3D gene of foot-and-mouth disease virus. Conservation by convergence of average sequences.

The nucleotide sequence of the 3D (polymerase) gene of eight epidemiologically related isolates of foot-and-mouth disease virus of serotype C1 is reported. The genetic heterogeneity of 3D RNA is compared with that of the VP1-coding RNA of the same viruses. Regression lines of substitutions per nucleotide that distinguish any pair of viruses as a function of the time interval between the corresponding isolations show: (1) the slope (substitutions/nucleotide per month) is 2.1 times larger for the VP1 RNA than for the 3D RNA region; (2) the intercept with the ordinate (substitutions/nucleotide) for VP1 RNA is indistinguishable from that for 3D RNA. Thus, the average heterogeneity of the VP1-coding region is very similar to that of the 3D-coding region only among co-circulating viruses. Nine mutations and points of heterogeneity occurred within nucleotide residues 883 to 1026, which encode an amino acid segment, extremely conserved among many different RNA viruses. The results suggest that, rather than due to inherently lower mutability, the conservation of 3D genes is caused by a limitation in the fixation of substitutions in viable genomes.

Internal ribosome entry site biology and its use in expression vectors.

Internal ribosome entry sites (IRESs) are cis-acting elements that recruit the small ribosomal subunits to an internal initiator codon in the mRNA with the help of cellular trans-acting factors. The recent discovery of the IRES recognition site of the eIF4G initiation factor is beginning to shed some light into how IRES elements are recognized by the translational machinery. Additionally, the progress made in the understanding of the parameters that influence start codon selection will be instrumental in establishing the rational design of bicistronic expression vectors.

Parameters influencing translational efficiency in aphthovirus IRES-based bicistronic expression vectors.

Initiation of translation in picornavirus RNAs occurs internally, mediated by an internal ribosome entry site (IRES) element. This property has been exploited to coexpress proteins from a single bicistronic transcription unit in eukaryotic cells. The region that separates the IRES element from the authentic initiator codon of the second gene plays an important role in the translation efficiency of this cistron. In the present report, we have analyzed the effect of sequence modifications in this region on the translation efficiency directed by the foot-and-mouth disease (FMDV) IRES in bicistronic expression vectors. Insertion of various sequences, which contained additional start codons and/or the capacity to form hairpins immediately downstream of the 3' border of the IRES, strongly reduced the translation efficiency of the second gene in bicistronic RNAs. Interestingly, an increase of distance per se did not have a deleterious effect on translation efficiency. The bicistronic vector studied here tolerated 95 nucleotides between the 3' border of the IRES and the authentic start codon, provided that out-of-frame AUG codons or hairpins were not present in this RNA segment. These results indicate that FMDV-derived bicistronic constructs are extremely well suited for use in eukaryotic expression vectors.

Sequence of the viral replicase gene from foot-and-mouth disease virus C1-Santa Pau (C-S8).

The nucleotide sequence of the region including the viral replicase gene, the carboxy terminus of protein P18, and the 3'-extracistronic region of foot-and-mouth disease virus (FMDV) type C1-Santa Pau (C-S8) has been determined from previously cloned cDNA fragments [Villanueva et al., Gene 23 (1983) 185-194]. The comparison with the corresponding gene segments of FMDV of serotypes A or O shows base substitutions in 7.2-8.6% of residues in the replicase gene with no insertions or deletions. This is about fourfold lower variation than found for the region encoding capsid protein VP1 of the corresponding viruses. Intermediate variability (substitution at 16.1-23.6% positions) exists in the 3'-extracistronic region, including point mutations, insertions and deletions. The predicted amino acid sequence of the replicase gene indicates that 75.5-82.6% of mutations are silent and that 93.4% of amino acids are conserved in the four FMDV replicases. The frequency of certain types of silent mutations and of rare codon usage is significantly lower for the replicase gene than for the protein VP1 coding region.

The quasispecies (extremely heterogeneous) nature of viral RNA genome populations: biological relevance--a review.

We review evidence that cloned (or uncloned) populations of most RNA viruses do not consist of a single genome species of defined sequence, but rather of heterogeneous mixtures of related genomes (quasispecies). Due to very high mutation rates, genomes of a quasispecies virus population share a consensus sequence but differ from each other and from the consensus sequence by one, several, or many mutations. Viral genome analyses by sequencing, fingerprinting, cDNA cloning etc. indicate that most viral RNA populations (quasispecies) contain all possible single and double genomic site mutations and varying proportions of triple, quadruple, etc. site mutations. This quasispecies structure of RNA virus populations has many important theoretical and practical implications because mutations at only one or a few sites may alter the phenotype of an RNA virus.

Heterotypic inhibition of foot-and-mouth disease virus infection by combinations of RNA transcripts corresponding to the 5' and 3' regions.

Strategies to inhibit RNA virus multiplication based on the use of interfering nucleic acids have to consider the high genetic polymorphism exhibited by this group of viruses. Here, we report high levels of heterotypic inhibition of foot-and-mouth disease virus (FMDV) infective particle formation in cotransfection experiments of susceptible cell lines with infections viral RNA and combinations of viral transcripts. The interfering molecules used include the following regions on type C FMDV RNA: (i) sequences from the 5' region, spanning the proximal part of the internal ribosome entry site element and the two functional initiator AUGs; and (ii) the 3' terminal region including the 3' end of 3D gene and the complete 3' non-coding region. Combination of 5' antisense RNA molecules with either sense or antisense RNA molecules from the 3' region resulted in inhibition of up to 90% of the infectivity of homologous type C FMDV RNA. The inhibition was dose-dependent and specific, as no reduction was observed in the plaque-forming units recovered from RNA of swine vesicular disease virus, a related picornavirus. Interestingly, high levels-of intertypic inhibition, about 60% or higher, were observed when viral RNAs of serotypes O and A were analysed. These levels of inhibition are consistent with the levels of nucleotide homology exhibited by the viruses analysed in the target sequences. Inhibition of virus yield was also observed in FMDV-infected cells transiently expressing the interfering RNAs. Thus, transcripts of the FMDV RNA corresponding to the 5' and 3' regions specifically inhibit FMDV particle formation in a serotype-independent manner.

Effect of expression of the aphthovirus protease 3C on viral infection and gene expression.

Cells transformed with specific regions of the foot-and-mouth disease virus (FMDV) genome have been constructed and analyzed with respect to viability and susceptibility to FMDV infection. Constitutive expression of an active protease 3C under the control of the tk promoter has been documented by the ability of transformed cells to catalyze the processing of a P1 capsid precursor. High-level, transient expression but not low-level, constitutive expression, of 3C caused a 10-fold reduction in the yield of FMDV and was detrimental to the expression of the cotransfected reporter luciferase gene. No such effect was observed in assays involving cells transfected with a deleted, inactive form of 3C. The negative effect of 3C was not observed when the same reporter gene was integrated and expressed in a constitutive fashion nor when its translation was directed by the internal ribosome entry site element of FMDV in transient expression assays. The results show that cells with a low level of expression of the aphthoviral 3C can be stably maintained and can provide a useful tool to study polyprotein processing.

Amber mutation affecting the length of Escherichia coli cells.

An amber mutation in a newly found gene (wee) of Escherichia coli has been isolated from strain OV-2, which harbors a temperature-sensitive suppressor. At 42 degrees C cells of the mutant, OV-25, increased in mass and deoxyribonucleic acid content and divided at normal rates, compared with the wild type under the same growth conditions. Total cell length increased under the restrictive conditions, although at a slightly lower rate. Values of mean cell length and cell volume, contrary to what would be expected from the increment in the rate of increase in particles, mass, and deoxyribonucleic acid, became at 42 degrees C smaller than those found in the wild type. A parallel increase in protein content per length and cell density and a loss of viability were found to occur after four generations at the restrictive temperature. The behavior of strain OV-25 in the absence of the wee gene product could be interpreted in terms of either a faulty regulation of the elongation processes or their abnormal coordination with the cell cycle. The genetic location of the wee gene has been found to be at 83.5 min on the E. coli genetic map.

Long-range RNA interactions between structural domains of the aphthovirus internal ribosome entry site (IRES).

Internal initiation of translation is promoted by internal ribosome entry site (IRES) cis-acting elements. Using transcripts that correspond to the structural domains of the foot-and-mouth disease virus (FMDV) IRES, we have identified RNA-RNA interactions between separated domains (1-2, 3, 4-5, or HH) of the IRES structure. All the assayed domains were able to interact with the full-length IRES as well as with domain 3, although to a different extent, with the most efficient interactions being those occurring between domains 3 and 4-5, and domains 3 and 1-2. RNA-RNA complexes were stable over 1 h of incubation at 37 degrees C, and depended on Mg2+ and RNA concentration. Neither the antisense domain 1-2 nor tRNA interacted with domain 3, providing experimental evidence of the specificity for the sense strand of the IRES sequence. Additionally, domain 1-2 did not interact with 4-5, leading to the suggestion that domain 3 acts as a scaffold structure where the other domains bind. The thermal disassociation profile of these complexes indicated different strength in these interactions. Whereas 50% of the complexes between domains 3 and 4-5 were destabilized at 45 degrees C, those formed by domain 1-2 and 3 required temperatures higher than 51 degrees C. Efficient self-dimerization of domains 3 and 4-5 was found in the absence of other transcripts. Formation of domain 3 homodimer competed with formation of heterocomplexes with other domains, and conversely, domain 3 homodimers were competed out by the presence of the other domains. RNA interactions were also observed at physiological concentrations of Mg2+ and K1+. The identification of the RNA-RNA complexes reported here provide direct experimental evidence of tertiary interactions within IRES elements.

Interaction of the eIF4G initiation factor with the aphthovirus IRES is essential for internal translation initiation in vivo.

The strategies developed by internal ribosome entry site (IRES) elements to recruit the translational machinery are poorly understood. In this study we show that protein-RNA interaction of the eIF4G translation initiation factor with sequences of the foot-and-mouth disease virus (FMDV) IRES is a key determinant of internal translation initiation in living cells. Moreover, we have identified the nucleotides required for eIF4G-RNA functional interaction, using native proteins from FMDV-susceptible cell extracts. Substitutions in the conserved internal AA loop of the base of domain 4 led to strong impairment of both eIF4G-RNA interaction in vitro and IRES-dependent translation initiation in vivo. Conversely, substitutions in the vicinity of the internal AA loop that did not impair IRES activity retained their ability to interact with eIF4G. Direct UV-crosslinking as well as competition assays indicated that domains 1-2, 3, and 5 of the IRES did not contribute to this interaction. In agreement with this, binding to domain 4 alone was as efficient as to the full-length IRES. The C-terminal fragment of eIF4G, proteolytically processed by the FMDV Lb protease, was sufficient to interact with the IRES or to its domain 4 alone. Additionally, we show here that binding of the eIF4B initiation factor to the IRES required domain 5 sequences. Moreover, eIF4G-IRES interaction was detected in the absence of eIF4B-IRES binding, suggesting that both initiation factors interact with the 3' region of the IRES but use different residues. The strong correlation found between eIF4G-RNA interaction and IRES activity in transfected cells suggests that eIF4G acts as a linker to recruit the translational machinery in IRES-dependent initiation.

Identification of an essential region for internal initiation of translation in the aphthovirus internal ribosome entry site and implications for viral evolution.

Translation of aphthovirus RNA is initiated at an internal ribosome entry site (IRES) element, preceding the first functional AUG initiation codon. The effect of mutations at the base of domain 3 of the aphthovirus IRES on translation activity has been analyzed by site-directed mutagenesis and expression of bicistronic RNAs in transfected cells. The results have shown that the enhanced IRES activity associated with a single pyrimidine transition fixed in a persistent aphthovirus variant (E. Martínez-Salas, J. C. Sáiz, M. Dávila, G. J. Belsham, and E. Domingo, J. Virol. 67:3748-3755, 1993) is base specific. Mutations predicted to destabilize the base of domain 3 were detrimental to IRES function, but subsequent restoration of the RNA structure gave rise to fully competent IRES. In contrast, single or multiple mutations that did not affect predicted helical structures modified the relative efficiency of translation by at most 10-fold, suggesting that primary sequence also plays a role in IRES activity. A correlation between the energy of stabilization of the IRES structure and the efficiency of translation has been noted. None of the 15 mutations studied reached a level of initiation of translation comparable to that of the IRES from the persistent variant. The results indicate a critical participation of the base of domain 3 in the activity of the aphthovirus IRES, with a strong effect of secondary or higher-order structures and minor effects of primary structure.

Stable expression of antisense RNAs targeted to the 5' non-coding region confers heterotypic inhibition to foot-and-mouth disease virus infection.

The antiviral potential of transcripts targeted to the non-coding regions (NCRs) of foot-and-mouth disease virus (FMDV) RNA have been studied during transient and constitutive expression in susceptible BHK-21 cells. Transient expression of antisense transcripts corresponding to the 5' and 3'NCRs, alone or in combination, confers specific inhibition of homologous (serotype C) virus infection in BHK-21 cells. Constitutive expression of antisense 5'NCR transcripts (5'AS) exerted higher levels of inhibition to homologous and heterologous (serotypes O, A, Asia, SAT 1, SAT 2 and SAT 3) FMDV infection, as estimated by a 10-fold reduction in virus titre in the supernatants from infected clones and by a plaque reduction assay. These inhibitions were also observed, albeit to a lesser extent, in clones stably expressing antisense 3'NCR transcripts. The antiviral response was specific for FMDV, as the picornavirus encephalomyocarditis virus was not inhibited in any of the transformed cell lines. In all cases, a correlation was found between the level of transcript expression and the extent of virus inhibition. The potential to efficiently inhibit FMDV, including isolates representing the seven serotypes, by expressing interfering 5'AS transcripts opens the possibility of developing transgenic animals with a reduced susceptibility to FMDV.

The need for enhancers is acquired upon formation of a diploid nucleus during early mouse development.

The activity of the polyoma virus (PyV) origin of DNA replication was used as a sensitive assay for enhancer function in one- and two-cell mouse embryos by injecting embryos with plasmid DNA containing different PyV ori configurations, allowing them to continue development in vitro, and then measuring plasmid DNA replication. Replication always required the PyV origin 'core' sequence in cis and PyV large tumor antigen (T-Ag) in trans. In developing two-cell embryos, DNA replication also required an enhancer in cis. Two copies of part of PyV enhancer 3 (beta element) was sevenfold better than one copy, and enhancer 3 was better than enhancer 1 + 2 (alpha element). Competition between ori configurations suggested that enhancers bound specific proteins required for replication and transcription. In contrast, DNA injected into one-cell embryos did not need an enhancer for replication, and no competition for replication factors was observed between different ori configurations. In fact, ori core replicated about ninefold better in one-cell embryos than the complete origin did in developing two-cell embryos. Therefore, core contains all the cis-acting information necessary to initiate DNA replication. Because one-cell embryos that replicated injected DNA retained their pronuclei and remained one-cell embryos, enhancers are not needed in mammalian development until a diploid nucleus is formed.

Involvement of the ftsA gene product in late stages of the Escherichia coli cell cycle.

Strain D-3 was shown, by genetic complementation with lambda phage vectors carrying either the wild type or a temperature-sensitive allele, to contain an ftsA mutation. The presence of this mutation rendered the strain unable to divide at 42 degrees C. The action of the ftsA gene product in strain D-3 takes place at a late stage of the cycle, starting near the end of the replication cycle and extending until cell division is completed. The action of the ftsA gene product seemed to take a constant time, independent of the duration of the cell division cycle. Experiments in which all other requirements for division except a "termination protein" were completed show that cells of strain D-3 can divide under these conditions only at the permissive temperature. We conclude that a likely role for the ftsA gene product is that of a termination protein.

Specific inhibition of aphthovirus infection by RNAs transcribed from both the 5' and the 3' noncoding regions.

RNA molecules containing the 3' terminal region of foot-and-mouth disease virus (FMDV) RNA in both antisense and sense orientations were able to inhibit viral FMDV translation and infective particle formation in BHK-21 cells following comicroinjection or cotransfection with infectious viral RNA. Antisense, but not sense, transcripts from the 5' noncoding region including the proximal element of the internal ribosome entry site and the two functional initiation AUGs were also inhibitory, both in in vitro translation and in vivo in comicroinjected or cotransfected BHK-21 cells. This effect was not observed with nonrelated RNA transcripts from lambda phage. The inhibitions found were permanent, sequence specific, and dose dependent; an inverse correlation between the length of the transcript and the extent of the antiviral effect was seen. In all cases, the extent of inhibition increased when viral RNAs and transcripts were allowed to reanneal before transfection, concomitant with a decrease in the doses required. The antiviral effect was specific for FMDV, since transcripts failed to inhibit infective particle formation by other picornavirus, such as encephalomyocarditis virus. These results indicate that the ability of RNA transcripts to inhibit viral multiplication depends on their efficient hybridization with target regions on the viral genome. Furthermore, cells transfected with the 5'1as transcript, which is complementary to the 5' noncoding region, showed a significant reduction of plaque-forming ability during the course of a natural infection. RNA 5'1as was able to inhibit FMDV RNA translation in vitro, suggesting that the inhibitions observed are mediated by a blockage of the viral translation initiation. Conversely, hybridization of short sequences of both sense and antisense transcripts from the 3' end induces distortion of predicted highly ordered structural motifs, which could be required for the synthesis of negative-stranded viral RNA, and correlates with inhibition of viral propagation.

Relationship of Escherichia coli density to growth rate and cell age.

The cell densities of Escherichia coli strains B/rA, BrF, and K-12 (OV-2) were measured at several growth rates and found to be very near 1.105 g/ml in all cases. Ninety percent of the cells of any exponentially growing population banded at densities differing less than 0.75% from the mean. Synchronized populations of B/rA selected as newborn cells were found to keep their density constant for longer than one generation time. However, if selection was based on cell size, by sedimentation through a sucrose gradient, cell density was found to be almost 2% lower than that of newborn cells, but it reached normal values before the first division had taken place. These results meant that mass and volume during the lifetime of the bacterial cell followed parallel kinetics. It was unlikely that density could regulate any event of the lifetime of a cell; on the contrary, density seemed to be a physical parameter that was well controlled during the bacterial growth.

Involvement of the aphthovirus RNA region located between the two functional AUGs in start codon selection.

Initiation of translation in picornavirus RNAs occurs internally, mediated by an element termed internal ribosome entry site (IRES). In the aphthovirus RNA, the IRES element directs translation initiation at two in-frame AUGs separated by 84 nucleotides. We have found that bicistronic constructs that contained the IRES element followed by the fragment including the aphthovirus start codons in front of the second gene mimicked the translation initiation pattern of viral RNA observed in infected cells. In those constructs, the frequency of initiation at the first AUG was increased by a sequence context that resembled the favorable consensus for cap-dependent translation, although initiation at the second site was always preferred. In addition, we have found that initiation at the second start codon was not diminished under conditions in which the first initiation codon was blocked by antisense oligonucleotide interference. Interestingly, mutations that positioned the second AUG out-of-frame with the first AUG did not interfere with the frequency of initiation at the second one. On the contrary, IRES-dependent translation initiation in bicistronic constructs lacking the sequences present between functional AUGs in the viral RNA was sensitive to the presence of out-of-frame initiator codons and hairpins in the spacer region. This remarkable difference in start codon recognition was due to the nucleotide composition of the RNA that separated the IRES from the initiator codon. Thus our results indicate that the region located in the aphthovirus RNA between functional AUGs is involved in start codon recognition, strongly favoring selection of the second start AUG as the main initiator codon.