Sequencing and analysis for the full-length genome RNA of foot-and-mouth disease virus China/99

The complete nucleotide sequence of genomic RNA of foot and mouth disease virus (FMDV) strain China/99 from infected bovine tongue epithelium is presented. The nucleotide sequence extending from the 5’ end of the genomic RNA to the 5’ end of poly (A) tail contains 8173 nucleotides (nt). Its open reading frame, which encodes a single polypeptide of 2332 amino acids, encompasses 6999 nt starting from the initiation codon AUG and terminating at the UAA codon 93 bases upstream from the 5’ end of poly (A) tract. The 5’ untranslated region (UTR) is composed of 1081nt. The consensus of the 1d gene of FMDV strain China/99 compared with that of UKG/6/2001, UKG/12/2001, China/99HN4 and China/3/Tibet is over 97%. The result showed the stains belong to the members of the Pan-Asia family. There is a remarkable differentiation in the function-unknown (FUR), p2 and p3 regions between FMDV isolates from infected cattle and swine, especially in 3a gene. No deletion was found in genes /, 1a, 1b, 2a, 2c, 3b, and 3d. These genes might be indispensable to the surviving of FMDV. The secondary structures of small (S) fragments, FUR and an internal ribosome entry site can be classified into three types, and the S fragment and 3’ UTR of the positive-sense RNA fold into stem-loop structures similar to the shape of clover.     

口蹄疫病毒O/NY00株基因组L片段的克隆及其基因特征

采用RT—PCR方法对口蹄疫病毒O／NY00株基因组L片段进行了分子克隆和序列测定。结果表明：获得的O／NY00株基因组L片段长7805nt,其中包括715nt的5’非编码区和6999nt的多聚蛋白编码区。将O／NY00株与其它参考毒株进行序列比较,结果显示：O／NY00株与O／SKR／2000、O／1JKG／3／2001遗传关系最近,而与其它毒株遗传关系较远,属于0型口蹄疫ME-SA拓扑型Pan／ksia株的成员。与Cathay拓扑型的代表毒株O／TAW／97比较,两者在主要抗原位点1和位点3呈现出明显的以拓扑型为特征的氨基酸差别,提示两拓扑型毒株之间有较高的抗原差异。     

Nucleotide sequence and genome organization of foot-and-mouth disease virus.

A continuous 7802 nucleotide sequence spanning the 94% of foot and mouth disease virus RNA between the 5'-proximal poly(C) tract and the 3'-terminal poly(A) was obtained from cloned cDNA, and the total size of the RNA genome was corrected to 8450 nucleotides. A long open reading frame was identified within this sequence starting about 1300 bases from the 5' end of the RNA genome and extending to a termination codon 92 bases from its polyadenylated 3' end. The protein sequence of 2332 amino acids deduced from this coding sequence was correlated with the 260 K FMDV polyprotein. Its processing sites and twelve mature viral proteins were inferred from protein data, available for some proteins, a predicted cleavage specificity of an FMDV encoded protease for Glu/Gly(Thr, Ser) linkages, and homologies to related proteins from poliovirus. In addition, a short unlinked reading frame of 92 codons has been identified by sequence homology to the polyprotein initiation signal and by in vitro translation studies.     

Control of translation by the 5'- and 3'-terminal regions of the dengue virus genome

The genomic RNAs of flaviviruses such as dengue virus (DEN) have a 5' m7GpppN cap like those of cellular mRNAs but lack a 3' poly(A) tail. We have studied the contributions to translational expression of 5'- and 3'-terminal regions of the DEN serotype 2 genome by using luciferase reporter mRNAs transfected into Vero cells. DCLD RNA contained the entire DEN 5' and 3' untranslated regions (UTRs), as well as the first 36 codons of the capsid coding region fused to the luciferase reporter gene. Capped DCLD RNA was as efficiently translated in Vero cells as capped GLGpA RNA, a reporter with UTRs from the highly expressed alpha-globin mRNA and a 72-residue poly(A) tail. Analogous reporter RNAs with regulatory sequences from West Nile and Sindbis viruses were also strongly expressed. Although capped DCLD RNA was expressed much more efficiently than its uncapped form, uncapped DCLD RNA was translated 6 to 12 times more efficiently than uncapped RNAs with UTRs from globin mRNA. The 5' cap and DEN 3' UTR were the main sources of the translational efficiency of DCLD RNA, and they acted synergistically in enhancing translation. The DEN 3' UTR increased mRNA stability, although this effect was considerably weaker than the enhancement of translational efficiency. The DEN 3' UTR thus has translational regulatory properties similar to those of a poly(A) tail. Its translation-enhancing effect was observed for RNAs with globin or DEN 5' sequences, indicating no codependency between viral 5' and 3' sequences. Deletion studies showed that translational enhancement provided by the DEN 3' UTR is attributable to the cumulative contributions of several conserved elements, as well as a nonconserved domain adjacent to the stop codon. One of the conserved elements was the conserved sequence (CS) CS1 that is complementary to cCS1 present in the 5' end of the DEN polyprotein open reading frame. Complementarity between CS1 and cCS1 was not required for efficient translation.     

Role of the 5' TAR stem--loop and the U5-AUG duplex in dimerization of HIV-1 genomic RNA

The HIV-1 genome consists of two identical RNAs that are linked together through noncovalent interactions involving nucleotides from the 5' untranslated region (5' UTR) of each RNA strand. The 5' UTR is the most conserved part of the HIV-1 RNA genome, and its 335 nucleotide residues form regulatory motifs that mediate multiple essential steps in the viral replication cycle. Here, studying the effect of selected mutations both singly and together with mutations disabling SL1 (SL1 is a 5' UTR stem-loop containing a palindrome called the dimerization initiation site), we have done a rather systematic survey of the 5' UTR requirements for full genomic RNA dimerization in grown-up (i.e., predominantly >/=10 h old) HIV-1 viruses produced by transfected human and simian cells. We have identified a role for the 5' transactivation response element (5' TAR) and a contribution of a long-distance base pairing between a sequence located at the beginning of the U5 region and nucleotides surrounding the AUG Gag initiation codon. The resulting intra- or intermolecular duplex is called the U5-AUG duplex. The other regions of the 5' UTR have been shown to play no systematic role in genomic RNA dimerization, except for a sequence located around the 3' end of a large stem-loop enclosing the primer binding site, and the well-documented SL1. Our data are consistent with a direct role for the 5' TAR in genomic RNA dimerization (possibly via a palindrome encompassing the apical loop of the 5' TAR).     

The 3' untranslated regions of influenza genomic sequences are 5'PPP-independent ligands for RIG-I

Retinoic acid inducible gene-I (RIG-I) is a key regulator of antiviral immunity. RIG-I is generally thought to be activated by ssRNA species containing a 5'-triphosphate (PPP) group or by unphosphorylated dsRNA up to ~300 bp in length. However, it is not yet clear how changes in the length, nucleotide sequence, secondary structure, and 5' end modification affect the abilities of these ligands to bind and activate RIG-I. To further investigate these parameters in the context of naturally occurring ligands, we examined RNA sequences derived from the 5' and 3' untranslated regions (UTR) of the influenza virus NS1 gene segment. As expected, RIG-I-dependent interferon-β (IFN-β) induction by sequences from the 5' UTR of the influenza cRNA or its complement (26 nt in length) required the presence of a 5'PPP group. In contrast, activation of RIG-I by the 3' UTR cRNA sequence or its complement (172 nt) exhibited only a partial 5'PPP-dependence, as capping the 5' end or treatment with CIP showed a modest reduction in RIG-I activation. Furthermore, induction of IFN-β by a smaller, U/A-rich region within the 3' UTR was completely 5'PPP-independent. Our findings demonstrated that RNA sequence, length, and secondary structure all contributed to whether or not the 5'PPP moiety is needed for interferon induction by RIG-I.     

A bulged stem-loop structure in the 3' untranslated region of the genome of the coronavirus mouse hepatitis virus is essential for replication.

The 3' untranslated region (UTR) of the positive-sense RNA genome of the coronavirus mouse hepatitis virus (MHV) contains sequences that are necessary for the synthesis of negative-strand viral RNA as well as sequences that may be crucial for both genomic and subgenomic positive-strand RNA synthesis. We have found that the entire 3' UTR of MHV could be replaced by the 3' UTR of bovine coronavirus (BCV), which diverges overall by 31% in nucleotide sequence. This exchange between two viruses that are separated by a species barrier was carried out by targeted RNA recombination. Our results define regions of the two 3' UTRs that are functionally equivalent despite having substantial sequence substitutions, deletions, or insertions with respect to each other. More significantly, our attempts to generate an unallowed substitution of a particular portion of the BCV 3' UTR for the corresponding region of the MHV 3' UTR led to the discovery of a bulged stem-loop RNA secondary structure, adjacent to the stop codon of the nucleocapsid gene, that is essential for MHV viral RNA replication.     

cis-acting sequences required for coronavirus infectious bronchitis virus defective-RNA replication and packaging

The parts of the RNA genome of infectious bronchitis virus (IBV) required for replication and packaging of the RNA were investigated using deletion mutagenesis of a defective RNA (D-RNA) CD-61 (6.1 kb) containing a chloramphenicol acetyltransferase reporter gene. A D-RNA with the first 544, but not as few as 338, nucleotides (nt) of the 5' terminus was replicated; the 5' untranslated region (UTR) comprises 528 nt. Region I of the 3' UTR, adjacent to the nucleocapsid protein gene, comprised 212 nt and could be removed without impairment of replication or packaging of D-RNAs. A D-RNA with the final 338 nt, including the 293 nt in the highly conserved region II of the 3' UTR, was replicated. Thus, the 5'-terminal 544 nt and 3'-terminal 338 nt contained the necessary signals for RNA replication. Phylogenetic analysis of 19 strains of IBV and 3 strains of turkey coronavirus predicted a conserved stem-loop structure at the 5' end of region II of the 3' UTR. Removal of the predicted stem-loop structure abolished replication of the D-RNAs. D-RNAs in which replicase gene 1b-derived sequences had been removed or replaced with all the downstream genes were replicated well but were rescued poorly, suggesting inefficient packaging. However, no specific part of the 1b gene was required for efficient packaging.     

口蹄疫病毒Asia1/YNBS/58株全基因组序列分析

口蹄疫是由口蹄疫病毒(Foot-and-mouth dis-ease virus,FMDV)感染引起的偶蹄动物(猪、牛、羊、骆驼等)共患的一种急性、烈性、接触性传染病。FMDV是小核糖核酸病毒科(Picornaviridae)口蹄疫病毒属(Aphthovirus)的成员,有7个血清型,分别为O、A、C、Asia1、SAT1、SAT2、SAT3,完整     

Initiation of Genomic Plus-Strand RNA Synthesis from DNA and RNA Templates by a Viral RNA-Dependent RNA Polymerase

In contrast to the synthesis of minus-strand genomic and plus-strand subgenomic RNAs, the requirements for brome mosaic virus (BMV) genomic plus-strand RNA synthesis in vitro have not been previously reported. Therefore, little is known about the biochemical requirements for directing genomic plus-strand synthesis. Using DNA templates to characterize the requirements for RNA-dependent RNA polymerase template recognition, we found that initiation from the 3' end of a template requires one nucleotide 3' of the initiation nucleotide. The addition of a nontemplated nucleotide at the 3' end of minus-strand BMV RNAs led to initiation of genomic plus-strand RNA in vitro. Genomic plus-strand initiation was specific since cucumber mosaic virus minus-strand RNA templates were unable to direct efficient synthesis under the same conditions. In addition, mutational analysis of the minus-strand template revealed that the -1 nontemplated nucleotide, along with the +1 cytidylate and +2 adenylate, is important for RNA-dependent RNA polymerase interaction. Furthermore, genomic plus-strand RNA synthesis is affected by sequences 5' of the initiation site.     

Position and stability are determining factors for translation repression by an RNA G-quadruplex-forming sequence within the 5' UTR of the NRAS proto-oncogene

Nucleic acid secondary structures in the 5' untranslated regions (UTRs) of mRNAs have been shown to play a critical role in translation regulation. We recently demonstrated that a naturally occurring, conserved, and stable RNA G-quadruplex element (5'-GGGAGGGGCGGGUCUGGG-3'), located close to the 5' cap within the 5' UTR of the NRAS proto-oncogene mRNA, modulates gene expression at the translational level. Herein, we show that the translational effect of this G-quadruplex motif in NRAS 5' UTR is not uniform, but rather depends on the location of the G-quadruplex-forming sequence. The RNA G-quadruplex-forming sequence represses translation when situated relatively proximal to the 5' end, within the first 50 nt, in the 5' UTR of the NRAS proto-oncogene, whereas it has no significant effect on translation if located comparatively away from the 5' end. We have also demonstrated that the thermodynamic stability of the RNA G-quadruplex at its natural position within the NRAS 5' UTR is an important factor contributing toward its ability to repress translation.     

In vitro, the major ribosome binding site on Rous sarcoma virus RNA does not contain the nucleotide sequence coding for the N-terminal amino acids of the gag gene product.

Ribosomes from the reticulocyte lysate bind strongly and mainly to a region located in the 5' end of the Rous sarcoma virus RNA molecule between residues 9 and 53. This binding involves the participation of initiator tRNA and is sensitive to inhibitors of initiation of protein synthesis such as 7-methyl-GMP and aurintricarboxylic acid. The nucleotide sequence of this ribosome binding site has been determined: it conatains a GUG codon centered at position 26 that is not in phase with any termination codon within the 5' end nucleotide sequence of the RNA that we have analyzed (101 residues). However, the predicted N-terminal amino acid sequence starting from this GUG codon (or even from any AUG or GUG codon in the 5' end of the RNA) does not coincide with that of the in vitro-synthesized product of the 5' end proximal gag gene. Nevertheless, inhibition of ribosome binding to this site is accompanied by an inhibition of the in vitro translation of the gag gene.     

Comparative genomics of foot-and-mouth disease virus

Here we present complete genome sequences, including a comparative analysis, of 103 isolates of foot-and-mouth disease virus (FMDV) representing all seven serotypes and including the first complete sequences of the SAT1 and SAT3 genomes. The data reveal novel highly conserved genomic regions, indicating functional constraints for variability as well as novel viral genomic motifs with likely biological relevance. Previously undescribed invariant motifs were identified in the 5' and 3' untranslated regions (UTR), as was tolerance for insertions/deletions in the 5' UTR. Fifty-eight percent of the amino acids encoded by FMDV isolates are invariant, suggesting that these residues are critical for virus biology. Novel, conserved sequence motifs with likely functional significance were identified within proteins L(pro), 1B, 1D, and 3C. An analysis of the complete FMDV genomes indicated phylogenetic incongruities between different genomic regions which were suggestive of interserotypic recombination. Additionally, a novel SAT virus lineage containing nonstructural protein-encoding regions distinct from other SAT and Euroasiatic lineages was identified. Insights into viral RNA sequence conservation and variability and genetic diversity in nature will likely impact our understanding of FMDV infections, host range, and transmission.     

Specific interaction between the classical swine fever virus NS5B protein and the viral genome.

The NS5B protein of the classical swine fever virus (CSFV) is the RNA-dependent RNA polymerase of the virus and is able to catalyze the viral genome replication. The 3' untranslated region is most likely involved in regulation of the Pestivirus genome replication. However, little is known about the interaction between the CSFV NS5B protein and the viral genome. We used different RNA templates derived from the plus-strand viral genome, or the minus-strand viral genome and the CSFV NS5B protein obtained from the Escherichia coli expression system to address this problem. We first showed that the viral NS5B protein formed a complex with the plus-strand genome through the genomic 3' UTR and that the NS5B protein was also able to bind the minus-strand 3' UTR. Moreover, it was found that viral NS5B protein bound the minus-strand 3' UTR more efficiently than the plus-strand 3' UTR. Further, we observed that the plus-strand 3' UTR with deletion of CCCGG or 21 continuous nucleotides at its 3' terminal had no binding activity and also lost the activity for initiation of minus-strand RNA synthesis, which similarly occurred in the minus-strand 3' UTR with CATATGCTC or the 21 nucleotide fragment deleted from the 3' terminal. Therefore, it is indicated that the 3' CCCGG sequence of the plus-strand 3' UTR, and the 3' CATATGCTC fragment of the minus-strand are essential to in vitro synthesis of the minus-strand RNA and the plus-strand RNA, respectively. The same conclusion is also appropriate for the 3' 21 nucleotide terminal site of both the 3' UTRs.