Virus Classification, Structure & Replication Flashcards
how is a virus different from a bacterium?
- virus: uses host machinery to make copies of viral DNA/RNA, smaller
- bacteria: asexual fission reproduction, larger
how is a virus different than a toxin?
a virus can make copies of its DNA/RNA a toxin is secreted and cannot replicate
how were viruses discovered based on size?
dimitri ivanofsky showed that Tobacco Mosaic Virus (TMV) was able to pass through a filter while bacteria could not (1892)
first electron micrograph of TMV in 1939
how were viruses discovered based on growth?
martinus beijerinck showed that the titer of TMV increased after infecting a plant, proving TMV was not a toxin (1898)
how were bacteriophages (bacteria viruses) discovered?
- discovered by frederick w. twort (1915) while trying to grow vaccina virus
- bacteriophage were instrumental in developing the field of virology and expanding the field of biology
how were animal viruses discovered?
- foot and mouth disease (first animal virus) discovered (1898)
- yellow fever virus (first human virus) discovered in 1901
describe general characteristic of viruses.
- smaller than bacteria, fungus, and other microorganisms
- replicate when provided a host
- viruses are obligate intracellular pathogens
- viruses are not autpoietic
- means “poison” in greek
- ubiquitous in nature
describe the theory of cellular virus origin.
proposes that viruses were once cellular components but over time they evolved separately
describe the theory of autopoietic virus origin.
proposes that viruses, once autopoietic entities, became dependent on cells for replication
name the attributes for virus classification
- virus particle structure
- genome
- replication features
- serology
- stability
what is meant by virus particle structure?
- composition
- shape
- size
- presence of nucleocapsid
- presence of envelopes
what is a nucleocapsid?
RNA or DNA in a core that is protected by a protein coat (capsid)
virus is defined by nucleocapsid structure
- helical
- pleomorphic
- icosahedral
what is a nucleocapsid comprised of?
repeating protein subunits called capsomeres
what is a viral envelope?
-virus-modified cellular membranes acquired upon exit from host
what renders enveloped viruses noninfectious?
exposure to lipid solvents in the lab (alcohol, ether, acetone, Freon)
can enveloped viruses have nucleocapsids?
yes, enveloped viruses may have nucleocapsids with different structures
what is the largest virus?
poxviridae
what is the smallest virus?
parvoviridae and circoviridae
describe virus composition
- DNA: ds or ss
- RNA: ds or ss
- ss: plus sense (+)ssRNA, minus sense (-)ssRNA, or ambisense (bidirectional + and - sense)
how is plus sense ssRNA used for translation?
directly
how is minus sense ssRNA used for translation?
needs to have a plus sense made of it before can cause translation protein from ribosome
how are viruses classified based on genome structure?
- linear
- circular
- segmented
- diploid
T/F: viruses are classified by gene arrangement
T
what is the ssDNA hairpin viral replication?
Rolling hairpin replicating genomes are single strand DNA with terminal hairpin structures. Upon infection, the 3’ hairpin serves as primer for host reparation enzymes to convert viral ssDNA into dsDNA form used both for transcription and replication.
what is the dsDNA rolling circle viral replication?
- DNA replication begins at specific locations in the genome, called “origins”. A viral endonuclease creates a nick in the origin of replication.
- The replication machinery assembles with the DNA polymerase on the 3’ extremity.
- The DNA polymerase and associated factors begins to proceed to a strand displacement synthesis, producing a concatemer linear single stranded DNA with one genome copy per turn of replication. On the concatemer strand, Okazaki fragments are elongated after sequencial RNA primer synthesis by the primase, theus turning it into dsDNA.
The concatemer strand RNA primer are removed and okazaki fragments ligated. - The replication forks go on and produces a long linear concatemer which will be processed into linear genomes and encapsidated.
describe (+)ssRNA replication
- positive strand enters membraneous vesicle in REG of double membrane vesicles
- 3’->5’ replication to dsRNA form
- protein synthesis
describe (-)ssRNA replication
- minus strand has a leader sequence where Pol complex binds
- creates antigenome positive strands simultaneously
- Pol complex binds to + stand trailer sequence and creates complement - strands simultaneoulsy
what advantage comes with a large genome?
resistance can package more “tools”/proteins
what disadvantages come with a large genome?
more material thru, requires more nutrients, easier to see, so takes longer to replicate
what do most DNA viruses need that RNA viruses do not?
for dsDNA requires access to a nucleus
what do RNA viruses need that DNA viruses do not?
need an RNA dependent RNA transcriptase
what are the steps of virus replication?
- attachment
- entry
- transcription
- translation
- replication
- assembly
- release
what happens in viral attachment?
-binding of a virus to a cellular receptor
viral attachment: what are functions of cellular receptors?
- signaling molecules: induced cellular response to binding
- cell adhesion
- transport
viral attachment: what are the functions of viral receptors?
- usually do not mimic cell receptor’s normal ligands
- typically are spike-like projections on particle surfaces
- may require a co-receptor (HIV: CD4, CXCR4)
viral attachment: describe what genetic engineering to modify receptor recognition can do?
- integrin RGD sequence: used by Ad, engineered into lambdaphage
- pseudotyping particle: improve retroviral entry, VSV, Ebola, LCMV
what is viral attachment a major determinant of?
virus tropism aka host range
can viruses infect essentially all known forms of life?
yes,
- specific host ranges, some can infect humans and animals (zoonoes)
- not shared across more divergent hosts (plants, bacteria, fungi)
what is host range a major factor of?
eradication
- small pox: only humans
- yellow fever: mosquitoes and humans
what are the pathways a virus can enter a host?
- receptor mediated endocytosis
- direct penetration of plasma membrane
how do non-enveloped viruses enter a host?
- not well understood
- create pores in cell membrane: picornavirus
- membrane disruption and allows particles to move thru: adenovirus, reovirus
how do enveloped viruses enter a host?
- membrane fusion
- best understood for influenza: HA protein-attachement & fusion
- receptor conformational change: low pH, receptor induced
how does nuclear replication uncoating occur?
- virus enters host thru plasma membrane acquiring coating
- genome and remaining nuclear protein coat (nucleocapsid) transported to the nuclear membrane
- delivery of genome to nucleus
how does cytoplasmic replication uncoating occur?
- virus enters host thru plasma membrane acquiring coating
- release of the genome in cytoplasm
- transportation of the genome to intracellular site of replication: many RNA viruses replicate in membrane assoc. complexes
- dsRNA viruses never release their genomic material from the entering particle= EXCEPTION
describe viral transcription
- production of mRNA templates for protein synthesis
- DNA viruses usually rely upon cellular RNA pols
- the genome of (+)ssRNA viruses can serve as mRNA: production of new transcripts can occur later using a (-)ssRNA template
what is the classification type for viral transcription?
baltimore classification
describe viral translation
- production of all proteins
- all viruses need the cell’s ribosomes to produce protein - no exceptions
- viral protein production can be regulated at the transcript (mRNA) level or translation level
- structural proteins are produced in high quantities
- non-structural proteins are only seen inside the infected cell
what is the objective of viral genome replication?
make additional genome copies…. duhhhh
T/F: the viral genome replication order of events depends on the virus genome itself
TRUE
what happens in (+)ssRNA genome replication?
- genome serves as template for translation
- polymerase makes (-)ssRNA copy as template for new genomes
what happens in (-)ssRNA genome replication?
- virus particle must include the viral Pol
- Pol makes mRNA for translation
- genome replicates through full-length (+)ssRNA intermediate
what happens in dsRNA genome replication?
- virus particle includes viral pol
- dsRNA induces innate immune response so genome stays inside particle
- mRNA synthesized in particle and exported to cytoplasm
- mRNA serves as (+) strand in virus genome, (-) strand synthesized during assembly
what happens in ssDNA and dsDNA genome replication?
- must gain access to the nucleus:
- EXCEPTION: poxvirus
- viron contains the necessary RNA pol and the genome encodes the DNA pol for replication
- prepare the cell for DNA replication
- growth phase, dNTP production, replication machinery
-ensure genome ends are copied
what is viral assembly?
package of new genomes into functional particles
how is viral assembly accomplished?
- localize structural proteins to aid assembly: cellular viral “factories”
- genome contains packaging signals
describe the adenovirus assembly mechanism
empty protein coat and imports genome
describe the reovirus assembly mechanism
RNA packaged during capsid assembly
describe the retrovirus assembly mechanism
preassembly on a membrane
what is virus release by lysis?
- best know for bacteriophage
- viral molecules that rupture cellular membrane
what is virus release by weak lysis?
depends on membrane breakdown after cell death
what is virus release by budding (enveloped only)?
enveloped viruses use cell membrane as the outer coat of the virus particle
what advantages come with targeting virus replication in the nucleus?
RNA has much higher mutation rate compared to DNA
what disadvantages come with targeting virus replication in the nucleus?
have to get thru nuclear pore complex
how does understanding a virus’ replication process help to design antivirals?
so you can block cellular receptors to shut it down or disrupt reverse transcriptase
what advantages/disadvantages come with lytic vs. non-lytic infections?
lytic: move faster in order to affect subsequent host
non-lytic: can have persistent membrane effect by having viruses bud off
what type of kinetic growth curve do viruses have?
one step growth curve: infect every cell at same time (MOI or multiplicity of infection >5), every cell dies at end of infection
what are the phases of viral replication?
- eclipse: attachment and uptake
- exponential growth: replication and assembly
- plateau: cell death
what is the virus replication growth curve useful for?
to asses mutations, cell entry, process design
how is the time for one step growth measured?
start of infection to beginning of plateau
what does the productivity of the one step growth curve measure?
amplification
STUDY TABLES ON SLIDES
STUDY TABLES ON SLIDES
where is initial viral detection at?
- disease in a host
- contaminant in cell culture
- molecular methods are useful for viruses that cannot be cultivated
how is viral infection confirmed?
- purification of virus
- confirmation of disease: animals, eggs, or cell culture
- cell culture is preferred
what are the 4 principles of detection and quantification methods?
- infectivity
- physical-particles (how many)
- genome
- serological
*just because genome doesn’t mean that it is infectious
name 4 infection assays and how they work
cytopathic effect (CPE)
- cell rounding
- syncytia formation
- inclusion bodies
fluorescent focus assay
- infect cells
- expose virus antigen
- stain with labeled antibody
- count areas that fluoresce
plaque assay
- cell monolayer
- inoculate with dilute virus
- infected cells die leaving a clear area = plaque
infectious dose
- systems: tissue culture, eggs, animals
- inoculate with different dilutions of virus
- calculate conc based on number infected
name 2 particle assays and how they work
electron microscopy
- direct image of virus particles
- calibrate with latex bead standard
hemagglutinin assay
- viruses that bind RBCs
- mix constant number of RBCs with various virus dilutions
- if virus conc sufficient, a matrix of RBCs and virus is formed
- matrix does not allow RBCs to pellet
- (no virus when red dot)
name 2 genome assays and how they work
PCR
- DNA primer specific to virus
- amplify the gene
- very sensitive
southern/norther DNA blots
- isolate DNA or RNA
- separate by electrophoresis
- used labeled DNA probe to detect
name 3 serological assays and how they work
virus neutralization
- antibody binding to virus can block infection
- virus conc determined by amnt of antibody needed
ELISA
- antibody recognition of virus
- amplification by enzyme linked to antibody
Western (Protein) Blot
- separate proteins by electrophoresis
- probe proteins using an antibody
T/F different methods of detection and quantification give different answers
true
how much infectious virus do you have?
particle to PFU ranges from 1:1 to 1:10,000
aka 10000 particles before seeing 1 infection
viruses are obligate _______ parasites; use host cell’s replication processes to duplicate themselves
intracellular
every virus has a _______ consisting of genetic material (RNA or DNA) and protein; some viruses are ________ (coated with a host cell membrane)
nucleocapsid
enveloped
viruses are “alive” only ______ cells
inside
b/c replicate only within cell
viruses are classified by the ______, __________, and __________
- genome
- virus particle structure
- replication strategy
