Four consensus sequences are conserved with the same linear arrangement in RNA-dependent DNA polymerases encoded by retroid elements and in RNA-dependent RNA polymerases encoded by plus-, minus- and double-strand RNA viruses. One of these motifs corresponds to the YGDD span previously described by Kamer and Argos (1984). These consensus sequences altogether lead to 4 strictly and 18 conservatively maintained amino acids embedded in a large domain of 120 to 210 amino acids. As judged from secondary structure predictions, each of the 4 motifs, which may cooperate to form a well-ordered domain, places one invariant amino acid in or proximal to turn structures that may be crucial for their correct positioning in a catalytic process. We suggest that this domain may constitute a prerequisite 'polymerase module' implicated in template seating and polymerase activity. At the evolutionary level, the sequence similarities, gap distribution and distances between each motif strongly suggest that the ancestral polymerase module was encoded by an individual genetic element which was most closely related to the plus-strand RNA viruses and the non-viral retroposons. This polymerase module gene may have subsequently propagated in the viral kingdom by distinct gene set recombination events leading to the wide viral variety observed today.

Identification of four conserved motifs among the RNA-dependent polymerase encoding elements.

Despite the rapid mutational change that is typical of positive-strand RNA viruses, enzymes mediating the replication and expression of virus genomes contain arrays of conserved sequence motifs. Proteins with such motifs include RNA-dependent RNA polymerase, putative RNA helicase, chymotrypsin-like and papain-like proteases, and methyltransferases. The genes for these proteins form partially conserved modules in large subsets of viruses. A concept of the virus genome as a relatively evolutionarily stable "core" of housekeeping genes accompanied by a much more flexible "shell" consisting mostly of genes coding for virion components and various accessory proteins is discussed. Shuffling of the "shell" genes including genome reorganization and recombination between remote groups of viruses is considered to be one of the major factors of virus evolution. Multiple alignments for the conserved viral proteins were constructed and used to generate the respective phylogenetic trees. Based primarily on the tentative phylogeny for the RNA-dependent RNA polymerase, which is the only universally conserved protein of positive-strand RNA viruses, three large classes of viruses, each consisting of distinct smaller divisions, were delineated. A strong correlation was observed between this grouping and the tentative phylogenies for the other conserved proteins as well as the arrangement of genes encoding these proteins in the virus genome. A comparable correlation with the polymerase phylogeny was not found for genes encoding virion components or for genome expression strategies. It is surmised that several types of arrangement of the "shell" genes as well as basic mechanisms of expression could have evolved independently in different evolutionary lineages. The grouping revealed by phylogenetic analysis may provide the basis for revision of virus classification, and phylogenetic taxonomy of positive-strand RNA viruses is outlined. Some of the phylogenetically derived divisions of positive-strand RNA viruses also include double-stranded RNA viruses, indicating that in certain cases the type of genome nucleic acid may not be a reliable taxonomic criterion for viruses. Hypothetical evolutionary scenarios for positive-strand RNA viruses are proposed. It is hypothesized that all positive-strand RNA viruses and some related double-stranded RNA viruses could have evolved from a common ancestor virus that contained genes for RNA-dependent RNA polymerase, a chymotrypsin-related protease that also functioned as the capsid protein, and possibly an RNA helicase.

Evolution and Taxonomy of Positive-Strand RNA Viruses: Implications of Comparative Analysis of Amino Acid Sequences

Hepatitis C virus (HCV) is a major causative agent of posttransfusion non-A, non-B hepatitis, which often develops into malignant chronic diseases, including liver cirrhosis and hepatocellular carcinoma. We have cloned from human carriers overlapping cDNAs (9,416 bp) covering the entire coding region of the HCV genome. The latter encodes a 3,010-amino-acid polyprotein. In addition, there are 332 and 54 bases of 5' and 3' noncoding sequences, respectively. Our HCV strain has a 77% nucleic acid identity to the HCV strain cloned by workers at Chiron Corporation. The hydrophobicity profile of the putative polyprotein is similar to those of flaviviruses, but it has limited amino acid homology to polyproteins of flaviviruses and other viruses, indicating that HCV is at most distantly related to flaviviruses.

https://jvi.asm.org/content/jvi/65/3/1105.full.pdf

Structure and organization of the hepatitis C virus genome isolated from human carriers

The potyvirus group [named after its type member, potato virus Y (PVY)] is the largest of the 34 plant virus groups and families currently recognized (Ward & Shukla, 1991). It contains at least 180 definitive and possible members (or 30% of all known plant viruses) which cause significant losses in agricultural, pasture, horticultural and ornamental crops (Ward & Shukla, 1991). These viruses are unique in the diversity of inclusion bodies that are formed during the infection cycle (see Lesemann, 1988). A feature shared by all potyviruses is the induction of characteristic pinwheel or scroll-shaped inclusion bodies in the cytoplasm of the infected cells (Edwardson, 1974). These cylindrical inclusion (CI) bodies are formed by a virus-encoded protein and can be considered as the most important phenotypic criterion for assigning viruses to the poty-virus group (Milne, 1988; Shukla et al., 1989; Ward & Shukla, 1991).

Highlights and prospects of potyvirus molecular biology

The RNA genome of tobacco etch virus (TEV), a plant potyvirus, functions as an mRNA for synthesis of a 346-kilodalton polyprotein that undergoes extensive proteolytic processing. The RNA lacks a normal 5' cap structure at its terminus, which suggests that the mechanism of translational initiation differs from that of a normal cellular mRNA. We have identified a translation-enhancing activity associated with the 144-nucleotide, 5' nontranslated region (NTR) of the TEV genome. When fused to a reporter gene encoding beta-glucuronidase (GUS), the 5' NTR results in an 8- to 21-fold enhancement over a synthetic 5' NTR in a transient-expression assay in protoplasts. A similar effect was observed when the 5' NTR-GUS fusions were expressed in transgenic plants. By using a cell-free translation system, the translation enhancement activity of the TEV 5' NTR was shown to be cap independent, whereas translation of GUS mRNA containing an artificial 5' NTR required the presence of a cap structure. Translation of GUS transcripts containing the TEV 5' NTR was relatively insensitive to the cap analog m7GTP, whereas translation of transcripts containing the artificial 5' NTR was highly sensitive. The 144-nucleotide TEV 5' NTR synthesized in vitro was shown to compete for factors that are required for protein synthesis in the cell-free translation reaction mix. Competition was not observed when a transcript representing the initial 81 nucleotides of the TEV 5' NTR was tested. These results support the hypothesis that the TEV 5' NTR promotes translation in a cap-independent manner that may involve the binding of proteins and/or ribosomes to internal sites within the NTR.

/pdf/cap-independent-enhancement-of-translation-by-a-plant-43wp2poosh.pdf

Cap-independent enhancement of translation by a plant potyvirus 5' nontranslated region.

The nucleotide sequence of the RNA of tobacco vein mottling virus, a member of the potyvirus group, was determined. The RNA was found to be 9471 residues in length, excluding a 3'-terminal poly(A) tail. The first three AUG codons from the 5'-terminus were followed by in-frame termination codons. The fourth, at position 206, was the beginning of an open reading frame of 9015 residues which could encode a polyprotein of 340 kDa. No other long open reading frames were present in the sequence or its complement. This AUG was present in the sequence AGGCCAUG, which is similar to the consensus initiation sequence shared by most eukaryotic mRNAs. The chemically-determined amino acid compositions of the helper component and coat proteins were similar to those predicted from the nucleotide sequence. Amino acid sequencing of coat protein from which an amino-terminal peptide had been removed allowed exact location of the coat protein cistron. A consensus sequence of V-(R or K)-F-Q was found on the N-terminal sides of proposed cleavage sites for proteolytic processing of the polyprotein.

Martin F.E. Siaw

Papers

The nucleotide sequence of tobacco vein mottling virus RNA

Identification of a protein covalently linked to the 5' terminus of tobacco vein mottling virus RNA