Host protein synthesis

About 1.5-2 hours after infection the first viral structural proteins can be detected in cells efficiently infected by SFV (see Kaariainen and S6-derlund, 1978). By 3-4 hours the synthesis of host cell proteins is shut off. The mechanism of host protein shutoff is not yet known, but studies by van Steeg (1982) suggest that it is the viral capsid protein itself which is responsible for the selective inhibition of host protein synthesis. The capsid protein seems to reduce the activity of the initiation factors elF-4B and CAP binding protein below levels necessary for the formation of the 80 S initiation complex from host mRNA. Translation of the viral 26 S RNA is, however, unaffected. More studies are clearly needed to substantiate this interesting possibility. 

Kerekatte, V., Keiper, B.D., Badorff, C., Cai, A., Knowlton, K.U., and Rhoads, R.E. (1999). Cleavage of poly(A)-binding protein by coxsackievirus 2A protease in vitro and in vivo another mechanism for host protein synthesis shutoff J Virol 73, 709-717. 

The secreted recombinant proteins are easily detected and purified from serum-free medium of infected cells late in infection when host protein synthesis is diminished. 

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... 

HOST-PROTEIN SYNTHESIS INHIBITION reproduced by permission of the publishers. 

HELENTJiEIS, T. and EHEEKPELD, E. Inhibition of host protein synthesis by W-inactivated poliovirus. J. Virol. (1977)> 21, 259-267. 

FERNANDEZ-MDNOZ, R. and DARNELL, J.E. Structural difference between the 5 termini of viral and cellular mRNA in poliovirus-infected cells Possible basis for the inhibition of host protein synthesis. J. Virol. (I976), 1 8, 719-726. 

RUSS, U.L., OPPEEMM, H. and KOCH, G. Selective blockage of initiation of host protein synthesis in ERA virus-infected cells. Proc. Natl. Acad. Sci. U.S.A. (1975), li, 1258-1262. 

The shut-off of protein synthesis in virus-infected cells is a phenomenon that has attracted the attention of several investigators. In an attempt to provide an explanation at the molecular level for the mechanism of shut-off, studies were initially carried out with virus-infected cells. Since the results of this kind of studies are extensively discussed in the preceeding chapter of this book, we will concentrate on a particular aspect, namely the role of initiation factors on the virus-induced inhibition of host protein synthesis. 

Inhibition of protein synthesis in intact HeLa cells infected with EMC virus was observed at the same time that viral RNA synthesis reached its peak (Figure l). The shut-off of host protein synthesis, however, preceded this general inhibition of protein synthesis (see below). When cell extracts were prepared at different times after infection and tested for endogenous protein synthesis, a progressive loss of activity with time of infection was observed. A similar result was obtained in L-cells infected with EMC (Figure 2). 

Under properly defined conditions picomaviruses interrupt host RNA and protein synthesis (1 ) and subvert the cellular machinery to production of viral protein and ENA. By feeding radiolabeled amino acids to virus-infected cells after cessation of host-protein synthesis, viral protein can be selectively labeled. In a pioneering study, which introduced the now widely used SDS-polyacrylamide gel electrophoresis technique. Summers et al. (2) identified some 14 different virus-specified polypeptides in extracts of poliovirus infected HeLa cells. The net mass of these polypeptides exceeded two-fold or more the known coding capacity of the viral genome. 

That the gene order and cleavage pattern of a rhinovirus is similar to that of poliovirus and cardiovirus was first shown by McLean and Rueckert (8) who found that rhinoviruses 1A and 2, like poliovirus and EMC virus, redirect host-protein synthesis to that of the virus and produce a cleavage pattern homologous to that of EMC virus. These results were confirmed by Butterworth (9) who carried out a usefiil comparative study on all three of these viruses (Figure I). 

In so far as such a sweeping generalisation can be accepted without qualifications, it appears that the reduction in host protein synthesis that follows virus infection of mammalian cells is not due to the destruction of the host mENA but is caused primarily by a decrease in the rate of initiation on host mENA (any changes in the rate of elongation being relatively minor). Host cell mENA extracted... 

There are two views currently in vogue to account for the shut-off. One proposes that as the translation of viral ENA in vitro is more resistant to high monovalent cation concentrations (particularly to added Na ) than is the translation of cellular mRNA, it might be the influx of Na into the infected cell (as a consequence of the membrane changes known to occur after viral infection) that suppresses host protein synthesis whilst viral protein synthesis is relatively unaffected or may even be enhanced (55> 54) It is generally the case that viral mRNA translation in infected cells is much more resistant to the inhibitory effects of additional extracellular NaCI (which causes inhibition of initiation) than is the translation of host cell mRNA either in infected or in uninfected cells (55) ... 

Both of these ideas have their attractions, but they seem unable to account for all virus-host systems, particularly those in which the reduction in host cell protein synthesis occurs soon after infection. The increased permeability of the membrane to monovalent cations is generally thought to occur too late in the infection, and the out-competition by viral mRNA can only cause a significant reduction in host cell protein synthesis when viral mRNA has been produced in sufficient amounts to suppress initiation on host mRNA. As a total explanation, the competition hypothesis seems limited to those systems where the overall rate of protein synthesis remains relatively constant after infection, the rate of host protein synthesis declining progressively as the rate of viral protein synthesis increases. It cannot, for instance, explain the rapid reduction in host protein synthesis which occurs shortly after... 

For the identification of the EMC proteins in HeLa cells, Butterworth t used pulse-chase experiments done at a time of infection when host protein synthesis is blocked, and only the viral proteins are being formed. The stable EMC proteins were identified after 1 h chase with cold amino acids. In an attempt to obtain a purified EMC replicase containing an assumed EMC stable protein we carried out a similar experiment. A suspension of 1.5 X 1q9 infected BHK cells was maintained for 4 h in a methionine-deficient medium, containing actinomycin I) (10 ng/ml). 

Despite the restrictive (abortive) infection described above, macromolecular events that lead to inhibition of host protein synthesis, and ultimately cell death still occur. This would suggest that one might need these specific macromolecules only in catalytic amounts in order to disrupt the cells. Since viral double-stranded RNA is synthesized in such cells, it becomes an ideal candidate as an effector of inhibition of host protein synthesis (see Lucas-Lenard, this volume). 

To analyze the protein products of 29 genes a culture of B. subtilis, irradiated with UV-light to inhibit host protein synthesis, is infected with 29 in the presence of radioactive amino acids. At the end of the labeling period the cells are harvested, lysed with lysozyme and subjected to polyacrylamide slab gel electrophoresis in the presence of the detergent sodium dodecylsulfate (SDS). After electrophoresis the gel slabs are dried and autoradiographed to determine the positions of the labeled proteins. This system of electrophoresis resolves the protein chains on the basis of size and allows an estimate of the molecular weight of each protein band. 

Bello, L. J., and Ginsberg, H. S., 1967, Inhibition of host protein synthesis in type 5 adenovirus-infected cells, J. Virol. 1 843. 

McAllister, P. E., and Wagner, R. R., 1976, Differential inhibition of host protein synthesis in L cells infected with RNA temperature-sensitive mutants of vesicular stomatitis virus, J. Virol. 18 550. 

Many cytopathogenic viruses induce a decline in the rate of host mRNA translation upon infection. Since this shut-off of host translation is accompanied by extensive synthesis of viral protein, and since the decline in host protein synthesis is not concomitant with degradation or inactivation of host mRNA, it is clear that translational control mechanisms are involved in this phenomenon. A number of explanations have been invoked to explain shut-off by various viruses. Here, a few of these will be examined that are related directly to the material already reviewed above, but for a more extensive treatment, surveys by Koch et al. (1982) and elsewhere in this volume should be consulted. 

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