Lecture 4: virus replication cycle Flashcards
describe the infectious virus replication cycle
begins at attachment of single virus particle
virus enters and genome is replicated then assembly
ends in release of many virions from cell by lyses
replication length varies
all events inside single infected cell
describe animal cell culture
not possible before 1949
poliovirus first
primary cells are used
-animals tissue (embryonic/ newborn/ foreskin)
-express contact inhibition
-cells have finite lifetime (one time use)
mice liver tissue extracted and digested with collagenase
- filter and obtain pellet
- wash pellet- differential centrifugation
- least dense (upper part) is added to culture enriched for slowly sedimenting single cells (primary cells form monolayer)
- more dense (pellet) added to culture for dense cell clumps
maintain diploidy
~100 divisions (DNA shortens)
human embryos
can be harvestes
diploid cell lines
aneuploid indefinite growth tumor tissue/ mutagens atypical can make own telomerase enzyme grow over eachother
continuous cell lines
advantages and disadvantages of live animal models
advantages
- only means until 1930s
- essential for some viruses (need host not cell culture)
- virus pathogenesis
disadvantages
- inconvenient and expensive
- variable responses
- mutants
- animal welfare issues
chorioallantoic membrane inoculation viruses in embryonic eggs
herpes simplex, poxvirus, rous sarcoma virus
amniotic inoculation viruses in embryonic eggs
influenza, mumps
yolk sac inoculation viruses in embryonic eggs
herpes simplex
allantoic inoculation viruses in embryonic eggs
influenza, mumps, newcastle disease, avian adenovisus
describe bacterial virus plaque assay
ellis and delbruck (1939)
- infect lawn of bacteria
- phage lyse host cell
- quantitative
describe animal virus plaque assay
dulbecco (1952)
- cultured cells form monolayer
- localized infection of neighboring cells= plaque
- staining necessary to visualize plaques (neutral red)
how many viruses needed to form a plaque
one hit (linear) kinetics
- number of plaques directly proportional to first power of [ ] virus is inoculated in
- if [ ] doubles then number of plaques doubles too
two hit (curved) kinetics -number of plaques directly proportional to the square of [ ] of the virus inoculated
-# of infectious particles added per cell
-infection depends on random collision of virions and cells
susceptible cells may remain uninfected or get one or more particles
MOI
multiplicity of infection
all cells infected at same time
synchronous infection
why use high inoculum dose
- damage particles
- defective genomes
- “empty” capsids
- antiviral properties
general stages of one step growth curve
attachment/penetration eclipse -uncoating- releasing genome -no progeny virus latent -intracellular virions maturation release -extracellular virions
step by step process of one step growth curve
- infect monolayers of tissue culture cells and allow infection to proceed in a CO2 incubator
- monitor experiments via inverted microscope
- collect infected cell lysates at various time points after infection
- perform serial dilutions on infected cell lysates and do plaque assay
- stain and analyze plaque assays. record results
starts when virus detected
produce another virus in cells
eclipse
typical virus replication cycle
- attachment- recognition and adsorption
- penetration- entry
- uncoating- disassembly and localization
- replication- transcription, translation, processing, duplication
- assembly
- maturation
- release
typical virus replication cycle
- attachment- recognition and adsorption
- penetration- entry
- uncoating- disassembly and localization
- replication- transcription, translation, processing, duplication
- assembly
- maturation
- release
how does a virus attach to host cell
-host cell surface receptors (essential)
-viral surface ligands (produced by virus)
proteins and glycoproteins
ability to infect animal/cell culture
host range
binding forces associated with interaction of cell receptors
electrostatic- form randomly and initial low affinity
hydrophobic- conformational changes and higher affinity
binding forces associated with interaction of cell receptors
electrostatic- form randomly and initial low affinity
hydrophobic- conformational changes and higher affinity
particle surrounded by plasma membrane and brought into cell
doesn’t happen with viruses
1-2um
phagocytosis
describe plasma membrane fusion entry
receptor mediated
glycoproteins attach to receptors on membrane
fusion of viral and cellular envelope
nucleocapsid released inside cell
viral envelope forms patch on plasma membrane
-viral glycoproteins from virus left on membrane (how we know someone has a virus)
describe plasma membrane fusion entry
receptor mediated
glycoproteins attach to receptors on membrane
fusion of viral and cellular envelope
nucleocapsid released inside cell
viral envelope forms patch on plasma membrane
-viral glycoproteins from virus left on membrane (how we know someone has a virus)
always recycling- virus takes advantage
lowers pH- virus rely on it
always with receptor mediated transport
endosomes
describe receptor mediated endocytic entry of enveloped virus
- cathrin coated pit forms; triggered by virion-ligand cell surface receptor interaction
- endocytic vesicle forms and becomes acidified
- pumping H+ ions in, converting ATP to ADP
- partial degradation of virion and potential expression of processed antigen
- cathrin released virion partially ‘‘opened’’
- capsid degraded and viral mRNA released in cytoplasm
how are endosomes transported
along microtubules
