More precise location of the polycytidylic acid tract in foot and mouth disease virus RNA.

The polycytidylic acid [poly(C)] tract in foot and mouth disease virus RNA has been located about 400 nucleotides from the 5' end of the RNA by analysis of the products from the digestion of the RNA with RNase H in the presence of oligodeoxyguanylic acid [oligo(dG)]. This treatment produces a small fragment (S) containing the small protein covalently linked to the RNA and a large fragment (L) that migrates faster than untreated RNA on low-percentage polyacrylamide gels, lacks the poly(C) tract as shown by RNase T1 digestion and oligo(dG)-cellulose binding, and is no longer infective. Polyacrylamide gel electrophoresis of fragment S suggests that it is about 400 nucleotides long, in agreement with the size estimated from the proportion of radioactivity in the fragment. Analysis of the RNase T1 digestion products of S shows that it contains only those oligonucleotides mapping close to the poly(C) tract that is situated near the 5' end of the virus RNA.     

The nucleotide sequence at the 5'' end of foot and mouth disease virus RNA.

Foot and mouth disease virus RNA has been treated with RNase H in the presence of oligo (dG) specifically to digest the poly(C) tract which lies near the 5' end of the molecule (10). The short (S) fragment containing the 5' end of the RNA was separated from the remainder of the RNA (L fragment) by gel electrophoresis. RNA ligase mediated labelling of the 3' end of S fragment showed that the RNase H digestion gave rise to molecules that differed only in the number of cytidylic acid residues remaining at their 3' ends and did not leave the unique 3' end necessary for fast sequence analysis. As the 5' end of S fragment prepared form virus RNA is blocked by VPg, S fragment was prepared from virus specific messenger RNA which does not contain this protein. This RNA was labelled at the 5' end using polynucleotide kinase and the sequence of 70 nucleotides at the 5' end determined by partial enzyme digestion sequencing on polyacrylamide gels. Some of this sequence was confirmed from an analysis of the oligonucleotides derived by RNase T1 digestion of S fragment. The sequence obtained indicates that there is a stable hairpin loop at the 5' terminus of the RNA before an initiation codon 33 nucleotides from the 5' end. In addition, the RNase T1 analysis suggests that there are short repeated sequences in S fragment and that an eleven nucleotide inverted complementary repeat of a sequence near the 3' end of the RNA is present at the junction of S fragment and the poly(C) tract.     

Sequence and location of the poly C tract in aphtho- and cardiovirus RNA.

The poly C tract in the RNA of the aphtho- and cardio viruses has been examined in several isolates of foot-and-mouth disease virus (FMDV) and encephalomyocarditis (EMC) virus. The length of the tract is variable, containing 100 to 170 bases in the FMDV isolates and 80 to 250 bases in the EMC virus isolates. Each poly C tract contains c. 10% A and U residues, located at the 5' end, i.e. most of the tract is a continuous run of C residues. The position of the tract on the genome was the same in each of the FMDV isolates, about 400 bases from the 5' end, whereas in the EMC virus isolates it was about 150 bases from the 5' end.     

Nucleotide and amino acid sequence coding for polypeptides of foot-and-mouth disease virus type A12.

The coding region for the structural and nonstructural polypeptides of the type A12 foot-and-mouth disease virus genome has been identified by nucleotide sequencing of cloned DNA derived from the viral RNA. In addition, 704 nucleotides in the 5' untranslated region between the polycytidylic acid tract and the probable initiation codon of the first translated gene, P16-L, have been sequenced. This region has several potential initiation codons, one of which appears to be a low-frequency alternate initiation site. The coding region encompasses 6,912 nucleotides and ends in a single termination codon, UAA, located 96 nucleotides upstream from a 3'-terminal polyadenylic acid tract. Microsequencing of radiolabeled in vivo and in vitro translation products identified the genome position of the major foot-and-mouth disease virus proteins and the cleavage sites recognized by the putative viral protease and an additional protease(s), probably of cellular origin, to generate primary and functional foot-and-mouth disease virus polypeptides.     

The sequence of foot-and-mouth disease virus RNA to the 5′ side of the poly(C) tract

The nucleotide sequence of foot-and-mouth disease virus (FMDV) RNA to the 5' side of the poly(C) tract (S fragment) has been determined for representatives of the A and O serotypes of the virus. The two S fragments differ in length by five nucleotides (nt), with 367 nt for O₁ compared with 362 nt for A₁₀, due to a number of insertions/deletions. However, the two sequences show 86% homology. There are no conserved open reading frames (ORFs). Secondary structure predictions reveal a high degree of potential base-pairing, such that the entire S fragment sequence can be folded into a hairpin structure.     

Sequence and translation of the murine coronavirus 5''-end genomic RNA reveals the N-terminal structure of the putative RNA polymerase.

A 28-kilodalton protein has been suggested to be the amino-terminal protein cleavage product of the putative coronavirus RNA polymerase (gene A) (M.R. Denison and S. Perlman, Virology 157:565-568, 1987). To elucidate the structure and mechanism of synthesis of this protein, the nucleotide sequence of the 5' 2.0 kilobases of the coronavirus mouse hepatitis virus strain JHM genome was determined. This sequence contains a single, long open reading frame and predicts a highly basic amino-terminal region. Cell-free translation of RNAs transcribed in vitro from DNAs containing gene A sequences in pT7 vectors yielded proteins initiated from the 5'-most optimal initiation codon at position 215 from the 5' end of the genome. The sequence preceding this initiation codon predicts the presence of a stable hairpin loop structure. The presence of an RNA secondary structure at the 5' end of the RNA genome is supported by the observation that gene A sequences were more efficiently translated in vitro when upstream noncoding sequences were removed. By comparing the translation products of virion genomic RNA and in vitro transcribed RNAs, we established that our clones encompassing the 5'-end mouse hepatitis virus genomic RNA encode the 28-kilodalton N-terminal cleavage product of the gene A protein. Possible cleavage sites for this protein are proposed.     

Genomic RNA of mengovirus. VI. Translation of its two cistrons in lysates of interferon-treated cells.

The addition of low levels (40 ng/ml) of the synthetic double-stranded polyribonucleotide poly I:C to lysates of interferon-treated L-cells resulted in a strong inhibition (70 to 75%) of the in vitro translation of mengovirus RNA. Under these conditions, the rates of incorporation of [35S]methionine or formyl-[35S]methionine were depressed to a comparable extent. The sequences of mengovirus RNA recognized by ribosomes of interferon-treated cells at initiation of translation were compared with those present in initiation complexes formed by ribosomes of untreated controls. Fingerprint analysis revealed that the same sequences of mengovirus RNA were protected against nuclease attack by the 80S and the 40S initiation complexes formed in vitro in lysates of control or interferon-treated L-cells. Mengovirus RNA-coded proteins were labeled at their N-terminal end with formyl-[35S]methionine and digested to completion with trypsin. The resulting fragments were separated by high-voltage paper electrophoresis. Two different formyl-[35S]methionine-labeled N termini were resolved. Further analyses supported the notion that the two radioactive peaks originated in the initiation of translation at two different sites. This pattern did not change when mengovirus RNA was translated in lysates of interferon-treated cells.     

A Recombinant Hepatitis C Virus RNA-Dependent RNA Polymerase Capable of Copying the Full-Length Viral RNA

All of the previously reported recombinant RNA-dependent RNA polymerases (RdRp), the NS5B enzymes, of hepatitis C virus (HCV) could function only in a primer-dependent and template-nonspecific manner, which is different from the expected properties of the functional viral enzymes in the cells. We have now expressed a recombinant NS5B that is able to synthesize a full-length HCV genome in a template-dependent and primer-independent manner. The kinetics of RNA synthesis showed that this RdRp can initiate RNA synthesis de novo and yield a full-length RNA product of genomic size (9.5 kb), indicating that it did not use the copy-back RNA as a primer. This RdRp was also able to accept heterologous viral RNA templates, including poly(A)- and non-poly(A)-tailed RNA, in a primer-independent manner, but the products in these cases were heterogeneous. The RdRp used some homopolymeric RNA templates only in the presence of a primer. By using the 3'-end 98 nucleotides (nt) of HCV RNA, which is conserved in all genotypes of HCV, as a template, a distinct RNA product was generated. Truncation of 21 nt from the 5' end or 45 nt from the 3' end of the 98-nt RNA abolished almost completely its ability to serve as a template. Inclusion of the 3'-end variable sequence region and the U-rich tract upstream of the X region in the template significantly enhanced RNA synthesis. The 3' end of minus-strand RNA of HCV genome also served as a template, and it required a minimum of 239 nt from the 3' end. These data defined the cis-acting sequences for HCV RNA synthesis at the 3' end of HCV RNA in both the plus and minus senses. This is the first recombinant HCV RdRp capable of copying the full-length HCV RNA in the primer-independent manner expected of the functional HCV RNA polymerase.     

Translation by Escherichia coli ribosomes of alfalfa mosaic virus RNA 4 can be initiated at two sites on the monocistronic message.

Substantial evidence is provided to corroborate our previous finding that Escherichia coli ribosomes recognize two binding sites on the 5' end of alfalfa mosaic virus (AMV) RNA 4 [for a preliminary report see Castel, A., Kraal, B., Kerklaan, P. R. M., Klok, J., and Bosch, L. (1977) Proc. Natl Acad. Sci. U.S.A. 74, 5509--5513]. Translation can start at either site using AcPhe-tRNA or fMet-RNA as initiator and takes place in the same reading frame along the monocistronic mRNA. The size and composition of the isolated extra NH2-terminal fragment of the acetylphenylalanyl product were found to be in agreement with the 5' non-coding region of the messenger. Removal of the 5'-terminal cap structure of AMV RNA 4 did not influence significantly both initiation reactions. Ribosomal protein S1 was essential for binding as well as incorporation of both fMet-tRNA and AcPhe-tRNA. A similar interaction on the ribosome was found for AcPhe-tRNA directed by AMV RNA 4 as for fMet-tRNA directed by either AMV RNA 4 or MS2 RNA with respect to the influence of initiation factors. It is concluded that the heterologous plant viral messenger is reliably translated in the E. coli system and that E. coli ribosomes recognize with high specificity an extra initiation site close to the 5' extremity of the messenger. The relationship of this site to a hypothetical entry site involved in the early recognition in the initiation mechanism between ribosome and messenger is discussed.     

Infectious foot-and-mouth disease virus derived from a cloned full-length cDNA.

A full-length cDNA plasmid of foot-and-mouth disease virus has been constructed. RNA synthesized in vitro by means of a bacteriophage SP6 promoter inserted in front of the cDNA led to the production of infectious particles upon transfection of BHK-21 cells. These particles were also found to be highly infectious for primary bovine kidney cells as well as for baby mice. The difficulty in cloning the foot-and-mouth disease virus cytidyl tract in Escherichia coli was circumvented by joining two separate cloned parts, representing the S and L fragments of the genome, and, in a second step, inserting a dC-dG homopolymer. Homopolymeric sequences of up to 25 cytidyl residues did not lead to the production of virus. Replicons containing poly(C) tracts long enough to permit virus replication were first established in yeast cells. One of these constructs could also be maintained in E. coli and was used to produce infectious RNA in vitro. The length of the poly(C) sequence in this cDNA plasmid was 32 nucleotides. However, the poly(C) tracts of two recombinant viruses found in transfected BHK-21 cells were 60 and 80 nucleotides long, respectively. Possible mechanisms leading to the enlargement of the poly(C) tract during virus replication are discussed.     

3' nontranslated RNA signals required for replication of hepatitis C virus RNA

We describe a mutational analysis of the 3' nontranslated RNA (3'NTR) signals required for replication of subgenomic hepatitis C virus (HCV) RNAs. A series of deletion mutants was constructed within the background of an HCV-N replicon that induces the expression of secreted alkaline phosphatase in order to examine the requirements for each of the three domains comprising the 3'NTR, namely, the highly conserved 3' terminal 98-nucleotide (nt) segment (3'X), an upstream poly(U)-poly(UC) [poly(U/UC)] tract, and the variable region (VR) located at the 5' end of the 3'NTR. Each of these domains was found to contribute to efficient replication of the viral RNA in transiently transfected hepatoma cells. Replication was not detected when any of the three putative stem-loop structures within the 3'X region were deleted. Similarly, complete deletion of the poly(U/UC) tract abolished replication. Replacement of a minimum of 50 to 62 nt of poly(U/UC) sequence was required for detectable RNA replication when the native sequence was restored in a stepwise fashion from its 3' end. Lengthier poly(U/UC) sequences, and possibly pure homopolymeric poly(U) tracts, were associated with more efficient RNA amplification. Finally, while multiple deletion mutations were tolerated within VR, each led to a partial loss of replication capacity. The impaired replication capacity of the deletion mutants could not be explained by reduced translational activity or by decreased stability of the RNA, suggesting that each of these mutations may impair recognition of the RNA by the viral replicase during an early step in negative-strand RNA synthesis. The results indicate that the 3'-most 150 nt of the HCV-N genome [the 3'X region and the 3' 52 nt of the poly(U/UC) tract] contain RNA signals that are essential for replication, while the remainder of the 3'NTR plays a facilitating role in replication but is not absolutely required.     

Comparison of the Nucleotide Sequence at the 5′ End of RNAs from Nine Aphthoviruses, Including Representatives of the Seven Serotypes

The sequence of about 70 nucleotides at the 5' end of the RNAs of nine different aphthoviruses (foot-and-mouth disease viruses), including representatives of the seven serotypes of the virus, has been determined by partial enzyme digestion of (32)P-end-labeled S fragment-that part of the RNA lying to the 5' side of the poly(C) tract and including the 5' end of the molecule. The S fragments were prepared from polyadenylated virus-specific RNA extracted from infected cells by digestion with RNase H in the presence of oligo(dG)(12-18). The first 27 nucleotides from the 5' end were highly conserved in all the RNAs. This region was followed by a more variable region of about 15 nucleotides, showing some length and sequence heterogeneity and including potential but probably nonutilized initiation codons. In agreement with previous homology studies, the sequencing results showed that the European serotypes A, O, and C form a group distinct from the SAT serotypes and that the Asia 1 serotype is closely related to the European group. The lengths of the S fragments of two different RNAs were confirmed as containing 360 to 400 nucleotides by gel electrophoresis with reference to nucleotide markers of known size.     

Cell-free translation of purified virion-associated high-molecular-weight RNA synthesized in vitro by vaccinia virus.

Virion-associated high-molecular-weight (HMW) RNA synthesized in vitro by purified vaccinia virus particles has been translated in a wheat germ cell-free protein synthesizing system. Purified HMW RNA directs the synthesis of translation products which are identical to the translation products made in response to in vitro-synthesized, virion-released 8 to 12S mRNA. The translation of HMW RNA proceeds exclusively through a 5'-terminal cap-mediated initiation step. Furthermore, only one coding sequence is translated per HMW RNA molecule, and that sequence is probably located near the 5' end of the molecule. These conclusions are based on the following results. (i) Sodium dodecyl sulfate--polyacrylamide gel electrophoresis patterns of translation products synthesized in response to HMW RNA and in response to 8 to 12S mRNA were qualitatively identical. (ii) On an equal weight basis, HMW RNA was 25 to 30% as active as 8 to 12S mRNA in stimulating in vitro protein synthesis. (iii) Unmethylated HMW RNA was translated at 10% the efficiency of the methylated form of this RNA. (iv) m7pG inhibited the translation of fully methylated HMW RNA by 90%. (v) After the initiation step of translation was blocked by aurintricarboxylic acid, the rate with which amino acids were incorporated into individual polypeptides decreased in a similar manner for the translation of both HMW RNA and 8 to 12S mRNA. Virion-released 8 to 12S mRNA derived from virion-associated HMW RNA during a chase in the presence of ATP, GTP, and S-adenosylmethionine was also translated. At low RNA concentrations, the derived RNA appeared to stimulate amino acid incorporation more efficiently than the HMW RNA precursor. However, at higher concentrations of this RNA, protein synthesis was severely inhibited.     

Gene Order of Encephalomyocarditis Virus as Determined by Studies with Pactamycin

Previous work has shown that translation of the encephalomyocarditis (EMC) viral ribonucleic acid (RNA) generates at least three primary products, polypeptides A, F, and C. The A and C polypeptides then undergo post-translational cleavages to complete the production of the stable viral polypeptides (delta, beta, gamma, alpha, G, I, F, H, and E). In this communication we show that A, F, and C are produced in equimolar amounts giving further support to the theory that the RNA of picornaviruses has only a single site for the initiation of protein synthesis. The biosynthesis of viral proteins in EMC virus-infected HeLa cells was studied in the presence of pactamycin at concentrations which preferentially inhibit the initiation of protein synthesis. The amount of each polypeptide formed during the residual period of protein synthesis observed after the addition of pactamycin was used as a criterion for ordering the genes on the viral RNA. The results obtained indicate that the primary gene products are ordered on the EMC viral RNA 5' --> 3' A-F-C and that the stable products are ordered delta-beta-gamma-alpha-G-I-F-H-E. Moreover, the intermediate chains B and epsilon map in the capsid region, whereas the intermediate chain D maps in the E region. This order is largely consistent with previously established relationships of the viral polypeptides and thus indicates that pactamycin is a valid tool for "genetic" mapping of polycistronic RNA molecules with single initiation sites.     

Genomic RNA of mengovirus V. Recognition of common features by ribosomes and eucaryotic initiation factor 2.

Binding of ribosomes to the 32P-labeled genomic RNA of mengovirus was studied in lysates of mouse L929 and Krebs ascites cells under conditions for initiation of translation. Upon total digestion with RNase T1, the 32P-labeled RNA protected in either 40S or 80S initiation complexes yielded four unique, large oligonucleotides. Each of these oligonucleotides occurred once in the viral RNA molecule. The same four oligonucleotides were recovered from 80S initiation complexes formed in lysates in which unlabeled mengovirus RNA had been translated extensively, indicating that recognition by ribosomes was not modulated detectably by a viral translation product. The recognition of intact, 32P-labeled mengovirus RNA by eucaryotic initiation factor 2 (eIF-2) was examined by direct complex formation. Fingerprint analysis of the RNA protected by eIF-2 against RNase T1 digestion yielded three T1 oligonucleotides that were identical to three of the four oligonucleotides protected in either 40S or 80S initiation complexes. A physical map of the large T1 oligonucleotides of the mengovirus RNA molecule was constructed, and the four protected oligonucleotides were found to map internally, within the region between the polycytidylate tract and the 3' end. For either ribosomes or eIF-2, the protected oligonucleotides could not be arranged in a continuous sequence, suggesting that they constitute at least two widely separated domains. These results show that ribosomes recognize and blind to more than a single sequence in mengovirus RNA, located internally in regions that are far removed from the 5' end of the molecule. eIF-2 itself binds with high specificity to mengovirus RNA, recognizing apparently three of the four sequences recognized by ribosomes.