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

Abstract

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.     

RNA helicase: a novel activity associated with a protein encoded by a positive strand RNA virus.

Most positive strand RNA viruses infecting plants and animals encode proteins containing the so-called nucleotide binding motif (NTBM) (1) in their amino acid sequences (2). As suggested from the high level of sequence similarity of these viral proteins with the recently described superfamilies of helicase-like proteins (3-5), the NTBM-containing cylindrical inclusion (CI) protein from plum pox virus (PPV), which belongs to the potyvirus group of positive strand RNA viruses, is shown to be able to unwind RNA duplexes. This activity was found to be dependent on the hydrolysis of NTP to NDP and Pi, and thus it can be considered as an RNA helicase activity. In the in vitro assay used, the PPV CI protein was only able to unwind double strand RNA substrates with 3' single strand overhangs. This result indicates that the helicase activity of the PPV CI protein functions in the 3' to 5' direction (6). To our knowledge, this is the first report on a helicase activity associated with a protein encoded by an RNA virus.     

A Novel Mycovirus That Is Related to the Human Pathogen Hepatitis E Virus and Rubi-Like Viruses

Previously, we reported that three double-stranded RNA (dsRNA) segments, designated L-, M-, and S-dsRNAs, were detected in Sclerotinia sclerotiorum strain Ep-1PN. Of these, the M-dsRNA segment was derived from the genomic RNA of a potexvirus-like positive-strand RNA virus, Sclerotinia sclerotiorum debilitation-associated RNA virus. Here, we present the complete nucleotide sequence of the L-dsRNA, which is 6,043 nucleotides in length, excluding the poly(A) tail. Sequence analysis revealed the presence of a single open reading frame (nucleotide positions 42 to 5936) that encodes a protein with significant similarity to the replicases of the “alphavirus-like” supergroup of positive-strand RNA viruses. A sequence comparison of the L-dsRNA-encoded putative replicase protein containing conserved methyltransferase, helicase, and RNA-dependent RNA polymerase motifs showed that it has significant sequence similarity to the replicase of Hepatitis E virus, a virus infecting humans. Furthermore, we present convincing evidence that the virus-like L-dsRNA could replicate independently with only a slight impact on growth and virulence of its host. Our results suggest that the L-dsRNA from strain Ep-1PN is derived from the genomic RNA of a positive-strand RNA virus, which we named Sclerotinia sclerotiorum RNA virus L (SsRV-L). As far as we know, this is the first report of a positive-strand RNA mycovirus that is related to a human virus. Phylogenetic and sequence analyses of the conserved motifs of the RNA replicase of SsRV-L showed that it clustered with the rubi-like viruses and that it is related to the plant clostero-, beny- and tobamoviruses and to the insect omegatetraviruses. Considering the fact that these related alphavirus-like positive-strand RNA viruses infect a wide variety of organisms, these findings suggest that the ancestral positive-strand RNA viruses might be of ancient origin and/or they might have radiated horizontally among vertebrates, insects, plants, and fungi.     

Genomic organization of GB viruses A and B: two new members of the Flaviviridae associated with GB agent hepatitis.

The genomes of two positive-strand RNA viruses have recently been cloned from the serum of a GB agent-infected tamarin by using representational difference analysis. The two agent, GB viruses A and B (GBV-A and GBV-B, respectively), have genomes of 9,493 and 9,143 nucleotides, respectively, and single large open reading frames that encode potential polyprotein precursors of 2,972 and 2,864 amino acids, respectively. The genomes of these agents are organized much like those of other pestiviruses and flaviviruses, with genes predicted to encode structural and nonstructural proteins located at the 5' and 3' ends, respectively. Amino acid sequence alignments and subsequent phylogenetic analysis of the RNA-dependent RNA polymerases (RdRps) of GBV-A and GBV-B show that they possess conserved sequence motifs associated with supergroup II RNA polymerases of positive-strand RNA viruses. On the basis of similar analyses, the GBV-A- and GBV-B-encoded helicases show significant identity with the supergroup II helicases of positive-strand RNA viruses. Within the supergroup II RNA polymerases and helicases, GBV-A and GBV-B are most closely related to the hepatitis C virus group. Across their entire open reading frames, the GB agents exhibit 27% amino sequence identity to each other, approximately 28% identity to hepatitis C virus type 1, and approximately 20% identity to either bovine viral diarrhea virus or yellow fever virus. The degree of sequence divergence between GBV-A and GBV-B and other Flaviviridae members demonstrates that the GB agents are representatives of two new genera within the Flaviviridae family.     

Identification of novel positive-strand RNA viruses by metagenomic analysis of archaea-dominated Yellowstone hot springs

There are no known RNA viruses that infect Archaea. Filling this gap in our knowledge of viruses will enhance our understanding of the relationships between RNA viruses from the three domains of cellular life and, in particular, could shed light on the origin of the enormous diversity of RNA viruses infecting eukaryotes. We describe here the identification of novel RNA viral genome segments from high-temperature acidic hot springs in Yellowstone National Park in the United States. These hot springs harbor low-complexity cellular communities dominated by several species of hyperthermophilic Archaea. A viral metagenomics approach was taken to assemble segments of these RNA virus genomes from viral populations isolated directly from hot spring samples. Analysis of these RNA metagenomes demonstrated unique gene content that is not generally related to known RNA viruses of Bacteria and Eukarya. However, genes for RNA-dependent RNA polymerase (RdRp), a hallmark of positive-strand RNA viruses, were identified in two contigs. One of these contigs is approximately 5,600 nucleotides in length and encodes a polyprotein that also contains a region homologous to the capsid protein of nodaviruses, tetraviruses, and birnaviruses. Phylogenetic analyses of the RdRps encoded in these contigs indicate that the putative archaeal viruses form a unique group that is distinct from the RdRps of RNA viruses of Eukarya and Bacteria. Collectively, our findings suggest the existence of novel positive-strand RNA viruses that probably replicate in hyperthermophilic archaeal hosts and are highly divergent from RNA viruses that infect eukaryotes and even more distant from known bacterial RNA viruses. These positive-strand RNA viruses might be direct ancestors of RNA viruses of eukaryotes.     

Novel catalytic activity associated with positive-strand RNA virus infection: nucleic acid-stimulated ATPase activity of the plum pox potyvirus helicaselike protein.

The cylindrical inclusion protein of potyviruses contains the so-called nucleoside triphosphate binding motif, an amino acid sequence motif present in proteins encoded by most positive-strand RNA viruses, some double-strand RNA viruses, apparently all groups of double-strand DNA viruses, and also several single-strand DNA viruses. Further sequence analysis has allowed to include the cylindrical inclusion protein of potyviruses as a member of a superfamily of helicaselike proteins. In this paper we show that the purified cylindrical inclusion protein of plum pox potyvirus interacts with RNA and ATP and copurifies with a nucleic acid-stimulated ATPase activity. To our knowledge, this is the first time that this kind of enzymatic activity has been experimentally associated with a positive-strand RNA virus-encoded protein.     

Flock House Virus RNA Replicates on Outer Mitochondrial Membranes in Drosophila Cells

The identification and characterization of host cell membranes essential for positive-strand RNA virus replication should provide insight into the mechanisms of viral replication and potentially identify novel targets for broadly effective antiviral agents. The alphanodavirus flock house virus (FHV) is a positive-strand RNA virus with one of the smallest known genomes among animal RNA viruses, and it can replicate in insect, plant, mammalian, and yeast cells. To investigate the localization of FHV RNA replication, we generated polyclonal antisera against protein A, the FHV RNA-dependent RNA polymerase, which is the sole viral protein required for FHV RNA replication. We detected protein A within 4 h after infection of Drosophila DL-1 cells and, by differential and isopycnic gradient centrifugation, found that protein A was tightly membrane associated, similar to integral membrane replicase proteins from other positive-strand RNA viruses. Confocal immunofluorescence microscopy and virus-specific, actinomycin D-resistant bromo-UTP incorporation identified mitochondria as the intracellular site of protein A localization and viral RNA synthesis. Selective membrane permeabilization and immunoelectron microscopy further localized protein A to outer mitochondrial membranes. Electron microscopy revealed 40- to 60-nm membrane-bound spherical structures in the mitochondrial intermembrane space of FHV-infected cells, similar in ultrastructural appearance to tombusvirus- and togavirus-induced membrane structures. We concluded that FHV RNA replication occurs on outer mitochondrial membranes and shares fundamental biochemical and ultrastructural features with RNA replication of positive-strand RNA viruses from other families.     

The rhinovirus type 14 genome contains an internally located RNA structure that is required for viral replication.

Cis-acting RNA signals are required for replication of positive-strand viruses such as the picornaviruses. Although these generally have been mapped to the 5' and/or 3' termini of the viral genome, RNAs derived from human rhinovirus type 14 are unable to replicate unless they contain an internal cis-acting replication element (cre) located within the genome segment encoding the capsid proteins. Here, we show that the essential cre sequence is 83-96 nt in length and located between nt 2318-2413 of the genome. Using dicistronic RNAs in which translation of the P1 and P2-P3 segments of the polyprotein were functionally dissociated, we further demonstrate that translation of the cre sequence is not required for RNA replication. Thus, although it is located within a protein-coding segment of the genome, the cre functions as an RNA entity. Computer folds suggested that cre sequences could form a stable structure in either positive- or minus-strand RNA. However, an analysis of mutant RNAs containing multiple covariant and non-covariant nucleotide substitutions within these putative structures demonstrated that only the predicted positive-strand structure is essential for efficient RNA replication. The absence of detectable minus-strand synthesis from RNAs that lack the cre suggests that the cre is required for initiation of minus-strand RNA synthesis. Since a lethal 3' noncoding region mutation could be partially rescued by a compensating mutation within the cre, the cre appears to participate in a long-range RNA-RNA interaction required for this process. These data provide novel insight into the mechanisms of replication of a positive-strand RNA virus, as they define the involvement of an internally located RNA structure in the recognition of viral RNA by the viral replicase complex. Since internally located RNA replication signals have been shown to exist in several other positive-strand RNA virus families, these observations are potentially relevant to a wide array of related viruses.     

A novel superfamily of nucleoside triphosphate-binding motif containing proteins which are probably involved in duplex unwinding in DNA and RNA replication and recombination

A statistically significant similarity was demonstrated between the amino acid sequences of 4 Escherichia coli helicases and helicase subunits, a family of non-structural proteins of eukaryotic positive-strand RNA viruses and 2 herpesvirus proteins all of which contain an NTP-binding sequence motif. Based on sequence analysis and secondary structure predictions, a generalized structural model for the ATP-binding core is proposed. It is suggested that all these proteins constitute a superfamily of helicases (or helicase subunits) involved in NTP-dependent duplex unwinding during DNA and RNA replication and recombination.     

Filling a GAP(DH) in asymmetric viral RNA synthesis

Host proteins, such as RNA-binding proteins, are involved in most steps of replication by positive-strand RNA viruses, including Tomato bushy stunt virus (TBSV). In this issue of Cell Host & Microbe, Wang and Nagy report that efficient replication of TBSV requires GAPDH, a host protein with glycolytic, RNA-binding, and other functions. GAPDH binds TBSV (-)RNA and promotes the normal excess of (+)RNA over (-)RNA products, possibly by selectively retaining (-)RNA templates for copying.     

Interaction of hepatitis C virus proteins with host cell membranes and lipids

For replication, viruses depend on specific components and energy supplies from the host cell. The main steps in the lifecycle of positive-strand RNA viruses depend on cellular membranes. Interest is increasing in studying the interactions between host cell membranes and viral proteins to understand how such viruses replicate their genome and produce infectious particles. These studies should also lead to a better knowledge of the different mechanisms underlying membrane-protein associations. The various molecular interactions of hepatitis C virus proteins with the membranes and lipids of the infected cell highlight how a virus can exploit the diversity of interactions that occur between proteins and membranes or lipid structures.     

An NTP-binding motif is the most conserved sequence in a highly diverged monophyletic group of proteins involved in positive strand RNA viral replication

Summary NTP-motif, a consensus sequence previously shown to be characteristic of numerous NTP-utilizing enzymes, was identified in nonstructural proteins of several groups of positive-strand RNA viruses. These groups include picorna-, alpha-, and coronaviruses infecting animals and como-, poty-, tobamo-, tricorna-, hordei-, and furoviruses of plants, totalling 21 viruses. It has been demonstrated that the viral NTP-motif-containing proteins constitute three distinct families, the sequences within each family being similar to each other at a statistically highly significant level. A lower, but still valid similarity has also been revealed between the families. An overall alignment has been generated, which includes several highly conserved sequence stretches. The two most prominent of the latter contain the socalled A and B sites of the NTP-motif, with four of the five invariant amino acid residues observed within these sequences. These observations, taken together with the results of comparative analysis of the positions occupied by respective proteins (domains) in viral multidomain proteins, suggest that all the NTP-motif-containing proteins of positive-strand RNA viruses are homologous, constituting a highly diverged monophyletic group. In this group the A and B sites of the NTP-motif are the most conserved sequences and, by inference, should play the principal role in the functioning of the proteins. A hypothesis is proposed that all these proteins posses NTP-binding capacity and possibly NTPase activity, performing some NTP-dependent function in viral RNA replication. The importance of phylogenetic analysis for the assessment of the significance of the occurrence of the NTP-motif (and of sequence motifs of this sort in general) in proteins is emphasized.     

Mutation of host DnaJ homolog inhibits brome mosaic virus negative-strand RNA synthesis

The replication of positive-strand RNA viruses involves not only viral proteins but also multiple cellular proteins and intracellular membranes. In both plant cells and the yeast Saccharomyces cerevisiae, brome mosaic virus (BMV), a member of the alphavirus-like superfamily, replicates its RNA in endoplasmic reticulum (ER)-associated complexes containing viral 1a and 2a proteins. Prior to negative-strand RNA synthesis, 1a localizes to ER membranes and recruits both positive-strand BMV RNA templates and the polymerase-like 2a protein to ER membranes. Here, we show that BMV RNA replication in S. cerevisiae is markedly inhibited by a mutation in the host YDJ1 gene, which encodes a chaperone Ydj1p related to Escherichia coli DnaJ. In the ydj1 mutant, negative-strand RNA accumulation was inhibited even though 1a protein associated with membranes and the positive-strand RNA3 replication template and 2a protein were recruited to membranes as in wild-type cells. In addition, we found that in ydj1 mutant cells but not wild-type cells, a fraction of 2a protein accumulated in a membrane-free but insoluble, rapidly sedimenting form. These and other results show that Ydj1p is involved in forming BMV replication complexes active in negative-strand RNA synthesis and suggest that a chaperone system involving Ydj1p participates in 2a protein folding or assembly into the active replication complex.     

Assembly of alphavirus replication complexes from RNA and protein components in a novel trans-replication system in mammalian cells

For positive-strand RNA viruses, the viral genomic RNA also acts as an mRNA directing the translation of the replicase proteins of the virus. Replication takes place in association with cytoplasmic membranes, which are heavily modified to create specific replication compartments. Here we have expressed by plasmid DNA transfection the large replicase polyprotein of Semliki Forest virus (SFV) in mammalian cells from a nonreplicating mRNA and provided a separate RNA containing the replication signals. The replicase proteins were able to efficiently and specifically replicate the template in trans, leading to accumulation of RNA and marker gene products expressed from the template RNA. The replicase proteins and double-stranded RNA replication intermediates localized to structures similar to those seen in SFV-infected cells. Using correlative light electron microscopy (CLEM) with fluorescent marker proteins to relocate those transfected cells, in which active replication was ongoing, abundant membrane modifications, representing the replication complex spherules, were observed both at the plasma membrane and in intracellular endolysosomes. Thus, replication complexes are faithfully assembled and localized in the trans-replication system. We demonstrated, using CLEM, that the replication proteins alone or a polymerase-negative polyprotein mutant together with the template did not give rise to spherule formation. Thus, the trans-replication system is suitable for cell biological dissection and examination in a mammalian cell environment, and similar systems may be possible for other positive-strand RNA viruses.     

Complex dynamic development of poliovirus membranous replication complexes

Replication of all positive-strand RNA viruses is intimately associated with membranes. Here we utilize electron tomography and other methods to investigate the remodeling of membranes in poliovirus-infected cells. We found that the viral replication structures previously described as "vesicles" are in fact convoluted, branching chambers with complex and dynamic morphology. They are likely to originate from cis-Golgi membranes and are represented during the early stages of infection by single-walled connecting and branching tubular compartments. These early viral organelles gradually transform into double-membrane structures by extension of membranous walls and/or collapsing of the luminal cavity of the single-membrane structures. As the double-membrane regions develop, they enclose cytoplasmic material. At this stage, a continuous membranous structure may have double- and single-walled membrane morphology at adjacent cross-sections. In the late stages of the replication cycle, the structures are represented mostly by double-membrane vesicles. Viral replication proteins, double-stranded RNA species, and actively replicating RNA are associated with both double- and single-membrane structures. However, the exponential phase of viral RNA synthesis occurs when single-membrane formations are predominant in the cell. It has been shown previously that replication complexes of some other positive-strand RNA viruses form on membrane invaginations, which result from negative membrane curvature. Our data show that the remodeling of cellular membranes in poliovirus-infected cells produces structures with positive curvature of membranes. Thus, it is likely that there is a fundamental divergence in the requirements for the supporting cellular membrane-shaping machinery among different groups of positive-strand RNA viruses.     

Examples of expression systems based on animal RNA viruses: alphaviruses and influenza virus.

Successful recovery of RNA viruses and functional RNA replicons from cDNA has greatly facilitated molecular genetic analyses of viral proteins and cis-regulatory elements. This technology allows the use of RNA virus replication machinery to express heterologous sequences. Both positive-strand and negative-strand animal RNA viruses have been engineered to produce chimeric viruses expressing protective epitopes from other pathogens and for transient expression of heterologous sequences.     

Specific binding of host cell proteins to the 3''-terminal stem-loop structure of rubella virus negative-strand RNA.

At the 5' end of the rubella virus genomic RNA, there are sequences that can form a potentially stable stem-loop (SL) structure. The complementary negative-strand equivalent of the 5'-end SL structure of positive-strand rubella virus RNA [5' (+) SL structure] is thought to serve as a promoter for the initiation of positive-strand synthesis. We screened the negative-strand equivalent of the 5' (+) SL structure (64 nucleotides) and the adjacent region of the negative-strand RNA for their ability to bind to host cell proteins. Specific binding to the 64-nucleotide-long potential SL structure of three cytosolic proteins with relative molecular masses of 97, 79, and 56 kDa was observed by UV-induced covalent cross-linking. There was a significant increase in the binding of the 97-kDa protein from cells upon infection with rubella virus. Altering the SL structure by deleting sequences in either one of the two potential loops abolished the binding interaction. The 56-kDa protein also appeared to bind specifically to an SL derived from the 3' end of positive-strand RNA. The 3'-terminal structure of rubella virus negative-strand RNA shared the same protein-binding activity with similar structures in alphaviruses, such as Sindbis virus and eastern equine encephalitis virus. A possible role for the host proteins in the replication of rubella virus and alphaviruses is discussed.     

Identification and study of tobacco mosaic virus movement function by complementation tests

The phenomenon of trans-complementation of cell-to-cell movement between plant positive-strand RNA viruses is discussed with an emphasis on tobamoviruses. Attention is focused on complementation between tobamoviruses (coding for a single movement protein, MP) and two groups of viruses that contain the triple block of MP genes and require four (potato virus X) or three (barley stripe mosaic virus) proteins for cell-to-cell movement. The highlights of complementation data obtained by different experimental approaches are given, including (i) double infections with movement-deficient (dependent) and helper viruses; (ii) infections with recombinant viral genomes bearing a heterologous MP gene; (iii) complementation of a movement-deficient virus in transgenic plants expressing the MP of a helper virus; and (iv) co-bombardment of plant tissues with the cDNAs of a movement-dependent virus genome and the MP gene of a helper virus.