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A gene homologous to topoisomerase II in African swine fever virus.

A putative topoisomerase II gene of African swine fever virus was mapped using a degenerate oligonucleotide probe derived from a region highly conserved in type II topoisomerases. The gene is located within EcoRI fragments P and H of the African swine fever virus genome. Sequencing of this region has revealed a long open reading frame, designated P1192R, encoding a protein of 1192 amino acids, with a predicted molecular weight of 135,543. Open reading frame P1192R is transcribed late after infection into a 4.6-kb RNA. The deduced amino acid sequence of this open reading frame shares significant similarity with topoisomerase II sequences from different sources, with percentages of identity between 23 and 29%. The evolutionary relationships among the topoisomerase II sequences of ASF virus, eukaryotes and prokaryotes were analyzed and a phylogenetic tree was established. The tree indicates that the ASF virus topoisomerase II gene was present in the virus genome before protozoa, yeasts, and metazoa diverged.

African Swine Fever Virus↗

The molecular basis of the evolution of sex.

Traditionally, sexual reproduction has been explained as an adaptation for producing genetic variation through allelic recombination. Serious difficulties with this explanation have led many workers to conclude that the benefit of sex is a major unsolved problem in evolutionary biology. A recent informational approach to this problem has led to the view that the two fundamental aspects of sex, recombination and outcrossing, are adaptive responses to the two major sources of noise in transmitting genetic information, DNA damage and replication errors. We refer to this view as the repair hypothesis, to distinguish it from the traditional variation hypothesis. On the repair hypothesis, recombination is a process for repairing damaged DNA. In dealing with damage, recombination produces a form of informational noise, allelic recombination, as a by-product. Recombinational repair is the only repair process known which can overcome double-strand damages in DNA, and such damages are common in nature. Recombinational repair is prevalent from the simplest to the most complex organisms. It is effective against many different types of DNA-damaging agents, and, in particular, is highly efficient in overcoming double-strand damages. Current understanding of the mechanisms of recombination during meiosis suggests that meiosis is designed for repairing DNA. These considerations form the basis for the first part of the repair hypothesis, that recombination is an adaptation for dealing with DNA damage. The evolution of sex can be viewed as a continuum on the repair hypothesis. Sex is presumed to have arisen in primitive RNA-containing protocells whose sexual process was similar to that of recombinational repair in extent segmented, single-stranded RNA viruses, which are among the simplest known organisms. Although this early form of repair occurred by nonenzymatic reassortment of replicas of undamaged RNA segments, it evolved into enzyme-mediated breakage and exchange between long DNA molecules. As some lines of descent became more complex, their genome information increased, leading to increased vulnerability to mutation. The diploid stage of the sexual cycle, which was at first transient, became the predominant stage in some lines of descent because it allowed complementation, the masking of deleterious recessive mutations. Out-crossing, the second fundamental aspect of sex, is also maintained by the advantage of masking mutations. However, outcrossing can be abandoned in favor of parthenogenesis or selfing under conditions in which the costs of mating are very high.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Ross River virus genetic variants in Australia and the Pacific Islands.

HaeIII and TaqI restriction digest profiles of cDNA to infected cell RNA or virion RNA were used as a guide to genetic relationships between fourteen isolates of Ross River virus (RRV) obtained from mosquitoes collected in various localities in eastern Australia where the virus is endemic. RRV isolates from Fiji, American Samoa, the Cook Islands and the Wallis Islands where major outbreaks of epidemic polyarthritis took place in 1979-1980 were also examined. Among these RRV isolates we have identified three genetic types (I-III) on the basis of differences between their restriction digest profiles. We estimate that 1.5-5% nucleotide sequence diversity exists between genetic types. Within each genetic type strain differentiation gave rise to small but significant differences in restriction digest profiles. No clear pattern of geographic distribution of RRV genetic types could be established from the limited number of RRV isolates examined. Genetic types I, II and III, respectively, were isolated from three, three and one different mosquito species, indicating there is no strong association between genetic type and the species of mosquito vector. HaeIII restriction digest analysis did not detect any genetic difference between the four Pacific Island isolates, suggesting that a single RRV variant was involved in the epidemics. Genetically, this variant was closely related to isolates of genetic type II. Virtually identical HaeIII restriction digest profiles were observed for isolates obtained at various stages of the Pacific Island epidemics, suggesting that extensive sequence evolution did not accompany Ross River virus spread.

Alphavirus↗

Nucleotide sequence of sweet clover necrotic mosaic dianthovirus RNA-1.

The complete nucleotide sequence of sweet clover necrotic mosaic dianthovirus (SCNMV) RNA-1 has been determined. RNA-1 consists of 3876 nucleotides in length, containing three large open reading frames (ORFs). The 5'-proximal, internal and 3'-terminal ORFs potentially encode 27-kDa, 57-kDa and 37-kDa proteins, respectively. The frameshift event between the C-terminus of the 27-kDa protein and extension of the N-terminus of the 57-kDa protein may result in the formation of a 88-kDa protein which is presumed to be a replicase. The 37-kDa coat protein ORF is located immediately downstream of the 57-kDa ORF. The same genome organization and high similarity (80-92%) of both the nucleotide sequences and the deduced amino acid sequences between red clover necrotic mosaic dianthovirus and SCNMV suggest that they originate from a common progenitor, but have divergent evolution later. Striking similarity was detected between the putative RNA-dependent RNA polymerase of SCNMV and that of the tombus-, carmo-, necro-, machlomo- and luteoviruses, supporting a proposal that they belong to the same virus supergroup although there is a relatively low degree of coat protein sequence similarity in these viruses.

Amino Acid Sequence↗

Host-induced avirulence of hibiscus chlorotic ringspot virus mutants correlates with reduced gene-silencing suppression activity.

Post-transcriptional gene silencing (PTGS) and virus-encoded gene-silencing suppressors are defence and counterdefence strategies developed by host and pathogens during evolution. Using a green fluorescence protein-based transient suppression system, the coat protein (CP) of Hibiscus chlorotic ringspot virus (HCRSV) was identified as a strong gene-silencing suppressor. CP suppressed sense RNA-induced but not dsRNA-induced local and systemic PTGS. This is different from another virus in the genus Carmovirus, Turnip crinkle virus (TCV), the CP of which strongly suppresses dsRNA-induced PTGS. HCRSV CP domain deletion mutants lost their suppression function, indicating that the complete CP is essential for suppression of PTGS. When CP was expressed from a Potato virus X (PVX) vector, it was able to enhance the symptom severity and to increase the accumulation of PVX RNA. Here, it is proposed that HCRSV CP suppresses PTGS at the initiation step, which is different from TCV CP. In addition, a previous study demonstrated that CP mutants resulting from serial passage of HCRSV in its local lesion host also showed a significantly reduced suppression function, indicating that host-induced mutations that lead to avirulence of HCRSV in kenaf correlate with its reduced ability to suppress PTGS.

Capsid Proteins↗

Computational design of antiviral RNA interference strategies that resist human immunodeficiency virus escape.

Recently developed antiviral strategies based upon RNA interference (RNAi), which harnesses an innate cellular system for the targeted down-regulation of gene expression, appear highly promising and offer alternative approaches to conventional highly active antiretroviral therapy or efforts to develop an AIDS vaccine. However, RNAi is faced with several challenges that must be overcome to fully realize its promise. Specifically, it degrades target RNA in a highly sequence-specific manner and is thus susceptible to viral mutational escape, and there are also challenges in delivery systems to induce RNAi. To aid in the development of anti-human immunodeficiency virus (anti-HIV) RNAi therapies, we have developed a novel stochastic computational model that simulates in molecular-level detail the propagation of an HIV infection in cells expressing RNAi. The model provides quantitative predictions on how targeting multiple locations in the HIV genome, while keeping the overall RNAi strength constant, significantly improves efficacy. Furthermore, it demonstrates that delivery systems must be highly efficient to preclude leaving reservoirs of unprotected cells where the virus can propagate, mutate, and eventually overwhelm the entire system. It also predicts how therapeutic success depends upon a relationship between RNAi strength and delivery efficiency and uniformity. Finally, targeting an essential viral element, in this case the HIV TAR region, can be highly successful if the RNAi target sequence is correctly selected. In addition to providing specific predictions for how to optimize a clinical therapy, this system may also serve as a future tool for investigating more fundamental questions of viral evolution.

Computational Biology↗

Sequence analysis of cloned dengue virus type 2 genome (New Guinea-C strain).

Sequences totalling 5472 nucleotides (nt) from four complementary DNA (cDNA) clones of the dengue virus type 2 (DEN-2) RNA (New Guinea strain, NGS-C) have been reported previously [Yaegashi et al., Gene 46 (1986) 257-267; Putnak et al., Virology 163 (1988) 93-103]. This report describes the complete nucleotide sequence, with the exception of about 7 nt at the 5'-noncoding region, of this RNA genome derived from several cDNA clones. It is 10,723 nt in length and contains a single long open reading frame of 10,173 nt, encoding a polyprotein of 3391 amino acids. The genomic organization is similar to that of other flaviviruses that have recently been reported. Among the three DEN-2 strains - the Jamaica genotype (DEN-2JAM), the DEN-2NGS-C, and the S1 candidate vaccine strain derived from Puerto Rico (PR)-159 isolate (DEN-2S1) - which have been sequenced to date, the amino acid sequences of the polyproteins bear 94%-99% similarity. When the amino acid sequences of DEN-2NGS-C are compared with those of the other two strains, the variations are greater in the DEN-2S1 than in the DEN-2JAM. When DEN-2 and DEN-4 are compared, the overall amino acid identities range from 30% to 80% in both the structural and nonstructural proteins; whereas between DEN-2 and DEN-1, they range from 68% to 79% in the region encoding the structural proteins and the nonstructural protein NS1.

Amino Acid Sequence↗

Bunyavirus-vector interactions.

Recent advances in the genetics and molecular biology of bunyaviruses have been applied to understanding bunyavirus-vector interactions. Such approaches have revealed which virus gene and gene products are important in establishing infections in vectors and in transmission of viruses. However, much more information is required to understand the molecular mechanisms of persistent infections of vectors which are lifelong but apparently exert no untoward effect. In fact, it seems remarkable that LAC viral antigen can be detected in almost every cell in an ovarian follicle, yet no untoward effect on fecundity and no teratology is seen. Similarly the lifelong infection of the vector would seem to provide ample opportunity for bunyavirus evolution by genetic drift and, under the appropriate circumstances, by segment reassortment. The potential for bunyavirus evolution by segment reassortment in vectors certainly exists. For example the Group C viruses in a small forest in Brazil seem to constitute a gene pool, with the 6 viruses related alternately by HI/NT and CF reactions, which assay respectively M RNA and S RNA gene products (Casals and Whitman, 1960; Shope and Causey, 1962). Direct evidence for naturally occurring reassortant bunyaviruses has also been obtained. Oligonucleotide fingerprint analyses of field isolates of LAC virus and members of the Patois serogroup of bunyaviruses have demonstrated that reassortment does occur in nature (El Said et al., 1979; Klimas et al., 1981; Ushijima et al., 1981). Determination of the genotypic frequencies of viruses selected by the biological interactions of viruses and vectors after dual infection and segment reassortment is an important issue. Should a virus result that efficiently interacts with alternate vector species, the virus could be expressed in different circumstances with serious epidemiologic consequences. Dual infection of vectors with different viruses is not unlikely, because many bunyaviruses are sympatric in nature. For example, the Ae. trivittatus-cottontail rabbit and the Ae. triseriatus-squirrel arbovirus cycles are sympatric in the ecotone between their respective grassland and forest ecosystems (LeDuc, 1979). Should a LaCrosse virus variant or reassortant evolve that was efficiently vectored by Ae. trivittatus mosquitoes, significantly more human infections with La Crosse virus would likely occur. Unlike Ae. triseriatus, Ae. trivittatus mosquitoes are not restricted to forested areas and consequently are more likely to encounter and to feed upon humans.(ABSTRACT TRUNCATED AT 400 WORDS)

Aedes↗

Genetic lesions associated with Muller's ratchet in an RNA virus.

The molecular basis of Muller's ratchet has been investigated using the important animal pathogen foot-and-mouth disease virus (FMDV). Clones from two FMDV populations were subjected to serial plaque transfers (repeated bottleneck events) on host BHK-21 cells. Relative fitness losses were documented in 11 out of 19 clones tested. Small fitness gains were observed in three clones. One viral clone attained an extremely low plating efficiency, suggesting that accumulation of deleterious mutations had driven the virus near extinction. Nucleotide sequence analysis revealed unique genetic lesions in multiply transferred clones that had never been seen in FMDVs isolated in nature or subjected to massive infections in cell culture. In particular, a frequent internal polyadenylate extension has identified a mutational hot spot on the FMDV genome. Furthermore, amino acid residue substitutions in internal capsid sites which are severely restricted during FMDV evolution, amounted to half of capsid replacements in the transferred clones. In addition, a striking dominance of non-synonymous replacements fixed upon large population infections of FMDV was not observed upon serial plaque transfers. The nucleotide sequence of the entire genome of a severely debilitated clone suggests that very few mutations may be sufficient to drive FMDV near extinction. The results provide an account of the molecular basis of Muller's ratchet for an RNA virus, and insight into the types of genetic variants which populate the mutant spectra of FMDV quasispecies.

Animals↗

Organization of the 3'-terminal half of beet yellow stunt virus genome and implications for the evolution of closteroviruses.

The 3'-terminal half of the beet yellow stunt virus (BYSV) genome 10,545 nt, has been cloned and sequenced. The sequenced portion of the BYSV genome encompasses 10 open reading frames (ORFs) and 241 nt of the 3' untranslated region. The sequence spans, in the 5' to 3' direction, the C-terminal region of the replication-associated polyprotein gene (ORF 1a) which includes the set of motifs typical of helicases (HEL), the entire 53-kDa polymerase (RdRp) gene (ORF 1b), and genes encoding 30-kDa (ORF 2), 6-kDa (ORF 3), 66-kDa (ORF 4), 61-kDa (ORF 5), 25-kDa (ORF 6), 23.7-kDa (coat protein, CP) (ORF 7), 18-kDa (ORF 8), and 22-kDa (ORF 9) proteins. The double-stranded RNA "replicative form" of the BYSV was demonstrated to have a nontemplate G residue at the 3' terminus of the (+) strand. The RdRp of BYSV is presumably expressed via a +1 ribosomal frameshift. The five-gene module conserved among closteroviruses was identified in BYSV; it includes a gene array coding for a 6-kDa small hydrophobic protein, a 66-kDa homolog of the cellular HSP70 heat shock proteins, a 61-kDa protein, and a 25-kDa diverged copy of the CP followed by the CP gene itself. Phylogenetic analysis of the replication-associated HEL and RdRp domains as well as proteins from the five-gene module demonstrated the closest relationship between BYSV and two other closteroviruses, beet yellows (BYV) and citrus tristeza (CTV) viruses. Like CTV, the BYSV genome contains a 30-kDa protein gene between the RdRp and the 6-kDa protein genes, and like BYV it has only two genes downstream of the CP gene. The organization of the BYSV genome appears to be intermediate between BYV and CTV, which suggests that these three viruses might represent three distinct but probably close stages in the closterovirus evolution.

Amino Acid Sequence↗

Efficiency of measles virus entry and dissemination through different receptors.

The efficiency with which different measles virus (MV) strains enter cells through the immune cell-specific protein SLAM (CD150) or other receptors, including the ubiquitous protein CD46, may influence their pathogenicity. We compared the cell entry efficiency of recombinant MV differing only in their attachment protein hemagglutinin (H). We constructed these viruses with an additional gene expressing an autofluorescent reporter protein to allow direct detection of every infected cell. A virus with a wild-type H protein entered cells through SLAM two to three times more efficiently than a virus with the H protein of the attenuated strain Edmonston, whereas cell entry efficiency through CD46 was lower. However, these subtle differences were amplified at the cell fusion stage because the wild-type H protein failed to fuse CD46-expressing cells. We also proved formally that a mutation in H protein residue 481 (asparagine to tyrosine) results in improved CD46-specific entry. To define the selective pressure exerted on that codon, we monitored its evolution in different H protein backgrounds and found that several passages in CD46-expressing Vero cells were necessary to shift it in the majority of the MV RNA. To verify the importance of these observations for human infections, we examined MV entry into peripheral blood mononuclear cells and observed that viruses with asparagine 481 H proteins infect these cells more efficiently.

Amino Acid Sequence↗

Selection of DNA aptamers that bind the RNA-dependent RNA polymerase of hepatitis C virus and inhibit viral RNA synthesis in vitro.

The RNA-dependent RNA polymerase (NS5B) of the hepatitis C virus (HCV) plays a key role in the life cycle of the virus. In order to find inhibitors of the HCV polymerase, we screened a library of 81 nucleotide (nt)-long synthetic DNA containing 35 random nucleotides by the Systematic Evolution of Ligands by Exponential enrichment (SELEX) approach. Thirty ligands selected for their binding affinity to the NS5B were classified into four groups on the basis of their sequence homologies. Among the selected molecules, two were able to inhibit in vitro the polymerase activity of the HCV NS5B. These aptamers appeared to be specific for HCV polymerase, as no inhibition of poliovirus 3D polymerase activity was observed. The binding and inhibitory potential of one aptamer (27v) was associated with the 35 nt-long variable region. This oligonucleotide displayed an apparent dissociation constant (K(d)) in the nanomolar range. Our results showed that it was able to compete with RNA templates corresponding to the 3'-ends of the (+) and the (-) HCV RNA for binding to the polymerase. The fact that a DNA aptamer could interfere with the binding of natural templates of the enzyme could help in performing structure-function analysis of the NS5B and might constitute a basis for further structure-based drug design of this crucial enzyme of HCV replication.

Base Sequence↗

[Comparative study of low molecular weight RNA in various taxonomic groups].

snRNAs of rat, mouse, hamster, chicken, frog, Drosophila and yeast were studied. Mammals are characterized by the highest content of snRNAs. Most evolutionary conservative are 8SII and 8SIII RNAs which were present in all organisms studied. On the contrary 4.5S RNA was found only in mammals and birds. These data accord with the notion that the eldest in the family of snRNAs are U3 RNA involved in pre-rRNA processing and 8S polysomal RNA, while U5-U6 RNAs are more recent evolutionary achievements. In the frog at different stages of development the relative amount and the number of fractions of snRNAs differ significantly. In embryonic and regenerating rat liver the differences are less pronounced. Extra fractions of snRNAs were found in virus transformed chick fibroblasts and in ascite hepatomas of rat.

Animals↗

Functional dissection of the eukaryotic-specific tRNA-interacting factor of lysyl-tRNA synthetase.

In the cytoplasm of higher eukaryotic cells, aminoacyl-tRNA synthetases (aaRSs) have polypeptide chain extensions appended to conventional prokaryotic-like synthetase domains. The supplementary domains, referred to as tRNA-interacting factors (tIFs), provide the core synthetases with potent tRNA-binding capacities, a functional requirement related to the low concentration of free tRNA prevailing in the cytoplasm of eukaryotic cells. Lysyl-tRNA synthetase is a component of the multi-tRNA synthetase complex. It exhibits a lysine-rich N-terminal polypeptide extension that increases its catalytic efficiency. The functional characterization of this new type of tRNA-interacting factor has been conducted. Here we describe the systematic substitution of the 13 lysine or arginine residues located within the general RNA-binding domain of hamster LysRS made of 70 residues. Our data show that three lysine and one arginine residues are major building blocks of the tRNA-binding site. Their mutation into alanine led to a reduced affinity for tRNA(3)(Lys) or minimalized tRNA mimicking the acceptor-TPsiC stem-loop of tRNA(3)(Lys) and a decrease in catalytic efficiency similar to that observed after a complete deletion of the N-terminal domain. Moreover, covalent continuity between the tRNA-binding and core domain is a prerequisite for providing LysRS with a tRNA binding capacity. Thus, our results suggest that the ability of LysRS to promote tRNA(Lys) networking during translation or to convey tRNA(3)(Lys) into the human immunodeficiency virus type 1 viral particles rests on the addition in evolution of this tRNA-interacting factor.

Amino Acid Sequence↗

Loss of antiretroviral drug susceptibility at low viral load during early virological failure in treatment-experienced patients.

BACKGROUND: Clinical studies have demonstrated a correlation between the response to second-line antiretroviral therapy and the number of drugs in the regimen to which the virus is susceptible. These studies have largely been performed in patients with viral loads over 1000 copies/ml. OBJECTIVES: To examine the evolution of resistance during early virological failure, and the potential role of susceptibility testing in patients with low viral loads (below 1000 copies/ml), in treatment-experienced patients. METHODS: Drug susceptibility and genotypes of HIV-1 from indinavir-experienced patients undergoing therapy with nelfinavir, saquinavir, abacavir and either a second nucleoside reverse transcriptase inhibitor (NRTI) or nevirapine were determined. RESULTS: Sixteen subjects were studied. Five of the ten subjects treated with nevirapine, and one of six treated with a second NRTI, achieved and maintained plasma HIV RNA < 500 copies/ml. Virus from the treatment failures lost susceptibility to one or more treatment drugs, including nelfinavir and/or saquinavir, after 4 to 36 weeks of treatment. In six of the ten failures, virus with new reductions in drug susceptibility was detected prior to failure. In five of the six failures who had at least one plasma sample with a viral load between 50 and 1000 copies/ml, reductions in susceptibility to one or more treatment drugs were detected (viral load range: 260 to 630 copies/ml). CONCLUSIONS: Drug resistance can be detected at viral loads below 1000 copies/ml which may be predictive of treatment failure. Failure of a second line regimen was typically associated with early evolution of resistance in HIV protease.

Anti-HIV Agents↗

Heterogeneous forms of polymerase proteins exist in influenza A virus-infected cells.

In influenza virus-infected cells a virus coded polymerase that consists of three polypeptide subunits, namely PB1, PB2 and PA, mediates both transcription and replication. Radioimmunoprecipitation with monospecific antisera to each of the polymerase proteins revealed additional forms of PB1 and PA proteins in infected cells. PA antiserum detected two additional proteins of 62k and 60k and PB1 antiserum recognized two additional proteins of 85k and 70k. Further investigation was carried out on the 62k PA and 85k PB1 related proteins. Limited proteolysis peptide mapping showed that these proteins are subsets of their normal counter-parts. These new forms of polymerase proteins are designated as "b" forms (PAb and PB1b) to distinguish them from the previously recognized forms designated as "a" forms (PAa and PB1a). Both PAb and PB1b proteins were found in cells infected with all the influenza type A viruses tested indicating that they are evolutionarily conserved. Pulse chase experiments showed that the "b" forms are not derived from "a" forms. This suggested that "b" forms are translated independently. The "b" forms were not detected in purified virus but were found to be associated with intracellular RNP templates, suggesting a role for these proteins in intracellular virus replication events.

Animals↗

Molecular biology of bovine viral diarrhea virus and its interactions with the host.

The contributions of pestivirus molecular biology research to our understanding of Bovine Viral Diarrhea Virus (BVDV) biology and disease have been remarkable. Completion of nucleotide sequence information for genomes of NCP and CP-BVDV isolates was an important milestone. Subsequent work on the protein map of BVDV and polyprotein processing pathways paved the way for the interpretation of many other virologic and immunologic studies. Discovery of a correlation between genotype II and virulence (hemorrhagic syndrome) will help to clarify previously controversial data and to improve disease control. Description of multiple pathways of p80 expression in CP-BVDV offered insight into the pathogenesis of mucosal disease. Identification of gp53/ E2 as the target of neutralizing antibodies and source of antigenic hypervariability helped us to understand immunity to BVDV. Collectively, the advances described contribute to the implementation of improved diagnostic and control strategies to reduce losses inflicted by the bovine pestivirus.

Animals↗