Lecture 2 Flashcards
What is the viral genetic code in viruses and when was it discovered?
The nucleic acid genome and it was found in the 1950s
What is the Hershey-Chase experiment?
They wanted to know what was the viral genetic code
-1st the viral protein was radio labeled and the virus infected the cell
radioactivity was mostly found in the supernatant fraction aka above the solid and no radioactivity was foiund in next gen of phages
-2nd the viral dna was radio labeles
radioactivity was mostly found in cell pellet and radioactivity was detected in next gen of phages
when did baltimore win his nobel and what did he dop
he won it in 1975
he made up the baltimore classification system and found reverse transcriptase
what is the universal feature of viral genomes?
their need to make proteins to undergo viral life cycle
how many types of viral genomes are there
7
why is there only 6 classes at first in the baltimore classification system?
because the 7th appeared when hep B was found
mRNA that is ribosome ready is always the positive or negative strand
positive
true or false: all + rna is mRNA
false some might not be ribosomal ready aka they are not all translated
the elegance of the Baltimore System
Knowing only the nature of the viral genome, one can deduce the basic steps that must take place to produce mRNA
What information is encoded in a viral genome?
gene products and regulatory signals for:
-Replication of the viral genome
-assembly and packaging pf the genome
-regulation and timing of the replication cycle
-modulation of host defenses
-spread to other cells and hosts
What information is not contained in viral genomes?
-no genes encoding the complete synthesis machinery aka no ribosomes
-no genes encoding proteins involved in energy production or membrane biosynthesis
-No classical centromeres (genome segregation) or telomeres (genome maintenance)
true or false we don’t really know what the genes in most of the giant viruses do
true
Largest known viral genomes
-Pandoravirus salinus (largest)
-Pandoravirus dulcis
-Megavirus chilensis
-Mamavirus
-Mimivirus
smallest known viral genomes
-Circovirus (smallest)
-Anellovirus
-Geminivirus
-Hep B
-Levivirus
what are the 7 classes of the baltimore classification
I: dsDNA viruses
II: ssDNA viruses (+)sense DNA
III: dsRNA viruses
IV: (+)ssRNA viruses (+)sense RNA
V: -ssrna viruses
VI: ssRNA-RT viruses (+)sense RNA with DNA intermediate in life-cycle
VII: dsDNA-RT viruses
dsDNA Genomes
-The host genetic system is based on DNA
-Many DNA viruses emulate the host
-However, almost all viral DNA genomes are NOT like cell chromosomes
-Unexpected tricks have evolved
-Genomes copied by host DNA polymerases
e.g. Polyomaviridae, Pappilomaviridae
-Genomes encode DNA polymerase
e.g. Adenoviridae, Poxviridae
ssDNA Genomes
-Can be circular or linear
e.g. Circoviridae (circular), Parvoviridae (linear)
-Must be converted to dsDNA to make mRNA
true or false: ssDNA viruses often infect humans
false: because we don’t have ssDNA in our DNA it looks foreign to our cells and we have a good system to kill them
ex: TT’virus’(ubiquitous human virus)
B19’parvovirus’(fifth disease)
True or false: mammalian cells do not have an RNA-dependant RNA polymerase aka RdRp
true
-RNA virus genomes have to encode for RdRp
-RdRp produce RNA genomes and mRNA from RNA templates
class 3: dsRNA genomes and examples
-dsRNA can’t be translated directly (ribosomes can’t access this) ribosomes only binds to ssrna
-d-sRNA must be copied to mRNA
- These viruses must carry a RdRp in the viral particle
-e.g. Reoviridae (segmented)
class 4: +ssrna genomes and examples
-Can be directly translated (+ stranded)
Don’t have to carry RdRp in the particle because it can be directly translated to make the RdRp
-e.g. Picornaviridae, Flaviviridae, Coronaviridae
Class 5 -ssrna genomes and examples
Complement to mRNA (can’t be translated)
-Must carry an RdRp in the viral particle to produce mRNA
-e.g. Orthomyxoviridae (segmented), Paramyxoviridae and Rhabdoviridae (non-segmented)
what is reassortment?
it is a consequence of a segmented genome
aka when a certain virus has different segments and like 2 infect a cell, when they get synthesized a crossover can happen
class 6: +ssrna-rt and examples
One viral family: Retroviridae Two human pathogens:
Human immunodeficiency virus (HIV)
Human T: lymphotropic virus(HTLV)
• Although it looks like an mRNA, it is not translated immediately upon entry into the cell
• Copied by reverse transcriptase (RT) to a (-) ssDNA and then to dsDNA
- RT is carried in the viral particle
• dsDNA integrates into the host DNA
(termed a provirus)
• Host polymerases then make the mRNA to make viral proteins
class 7: dsDNA rt and examples
•Partially dsDNA (gapped DNA)
Note: this genome cannot be copied to mRNA
-partically dsdns is converted to fully dsdna which can then be transcribed into mRNA
ex: hep B
what does ambisense ssrna mean and they are a part of which calss?
it is when a strand of ssrna has both a positive end and a negative one
class 3
In the 1900s viral stocks were maintained by…
continual passage from animal to animal
true or false: Experimental infection of laboratory animals is not obligatory for studying the processes by which viruses cause disease
false: it is obligatory
Before cell culture, many viruses were propagated in …
embryonated chicken eggs
in 1949, what did Enders, Wekker and Robbins discovered
poliovirus could multiply in cultured cells = nobel prize in 1954
what are the2 main types of cell cultures
- Primary cell cultures: prepared from animal
tissues; limited life span (5-20 cell divisions) usually from human foreskin - Continuous cell lines: single cell type that can be propagated indefinitely in culture
‣ Tumor tissue or immortalized primary cells
‣ May NOT resemble the cell of origin
what is a great evidence of viral grownth in cultured cells?
Cytopathic effects
-e.g. rounding up of cells, detachment, cell lysis, syncytium
formation cell fusion), nuclear shrinking/swelling, accumulation of
virions/viral proteins, membrane alterations, etc.
what is the virus titer?
concentration of a virus in a smaple
What is a plaque assay and what does it allow us to do?
-The plaque assay - quantitative measure of infectious units (a.k.a. plaque forming units, PFU)
-Allows scientists to count the # of infectious virus particles in a suspension with a high degree of precision and reproducibility
What is the focus-forming unit (FFU) assay? and why do we use it
•-Modification of a plaque assay
-After viral infection, cells are permeabilized and stained with an antibody against a viral protein
-we use it if the virus does not lyse the cells
What does endpoint dilution assay looks for
cytopathic effects usually we look for like a 50% clearing
True or false: ration of physical particles to infectious particles =1
false
Ratio of physical particles to
infectious particles not = 1
Particle-to-PFU ratio = # of physical particles ➗ # of infectious particles
-a single particle can initiate an infection
-not all virus particles are successful
Damaged particles, mutations,
“empty” particles, complexity of viral life cycles, antiviral defense mechanisms, multicomponent viruses
What is Multiplicity of infection?
-# of infectious particles ADDED per susceptible cell (Not the # of infectious particles each cell receives)
-If you add 10^7 PFUs to 10^6 cells (MOI = 10), each cell does NOT receive 10 virions:
- Infection depends on a random collision of virions and cells
- When susceptible cells are mixed with virus, some cells are
uninfected, some receive 1, 2, 3 or more particles
- Distribution of virus particles per cell is best described by the Poisson distribution
if there is an MOI of 1 what are the ratios of cells affected and etc?
36.8% of cells are uninfected
36.8% of cells receive 1 particle
26.4% receive >1 particle
Physical Measurements
of Virus Particles
• Hemagluttination Assay
-Imaging particles
Electron Microscopy
Assaying viral proteins or nucleic acids
Serology:
-immunistaining
-ELISA
Detecting viral nucleic acids
plaque assay allowed…
allowed the application of genetics to animal viruses
- Select viruses with altered properties and purify them by plaquing
Today, we engineer mutations into viral genomes using….
infectious DNA clones
-A modern validation of the Hershey-Chase experiment (1952)
-Deletion, insertion, substitution, nonsense, missense, etc
What is transfection?
-Transformation-infection = Transfection
First for production of infectious virus after transformation of cells by viral DNA (or RNA)
First done with bacteriophage lambda
Unfortunately, the term has become synonymous with the introduction of any RNA or DNA into cells
true or false: you can only use the cDNA and still be able to transfect a cell
true
