steitz et al., 1970 Hsu and Weiss, 1969 Klem et al, 1970). If ribosomes from infected cells are washed in 2.0 M NH,C1 and then supplemented with initiation factors from normal ribosomes they are once more able to translate phage RNA. On the other hand, the wash fraction from infected ribosomes prevents, and even reverses, the formation of initiation complexes between normal ribosomes and MS2 RNA. These observations suggest that after T4 infection E. coli initiation factors are selectively... 

As shown in Figure 5f much less of the protease inhibitor is bo md to the ribosomes from infected cells which correlates with their reduced proteolytic activity. It is not clear whether the lack of binding is due to specific loss of the protease polypeptide, or to a more general effect of the infection on ribosome structure (29). 

In a sensitive, solid-phase assay for endo-proteases (27, 54), the activity of polysomes and monosomes were compared. Shown in Table 1 is the loss of activity of SOS ribosomes from infected cells, but the retention of activity on polysomes. This data suggests continuing participation of a polysomal protease activity in viral... 

Sagot, J., and Beaud, G., 1979, Phosphorylation in vivo of vaccinia-virus structural protein found associated with the ribosome from infected cells, Eur. J. Biochem. 98 131. 

Recent evidence points to the presence of protease activity-associated with polysomes and ribosomes when extracts of uninfected cells are assayed (refs. 27 32, Figure j). Characteristic of infection of cells by poliovirus is drastic, rapid inhibition of protein synthesis. Poliovirus infection also depresses the ribosomal protease activity (27, 29, 55) Ribosomes from uninfected cells have been reported to possess an autoproteolytic activity (31, 32), and this has been confiimed by two-dimensional gel analysis (Figure 4) Poliovirus infection of HeLa cells reduces the autoproteolysis of isolated 808 ribosomes markedly (not shown). The inhibition of HeLa cell ribosomal protease activity requires protein synthesis, but proceeds in the presence of guanidine (55) ... 

In order to stimulate fmet incorporation by cell-free extracts, crude preparations of initiation factors prepared from the 0.5 M KGI wash of HeLa cell ribosomes were added to the protein-synthesizing reactions. The ribosomal salt wash from uninfected cells stimulated total fmet incorporation about two-fold at the concentrations utilized, and stimulated initiation at both sites eq ially. Ribosomal salt wash prepared from infected cells also stimulated total fmet incorporation to about the same extent as the uninfected cell preparation however, SDS-PAGE analysis of the fmet-labeled products revealed that only synthesis of the smaller polypeptide was increased. No stimulation of NCVPla synthesis occurred (Knauert and Ehrenfeld, submitted). 

In a comparison of supernatant and ribosomal tRNA populations, Wettstein [55] had found that Leu-tRNAi, which accounts for more than 50% of coli Leu-tRNA, is present much less frequently in ribosomal fractions than is the second fraction (MAK peak 2) and that, in T4-infected cells, it is largely excluded from the ribosomal fraction. In an extension of this work, Sueoka and Kano-Sueoka [56] obtained polysomal, ribosomal, and supernatant fractions from noninfected cells, as well as from infected cells, 1.5, 3, 6, and 10 minutes after T2 infection. Transfer RNA was then isolated from the various fractions, deacylated, and reacylated with radioactive leucine. The proportion of the various Leu-tRNA species in each preparation was analyzed by MAK chromatography. As shown in Table I, the Leu-tRNAi content of the polysomal fraction, which amounted to 20% of total Leu-tRNA before infection, fell to 6% within 1.5 minutes after infection and then remained essentially constant up to 10 minutes, suggesting a reduced usage of this species and consequently of the CpUpG codon. 

Efforts to identify the inhibitory component in infected cell ribosomal salt wash yielded a protein of Mr 33,0(X) daltons, which comigrated with the SFV capsid protein on SDS-polyacrylamide gels and which reacted with antibodies raised against viral capsid protein (H. Van Steeg, personal communication). This protein, purified from infected cell ribosomal salt wash, selectively inhibited translation in vitro of host and early viral mRNA, but had no effect on translation of late viral mRNA or EMC virus RNA. The mechanism by which SFV capsid protein interferes with translation initiation of some mRNAs is not understood, nor is it clear how SFV late mRNA eludes the inhibition, or what role cap recognition plays in this scheme. However, the overall strategies followed by SFV and poliovirus may be quite similar, despite differences in underlying mechanisms. 

In recent studies, Lemieux and Millward (Zarbl and Millward, 1983) have examined fractionated translation system lysates for their ability to stimulate the translation of uncapped reovirus mRNAs in unfractionated lysates prepared from uninfected cells. Fractions from infected but not uninfected cells could stimulate this translation, indicating that a viral factor stimulates the translation. The bulk of the stimulation activity was present equally in the S-200 supernate and the ribosomal salt wash. The specific activity of the stimulatory factor was greatest in the ribosomal salt wash, that appears to be enriched for the a3 outer capsid polypeptide. A monoclonal antibody to a3 could specifically inhibit the translation of uncapped reovirus mRNAs in lysates prepared from infected cells. 

A. Characteristics. Ricin is a glycoprotein toxin from the seed of the castor bean plant. Altering ribosomal RNA blocks protein synthesis, thereby killing infected cells. Ricin s significance as a potential biological warfare agent relates to its availability worldwide, ease of production, and extreme pulmonary toxicity when inhaled. 

Another theory to explain the shut-off, proposed by Cooper et al. (4Q) involves the synthesis of viral proteins which have an affinity for the small ribosomal subunit and the 5 end of viral mENA. The viral protein would repress the synthesis of cellular proteins by combining with the 4OS subunit, thereby blocking its link with host mENA. By also binding to the 5 end of viral ENA, the proteins would facilitate the attachment of viral ENA to the 40s subunit and increase the translation of viral ENA. Viral proteins have been found to co-sediment with the 4OS subunits of HeLa cells infected with poliovirus (49)> of Ehrlich ascites tumor cells infected with EMC virus (50) and of L-cells infected with mengovirus (5 ) poliovirus infected cells, the viral proteins co-sedimenting with ribosomes were identified as VPO, VP1 and VP3, all structural proteins (49) Both structural and non-structural proteins were found associated with ribosomes from EMC... 

Figure 6. Electrophoretic analysis of proteins associated with native 40 S ribosomal subunits of control and EMC-infected HeLa cells. Extracts were prepared from mock-infected and infected cells as described in Figure 2 0.5 ml of extract from control cells (b) or from cells infected 2 hr (a) or 3 hr (c) with EMC were layered on 15-50% sucrose gradients in 30 mM KC), 2mM Mg(OAc)p, 20 mM HHPES-KOH, pH 7 4i 1 mM dithiothreitol, and centrifuged 17 hr at 50f000 rpm. Fractions corresponding to the 40 S peak were combined, precipitated with 10% trichloroacetic acid, washed with acetone/ether (2/3) and ether, dissolved in sample buffer and fractionated on 12,5% polyacrylamide gels as previously described (14) The gels were stained with Goomassie Blue. Markers were run in parallel to assign M to the bands indicated by arrows.

If idENA has the same nucleotide sequence as virion MA, why-are mRNA strands not encapsidated into virions A trivial explanation would be that viral MA, once it has combined with ribosomes, may subsequently not exist free of them. However, free viral MA (EHA not bound to ribosomes) that is 5 "terminated with pUp has been found in cytoplasmic extracts of infected cells (21). Thus there exists the intriguing possibility that, in addition to the role in replication, VPg linked to the 5 end of plus-strand EHA is also a prerequisite for virion formation. For example, the newly synthesized Pg-MA may associate with one or several capsid protomers (42) prior to encapsidation. This protomer/VPg-EHA complex could then protect the YPg(Tyr)-0 pU linkage from cleavage by the host cell enzyme. Consequently, the concentration of protomers would determine how much of the newly synthesized VPg-RNA is "processed" to mENA, that is, early in infection most if not all progeny EHA may be cleaved at the 5 end. If VPg linked to EHA is a prerequisite for encapsidation, why do virions not contain minus strands A simple explanation could be provided by the observation that newly synthesized minus strands are rapidly incorporated into El and RP structures inside the membrane-bound replication complex minus strands are not found as free ENA in the cytoplasm (5) and are thus neither cleaved at the 5 end nor encapsidated. 

Other investigators have studied translation of poliovirus ENA in non-preincubated cell-free systems prepared from infected HeLA cells, in which ribosomes initiate and elongate on endogenous viral mRNAs which were in the cell at the time of extract preparation (32, 33) These extracts synthesize polypeptides which show excellent correlation with those made in infected cells (32, 34, Ehrenfeld, unpublished observations). Neither detectable premature termination nor cleavage deficiencies are apparent. 

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