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We have characterized by T1 fingerprint analysis several defective interfering (DI) double-stranded RNAs of the simple yeast virus ScV. A common sequence of about 0.5 to 0.6 kilobase pairs, including both 3' termini of the parental RNA, was present in each DI RNA. Several DI RNAs had novel T1 oligonucleotides not present in their parental RNA.
Infection with human hepatitis C virus (HCV) as a result of a bilateral process of host-virus interactions. There are factors on both sides that contribute to clearance and to chronicity. Virus strategy to survive is built on several basic features. The first, recently recognized, is a wide cell tropism. HCV can infect not only hepatocytes, but also cells of immune system (B-cells, monocytes, macrophages, dendritic cells), epithelium, and immunologically privileged sites such as the central nervous system. Dendritic cells and platelets can also be passive virus carriers. Possibilities of virus clearance or abortive inapparent HCV infection at the stage of adsorption are considered. The second feature is rapid error-prone replication that leads to accumulation within one host of multiple virus variants (quasispecies). Viral heterogeneity could be multiplied by recombination of HCV genomic/subgenomic RNA molecules. Quasispecies nature gives virus an advantage in adaptation to varying host environment including availability of permissive cells, the presence of innate and adaptive immune response, and antiviral treatment. Analysis of HCV polymorphisms and their evolution rates may pinpoint the molecular (sequence) correlates of HCV clearance. The third feature is the capacity to modify or adapt host milieu. HCV core, envelope E2 and nonstructural NS2, NS3, NS5A proteins seem to hold a grip over the host cellular functions by down-regulating processes unfavorable and up-regulating processes favorable for virus replication and persistence. The relevance of the latter interactions to HCV infection outcome remains to be demonstrated. This review discusses recent developments in this area of HCV research.
The translational control involving internal ribosome binding occurs in poliovirus (PV), human rhinoviruses (HRV), encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), and hepatitis A virus (HAV). Internal ribosome binding utilizes cis-acting genetic elements of approximately 450 nucleotides (nt) termed the internal ribosome entry sites (IRES) found in these picornaviral 5'-untranslated region (5'UTR). Although these IRES elements are quite different in their primary sequence, a similar folding structure with a conserved 3' structural core exists in the IRES. Phylogenetic analysis and RNA folding of the 5' UTR of picornaviruses, including PV types 1-3, coxsackievirus types A and B, swine vesicular disease virus, echoviruses, enteroviruses (human and bovine), HRV, HAV, EMCV, mengovirus, Theiler's murine encephalomyelitis viruses, FMDV, and equine rhinoviruses, indicates that the predicted conserved structural core is indeed a general structural feature for all members of the picornavirus family. The evolution of a common structural core likely occurred by the gradual addition or deletion of structural domains and elements to preserve a similar tertiary structure that facilitates the utilization of the IRES in specific host-cell environments.
The molecular evolution of HCV 2c in a case of vertical transmission was studied by comparing the virus quasispecies in the sera from the mother and from the child in a two-year follow-up. The positivity of HCV-RNA since the delivery accounted for an in-utero infection. The Core-E1 genome region (nt 928-1225) was amplified by polymerase chain reaction (PCR) from serum samples collected at delivery and at 3, 9, 18 and 24 months after birth. The RIBA pattern was characterised by isolated anti-c22 positivity in the serum from mother and in sera from the child during the first 9 months. Additional presence of anti-c33 was observed afterwards. Genetic relatedness among isolates and with a mother minor variant serum (Mo1. 13) was found (mean variability ranged between 0.79% and 1.20%). From phylogenetic analysis this variant was identified as the origin of one of the two main lineages that included all isolates from child sera at 9, 18 and 24 months. The variability analysis has shown that high viral heterogeneity is present in the child serum collected at birth (3.16%). In this phase the dn/ds index (1.26%) indicates the presence of strong selective pressures. The development of child specific immune response at 9th month was concurrent with the disappearance of two mutants at positions 11 and 104 of E1. This rare case of in-utero mother-to-infant transmission can be considered as a model to elucidate the HCV quasispecies diversification during the first stage of infection.
Porcine reproductive and respiratory syndrome viruses (PRRSV) are divided into North American and European types, which show about 40% difference in their amino acid sequences. The divergence time of these two types has been estimated to be about 1980 from epidemiological data. This suggested that PRRSV have evolved at a higher evolutionary rate (order of 10(-2)/site/year) compared with other RNA viruses of 10(-3) to 10(-5)/site/year. Here, to test the evolutionary history of PRRSV speculated by the epidemiological background, we estimated the divergence time and evolutionary rate of PRRSV with molecular evolutionary analysis. Estimated divergence time (1972-1988) corresponded well to that estimated by the epidemiological data, and the evolutionary rate (4.71-9.8) x 10(-2) of PRRSV was indeed the highest among RNA viruses so far reported. Furthermore, we inferred important sites for the adaptation in order to examine how PRRSV have adapted to swine since they emerged. The adaptive sites were located not only in the epitopes related to immunity but also in the transmembrane regions including a signal peptide. In particular, the adaptive sites in the transmembrane regions were considered to affect compatibility to the host cell membrane. We conclude that PRRSV were transmitted from another host species to swine in about 1980 and have adapted to swine by altering the transmembrane regions.
The globin genes represent a complex set of sequences that are expressed in a coordinate fashion during the development of red blood cells. while this complex family of genes may consist of as many as ten to fourteen members [34], three of these genes have now been cloned and their entire nucleotide sequence determined. As was initially observed in the case of beta globin major gene, all are encoded in three distinct coding blocks separated by two intervening sequences of DNA. Their intervening sequences of DNA are preserved, with respect to location, but are widely divergent, with respect to size and sequence. The divided information in each gene is edited and spliced together at the level of its initial RNA transcript which is complementary to the entire gene sequence including its intervening sequences. Structural correlation analyses have allowed us to identify sites in all three genes that might be responsible for the initiation of transcription, RNA splicing, and poly A addition. The function of these sites has been tested by cloning these genes in an animal virus vector SV40. Such animal virus hybrids have been used to infect tissue culture cells and have directed the synthesis of both alpha and beta mouse globin in cells of monkey origin. These studies indicate that such signals operate across species barriers and further indicate that the animal virus vector system will be useful in elucidating their function.
In the context of yet unclarified issues of RNA interference (RNAi), it is discussed that RNAi-induced histone modification may not only have the purpose of inactivating native genes by blocking their transcription in the sense direction but may also simultaneously trigger transcription of the corresponding antisense strand to form double-stranded RNA for posttranscriptional gene-silencing in cells lacking RNA replicase activities. Invading foreign genetic traits may be posttranscriptionally silenced through complementary transcripts from specific, highly variable genomic regions, which are able to finally match any given sequence by the appropriate recombination and processing of their transcripts. The information to fight these traits may additionally become anchored in the genome, to provide at least a temporary "immunity" and may be inherited at least for a few generations. It is further proposed that: (1) RNA viruses evolved from constituents of the RNAi machinery through the capture of functions essential for their maintenance and replication and (2) viruses and RNAi are mutually interacting components of a universal and predominant genetic steering system that is involved in the modulation of gene expression on the cellular level and simultaneously constitutes a driving force for evolution, particularly in imperfect organisms. Such a model would deliver explanations for yet unresolved issues of RNAi, the clarification of which will have a significant impact on its future medical and biotechnological application.
The complete sequence (28580 nt) of the PUR46-MAD clone of the Purdue cluster of transmissible gastroenteritis coronavirus (TGEV) has been determined and compared with members of this cluster and other coronaviruses. The computing distances among their S gene sequences resulted in the grouping of these coronaviruses into four clusters, one of them exclusively formed by the Purdue viruses. Three new potential sequence motifs with homology to the alpha-subunit of the polymerase-associated nucleocapsid phosphoprotein of rinderpest virus, the Bowman-Birk type of proteinase inhibitors, and the metallothionein superfamily of cysteine rich chelating proteins have been identified. Comparison of the TGEV polymerase sequence with that of other RNA viruses revealed high sequence homology with the A-E domains of the palm subdomain of nucleic acid polymerases.
Reverse transcription of the Human Immunodeficiency Virus type I (HIV-1) RNA genome is primed by a cellular tRNA-lys3 molecule that binds to the primer binding site (PBS). The PBS is predicted to be part of an extended RNA structure, consisting of a small U5-PBS hairpin and a large U5-leader stem. In this study we stabilized the U5-leader stem of HIV-1 to study its role in reverse transcription. We tested in vitro synthesized wild-type and mutant templates in primer annealing, initiation and elongation assays. Stabilization of the stem inhibits the initiation of reverse transcription, but not the annealing of the tRNA primer onto the PBS. These results suggest that stabilization of the stem results in occlusion of a sequence motif that is involved in an additional interaction with the tRNA-lys3 primer and that is needed to trigger the initiation of reverse transcription. The stable structure was also found to affect the elongation of reverse transcription, causing the RT enzyme to pause upon copying 7-8 bases into the extended base paired stem. The stabilizing mutations were also introduced into proviral constructs for replication studies, demonstrating that the mutant viruses have a reduced replication capacity. Analysis of a revertant virus demonstrated that opening of the stabilized U5-leader stem can restore both virus replication and reverse transcription.
Seoul (SEO) viruses belong to the Hantavirus genus (family Bunyaviridae), cause the moderate form of haemorrhagic fever with renal syndrome, and have been associated with laboratory-acquired infections in many countries. To investigate the pedigree of an isolate, designated IR461, which was obtained from a laboratory-acquired infection in a UK research institute, we determined the nucleotide sequences of the small (S) and medium (M) genome segments. In addition, we determined the sequences of the S segments of two Chinese isolates (R22 and L99) and an American isolate (Tchoupitoulas [TCH]). The S segments range within 1769-1785 nucleotides in length and showed identities of >88% in nucleotide sequence and 97% in amino acid sequence to those of published S segment sequences. The M RNA segment of IR461 is 3651 nucleotides long and shows >84% identity at the nucleotide level and >98% at the amino acid level to the M segments of other SEO viruses. These data confirm that SEO viruses show the least diversity within the Hantavirus genus. Phylogenetic analyses of the sequences showed geographic clustering of the Chinese SEO viruses, and that IR461 was more closely related to SEO viruses isolated in the New World than to those from the Far East.
Foot-and-mouth disease virus (FMDV) is an aphthovirus of the family Picornaviridae and the etiological agent of the economically most important animal disease. As a typical picornavirus, FMD virions are nonenveloped particles of icosahedral symmetry and its genome is a single stranded RNA of about 8500 nucleotides and of positive polarity. FMDV RNA is infectious and it replicates via a complementary, minus strand RNA. FMDV RNA replication is error-prone so that viral populations consist of mutant spectra (quasispecies) rather than a defined genomic sequence. Therefore FMDV in nature is genetically and antigenically diverse. This poses important challenges for the diagnosis, prevention and control of FMD. A deeper understanding of FMDV population complexity and evolution has suggested requirements for a new generation of anti-FMD vaccines. This is relevant to the current debate on the adequacy of non-vaccination versus vaccination policies for the control of FMD.
Outbreaks of gastroenteritis are a major public health problem in New Zealand. The introduction of molecular detection methods has now shown that the 'Norwalk-like viruses' (NLVs) are the major cause of food and waterborne nonbacterial gastroenteritis. Reverse transcription and polymerase chain reaction (RT-PCR) were used to determine the presence of NLVs in faecal specimens from 83 nonbacterial gastroenteritis outbreaks occurring in New Zealand between August 1995 and July 1999. Further characterisation of the NLVs for epidemiological purposes was carried out by dot blot DNA hybridisation and DNA sequencing of representative outbreak strains. The majority of NLV strains occurring in New Zealand since August 1995 are similar to those occurring overseas. The predominant New Zealand strain is genetically similar to the Bristol/Lordsdale virus group. Several New Zealand outbreaks were attributed to Auckland virus, a Mexico-like NLV strain identified as the most likely cause of gastroenteritis after consumption of contaminated oysters in 1994. A new strain, designated Napier virus, has been identified in six outbreaks since 1996. A number of strains closely resembling internationally recognised strains, including Southampton virus, Saratoga virus; Desert Shield virus and Melksham virus have been associated with gastroenteritis outbreaks across New Zealand. Application of these typing methods has provided information on disease transmission for epidemiological investigations of public health significance.
According to phylogenetic data, about 100 years ago an avian influenza virus passed the species barrier (possibly first) to pigs and (possibly from there) to humans. In 1979 an avian influenza A virus (as a whole, without reassortment) again entered the pig population in northern Europe, forming a stable lineage. Here it is shown that the early North European swine viruses exhibit higher than normal evolutionary rates and are highly variable with respect to plaque morphology and neutralizability by monoclonal antibodies. Our results are consistent with the idea that, in order to pass the species barrier, an influenza A virus needs a mutator mutation to provide an additional number of variants, from which the new host might select the best fitting ones. A mutator mutation could be of advantage under such stress conditions and might enable a virus to pass the species barrier as a whole even twice, as it seems to have happened about 100 years ago. This stressful situation should be over for the recent swine lineage, since the viruses seem to be adapted already to the new host in that the most recent isolates--at least in northern Germany--are genetically stable and seem to have lost the putative mutator mutation again.
A wide variety of different genetic elements and DNA sequences have used reverse transcription in their replication or transposition. Some of these elements may be evolutionarily related and show a continued evolution to autonomy from the cell genome. The evolution of a cancer reflects a similar drive for autonomy from the organism.
The genome sequence of tick-borne encephalitis (TBE) virus (Western subtype vaccine strain Neudoerfl) was determined. This extends the previously published sequence of the structural proteins to the nonstructural protein region and noncoding sequences at the 5'- and 3'-termini. The amino-termini of the individual proteins were assigned by comparison with other flavivirus sequences. Amino acid homology calculations between TBE virus and mosquito-borne flaviviruses were performed for all nonstructural proteins. An evolutionary tree based on protein NS1 is presented that reveals the molecular basis of relationships among flaviviruses. Tick-borne and mosquito-borne flaviviruses share a common hydrophilicity profile and also other features of their primary sequences, such as the presumably functional Gly-Asp-Asp sequence element within protein NS5. Other characteristics, such as the potential N-glycosylation sites of protein NS1 and a potential proteolytic cleavage site within protein NS4B, are conserved within the mosquito-borne group, but differ in the TBE virus sequence.
In vitro virus is a molecular construct for in vitro protein evolution, which requires some mechanism to link phenotype to genotype. The first in vitro virus was realized by bonding a nascent protein with its coding mRNA via puromycin in in vitro translation. We report a new construct of in vitro DNA virus. The virion was a covalent cDNA-protein fusion, and virion formation did not require any modification of mRNA. Due to intactness of mRNA, this type of in vitro DNA virus will take the next step toward in vitro autonomous evolution, just like in vivo viral evolution in a cellstat.
By capitalizing on the initially puzzling observations of unpredictable transgene silencing and variable expression, plant scientists have pioneered research into a novel type of epigenetic regulation, termed homology-dependent gene silencing. This silencing process has implications for natural mechanisms of gene expression in plants and other eukaryotes, and has branched out into studies of reversible DNA modifications; RNA metabolism, transport and processing; and host responses to plant viruses, viroids and transposable elements. The analysis of transgene silencing systems has enriched our understanding of other epigenetic phenomena, including paramutation, as well as heterosis and genome evolution. This research is also highly relevant to the biotechnology industry, which is interested in avoiding unwanted transgene silencing in genetically engineered lines and in exploiting various types of silencing to inactivate specific genes. Homology-dependent gene silencing can also be used in high-throughput approaches for functional genomics.
One of the most important families of antibiotics are the aminoglycosides, including drugs such as neomycin B, paromomycin, gentamicin and streptomycin. With the discovery of the catalytic potential of RNA, these antibiotics became very popular due to their RNA-binding capacity. They serve for the analysis of RNA function as well as for the study of RNA as a potential therapeutic target. Improvements in RNA structure determination recently provided first insights into the decoding site of the ribosome at high resolution and how aminoglycosides might induce misreading of the genetic code. In addition to inhibiting prokaryotic translation, aminoglycosides inhibit several catalytic RNAs such as self-splicing group I introns, RNase P and small ribozymes in vitro. Furthermore, these antibiotics interfere with human immunodeficiency virus (HIV) replication by disrupting essential RNA-protein contacts. Most exciting is the potential of many RNA-binding antibiotics to stimulate RNA activities, conceiving small-molecule partners for the hypothesis of an ancient RNA world. SELEX (systematic evolution of ligands by exponential enrichment) has been used in this evolutionary game leading to small synthetic RNAs, whose NMR structures gave valuable information on how aminoglycosides interact with RNA, which could possibly be used in applied science.