Svetlana V. Maslova

Prokaryotic-like Cis Elements in the Cap-Independent Internal Initiation of Translation on Picornavirus RNA

Initiation of translation on picornavirus RNAs is accomplished through internal binding of ribosomes to a complex cis-acting element. Here we show that efficient function of this element involves two appropriately spaced smaller elements: UUUCC and an AUG. This conclusion emerged from analysis of the genome structures of spontaneous revertants of mutant polioviruses with extended insertions between the UUUCC and AUG motifs. It was confirmed by the results obtained with specially designed constructs. A similarity to the prokaryotic translation initiation mechanism, which involves the Shine-Dalgarno sequence, is emphasized, but in the picornavirus system the position of the UUUCC must be strictly fixed relative to upstream cis-acting elements, and the AUG may not necessarily serve as an initiation codon.

The Genomes of attenuated and virulent poliovirus strains differ in their in vitro translation efficiencies

In mRNA-dependent extracts of Krebs-2 cells, RNAs from attenuated strains of poliovirus type 1 and type 3 exhibited diminished template activity as compared to RNAs from the respective virulent counterparts. This defect appeared to be due to the impaired initiation of viral polyprotein synthesis as evidenced by a relatively low level of accumulation of polypeptide 1a (which corresponds to an NH2-terminal region of the polyprotein) in samples programmed with RNAs from attenuated strains. In reticulocyte lysates, where poliovirus RNA is translated predominantly from abnormal (internal) sites [Dorner et al. (1984) J. Virol. 50, 507-514], this difference in the overall template activity of the attenuated and virulent poliovirus genomes was less pronounced, but the correct initiation (as judged by polypeptide 1a accumulation) was again more efficient on RNAs from virulent strains. It is suggested that template deficiency is a factor contributing to the attenuated phenotype of poliovirus strains studied. A possible involvement of nucleotide sequences located far upstream from the initiator codon in the control of translation of poliovirus genome is briefly discussed.

Coupled mutations in the 5'-untranslated region of the Sabin poliovirus strains during in vivo passages: structural and functional implications

All entero- and rhinovirus RNAs sequenced thus far possess A and U residues at positions corresponding to nucleotides 480 and 525, respectively, of poliovirus type 1. These two nucleotides have been proposed previously to form a base pair. The single exception to this rule appears to be the Sabin type 1 strain, which has a G480. Isolates of the Sabin 1 virus from healthy vaccinees were shown to have either a reversion to A480 or a second-site mutation U525----C, both restoring a potential for efficient base pairing. In vitro translation experiments demonstrated that poliovirus type 1 RNAs with either A480-U525 or G480-C525 are more efficient in promoting translation initiation as compared with the Sabin 1 RNA (G480-U525). The Sabin 2 strain has a U and an A at position 398 and 481, respectively, while its predecessor, strain P712, is shown to have C398 and G481. All the derivatives of the Sabin 2 isolated from vaccine-associated paralytic poliomyelitis cases shown reversion to G481, and most of them reverted also to C398. It is proposed that bases at positions 398 and 481 may be involved in a tertiary interaction. The in vitro template activity of the Sabin type 2 RNA (A481) is significantly lower than that of the isolate RNAs with G481, thus confirming the relation between attenuation and translation efficiency demonstrated previously for the type 1 and type 3 Sabin strains. The C----U change at position 398 exerted only a minor effect on the RNA template activity.

Geographical genotypes (geotypes) of poliovirus case isolates from the former Soviet Union: relatedness to other known poliovirus genotypes

A 150 nucleotide long region corresponding to adjoining segments of the genes encoding polypeptides VP1 and 2A of 84 poliovirus strains recently isolated from patients with paralytic poliomyelitis over the territory of the former Soviet Union (FSU) were characterized by sequencing and/or PCR amplification using specially designed primers. Eighteen isolates were found to be very closely related to one or another of the three Sabin vaccine strains. Three distinct classes of geographical genotypes (geotypes) were discerned among 42 wild-type (non-Sabin) strains of serotype 1. One such geotype (called A) was widely circulating in 1990-91 in the Caucasian (Azerbaijan and Georgia) as well as Asian (Kyrgyzstan and Turkmenistan) Republics; this geotype exhibited only weak relatedness to known strains isolated outside the FSU. On the other hand, a subset of strains belonging to another geotype (T) of serotype 1, which circulated in 1991 in Tajikistan, demonstrated very close relatedness to contemporaneous strains isolated in Pakistan, India and Jordan. Strains that were somewhat different, but belonging to the same T-geotype, were found also in Moldova and Georgia. Strikingly, the primary structure of the VP1/2A junction of certain T-geotype isolates differed from the corresponding region of Sabin 1 only in 13-15% of positions, thereby not reaching the upper limit accepted for a geotype. This observation raises, though does not prove, the possibility that at least the relevant segment of the T-geotype RNA originated from the vaccine strain. The third geotype of serotype 1 was represented by a single, perhaps imported, isolate. Four distinct subsets of a common geotype (C) were discerned among 24 wild-type isolates belonging to serotype 3. These strains exhibited a broad geographical distribution being found, in particular, in Armenia, Azerbaijan, Georgia, Turkmenistan and Tajikistan; on the other hand, the C-geotype strains exhibited only a relatively distant relatedness to a strain isolated outside of the FSU (in Oman).

Mutants of encephalomyocarditis virus requiring a hypertonic environment for optimal growth in HeLa cells

Abstract A novel class of mutants, hypertonic ( hyp ) mutants, of encephalomyocarditis (EMC) virus, which require media with a higher than usual NaCl concentration for optimal growth, is described. One of these mutants, hyp -101, when grown in HeLa cells, produced 10- to 100-fold less infectious progeny in an isotonic medium than in a medium with a doubled NaCI concentration. In contrast, the yield of wild-type virus was 10- to 100-fold higher in the former conditions compared to the latter. Under their respective restrictive conditions, both viruses synthesized a normal amount of the apparently full complement of virus-specific polypeptides and only a slightly (2- to 3-fold) diminished level of virus-specific RNA but failed to produce viral particles; this suggests that there was a defect in virion maturation. Reproduction of hyp -101 and wild-type virus in Krebs-II cells, however, was nearly equally inhibited upon an increase in NaCl concentration in the medium. The possibility is considered that maturation of picornavirus virions may involve an interaction of viral and cellular components that requires a more or less narrow range of ionic conditions; this range may be altered as a result of mutations in the viral genome.