Some problems of molecular biology of poliovirus infection relevant to pathogenesis, viral spread and evolution.

Molecular mechanisms of poliovirus reproduction in the human gut remain largely unexplored. Nevertheless, there are grounds to believe that the virus spreads from cell to cell, like that from person to person during natural circulation, and involves a relatively small proportion of the highly heterogeneous viral population generated by the previous host. This mechanism of random sampling is responsible for the majority of fixed mutations, and contributes to the maintenance of a certain level of viral fitness (virulence). In the long term, random sampling may lead to the decrease in fitness and even to extinction of some viral evolutionary branches, explaining cases of self-limiting poliovirus infection in immunodeficient patients. A low propensity of the Sabin viruses for natural circulation may also be a related phenomenon. The trend to decrease in fitness may be interrupted by the appearance of rare, fitter (more virulent) variants, which may be responsible for poliomyelitis outbreaks caused by wild type virus, and for the development of paralytic disease in chronic carriers of the Sabin vaccine. All these evolutionary events are largely stochastic and hence are unpredictable in principle.

Early alteration of nucleocytoplasmic traffic induced by some RNA viruses.

A HeLa cell line expressing the green fluorescent protein fused to the SV40 T-antigen nuclear localization signal (EGFP-NLS) was established. Fluorescence in these cells was confined to the nuclei. After poliovirus infection, cytoplasmic fluorescence in a proportion of cells could be detected by 1 h postinfection (p.i.) and in virtually all of the fluorescent cells by 2 h p.i. The relocation could be prevented by cycloheximide but not by inhibition of poliovirus replication by guanidine. HCl. Nuclear exit of a protein composed of three copies of GFP fused to the NLS also occurred upon poliovirus infection. A similar redistribution of EGFP-NLS took place upon infection with coxsakievirus B3 and, to a lesser extent, with vesicular stomatitis virus. The EGFP-NLS efflux was not due to the loss of NLS. Thus, some positive-strand and negative-strand RNA viruses trigger a rapid nonspecific relocation of nuclear proteins.

Competing death programs in poliovirus-infected cells: commitment switch in the middle of the infectious cycle.

Productive poliovirus infection of HeLa cells leads to the canonical cytopathic effect (CPE), whereas certain types of abortive infection result in apoptosis. To define the time course of commitment to the different types of poliovirus-induced death, inhibitors of viral replication (guanidine HCl) or translation (cycloheximide) were added at different times postinfection (p.i.). Early in the infection (during the first approximately 2 h p.i.), predominantly proapoptotic viral function was expressed, rendering the cells committed to apoptosis, which developed several hours after viral expression was arrested. In the middle of infection, concomitantly with the onset of fast generation of viral progeny, the implementation of the viral apoptotic program was abruptly interrupted. In particular, activation of an Asp-Glu-Val-Asp (DEVD)-specific caspase(s) occurring in the apoptosis-committed cells was prevented by the ongoing productive infection. Simultaneously, the cells retaining normal or nearly normal morphology became committed to CPE, which eventually developed regardless of whether or not further viral expression was allowed to proceed. The implementation of the poliovirus-induced apoptotic program was suppressed in HeLa cells overexpressing the Bcl-2 protein, indicating that the fate of poliovirus-infected cells depends on the balance of host and viral pro- and antiapoptotic factors.

Prothymosin alpha fragmentation in apoptosis.

We observed fragmentation of an essential proliferation-related human nuclear protein prothymosin alpha in the course of apoptosis induced by various stimuli. Prothymosin alpha cleavage occurred at the DDVD(99) motif. In vitro, prothymosin alpha could be cleaved at D(99) by caspase-3 and -7. Caspase hydrolysis disrupted the nuclear localization signal of prothymosin alpha and abrogated the ability of the truncated protein to accumulate inside the nucleus. Prothymosin alpha fragmentation may therefore be proposed to disable intranuclear proliferation-related function of prothymosin alpha in two ways: by cleaving off a short peptide containing important determinants, and by preventing active nuclear uptake of the truncated protein.

Two types of death of poliovirus-infected cells: caspase involvement in the apoptosis but not cytopathic effect.

The death of poliovirus-infected cells may occur in two forms: canonical cytopathic effect (CPE) (on productive infections) or apoptosis (when the viral reproduction is hindered by certain drugs or some other restrictive conditions). Morphological manifestations of the CPE and apoptosis, being distinct, share some traits (e.g., chromatin condensation and nuclear deformation). It was shown here that a permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (zVAD.fmk), prevented the development of the poliovirus-induced apoptosis on abortive infection. The apoptotic pathway could be dissected by an inhibitor of chymotrypsin-like serine proteases, N-tosyl-l-phenylalanine chloromethyl ketone (TPCK), which prevented the cleavage of DNA to oligonucleosome-sized pieces and nuclear fragmentation but did not suppress cellular shrinkage, cytoplasmic blebbing, and partial chromatin condensation. These results demonstrate that caspase activation is involved in the execution phase of the viral apoptosis and suggest that a nuclear subset of the apoptotic program is under a separate control, involving a TPCK-sensitive event. Neither zVAD.fmk nor TPCK, at the concentrations affecting the apoptotic response, exerted appreciable influence on the virus growth or cellular pathological changes on productive infection, indicating that the pathways leading to the poliovirus-evoked CPE and apoptosis are different.

[The critical significance of a conserved single-stranded interdomain linker in the 5'-untranslated region of poliovirus RNA]. / Kriticheskoe znachenie konservativnogo odnotsepochechnogo mezhdomennogo linkera v 5'-netransliruemoi oblasti poliovirusnoi RNK.

Translation of polioviral RNA is initiated by interaction of a small ribosomal subunit with internal segments of the 5'-untranslated region (5'UTR). Several mutations were constructed within 5'UTR segment 425-449. All of them (including a single C444-->U replacement) inhibited in vitro translation, which decreased about 10-fold. Two mutant constructs, pPV12-05 (C444-->U) and pPV12K (containing also an AAUU insert between positions 441 and 442) produced plaques on monolayers of susceptible cells. All the viruses isolated from these plaques exhibited a reversion at position 444; the template activities of the revertant RNAs were restored completely or significantly. The results show the importance of the relevant 5'UTR segment for the initiation of polioviral RNA translation.

Starting window, a distinct element in the cap-independent internal initiation of translation on picornaviral RNA.

Initiation of translation on picornaviral RNA templates occurs via cap-independent ribosome binding to a cis-acting element, internal ribosome entry site (IRES). Mapping of the starting point of translation relative to the IRES was attempted using Theiler's murine encephalomyelitis virus (TMEV) RNA as a model. The possibility that the starting point is determined by the conserved oligopyrimidine upstream of the initiator codon was studied. In contrast to poliovirus, neither the conserved oligopyrimidine nor an AUG at a fixed distance downstream of this oligopyrimidine are required for efficient translation of the TMEV RNA in Krebs-2 extracts or reticulocyte lysates or for viral infectivity; mutants lacking the oligopyrimidine/AUG tandem were stable upon passage in BHK-21 cells. A short template segment, the starting window, was defined, wherefrom ribosomes begin translation or downstream scanning depending, respectively, on the presence or absence of a good-context AUG within this window. Using a collection of the engineered TMEV mutant RNAs, the starting window was mapped to 16-17 nt downstream of the IRES and was found to be approximately a dozen nt long. The efficiency of translation initiation from an AUG linearly increased upon the 5'-->3' displacement of the initiator codon within the window. The competence of the starting window did not appear to depend markedly on its primary structure; however, it was completely inactivated ("closed") with concomitant dramatic inhibition of total protein synthesis upon conversion of the corresponding RNA segment into a double-stranded form.

Functional and genetic plasticities of the poliovirus genome: quasi-infectious RNAs modified in the 5'-untranslated region yield a variety of pseudorevertants.

Poliovirus RNA species with nucleotides 564 to 571 deleted or with a secondary structure domain (positions 564 to 629) replaced by a shorter irregular oligonucleotide have been engineered previously; these RNAs have been considered quasi-infectious (yielding a single late revertant plaque) and dead, respectively (E. Pilipenko, A. Gmyl, Y. Svitkin, S. Maslova, A. Sinyakov, and V. Agol, Cell 68:119-131, 1992). By using large amounts of these RNAs for transfections, revertant clones with a great variety of genetic changes (point mutations, insertions of foreign sequences, short or extended deletions) were isolated. The pattern of these changes supported the notion that an appropriately spaced oligopyrimidine-AUG tandem is important for efficient poliovirus RNA translation. Structural features within and around this tandem modulated the initiation efficiency. The functional and genetic plasticities of the poliovirus genome are briefly discussed.