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1.
Stéphanie Beaucourt Antonio V. Bordería Lark L. Coffey Nina F. Gn?dig Marta Sanz-Ramos Yasnee Beeharry Marco Vignuzzi 《Journal of visualized experiments : JoVE》2011,(52)
RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7. 相似文献
2.
LOIS ANN SALZMAN 《Nature: New biology》1971,231(23):174-176
RNA tumour viruses contain an enzyme which can transcribe DNA from an RNA template1,2, an endonuclease and a DNA-dependent DNA polymerase activity3,4. RNA polymerase has been reported in vaccinia virus5,6, reovirus7,8 and cytoplasmic polyhidrosis virus9. I wish to describe a DNA polymerase activity associated with a highly purified preparation of the parvovirus, Kilham rat virus (KRV), which is thus the first report of a DNA polymerase associated with a DNA virus. KRV, a small virus first isolated from a rat sarcoma10, is antigenically related to the H viruses isolated from human transplantable tumours11. Those parvoviruses which have been characterized all contain single stranded DNA with molecular weights of 1.5 to 2.5 × 106 (refs. 12,13 and 14). 相似文献
3.
W D Graziadei H Weideli P Lengyel 《Biochemical and biophysical research communications》1973,54(1):40-46
We find an endonuclease of high specific activity in a purified mouse interferon preparation. The interferon was purified from Ehrlich ascites tumor cultures which were induced with Newcastle disease virus. It has a higher specific activity (1.5 × 109 NIH mouse reference standard interferon units/mg protein) than reported for any interferon preparation but is not homogeneous. We do not know if the endonuclease activity is due to a contaminating protein or to interferon. The endonuclease does not degrade in our conditions polyuridylic acid or double stranded reovirus RNA and does not inactivate the tRNA2Gln species from , or tRNAVal species or polysomes from mouse L cells. Endonuclease in as little as 0.5 ng protein of the interferon preparation degrades μg quantities of messenger RNA from mouse L cells, of encephalomyocarditis virus RNA and of -synthesized reo-virus messenger RNA at 37° in 1 hour. Further characteristics of the endonuclease and its possible relationship (if any) to interferon remain to be established. 相似文献
4.
J M Taylor H E Varmus A J Faras W E Levinson J M Bishop 《Journal of molecular biology》1974,84(1):217-221
The complexity of Rous sarcoma virus RNA has been determined using molecular hybridization. Relative to poliovirus RNA, the complexity of Rous sarcoma virus is 9·3 × 106 daltons, a value close to its physically-determined molecular weight of about 107. Our interpretation is that the 35 S RNA subunits of the 70 S virus genome are non-repetitive, that is, each possesses a unique nucleotide sequence, although a limited amount of redundancy cannot be excluded. 相似文献
5.
Summary The error frequency during the RNA replication of alfalfa mosaic virus (AMV) was calculated to be significantly higher than
10−5. It may be expected that RNA synthesis in general will have low fidelity compared to DNA synthesis. The low fidelity of RNA
replication will severely restrict the usefulness of vectors for genetic engineering which are based on RNA viruses, viroids
or DNA viruses which are replicated via an RNA intermediate (e.g. caulimoviruses). Spontaneous mutants selected by host shift
were found to be much less stable than UV-induced mutants. This difference points to variations in fidelity during RNA synthesis,
probably due to the local sequence of the template. 相似文献
6.
RUDOLF JAENISCH 《Nature: New biology》1972,235(54):46-47
AFTER infection of monkey kidney cells with simian virus 40 (SV40), several species of SV40 specific RNA are synthesized1. Most SV40 RNA have a molecular weight of about 6×105 and 8×105 as measured by polyacrylamide gel electrophoresis1. In addition to these classes of RNA, a large heterogeneous SV40 specific RNA species of up to three times the length of the monomeric SV40 DNA molecule has been observed1–4. Nothing is known about the structure of this large heterogeneous virus specific RNA. 相似文献
7.
Long-Term Inactivation Study of Three Enteroviruses in Artificial Surface and Groundwaters, Using PCR and Cell Culture
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A. M. de Roda Husman W. J. Lodder S. A. Rutjes J. F. Schijven P. F. M. Teunis 《Applied microbiology》2009,75(4):1050-1057
Since the transmission of pathogenic viruses via water is indistinguishable from the transmission via other routes and since the levels in drinking water, although significant for health, may be too low for detection, quantitative viral risk assessment is a useful tool for assessing disease risk due to consumption of drinking water. Quantitative viral risk assessment requires information concerning the ability of viruses detected in drinking water to infect their host. To obtain insight into the infectivity of viruses in relation to the presence of virus genomes, inactivation of three different enteroviruses in artificial ground and surface waters under different controlled pH, temperature, and salt conditions was studied by using both PCR and cell culture over time. In salt-peptone medium, the estimated ratio of RNA genomes to infectious poliovirus 1 in freshly prepared suspensions was about 100. At 4°C this ratio was 103 after 600 days, and at 22°C it was 104 after 200 days. For poliovirus 1 and 2 the RNA/infectious virus ratio was higher in artificial groundwater than in artificial surface water, but this was not the case for coxsackievirus B4. When molecular detection is used for virus enumeration, it is important that the fraction of infectious virus (based on all virus genomes detected) decays with time, especially at temperatures near 22°C. 相似文献
8.
9.
Arthur L Koch 《Virology journal》2008,5(1):1-11
Background
Recent reports have indicated that single-stranded DNA (ssDNA) viruses in the taxonomic families Geminiviridae, Parvoviridae and Anellovirus may be evolving at rates of ~10-4 substitutions per site per year (subs/site/year). These evolution rates are similar to those of RNA viruses and are surprisingly high given that ssDNA virus replication involves host DNA polymerases with fidelities approximately 10 000 times greater than those of error-prone viral RNA polymerases. Although high ssDNA virus evolution rates were first suggested in evolution experiments involving the geminivirus maize streak virus (MSV), the evolution rate of this virus has never been accurately measured. Also, questions regarding both the mechanistic basis and adaptive value of high geminivirus mutation rates remain unanswered.Results
We determined the short-term evolution rate of MSV using full genome analysis of virus populations initiated from cloned genomes. Three wild type viruses and three defective artificial chimaeric viruses were maintained in planta for up to five years and displayed evolution rates of between 7.4 × 10-4 and 7.9 × 10-4 subs/site/year.Conclusion
These MSV evolution rates are within the ranges observed for other ssDNA viruses and RNA viruses. Although no obvious evidence of positive selection was detected, the uneven distribution of mutations within the defective virus genomes suggests that some of the changes may have been adaptive. We also observed inter-strand nucleotide substitution imbalances that are consistent with a recent proposal that high mutation rates in geminiviruses (and possibly ssDNA viruses in general) may be due to mutagenic processes acting specifically on ssDNA molecules. 相似文献10.
Molecular Weight Determination of Sendai RNA by Dimethyl Sulfoxide Gradient Sedimentation 总被引:12,自引:10,他引:2
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The molecular weight of the large RNA of Sendai virus has been determined by sedimentation analysis in sucrose gradients containing 99% dimethyl sulfoxide (DMSO) to be 2.3 × 106. Sendai RNA recovered from 99% DMSO was found to cosediment with nondenatured Sendai RNA at 46 to 48s in ordinary sucrose gradients. The molecular weight value of 2.3 × 106 is considerably smaller than the estimates of 6 × 106 to 7 × 106 determined under nondenaturing conditions, suggesting a unique structure for Sendai RNA. 相似文献
11.
Synthesis of Double-Stranded RNA in a Virus-Enriched Fraction from Agaricus bisporus 总被引:1,自引:1,他引:0
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Partially purified virus preparations from sporophores of Agaricus bisporus affected with LaFrance disease had up to a 15-fold-higher RNA-dependent RNA polymerase activity than did comparable preparations from healthy sporophores. Enzyme activity was dependent upon the presence of Mg2+ and the four nucleoside triphosphates and was insensitive to actinomycin D, α-amanitin, and rifampin. The 3H-labeled enzyme reaction products were double-stranded RNA (dsRNA) as indicated by CF-11 cellulose column chromatography and by their ionic-strength-dependent sensitivity to hydrolysis by RNase A. The principal dsRNA products had estimated molecular weights of 4.3 × 106 and 1.4 × 106; they corresponded in size and hybridized to the major dsRNAs detected in the virus preparation by ethidium bromide staining. Cs2SO4 equilibrium centrifugation of the virus preparation resolved a single peak of RNA polymerase activity that banded with a 35-nm spherical virus particle containing dsRNAs with molecular weights of 4.3 × 106 and 1.4 × 106. The data suggest that the RNA-dependent RNA polymerase associated with the 35-nm spherical virus is a replicase which catalyzes the synthesis of the genomic dsRNAs. 相似文献
12.
M.G. Munowitz C.M. Dobson R.G. Griffin S.C. Harrison 《Journal of molecular biology》1980,141(3):327-333
The motional state of RNA in tomato bushy stunt virus, both in the crystalline state and in solution, has been investigated using 31P nuclear magnetic resonance methods. It has been found that the RNA is highly immobile in the native virus and it is suggested that the lack of a high-resolution X-ray diffraction pattern for either the RNA or the N-terminal regions of the protein coat molecules (Harrison et al., 1978) is due to static disorder in the crystals. Dynamic disorder has been detected in the virus after treatment with EDTA, which causes a structural change and an increase in particle size. 相似文献
13.
MESSENGER-SPECIFIC initiation factors, capable of discriminating between classes of messenger RNAs (mRNAs) or different cistrons in viral RNA, have been implicated in the regulation of protein synthesis in bacteria1–5. Comparable but less detailed observations have also been made in eukaryotic systems6–10. For example, RNA extracted from a mammalian virus (encephalomyocarditis virus, EMC) cannot be translated in a reticulocyte cell-free system unless the system is fortified with an extract from responsive cells—in this case, Krebs II ascites cells6. Such results imply the existence of tissue-specific factors and lead to questions whether this incompatibility is reciprocated by an inability of the Krebs II ascites cell system to respond to the mRNA for globin. 相似文献
14.
GB virus C/hepatitis G (GBV-C) is an RNA virus of the family Flaviviridae. Despite replicating with an RNA-dependent RNA polymerase, some previous estimates of rates of evolutionary change in GBV-C
suggest that it fixes mutations at the anomalously low rate of ∼10−7 nucleotide substitution per site, per year. However, these estimates were largely based on the assumption that GBV-C and
its close relative GBV-A (New World monkey GB viruses) codiverged with their primate hosts over millions of years. Herein,
we estimated the substitution rate of GBV-C using the largest set of dated GBV-C isolates compiled to date and a Bayesian
coalescent approach that utilizes the year of sampling and so is independent of the assumption of codivergence. This revealed
a rate of evolutionary change approximately four orders of magnitude higher than that estimated previously, in the range of
10−2 to 10−3 sub/site/year, and hence in line with those previously determined for RNA viruses in general and the Flaviviridae in particular. In addition, we tested the assumption of host-virus codivergence in GBV-A by performing a reconciliation analysis
of host and virus phylogenies. Strikingly, we found no statistical evidence for host-virus codivergence in GBV-A, indicating
that substitution rates in the GB viruses should not be estimated from host divergence times. 相似文献
15.
Effects of Actinomycin D and Ultraviolet and Ionizing Radiation on Pichinde Virus 总被引:8,自引:7,他引:1
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Michael F. Carter Frederick A. Murphy J. Pierre Brunschwig Christine Noonan William E. Rawls 《Journal of virology》1973,12(1):33-38
Actinomycin D (0.05 μg/ml) suppresses the synthesis of ribosomal RNA of baby hamster kidney (BHK21) cells. The production of infectious Pichinde virus was enhanced in the presence of actinomycin D, although the production of virus particles was not substantially different from cultures inoculated in the absence of the drug. By prelabeling BHK21 cells with 3H-uridine and then allowing the virus to replicate in the presence of actinomycin D, it was possible to show that ribosomal RNA synthesized prior to infection was incorporated into the virion. A single-hit kinetics of inactivation of Pichinde virus was observed with ultraviolet light, suggesting that the virus contains only a single copy of genome per virion. Comparison of the inactivation kinetics by gamma irradiation of Pichinde virus with Sindbis and rubella virus indicated that the radiosensitive genome of Pichinde virus was about 6 × 106 to 8 × 106 daltons. This value is greater than the 3.2 × 106 daltons which was estimated by biochemical analysis. One possible explanation considered is that the ribosomal RNA of host cell origin is functional and accounts for the differences in genome size estimated by the two methods. 相似文献
16.
H. C. T. Ribeiro O. H. O. Pavan A. R. Muotri 《Entomologia Experimentalis et Applicata》1997,83(2):233-237
The susceptibility of third instar larvae of Anticarsia gemmatalis (Lepidoptera: Noctuidae) and Diatraea saccharalis (Lepidoptera: Pyralidae) to ten distinct plaque purified genotypic variants of a selected isolate of the Anticarsia gemmatalis multiple-embedded nuclear polyhedrosis virus (AgMNPV), was compared. Despite the fact that this isolate, AgMNPV-Ds20, represents a wild strain of the AgMNPV selected for higher virulence to D. saccharalis, an alternate host, most of the variants are much more virulent to the original host Anticarsia than to Diatraea. Bioassays have shown an over one hundred-fold variation in LD50 values ranging from 1700 polyhedron inclusion bodies (PIBs) to more than 200 000 PIBs/larva. The PIB production in infected larvae increased with the pathogenicity of the variant to the host, showing an average ten-fold reduction in Diatraea when compared to Anticarsia for the same variant. The virus particle yield ranged from 6×107 to more than 109 PIBs/g of infected larvae in Diatraea and from 8×108 to more than 1010 PIBs/g of infected Anticarsia larvae. The data show a clear difference of the pathogenicity of the genotypic variants of AgMNPV in vivo both between the original and alternate host and between the individual variants for the same host. These differences found in vivo indicate that monitoring of shifts in variant frequency of wild and laboratory-propagated viral isolates in these highly heterogeneous populations would help ensure the efficacy of biological control programs. 相似文献
17.
Shashi Sharma Paban Kumar Dash S. R. Santhosh Jyoti Shukla Manmohan Parida P. V. Lakshmana Rao 《Molecular biotechnology》2010,45(1):49-55
Chikungunya is one of the most important emerging arboviral infections of public health significance. Due to lack of a licensed
vaccine, rapid diagnosis plays an important role in early management of patients. In this study, a QC-RT–PCR assay was developed
to quantify Chikungunya virus (CHIKV) RNA by targeting the conserved region of E1 gene. A competitor molecule containing an
internal insertion was generated, which provided a stringent control of the quantification process. The introduction of 10-fold
serially diluted competitor in each reaction was further used to determine sensitivity. The applicability of this assay for
quantification of CHIKV RNA was evaluated with human clinical samples, and the results were compared with real-time quantitative
RT–PCR. The sensitivity of this assay was estimated to be 100 RNA copies per reaction with a dynamic detection range of 102 to 1010 copies. Specificity was confirmed using closely related alpha and flaviviruses. The comparison of QC-RT–PCR result with real-time
RT–PCR revealed 100% concordance for the detection of CHIKV in clinical samples. These findings demonstrated that the reported
assay is convenient, sensitive and accurate method and has the potential usefulness for clinical diagnosis due to simultaneous
detection and quantification of CHIKV in acute-phase serum samples. 相似文献
18.
Joy Gardner Penny A. Rudd Natalie A. Prow Essia Belarbi Pierre Roques Thibaut Larcher Lionel Gresh Angel Balmaseda Eva Harris Wayne A. Schroder Andreas Suhrbier 《PloS one》2015,10(10)
Chikungunya virus (CHIKV) is a reemerging, ordinarily mosquito-transmitted, alphavirus that occasionally produces hemorrhagic manifestations, such as nose bleed and bleeding gums, in human patients. Interferon response factor 3 and 7 deficient (IRF3/7-/-) mice, which are deficient for interferon α/β responses, reliably develop hemorrhagic manifestations after CHIKV infection. Here we show that infectious virus was present in the oral cavity of CHIKV infected IRF3/7-/- mice, likely due to hemorrhagic lesions in the olfactory epithelium that allow egress of infected blood into the nasal, and subsequently, oral cavities. In addition, IRF3/7-/- mice were more susceptible to infection with CHIKV via intranasal and oral routes, with IRF3/7-/- mice also able to transmit virus mouse-to-mouse without an arthropod vector. Cynomolgus macaques often show bleeding gums after CHIKV infection, and analysis of saliva from several infected monkeys also revealed the presence of viral RNA and infectious virus. Furthermore, saliva samples collected from several acute CHIKV patients with hemorrhagic manifestations were found to contain viral RNA and infectious virus. Oral fluids can therefore be infectious during acute CHIKV infections, likely due to hemorrhagic manifestations in the oral/nasal cavities. 相似文献
19.
20.
It is generally accepted that mutation rates of RNA viruses are inherently high due to the lack of proofreading mechanisms. However, direct estimates of mutation rate are surprisingly scarce, in particular for plant viruses. Here, based on the analysis of in vivo mutation frequencies in tobacco etch virus, we calculate an upper-bound mutation rate estimation of 3×10−5 per site and per round of replication; a value which turns out to be undistinguishable from the methodological error. Nonetheless, the value is barely on the lower side of the range accepted for RNA viruses, although in good agreement with the only direct estimate obtained for other plant viruses. These observations suggest that, perhaps, differences in the selective pressures operating during plant virus evolution may have driven their mutation rates towards values lower than those characteristic of other RNA viruses infecting bacteria or animals. 相似文献