Host Cell RNA Synthesis

Vaccinia virus infection inhibits the transport of newly synthesized ribosomal RNA (rRNA) to the cytoplasm (Becker and Joklik, 1964 Salzman et al., 1964). This inhibition was studied in HeLa cells and it was detected 3-4 hr after infection. In another study where L cells were used, inhibition of rRNA synthesis occurred more rapidly (Jefferts and Holowczak, 1971). These authors showed that the cleavage of the 45 S ribosomal precursor RNA to 32 S and 18 S was inhibited by 2 hr after infection and the transport of rRNA to the cytoplasm was curtailed by 3 hr after infection. The methylation of the 45 S RNA was reduced in infected cells but was not completely abolished. Furthermore, inhibition of ribosomal protein synthesis occurred before a decline in the synthesis of rRNA. The product responsible for these changes has not been determined however, it may 

---

These data lead to the conclusion that inhibition of host cell RNA synthesis occurs concomitantly with the increase in virus-specific RNA synthesis. Further evidence for this conclusion was obtained from the following experiments (1) in interferon-treated CEF cells, SF virus-specific RNAs are not detected, and no inhibition of host cell RNA synthesis is observed (Taylor, 1965), and (2) ultraviolet-irradiated WEE virus loses the ability to inhibit host cellular RNA synthesis (M. Wagatsuma and B. Simizu, unpublished data). These data suggest that viral replication is necessary for the inhibition of host RNA synthesis, and that components of infecting virions are insufficient for this effect. 

These data suggest that the alphavirus-induced inhibition of host cell RNA synthesis occurs at the transcriptional level. To test this possibility, the cell-free transcription system (Manley et al., 1980) which has been developed for studying an inhibitory factor present in poliovirus-infected cells (Baron and Baltimore, 1982) would be useful. With such a system, it would be possible to determine whether inhibitory factor(s) are induced in alphavirus-infected cells or whether the inhibition results from a competition for the available pool of RNA precursors. The genetic approach is not possible at this time since no conditional lethal alphavirus mutant has been described which affects the regulation of host cell RNA synthesis. 

Sarver and Stollar (1977) compared host cell RNA synthesis in CPE-sensitive and CPE-resistant mosquito cells infected with SIN virus. Early in infection, prior to observed CPE, there was a marked depression of RNA synthesis in the CPE-susceptible cells, while CPE-resistant cultures showed only a slight transient decrease in host cell RNA synthesis. WEE and SF virus infection of CPE-susceptible cells also caused an extensive inhibition of host protein and DNA synthesis (Logan, 1979 Simizu and Maeda, 1981 Tooker and Kennedy, 1981). 

Preincubation of HeLa cells with actinomycin (0.4 to 2.0 (xg/ml) for 150 min at 37° C before exposure to polycations increases the infectivity of viral RNA up to 3-fold (Koch et al., 1967) (Table2). Inhibition of host cell RNA synthesis results in an accumulation of free ribosomes inside the cells which provide the invading viral RNA with a greater chance of finding free ribosomes and thus of acting as mRNA. Incubation of cells with actinomycin has the opposite effect on the infectivity of RF-RNA. The polycation-induced competence of actinomycin-treated cells for RF-RNA is only 10% that of normal cells. This agrees with the h q)othesis that host cell functions are required to melt double-stranded RNA or to use it as a template for the synthesis of new viral RNA. 

One subunit (Mr 65,000) is the product of the replicase gene encoded by the viral RNA and has the active site for replication. The other three subunits are host proteins normally involved in host-cell protein synthesis the E. coli elongation factors Tu (Mt 30,000) and Ts (Mr 45,000)... 

Poliovirus is an RNA-RNA virus that redirects the host-cell protein synthesis machinery toward translation of its own RNA genome. The poliovirus polypeptide is cleaved into active subunits by the proteolytic action of virally encoded proteases. Poliovirus RNA replication utilizes a novel protein-uridine primer to initiate RNA synthesis using its own RNA-dependent RNA polymerase. 

The other view, which is not necessarily in contradiction,stems from the observation that in a competitive situation the translation of EMC RNA outcompetes the translation of cellular mRNA in vitro under virtually all experimental conditions (56). The only exception is that the addition of extra eIF-4B tends to diminish the competitive advantage of the viral RNA (18). (According to the Basel group there is a requirement for eIF-4B for initiation on EMC RNA (17) Ihe conclusion which is the most consistent with all these observations is therefore that the optimiam ratio of eIF-4B to other factors may be lower for EMC ENA than for cellular mRNAs), The shut-off of host cell protein synthesis in this view is due to the competition by the viral RNA at the initiation step. The competitive effect might be enhanced if viral infection resulted in some inactivation or modification of eIP-4B, and some compelling evidence for this has recently been found (19)> althou the nature of the change in eIP-4B remains unknown. 

CELMA, M.L. and EHEIENEELD, E. Effect of poliovirus double-stranded RNA on viral and host cell protein synthesis. Proc. Nat. Acad. Sci. U.S.A. (1974), H, 2440-2444. 

Baglioni, C., Simili, M., and Shafritz, D. A., 1978, Initiation activity of EMC virus RNA, binding to eIF-4B and shut-off of host cell protein synthesis. Nature 275 240. 

Infection of cultured cells with many lytic viruses results in a marked decrease in the rate of cellular protein synthesis. Usually, this decrease is accompanied by increasing rates of viral protein synthesis, marked cytopathic effects, and ultimately cell death. In most cases, it is not known whether the shut-off of host cell protein synthesis results from an active process induced by the virus evolved for that (or some other) purpose, or whether it is merely a passive result of another viral function, such as production of large quantities of viral mRNA which compete effectively with their cellular counterparts. In the case of poliovirus, however, three types of studies suggested that the former, active type of mechanism was at work. Kinetic analysis of the rate of protein synthesis in cells synchronously infected with high multiplicities of virus showed that cellular protein synthesis could be virtually completely inhibited prior to the synthesis of significant quantities of viral RNA and protein (Summers et ai, 1965). In addition, infection in the presence of 1-3 mM guanidine, which prevents detectable replication of viral RNA, nevertheless results in viral inhibition of host cell protein synthesis (Holland, 1%4 ... 

One approach was to prepare extracts of poliovirus-infected HeLa cells and to assay for an inhibitor of initiation by addition to a reticulocyte lysate translation reaction. The reticulocyte lysate had been well-characterized as a system capable of efficient initiation in vitro. A potent inhibitor of initiation was indeed found in the cytoplasm of poliovirus-infected cells, which was not present in similar preparations from uninfected HeLa cells (Hunt and Ehrenfeld, 1971), and the inhibitor was subsequently identified as double-stranded poliovirus RNA (Ehrenfeld and Hunt, 1971). In order to qualify as a specific inhibitor of host cell protein synthesis, however, it was necessary to demonstrate that viral protein synthesis was immune to the inhibitory effects of the putative shut-off factor. When double-stranded RNA was put to this test, no specificity between cellular and viral protein synthesis could be demonstrated (Celma and Ehrenfeld, 1974) rather, all protein synthesis was inhibited equally. In addition, concentrations of viral double-stranded RNA required to... 

An alternative type of explanation for the specific discrimination against host cell protein synthesis in poliovirus-infected cells stemmed from the observation that initiation of protein synthesis could be selectively inhibited in HeLa cells and in poliovirus-infected HeLa cells by increasing the osmolarity of the growth medium (Sa-borio et al., 1974). The inhibition was independent of the solute used to increase the osmolarity. However, virus-directed protein synthesis was observed to be relatively more resistant to inhibition by hypertonic medium than was cellular protein synthesis, a fact which was interpreted as indicating that initiation of viral RNA translation was intrinsically more efficient than that of cellular mRNA (Nuss et al., 1975). These workers, therefore, proposed that the virus-specific or virus-induced factor involved in suppression of host protein synthesis could function by indiscriminantly lowering the rate of peptide chain initiation. Under such conditions, translation of viral mRNA, when it was synthesized, could occur due to its inherently strong affinity... 

Week and Wagner (1979a) analyzed RNA metabolism in MPC-11 cells infected with various temperature-sensitive (ts) mutants and 5 -defective-interfering particles which cannot synthesize mRNA. A group I mutant, /sG114, restricted in transcriptional activities (Hunt et al., 1976), failed to shut-off host cell RNA metabolism in MPC-11 cells incubated at the restrictive temperature of 39°C for 4 hr. At the permissive temperature (3rC), all mutants (including /5G114) were as effective as the wild-type virus in the shut-off of RNA synthesis. Because tjG 114(1) did not inhibit cell RNA metabolism at the non-permissive temperature, it was used to test for a virion structural... 

REOVIRUS EFFECTS ON HOST CELL RNA AND PROTEIN SYNTHESIS... 

All of the aforementioned studies examined total rather than host-specific protein synthesis. Joklik and his colleagues studied host cell-specific protein synthesis in mouse L cells and found that host cell protein synthesis continues relatively undiminished at early times after infection but gradually falls such that host-specific protein synthesis is inhibited 3-6 hr prior to the inhibition of total protein synthesis in suspension cultures (Zweerink and Joklik, 1970). This inhibition is dependent upon the multiplicity of infection and on the temperature of incubation. It correlates closely with the onset of viral messenger RNA (mRNA) synthesis, suggesting that host protein synthesis might be inhibited by direct competition between host and viral RNA for some component of the cell translational machinery. Other... 

Few studies have examined the effects of reovirus type 1 and 2 on host cell macromolecular synthesis. Loh and Soergel (1965) observed that type 2 reovirus inhibits protein and DNA synthesis in human amnion cells but has little effect on RNA synthesis in these cells. Sharpe and Fields (1982) demonstrated that type 2 reovirus produces a decrease in the rate of total RNA synthesis in mouse L cell, whereas, type 3 causes little or no alteration in the rate of RNA... 

The suggestion has been made (Doi and Spiegelman, 1963) that virus RNA, when it has penetrated into the host cell, programs synthesis of a special RNA-polymerase for use in synthesis of virus RNA just as in the case of RNA-dependent complementary RNA synthesis. This hypothesis has so far been confirmed for TMV only by the work of Karasek and Schramm (1962), who isolated a special polymerase, catalyzing incorporation of C -adenine into... 

It has been shown, for example, that in cells in which the proliferation of virus RNA is beginning, synthesis of host cell RNA or DNA templates in the nucleus comes to an end (Bukrinskaya et al., 1964 Schafer, 1963). Inhibition of RNA synthesis in the nuclei after virus infection was also demonstrated by earlier studies (Luria, 1958 Franklin, Wecker, and Henry, 1959). In connection with this, S5mthesis of cell proteins also is inhibited. This inhibi-... 

By cell-free enzyme synthesis, the host shut-off was analysed and two mechanisms distinguished Shut-off of host translation and inhibition of host messenger RNA synthesis. 

---