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PubMed1978

Immunologic approaches toward detection of type C viral expression in man.

Type C RNA viruses have been isolated from a large number of mammalian species. These agents may be horizontally transmitted as infectious cancer-inducing agents, or vertically transmitted from one generation to the next, often in an unexpressed form, within the host genome. To date, the translational products of three viral genes have been identified. With purified virus-coded proteins as probes, sensitive and highly specific radioimmunologic assays have been developed for the detection of antibodies and antigens related to the known type C viruses. These techniques have proved valuable in sero-epidemiologic studies of the horizontally transmitted oncogenic viruses of cats, cattle, and gibbons, and have been used to detect translational products of endogenous viruses in tissues of species from which complete virus has yet to be isolated. This review describes the application of radioimmunoassays in the search for immunologic evidence of type C virus expression in man.

Animals↗
PubMed1999

Lack of evolutionary stasis during alternating replication of an arbovirus in insect and mammalian cells.

The evolution of vesicular stomatitis virus (VSV) in a constant environment, consisting of either mammalian or insect cells, has been compared to the evolution of the same viral population in changing environments consisting in alternating passages in mammalian and insect cells. Fitness increases were observed in all cases. An initial fitness loss of VSV passaged in insect cells was noted when fitness was measured in BHK-21 cells, but this effect could be attributed to a difference of temperature during VSV replication at 37 degrees C in BHK-21 cells. Sequencing of nucleotides 1-4717 at the 3' end of the VSV genome (N, P, M and G genes) showed that at passage 80 the number of mutations accumulated during alternated passages (seven mutations) is similar or larger than that observed in populations evolving in a constant environment (two to four mutations). Our results indicate that insect and mammalian cells can constitute similar environments for viral replication. Thus, the slow rates of evolution observed in natural populations of arboviruses are not necessarily due to the need for the virus to compromise between adaptation to both arthropod and vertebrate cell types.

Adaptation, Physiological↗
PubMed2005

[Studies on construction of artificial mutants of Cucumber mosaic virus satellite RNA and their biological activity].

Based on the full length cDNA clone of a Cucumber mosaic virus satellite RNA, which was 369nt in size, artificial mutants were developed by the method of error-prone PCR and DNA shuffling. The new satellite cDNAs were transcribed in vitro into ssRNA and pseudo-recombined with a helper Cucumber mosaic virus, which contains no satellite RNA. Sequence analysis showed that A to T/G or G to A replacement all the four mutants, named MS1, MS5, MS6 and MS11 respectively, and there is no C to G or G to C replacement, but amongst, only the mutants MS11 could replicated when recombined with the helper virus strain. No satellite RNA could be detected by RT-PCR amplification and double-stranded RNA analysis for those pseudo-recombination constitution of Cucumber mosaic virus strain with mutants MS1, MS5 and MS6.Sequence homological comparison showed that the single replacement of mutants MS1, MS5 and MS6 occurred in the highly conservative regions and the T to A replacement of mutant MS11 was located in the normal-variation region. This is the first artificial mutation of satellite RNA of plant RNA viruses. The results indicated that single base in the region of satellite RNA maybe important to maintaining the biological activity of satellite RNA for its replication and stability. The variation and evolution of satellite RNA could be hopefully studied through combination directed evolution by DNA shuffling with pseudo-recombination in vitro.

Cucumber Mosaic Virus Satellite↗
PubMed1988

Measles virus L protein evidences elements of ancestral RNA polymerase.

We have determined the nucleotide sequence of the measles virus (MV) L gene using a cDNA library encompassing the entire MV genome (J. Crowley et al. (1987) Intervirology, 28, 65-77). The L gene is 6639 nucleotides in length, and contains a single long open reading frame that could code for a protein of 247,611 kDa. Both the L gene and in particular the predicted L protein of MV bear substantial homology to their counterparts in Sendai virus and Newcastle disease virus, suggesting that the multifunctional nature of paramyxovirus L proteins imposes strong evolutionary constraints. The predicted MV L protein also contains distinct elements of a postulated ancestral RNA polymerase.

Amino Acid Sequence↗
PubMed1997

Is group selection a factor modulating the virulence of RNA viruses?

RNA viruses consist of populations of extremely high genetic heterogeneity called quasispecies. Based on theoretical considerations, it has been suggested that the unit of selection in such complex genetic populations is not the single viral particle but a set of genetically related particles which form the quasispecies. In the present study we carried out a set of experiments with the vesicular stomatitis virus (VSV) dealing with the evolution of life-history characters under selection acting at two factors either in the same or in opposite directions. The two factors at which selective pressure is applied are the individual and the group. We show evidence that group selection modulates the virulence of VSV populations, in opposition to an unlimited increase in virulence by competitive optimization promoted by individual selection. The results are of relevance for understanding the evolution of parasite virulence.

Genetics, Population↗
PubMed2001

Functional specialization and evolution of leader proteinases in the family Closteroviridae.

Members of the Closteroviridae and Potyviridae families of the plant positive-strand RNA viruses encode one or two papain-like leader proteinases. In addition to a C-terminal proteolytic domain, each of these proteinases possesses a nonproteolytic N-terminal domain. We compared functions of the several leader proteinases using a gene swapping approach. The leader proteinase (L-Pro) of Beet yellows virus (BYV; a closterovirus) was replaced with L1 or L2 proteinases of Citrus tristeza virus (CTV; another closterovirus), P-Pro proteinase of Lettuce infectious yellows virus (LIYV; a crinivirus), and HC-Pro proteinase of Tobacco etch virus (a potyvirus). Each foreign proteinase efficiently processed the chimeric BYV polyprotein in vitro. However, only L1 and P-Pro, not L2 and HC-Pro, were able to rescue the amplification of the chimeric BYV variants. The combined expression of L1 and L2 resulted in an increased RNA accumulation compared to that of the parental BYV. Remarkably, this L1-L2 chimera exhibited reduced invasiveness and inability to move from cell to cell. Similar analyses of the BYV hybrids, in which only the papain-like domain of L-Pro was replaced with those derived from L1, L2, P-Pro, and HC-Pro, also revealed functional specialization of these domains. In subcellular-localization experiments, distinct patterns were observed for the leader proteinases of BYV, CTV, and LIYV. Taken together, these results demonstrated that, in addition to a common proteolytic activity, the leader proteinases of closteroviruses possess specialized functions in virus RNA amplification, virus invasion, and cell-to-cell movement. The phylogenetic analysis suggested that functionally distinct L1 and L2 of CTV originated by a gene duplication event.

Amino Acid Sequence↗
PubMed1977

Distribution and expression in mammals of genes ralated to an endogenous type C RNA virus of Odocoileus hemionus.

An endogenous type C virus recently isolated from the Columbian black-tailed deer (Odocoileus hemionus) was used as a molecular probe to study the distribution of virus-related nucleotide sequences in cellular DNAs of mammalian species. By DNA-DNA hybridization, the most extensive homology was demonstrated between the viral complementary DNA and cellular DNA isolated from Odocoileus species. DNAs of representatives of other genera within the same family, Cervidae, were partially related to the virus, consistent with the phylogenetic relationship of these species to Odocoileus. O. hemionus viral sequences were also detected within cellular DNAs of members of a more distantly related artiodactyl family, Bovidae. These findings suggest the genetic transmission of type C viral genes within cervids and bovids for a period of at least 25 to 30 million years. There was no detectable nucleotide sequence homology between O. hemionus virus and representatives of other major groups of mammalian type C viruses. These results indicate that despite the known antigenic relatedness of mammalian type C viruses, the O. hemionus virus has diverged sufficiently to be considered the prototype of a separate group. By radioimmunological techniques, it was possible to detect and partially purify, from normal tissues of cervid species, antigens related to the major structural protein of the O. hemionus virus. The present findings, that O. hemionus virus has been genetically transmitted for millions of years and yet has maintained the ability to be expressed as infectious virus, argue for positive evolutionary selective pressures for the maintenance of type C viral genes.

Animals↗
PubMed2003

Early viral load and CD4+ T cell count, but not percentage of CCR5+ or CXCR4+ CD4+ T cells, are associated with R5-to-X4 HIV type 1 virus evolution.

HIV-1 infection is established by CCR5-utilizing (R5) variants, and CXCR4-utilizing (X4) variants emerge in approximately 50% of infected patients. We studied the role of CCR5 and CXCR4 expression before and 1 and 5 years after seroconversion in HIV-1 disease in a prospective study of 102 seroconverters. High percentages of CCR5(+) cells among total cells (relative hazard [RH], 2.55; 95% confidence interval [95% CI], 0.99-6.52), but not among CD45RO(-)CD4(+) and CD45RO(+)CD4(+) cells preseroconversion and among total cells and CD45RO(-)CD4(+) cells (RH, 2.70; 95% CI, 1.06-6.92 and RH, 3.54; 95% CI, 1.27-9.90, respectively) 5 years after seroconversion were associated with more rapid progression to AIDS. One year after seroconversion, high percentages of CXCR4(+) cells among total and CD45RO(-)CD4(+) cells were associated with delayed development of X4 variants (RH, 0.49; 95% CI, 0.20-1.21 and RH, 0.41; 95% CI, 0.17-1.02, respectively), whereas no association was observed for the percentage of CCR5(+) cells. In a larger study population, high early serum viral RNA and low CD4(+) T cell numbers were associated with more rapid development of X4 variants. Our results exclude target cell availability as a driving force for R5-to-X4 virus phenotype evolution.

Adult↗
PubMed1989

High nucleotide substitution error frequencies in clonal pools of vesicular stomatitis virus.

Nucleotide substitution error frequencies were determined for several specific guanine base positions in the genomes of cloned vesicular stomatitis virus populations. Predetermined sites were examined in coding regions for the N, M, and L proteins and at a site in the genome 5'-end regulatory region. Misincorporation frequencies were estimated to be on the order of 10(-3) to 10(-4) at all positions analyzed. Isolates taken from virus populations after disruption of equilibrium conditions displayed replicase fidelity similar to that of cloned wild-type vesicular stomatitis virus. These mutation frequencies apply to all virus genomes present, including viruses rendered nonviable by lethal mutations. At one selected site in the N gene, two of three G----N base substitutions generated lethal nonsense mutations, yet their frequency was also very high. Biological implications for rapid virus evolution are discussed.

Animals↗
PubMed2001

Human immunodeficiency virus seroconversion and evolution of the hepatitis C virus quasispecies.

When chronic hepatitis C virus (HCV) infections are complicated by acquisition of human immunodeficiency virus (HIV), liver disease appears to accelerate and serum levels of HCV RNA may rise. We hypothesized that HIV might affect the HCV quasispecies by decreasing both complexity (if HIV-induced immunosuppression lessens pressure for selecting HCV substitutions) and the ratio of nonsynonymous (d(N)) to synonymous (d(S)) substitutions, because d(N) may be lower (if there is less selective pressure). To test this hypothesis, we studied the evolution of HCV sequences in 10 persons with chronic HCV infection who seroconverted to HIV and, over the next 3 years, had slow or rapid progression of HIV-associated disease. From each subject, four serum specimens were selected with reference to HIV seroconversion: (i) more than 2 years prior, (ii) less than 2 years prior, (iii) less than 2 years after, and (iv) more than 2 years after. The HCV quasispecies in these specimens was characterized by generating clones containing 1 kb of cDNA that spanned the E1 gene and the E2 hypervariable region 1 (HVR1), followed by analysis of clonal frequencies (via electrophoretic migration) and nucleotide sequences. We examined 1,320 cDNA clones (33 per time point) and 287 sequences (median of 7 per time point). We observed a trend toward lower d(N)/d(S) after HIV seroconversion in 7 of 10 subjects and lower d(N)/d(S) in those with rapid HIV disease progression. However, the magnitude of these differences was small. These results are consistent with the hypothesis that HIV infection alters the HCV quasispecies, but the number of subjects and observation time may be too low to characterize the full effect.

Adult↗
PubMed1989

Characterization of a minimal simian virus 40 late promoter: enhancer elements in the 72-base-pair repeat not required.

A 272-base-pair (bp) portion of the simian virus 40 regulatory region containing the replication origin, Sp1-binding region, and part of the 72-bp direct repeats makes up a minimal late promoter that is able to direct late-direction RNA synthesis in vivo and in vitro. Fourteen linker-scan mutants within this region were characterized. Mutations in the Sp1-binding region decreased late expression both in vivo and in vitro. By contrast, mutations that eliminate genetically defined elements of the early transcriptional enhancer or that prevent binding of the transcription factors AP-1, AP-2, and AP-3 in the 72-bp repeat region had little or no effect on late-direction expression. These results argue that, at least under certain circumstances, the early transcriptional enhancer sequences are not required for simian virus 40 late gene expression.

Biological Evolution↗
PubMed2005

Editing at the crossroad of innate and adaptive immunity.

Genetic information can be altered through the enzymatic modification of nucleotide sequences. This process, known as editing, was originally identified in the mitochondrial RNA of trypanosomes and later found to condition events as diverse as neurotransmission and lipid metabolism in mammals. Recent evidence reveals that editing enzymes may fulfill one of their most essential roles in the defense against infectious agents: first, as the mediators of antibody diversification, a step crucial for building adaptive immunity, and second, as potent intracellular poisons for the replication of viruses. Exciting questions are raised, which take us to the depth of the intimate relations between vertebrates and the microbial underworld.

Animals↗
PubMed1988

[Killer systems of Saccharomyces cerevisiae yeasts].

The killer systems of Saccharomyces cerevisiae are a peculiar group of cytoplasmic symbionts of primitive eukaryotes. The genetic material of these symbionts is double-stranded RNA. Their basic properties are linearity of genome, its fragmentation, resulting in two separately replicating major and minor segments, and the ability to control the synthesis of secretory proteins--mycocins which can kill the taxonomically related strains. Secretion of mycocins also confers immunity to their action. The strains containing killer symbionts are toxigenic and resistant to their own toxins, while those with no killer double-stranded RNA are sensitive to mycocins. The killer systems of Saccharomyces cerevisiae possess some properties relevant to viruses and evidently are evolved during the evolution of infectious viruses. Occurrence of such systems in monocellular eucaryotic organisms is an example of genome complication in the course of putting together the virus-like components. The peculiarities of replication and expression of killer systems and their utilization for the construction of vector molecules are discussed.

Cytoplasm↗
PubMed1983

On the evolution of orbiviruses.

The genomes of orbiviruses consist of 10 segments of double-stranded RNA. In cells simultaneously infected with two or more related viruses, recombinants are derived by independent reassortment of parental genes. The process is analogous to sexual reproduction in higher organisms and provides a mechanism for generating extensive diversity within this group of viruses. This genetic diversity can be explained by reference to modern concepts of the structure of natural populations of organisms. A species in the phylogenetic sense is the largest aggregate of individual organisms that evolves as a unit. The biological species concept stresses the community gene pool and reproductive isolation. The orbiviruses are now classified into 13 distinct serological groups, but confusion exists in defining species. Classification by reference to concepts of evolutionary species would define genetically interacting groups, estimate the extent of diversity within these groups, and establish phylogenetic relationships between species.

Biological Evolution↗
PubMed1997

Nutrition and newly emerging viral diseases: an overview.

Infectious diseases are on the increase worldwide. When discussing interactions of nutrition and infection, nutritionists have traditionally considered only the effects of diet on the host. Recent data, however, indicate that, at least for an RNA virus, host nutriture can influence the genetic make-up of the pathogen and thereby alter its virulence. This symposium was organized to alert the nutrition community to this discovery and its possible implications for the investigation of nutrition-infection interrelationships. Topics covered in the symposium include the following: the public health threat of emerging viral diseases; the rapid evolution of viral RNA genomes; oxidants and antioxidants in viral diseases-disease mechanisms and metabolic regulation; and increased virulence of coxsackievirus B3 due to vitamin E or selenium deficiency. If the findings with coxsackievirus are more broadly applicable to other RNA viruses, the results could be of great public health significance because RNA viruses constitute the majority of all plant, animal and human viruses.

Antioxidants↗
PubMed2005

Population dynamics of RNA viruses: the essential contribution of mutant spectra.

Cells and their viral and cellular parasites are genetically highly diverse, and their genomes contain signs of past and present variation and mobility. The great adaptive potential of viruses, conferred on them by high mutation rates and quasispecies dynamics, demands new strategies for viral disease prevention and control. This necessitates a more detailed knowledge of viral population structure and dynamics. Here we review studies with the important animal pathogen Foot-and-mouth disease virus (FMDV) that document modulating effects of the mutant spectra that compose viral populations. As a consequence of interactions within mutant spectra, enhanced mutagenesis may lead to viral extinction, and this is currently investigated as a new antiviral strategy, termed virus entry into error catastrophe.

Animals↗
PubMed1994

Nonhomologous RNA recombination in tombusviruses: generation and evolution of defective interfering RNAs by stepwise deletions.

We used a protoplast system to study the mechanisms involved in the generation and evolution of defective interfering (DI) RNAs of tomato bushy stunt tombusvirus (TBSV). Synthetic transcripts corresponding to different naturally occurring TBSV DI RNAs, or to various artificially constructed TBSV defective RNAs, were analyzed. The relative levels of competitiveness of different DI RNAs were determined by coinoculating their corresponding transcripts into protoplasts along with helper genomic RNA transcripts and monitoring the level of DI RNA accumulation. Further studies were performed to assess the contribution of naked DI RNA stability and DI RNA encapsidation efficiency to the observed levels of competitiveness. In addition, the ability of various defective RNAs to evolve to alternative forms was tested by serially passaging protoplast infections initiated with transcripts corresponding to helper genomic RNA and a single type of defective RNA. These studies, and the analysis of the sequences of observed recombinants, indicate that (i) replication competence is a major factor dictating DI RNA competitiveness and is likely a primary determinant in DI RNA evolution, (ii) DI RNAs are capable of evolving to both smaller and larger forms, and the rates at which various transitions occur differ, (iii) DI RNA-DI RNA recombination and/or rearrangement is responsible for the formation of the evolved RNA molecules which were examined, and (iv) sequence complementarities between positive- and negative-sense strands in the regions of the junctions suggest that, in some cases, base pairing between an incomplete replicase-associated nascent strand and acceptor template may mediate selection of recombination sites. On the basis of our data, we propose a stepwise deletion model to describe the temporal order of events leading to the formation of tombusvirus DI RNAs.

Base Sequence↗