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1、 Academic Press, 2000.,Replication,Learning Objectives: On completing this session, you should be able to:Understand how the nature of a virus genome determines its pattern of replication. Describe a typical, generalized replication cycle of a virus. Compare the patterns of replication of each of th
2、e seven major virus groups., Academic Press, 2000.,Classification of Viruses,The way in which viruses are classified has altered as our perception of them has changed:By disease: early civilizations By morphology: 1930s-1950s Functional classification: In recent years, more emphasis has been placed
3、on the replication strategy of the virus. This is particularly true for the composition and structure of the virus genome and the constraints which this imposes on replication., Academic Press, 2000.,Investigation of Virus Replication,Bacteriophages have long been used by virologists as models to un
4、derstand the biology of viruses. Two particularly significant experiments which illustrate the fundamental nature of all viruses were performed on bacteriophages:Ellis and Delbruck in 1939 Hershey and Chase in 1952, Academic Press, 2000.,Ellis and Delbruck (1939) The Single-Burst Experiment or One-S
5、tep Growth Curve,This was the first experiment to show the three essential phases of virus replication: Initiation of infection Replication and expression of the virus genome Release of mature virions from the infected cell Bacteriophages were added to a culture of rapidly growing bacteria: After a
6、few minutes, the culture was diluted, effectively preventing further interaction between the phage particles and the cells. This step is the key to the entire experiment, since it effectively synchronizes the infection of the cells and allows the subsequent phases of replication in a population of i
7、ndividual cells and virus particles to be viewed as if it were a single interaction. Repeated samples of the culture were taken at short intervals and analysed for bacterial cells by plating onto agar plates and for phage particles by plating onto lawns of bacteria., Academic Press, 2000.,The Single
8、-Burst Experiment, Academic Press, 2000.,The Single-Burst Experiment,There is a stepwise increase in the concentration of phage particles with time, each increase in phage concentration representing one replicative cycle of the virus. However, the data from this experiment can also by analysed in a
9、different way, by plotting the number of plaque-forming units (p.f.u.) per bacterial cell against time. In this type of assay, a plaque-forming unit can be either a single extracellular virus particle or an infected bacterial cell. These two can be distinguished by disruption of the bacteria with ch
10、loroform before plating, which releases any intracellular phage particles, giving the total virus count, i.e. intracellular plus extracellular particles., Academic Press, 2000.,The Single-Burst Experiment, Academic Press, 2000.,The Single-Burst Experiment,Immediately after dilution of the culture, t
11、here is a phase of 10-15 min when no phage particles are detectable - the eclipse period.This represents a time when virus particles have broken down after penetrating cells, releasing their genomes as a prerequisite to replication and therefore cannot be detected by the plaque assay. The latent per
12、iod is the time before the first new extracellular virus particles appear, 20-25 min for most bacteriophages. About 40 min after the cells were infected, the curves for the total number of virus particles and for extracellular virus merge because the infected cells have lysed and released any intrac
13、ellular phage particles by this time. The yield (i.e. number) of particles produced per infected cell can be calculated from the overall rise in phage titre., Academic Press, 2000.,Virus Replication,Following the development of plaque assays for animal viruses in the 1950s, single burst experiments
14、have now been performed for eukaryote viruses, with similar results. The major difference between these viruses and bacteriophages is the much longer time interval required for replication, which is measured in terms of hours or days rather than minutes after infection. This difference reflects the
15、much slower growth rate of eukaryotic cells, and in part, the complexity of virus replication in compartmentalized cells., Academic Press, 2000.,Virus Replication, Academic Press, 2000.,Biochemical Analysis of Virus Infection, Academic Press, 2000.,The Hershey-Chase Experiment (1952),Bacteriophage T
16、2 was propagated in Escherichia coli cells which had been labelled with one of two radioisotopes: either 35S, which is incorporated into sulphur-containing amino acids in proteins or 32P, which is incorporated into nucleic acids (which do not contain any sulphur) Particles labelled in each of these
17、ways were used to infect bacteria. After a short period to allow attachment to the cells, the mixture was homogenized briefly in a blender which did not destroy the bacterial cells but was sufficiently vigorous to knock the phage coats off the outside of the cells. Analysis of the radioactive content in the cell pellets and culture supernatant (containing the empty phage coats) showed that most of the radioactivity in the 35S-labelled particles remained in the supernatant, while in the 32P-labelled particles, most of the radiolabel had entered the cells.,
