Picornavirus synthesis

Diana GD, Cutcliffe D, Oglesby RC, Otto MJ, Mallamo JP, Akullian V, McKinlay MA. Synthesis and structure-activity studies of some disubsti-tuted phenylisoxazoles against human picornavirus. J Med Chem 1989 32 450-455. 

Carrasco L, Smith AE (1976) Sodium ions and the shut-off of host ceU protein synthesis by picornaviruses. Nature 264 807-809... 

In considering the function of poly(A), its presence within picornavirus ENAs is of particular interest, firstly since its mode of synthesis differs from that of cellular mENAs, and secondly since, unlike in cellular mENAs, a functional poly(A) requirement has now been established, althougji the precise details are not yet known. 

In this review I have outlined several theories that have been proposed to explain the mechanism by which picornaviruses inhibit cellular protein synthesis. Some theories seem less likely than others. Inhibition by ds ENA, for example, is no longer thought to be a likely possibility. In cell-free extracts ds ENA inhibits both cellular and viral mRNA translation (61). The inhibitor of cellular protein synthesis would be expected to be selective in its inhibitory activity. It is also apparent that picornavirus infection does not result in the degradation or alteration of cellular mENA (9> 27, 29 51). So,.too, experiments demonstrating that protein synthesis inhibition takes place in the absence of significant viral ENA synthesis (I4) tend to weaken the argument that protein synthesis inhibition results from direct competition of viral mENA with cellular ENA for initiation factor eIE-4D (47) As mentioned earlier, superinfection with poliovirus of cells infected with VSV prevents VSY mENA translation (J2, 56). In lysates from uninfected HeLa cells, however, 7SY mENA translation is favored over poliovirus mENA translation when both mENA species are present in equimolar saturating concentrations (55) If competition were a major cause of cellular protein synthesis inhibition, one would have expected poliovirus mENA to out-compete VSV mENA in cell-free translation, not the contrary. 

OBERG, B.F. and SHATKIN, A.J. Initiation of picornavirus protein synthesis in ascites cell extracts. Proc. Natl. Acad. Sci. U.S.A. (1972), 62, 5589-5593. 

Viral ERA synthesis in the picornavirus group is associated with membrane structirres (25, 26) and can be isolated in the form of a replication complex by centrifugation of the cytoplasmic extracts at 20,000 g (27). The phosphorylase activity was shown to be associated with the replication complexes (Table 7)> result expected, as dsRRA structures were in the P-20 fraction. 

The process of replication of the viral RNA is probably the most extensively studied aspect of the molecular biology of picornaviruses. Beginning (just to set a date) in the early sixties with the reports by Sanders (l) and Darnell (2) on the time-course of RNA synthesis, experimental data have accumulated at such a pace that an attempt to provide the reader with a more or less complete list of references would prove not only impossible (because of the mavoidable omission of important contributions), but would also be out of the scope of this chapter. The purpose of this presentation being to review our current understanding of the mechanism of RNA synthesis in picornavirus-infected cells, I shall only refer to those few papers strictly pertinent to the problems inder consideration. 

The genome of picornaviruses is a single-stranded RNA molecule about 7000 nucleotides long (see chapter 2). The synthesis of such a macromolecule proceeds by sequential condensation of nucleoside-5 -triphosphates in the 5 "to 5 direction (Figure 1). 

Infection with picornaviruses results in a strong (though selective) inhibition of the cellular ENA synthesis (5, 4> 85> 84), and the appearance of a new, virus-induced ENA synthesizing activity. The latter was soon shown to be insensitive to the action of Actinomycin D (5), an antibiotic which prevent DNA-dependent ENA synthesis by intercalating in the dG-dC sequences of the template MA (6, 7) These observations suggested the possibility of treating infected cultures with the antibiotic in order to suppress host-cell activity and measxiring the incorporation of radioactive precursors into viral ENA. Since the precursors added to the medium do not equilibrate instantaneously with the intracellular pool of nucleotides, a correction for this fact must be introduced, at least for the very early times. 

ENA synthesis in picornavirus-infected cells proceeds according to a well defined temporal pattern (Figure 2) Beginning about one hour after infection, the synthesis of the viral ENA proceeds at an exponential rate for about two hours. Then, when 20-25% of the total viral ENA has been produced, there is an abrupt change and for 6O-9O minutes viral ENA accumulates at a linear rate. At the end of this second phase (4-5 hours after infection, see below), the bulk of the viral ENA has been synthesized and thereafter the rate of synthesis declines. 

Figure 2. Time-course of RNA synthesis in cells infected with a temperature-sensitive mutant of a picornavirus at permissive ( —— ) and non-permissive temperature (o-----------------o).

It was soon realized, however, that some kind of regulatory mechanism must intervene, because the synthesis of virus-induced RNAs was shown to be quite an asymmetrical process The bulk of the RNA found in the cytoplasm of picornavirus-infected cells is virion-like (i.e. "plus" strand), and only a very minor fraction of the newly synthesized RNA would hybridize to the RNA extracted from virions. Several explanations of this phenomenon have been offered ... 

All viral structures involved in picornavirus RNA synthesis are tightly associated with the smooth cytoplasmic membranes (50 Synthesis of viral RNA occurs exclusively in the replication complex (RC), a complex structure including the RNA template and a virus-coded RNA polymerase (52). 

The synthesis of picornavirus RNA strictly depends on the concomitant synthesis of viral proteins so much so that inhibition of the latter results in an almost immediate decline of the production of viral RNA. 

The search for substances which could distinguish between cell- and virus-directed synthesis led eventually to the identification of a group of compounds able to inhibit specifically the replication of the RNA of some picornaviruses. Guanidine and (to a certain extent) hydroxy-benzyl-benzimidazol (HBB) are the best studied among them (dj-d f 101). 

The replication complex can be isolated with the particulate fraction of the cytoplasm of picornavirus-infected cells (71) Under proper conditions the ECs are able to continue vitro the synthesis of ENA, providing a unique tool to study the mechanism of ENA synthesis with all the advantages (and all the limitations, too) of an m vitro cell-free system. 

Because restriction begins soon after infection, both viral RNA and protein synthesis are markedly reduced. The resulting low level of viral macromolecular synthesis delays the inhibition of host functions, impairs virion formation, and prevents physical particle accumulation. Although it is not known which of the three major picornavirus synthetic activities - translation, transcription, or protein processing - is the primary site of restriction, the hypothesis is that at least one of these is impaired. 

Picornavirus Inhibition of Host Cell Protein Synthesis... 

The suggestion that viral mRNA is an inherently efficient mRNA and can effectively compete with cellular mRNAs for some limiting component of the protein-synthesizing machinery has acquired experimental support for picornaviruses other than polioviruses. A direct competition model for shut-off of host cell translation has been proposed, specifically, to describe the shut-off induced by the car-dioviruses, EMC, and mengo (see Section 7, below). However, these models do not apply to poliovirus-induced inhibition because, as noted above, cessation of protein synthesis in poliovirus-infected cells occurs before detectable viral RNA is synthesized and shut-off does occur after infection in the presence of guanidine or with a mutant virus temperature-sensitive for RNA synthesis at restrictive temperature. 

The above sections have described several different mechanisms which have been proposed and explored during the last decade to explain the selective inhibition of host cell protein synthesis in poliovirus-infected cells. Admittedly, this author s bias has presented each mechanism as a straw man, requiring the reader to await what is perceived at this time to be the correct explanation for this aspect of the regulation of protein synthesis in poliovirus-infected cells. The favored model will be discussed in this and subsequent sections. It is important to state, however, that there is no convincing evidence that other picornaviruses are necessarily similar to poliovirus in the mechanism(s) utilized for host protein synthesis inhibition and that the mechanisms described above, as well as others, cannot all be dismissed in every case of picorna virus-induced protein synthesis inhibition. Thus, the data for other picornaviruses will be reviewed separately. 

Virtually no studies of host cell protein synthesis inhibition have been conducted for other picornaviruses such as rhino virus, foot-and-mouth disease virus, Theiler s virus, coxsackie, echo, or hepatitis A... 

Many lytic viruses, other than picornaviruses, markedly inhibit host cell protein synthesis during the course of the infectious cycle. None have been investigated to the same extent as poliovirus with respect to the mechanism of this function. However, there are a few preliminary studies which might be interpreted as indications of similar effects on initiation factor activity. 

Although the biochemistry of translational initiation is steadily being unraveled, and the block imposed by poliovirus in HeLa cells is simultaneously becoming clarified, other picornaviruses may interact with other cell types in different ways. Infection of at least some cells with the cardioviruses, EMC or mengovirus, does not appear to produce the same initiation factor inactivation as does poliovirus in HeLa cells, and the regulation of protein synthesis in such cells is not well understood. The majority of picornaviruses, in natural host tissue or in cultured cells, have not been studied at all. Thus, despite a long-standing interest in the phenomenon of virus-induced interference with host cell protein synthesis, many questions remain to be answered. 

---