Genetic analysis of an NTP-binding motif in poliovirus polypeptide 2C.

Poliovirus polypeptide 2C is a nonstructural protein involved in replication of the viral genome. Analysis of the primary amino acid sequence of 2C shows homology to a family of proteins which contain a nucleoside-triphosphate (NTP)-binding motif. This motif consists of elements "A" (2/5 hydrophobic stretch) G/AXXGXGKS/T, where X stands for any amino acid, and "B" (3/5 hydrophobic stretch) D or DD/E. To assess the significance of the consensus sequence in 2C, we have engineered point mutations into the most conserved residues in the A and B sites and tested their effect on viral RNA replication in vivo and translation in vitro. Whereas in vitro translation of synthetic RNAs carrying mutations in the NTP-binding motif showed efficient processing of all viral proteins, indistinguishable from that of the parental strain, transfection of the RNAs into HeLa cells did not give rise to infectious virus. No viral RNA replication could be detected in cells transfected with mutant RNAs. However, revertants to the wild-type genotype in the A and B sites were obtained which gave rise to wild-type RNA synthesis, but pseudorevertants or second-site suppressors were not observed. Thus, viral RNA synthesis is greatly reduced but not entirely abolished in cells transfected with mutant RNAs. These results strongly suggest a functional role for the proposed NTP-binding motif of 2C in RNA replication and proliferation of poliovirus.     

Pyrimidine-rich region mutations compensate for a stem-loop V lesion in the 5' noncoding region of poliovirus genomic RNA

Five revertants of a linker-scanning mutation adjacent to the stem-loop V attenuation determinant (X472) in the 5' noncoding region of poliovirus RNA were independently isolated from neuroblastoma cells and contained RNAs with seven nucleotide changes in the pyrimidine-rich region. Generation of the identical rare second-site mutations suggests the existence of a replicase-dependent mutagenesis mechanism during poliovirus replication. Enzymatic structure probing of the mutated pyrimidine-rich domain identified secondary structure changes between stem-loops V and VI. A consensus secondary structure model is presented for wild-type stem-loops V and VI and the pyrimidine-rich region located in the 5' noncoding region of poliovirus RNA. A pyrimidine-rich region mutant (X472-R4N) produced large plaques in neuroblastoma cells and small plaques in HeLa cells, but the plaque size differences were not due to cell-type differences in viral translation or RNA replication. Release of X472-R4N from HeLa cells was 10-fold lower than release from neuroblastoma cells, which may explain the small plaque phenotype of X472-R4N in HeLa cells. Wild-type poliovirus was also released more efficiently from neuroblastoma cells (approximately 4-fold increase compared with release from HeLa cells), indicating that poliovirus neurotropism may be influenced by the cell-type efficiency of virus release. Thermal treatment increased the levels of infectious X472-R4N virions but not wild-type virus particles; thus RNA sequence and structural changes in the mutated 5' noncoding region of X472-R4N may have altered RNA-protein interactions necessary for virus infectivity.     

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.     

Hepatitis B virus proteins expressed by recombinant vaccinia viruses: influence of preS2 sequence on expression surface and nucleocapsid proteins in human diploid cells

Summary Translation of poliovirus RNA is initiated by entry of ribosomes into the nucleotide sequence (internal ribosomal entry site; IRES) within the 5-untranslated region (5-UTR). Efficiency of this translation initiation in rabbit reticulocyte lysates (RRL) was very low and was greatly enhanced by addition of the ribosomal salt-wash fraction (RSW) prepared from HeLa cells. This stimulating activity in the RSW was partially purified by gel-filtration column chromatography and its molecular weight was estimated to be higher than 240 000. Several proteins that bind specifically to the poliovirus IRES were detected in the active fraction. Among those, a 57 kDa protein, recognized by antibodies against polypyrimidide tract-binding protein (PTB), was found. In addition, La protein (52 kDa) which is a human antigen recognized by antibodies from patients with autoimmune disorders was also detected. Further purification on a hydroxylapatite column resulted in considerable loss of the stimulatory activity, accompanied by a reduction of the apparent molecular weight of active component(s). These results suggest that fully active HeLa cell stimulatory factors for the translation initiation on poliovirus RNA function in RRL as a large complex consisted of several components including PTB and La protein.     

Differential roles of the 5' untranslated regions of cucumber mosaic virus RNAs 1, 2, 3 and 4 in translational competition

RNA species of plant tripartite RNA viruses show distinct translational activities in vitro when the viral RNA concentration is high. However, it is not known what causes the differential translation of virion RNAs. Using an in vitro wheat germ translation system, we investigated the translation efficiencies and competitive activities of chimeric cucumber mosaic virus (CMV) RNAs that contained viral untranslated regions (UTRs) and a luciferase-coding sequence. The chimeric RNAs exhibited distinct translation efficiencies and competitive activities. For example, the translation of chimeric CMV RNA 4 was about 40-fold higher than that of chimeric CMV RNA 3 in a competitive environment. The distinct translation resulted mainly from differences in competitive activities rather than translation efficiencies of the chimeric RNAs. The differential competitive activities were specified by viral 5 UTRs, but not by 3 UTRs or viral proteins. The competitive translational activities of the 5 UTRs were as follows: RNA 4 (coat protein)>RNAs 2 and 1 (2a and 1a protein, or replicase )> RNA 3 (3a protein).     

Multiple mutations involved in the phenotype of a temperature-sensitive small-plaque mutant of poliovirus

A temperature-sensitive small-plaque mutant of poliovirus type 1, ts247, has been analyzed previously. Several mutations were detected in the P3 region of the genome by analysis of proteins and by T1 oligonucleotide mapping of viral RNA. We have now studied spontaneous reversion of ts247 to the wild-type phenotype. This was found to be a two-step event, reversion to a ts+ phenotype (revertant R247-51) being distinct from acquisition of normal plaque size (revertant R247-12). The mutation responsible for the ts phenotype of ts247, implicated also in virus aggregation and heat lability, could not be detected by biochemical studies. Analysis of homotypic recombinants obtained by crossing ts247 with a guanidine-resistant derivative of a temperature-sensitive replicase mutant mapped this mutation to the P1 region or to the 5' end of the P2 region of the genome. The small-plaque phenotype of ts247 and R247-51 was correlated with an abnormality in polypeptide 3C (protease); direct sequencing of viral RNA revealed a U to C change at nucleotide 5658, which altered an isoleucine to threonine in the protease of ts247 and R247-51 but not of R247-12. Two other mutations were present in the region of the genome coding for polypeptide 3D of ts247 and of both classes of revertants. They thus seemed to play no role in the phenotype of ts247. One mutation, an A to G change at nucleotide 7135, was silent at the protein level, whereas the other, an A to G change at nucleotide 6264, determined a major amino acid change from glutamate to glycine in the viral replicase.     

Role of the RNA2 3' non-translated region of Blackcurrant reversion nepovirus in translational regulation

The 3' non-translated regions (NTRs) of mRNAs of eukaryotes and their viruses often contain translational enhancers (TEs). Blackcurrant reversion nepovirus (BRV) has a genome composed of two uncapped polyadenylated RNAs with very long 3' NTRs, nucleotide sequences of which are very conserved between different BRV isolates. In this work, we studied a role of the RNA2 3' NTR in translation, using mutagenesis of the firefly luciferase reporter mRNA, in protoplasts of Nicotiana benthamiana. The RNA2 3' NTR was found to contain a cap-independent TE (3' CITE), which must base pair with the 5' NTR to facilitate translation. The BRV 3' CITE and poly(A) tail provided a major contribution to translational efficiency, with less input from other 3' NTR parts. The BRV 3' CITE does not share similarity in nucleotide sequence and secondary structure with other viruses and thus represents a new class of 3' CITE.     

The formation of arenaviruses that are genetically diploid

Analyses of RNA extracted from preparations of arenaviruses indicate that the relative molar proportions of the genomic L and S RNA species are frequently far from equal. In order to investigate the genetic significance of this observation temperature-sensitive (ts) mutants of two lymphocytic choriomeningitis (LCM) virus strains (ARM and WE) have been recovered and categorized into recombination groups (Groups I and II). Fingerprint analyses of wild-type progeny viruses obtained from dual infections with ARM Group II and WE Group I ts viruses indicate that they have L/S RNA genotypes of WE/ARM. It is concluded that the ARM Group II ts viruses have mutations in their L RNA species and that the WE Group I ts viruses have mutations in their S RNA species. Correspondingly it is deduced that the ARM Group I ts viruses have S RNA mutations and the WE Group II ts viruses mutations in their L RNA species. Cells coinfected with certain WE Group I mutants, or an ARM Group I and certain WE Group I ts mutants, have also yielded wild-type viruses. Fingerprint analyses have shown that the wild-type viruses obtained from the latter crosses are diploid with respect to their S RNA species. On subsequent passage these wild-type viruses shed high proportions of ts mutants. We interpret the data to indicate that the original Group I ts mutants that yielded the diploid viruses have mutations in different S RNA gene products so that the progeny produce plaques at the nonpermissive temperature by gene product complementation. No wild-type recombinant viruses have been obtained from crosses involving Pichinde and LCM ts mutants.     

Evidence that PTB does not stimulate HCV IRES-driven translation

It is now well established that Hepatitis C Virus (HCV) translation is driven by an Internal Ribosome Entry Site (IRES) resulting in cap-independent translation. Such a mechanism usually occurs with the help of IRES Associated Factors (ITAFs). Moreover, an important translational feature is likely conserved from the model of classical mRNA circularisation (5'-3' cross-talk), involving the HCV RNA highly structured 3' extremity called the 3'X region. This could bind several cellular factors and modulate the translation efficacy, at least in Rabbit Reticulocyte Lysate (RRL). In particular, polypyrimidine-binding proteins have been proposed to be potential HCV ITAFs, such as Polypyrimidine Tract Binding protein (PTB). However, contradictions still exist as to the role of PTB: its ability to bind both the HCV IRES and the 3'X region leads to the hypothesis that it could positively modulate IRES-driven translation in the presence of the X structure. Results of translational and PTB-binding studies of X mutant sequences led us to discredit PTB as protagonist of 3'X region stimulation on HCV IRES-driven translation. Moreover, competition assays of X RNA in trans on IRES-driven translation demonstrate the involvement of at least two stimulating factors and led to the conclusion that this mechanism is more complex than initially thought. Although we did not identify these factors, it is no longer doubtful that there is effectively a stimulating functional interaction between the HCV IRES and the 3'X region in RRL.     

A point mutation in the poliovirus polymerase gene determines a complementable temperature-sensitive defect of RNA replication

We have previously described the isolation of a RNA- temperature-sensitive (ts) mutant of poliovirus type 1, ts035, after chemical mutagenesis by 5-fluorouracil. The ts defect of ts035 correlated with defective RNA replication, since the two characters corevert in the case of spontaneous revertants. The alteration of a trans-acting replication function of ts035 was suggested by significant rescue following mixed infection with another ts mutant, ts221, or with wild-type virus. Protein synthesis appeared normal at 39 degrees (nonpermissive temperature) in shift-up experiments and no defect of RNA elongation was evidenced when the activity of replication complexes or purified polymerase was measured at 39 degrees. These results provide circumstantial evidence that the initiation of ts035 RNA synthesis at 39 degrees is impaired. Molecular cloning of the ts035 genome allowed us to construct a recombinant virus with the same ts phenotype as ts035, by the transfer of a fragment of the mutant polymerase gene into the wild-type genome. Two mutations were present in this region of the ts035 genome but the determination of nucleotide sequences in the case of ts035 revertants indicated that only the substitution from A to G at nucleotide 7256 was necessary for the ts phenotype. This mutation replaces Asn 426 by an Asp in polypeptide 3D, the viral polymerase.     

Altered translation of the matrix genes in Niigata and Yamagata neurovirulent measles virus strains

Niigata and Yamagata strains measles virus were isolated from subacute sclerosing panencephalitis patients. These viruses were defective in virion production and expression of matrix (M) protein. The Niigata M protein-coding frame was interrupted by an in-frame termination codon, whereas the Yamagata M gene lacked the normal translational initiation codon. These mutations prevented translation of a normal M protein. However, RNA derived from the cloned Niigata and Yamagata M genes was translatable in vitro into low levels of aberrant proteins that reacted with M-specific antiserum. These proteins were also translated from poly(A)+ RNA from cells infected by Niigata and Yamagata virus strains. The aberrant M protein of Niigata virus was initiated at a downstream AUG codon created by a second mutation. The Yamagata M gene produced two aberrant proteins: one initiated mainly in vitro at an ACG codon, and a second species initiated at a downstream site both in vitro and in vivo. These results define the abnormal translational functions of the Niigata and Yamagata M genes, and further implicate the involvement of M protein defects in chronic central nervous system infections by measles virus.     

Initiation factor preparations from poliovirus-infected cells restrict translation in reticulocyte lysates

The translation of poliovirus RNA and vesicular stomatitis virus (VSV) mRNAs was studied in reticulocyte lysates supplemented with uninfected HeLa cell or polio-infected HeLa cell ribosomal salt wash as a source of initiation factors. Ribosomal salt wash from uninfected HeLa cells supported translation of both RNAs. Similar preparations from polio-infected HeLa cells stimulated translation of polio RNA; however, the translation of VSV mRNAs was completely restricted. Lysates programmed simultaneously with polio RNA and VSV mRNA translated both RNAs, although VSV translation was predominant. Addition of polio-infected cell ribosomal salt wash resulted in translation of polio RNA exclusively. The infected cell salt wash did not prevent formation of 40 S·Met-tRNA_f^Met complexes, but did prevent binding of labeled VSV mRNA to 40 S initiation complexes. The ability to restrict VSV mRNA translation resided in the 40% ammonium sulfate fractional precipitate prepared from infected cell salt wash. Similar fractionation of ribosomal salt wash mixed with labeled poliovirus double-stranded RNA indicated that this component was also found in the 40% ammonium sulfate fraction. The effects on translation of double-stranded RNA and the restrictive activity of infected cell salt wash were compared: mRNA specificity, kinetics of inhibition, nuclease sensitivity, and relief of inhibition by excess double-stranded RNA differed. From these comparisons, the possibility that double-stranded RNA caused the restriction of VSV mRNA translation was excluded.     

Point mutations modify the response of poliovirus RNA to a translation initiation factor: a comparison of neurovirulent and attenuated strains

Upon translation of poliovirus RNA in reticulocyte lysates, initiation occurs largely "incorrectly," that is, at sites in the middle of the viral genome rather than at the beginning of the polyprotein reading frame; the anomaly appears to be due to an initiation factor deficiency. Here, a fraction which stimulated initiation at the correct site, provisionally called "initiation correcting factor" (ICF), was partially purified from Krebs-2 cells. The ICF activity appeared to copurify with a complex of initiation factors eIF-2 and eIF-2B. The ability of ICF to stimulate, in reticulocyte lysates, the correct initiation of translation on the RNAs from neurovirulent and attenuated type 1 and type 3 poliovirus strains was investigated. Like crude initiation factor preparations, ICF appeared to be relatively less active with the RNAs from attenuated strains, the difference being especially pronounced for the type 3 strains. For the latter strains, the data suggested an important role of the nucleotide at, and perhaps around, position 472 in determining a response to the addition of ICF. It is proposed that interaction of a specific segment of the viral RNA with one or more of initiation factors plays an important part in the mechanism of translation of the picornavirus genomes, poliovirus attenuation, and, possibly, pathogenesis of poliomyelitis.     

Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition

The widespread class of RNA viruses that utilize internal ribosome entry sites (IRESs) for translation include poliovirus and Hepatitis C virus. To identify host factors required for IRES-dependent translation and viral replication, we performed a genome-wide RNAi screen in Drosophila cells infected with Drosophila C virus (DCV). We identified 66 ribosomal proteins that, when depleted, specifically inhibit DCV growth, but not a non-IRES-containing RNA virus. Moreover, treatment of flies with a translation inhibitor is protective in vivo. Finally, this increased sensitivity to ribosome levels also holds true for poliovirus infection of human cells, demonstrating the generality of these findings.     

Comparative studies on the genomes of some picornaviruses: denaturation mapping of replicative form RNA and electron microscopy of heteroduplex RNA.

Methods for denaturation mapping of double-stranded RNA and for heteroduplex RNA analysis are described. Denaturation maps of encephalomyocarditis (EMC) virus, poliovirus type 1, and poliovirus type 3 replicative form RNAs are presented. The three maps possess distinct features. Electron microscopy of heteroduplex RNA molecules constructed from poliovirus type 1 and poliovirus type 3 complementary RNA chains reveals two regions of considerable nonhomology as well as relatively conserved regions. The position of loops in this heteroduplex roughly corresponds to the location of regions where the denaturation maps of two polioviruses differ from each other. There is no evidence indicating that differences in the RNA sequences of poliovirus type 1 and poliovirus type 3 are due to extensive deletions (insertions) of genetic material; rather, the evolutionary divergence of these viruses seems to result, at least largely, from point mutations or equal substitutions.     

A shine-dalgarno-like sequence mediates in vitro ribosomal internal entry and subsequent scanning for translation initiation of coxsackievirus B3 RNA

Translation initiation of coxsackievirus B3 (CVB3) RNA is directed by an internal ribosome entry site (IRES) within the 5' untranslated region. However, the details of ribosome-template recognition and subsequent translation initiation are still poorly understood. In this study, we have provided evidence to support the hypothesis that 40S ribosomal subunits bind to CVB3 RNA via basepairing with 18S rRNA in a manner analogous to that of the Shine-Dalgarno (S-D) sequence in prokaryotic systems. We also identified a new site within both the 18S rRNA and the polpyrimidine-tract sequence of the IRES that allows them to form stronger sequence complementation. All these data were obtained from in vitro translation experiments using mutant RNAs containing either an antisense IRES core sequence at the original position or site-directed mutations or deletions in the polypyrimidine tract of the IRES. The mutations significantly reduced translation efficiency but did not abolish protein synthesis, suggesting that the S-D-like sequence is essential, but not sufficient for ribosome binding. To determine how ribosomes reach the initiation codon after internal entry, we created additional mutants: when the authentic initiation codon at nucleotide (nt) 742 was mutated, a 180-nt downstream in-frame AUG codon at nt 922 is able to produce a truncated smaller protein. When this mutation was introduced into the full-length cDNA of CVB3, the derived viruses were still infectious. However, their infectivity was much weaker than that of the wild-type CVB3. In addition, when a stable stem-loop was inserted upstream of the initiation codon in the bicistronic RNA, translation was strongly inhibited. These data suggest that ribosomes reach the initiation codon from the IRES likely by scanning along the viral RNA.