Midterm 1 Material Flashcards
Give three reasons why we study viruses and sub-viral agents and their respective importance
- medical, veterinary and agricultural importance
- some are very common (e.g. cold)
- some are highly lethal (e.g. HIV/AIDS)
- some cause dread, major disruption, economic loss (e.g. Sars-CoV2)
- some infect animals and plants leading to econmic loss (e.g. foot mouth disease) - source of fundamental info about biology and medicine
- flow of genetic information
- mechanisms of DNA rep., transcription, translation, RNA splicing, and protein localization
- cell cycle and cancer
- immunology - industrial and therapeutic applications
- recombination DNA tech (e.g. using reverse transcription to make RNA into DNA)
- gene therapy
- modulation of the immune system
- treatment of cancer
Give three reasons why viruses are distinct from other biological entities & name which one is the most important factor
- obligate intracellular parasites
- replicate by assembly (most important factor)
- depend of HC for energy, metabolic precursors, translation machinery
What is the viral genome comprised of?
DNA or RNA, but never both
What is the capsid?
protective protein shell that consists of smaller subunits, capsomeres that are arranged in a symmetrical pattern
What is the viral envelope?
- lipid bilayer which contains viral proteins (some viruses, not all) where the membrane belongs to the host membrane
- without the envelope, the virus is no longer infectious (given that it is an enveloped virus)
What are the eight steps in the viral life cycle? Name and describe each step
- attatchment - viral attatchment proteins bind to a specific cellular receptor
- penetration - getting across the cell membrane
- uncoating - release of the viral genome from the capsid and delivery to the correct cellular location
- transcription - production of viral mRNA
- translation - production of structural and non-structural proteins
- genome replication - production of multiple progeny virus genomes
- assembly - assembling genomes and structural proteins into progeny virions
- release - release of progeny virions by cell lysis (non-env) or budding (env.)
What three factors classify viruses as biological systems?
- contain a nucleic acid which replicates
- encode more than one protein
- undergo mutations and natural selection (e.g. emergence of covid-19 variants)`
What do all viruses rely on the HC for?
- biosynthetic precursors (a.a and nt)
- energy
- machinery to translate mRNA
What do some viruses depend on the HC for?
- enzymes that replicate the viral genome
- enzymes that transcribe viral mRNAs
Which viruses have been associated with humans since specification, and how do we know that humans have been associated with viruses since speciation?
viruses: herpesvirus and endogenous retroviruses
how: 10% of the human genome consists of retrovirus proviral genomes
Which viruses have been recognized since antiquity, and what is one piece of evidence?
viruses: polio and smallpox
evidence: ancient Egyptian tablets depicting an injury caused by polio
Which viruses have been associated with humans more recently?
HIV, ebola, sars-cov2
which viruses are zoonotic and when did they jump?
HIV-1 -> early 20th century
Sars-Cov2 -> 2019
How did the move from hunter/gatherer societies to agricultural societies effect viral spread?
increased population density -> greater number of hosts for viruses
What are the properties of a virus that can maintain stablitity in a small population vs that of a large population? Name an example of each
small population: viruses that establish life-long latent infections (e.g. herpesvirus)
large population: acute viruses (e.g smallpox)
Why did variolization come about?
Life long immunity from smallpox was evident which led to deliberate immunization because the mortality rate of variolization was much smaller than that of getting smallpox without variolization
What is variolization?
deliberate infection with variola virus to generate mild disease with protective immunity
What are Koch’s postulates?
- the organism must always be present in diseased people or animals
- it must be isolated as a pure culture in vitro, say by growth on nutrient agar plates
- the isolated organism must cause the same disease when injected into a healthy host
- the organism must be reproducibly isolated from the diseased test hosts
How did vaccination arise?
Jenner used cowpox scabs instead of smallpox scabs to deliberately immunize people, which was just as effective as variolization but was much safer
What are the three revolutionary changes in our understanding of infectious diseases?
- Pasteur discovered that cellular life forms do no arise by spontaneous generation
- Koch discovered that infectious diseases are caused by bacteria
- Beijernik established the existence of acellular infectious agents (viruses)
What were Beijernik’s major observations of the agent that disfigured tobacco leaves?
- agent passed through filters that retained the smallest known bacteria
- the filtrate could be diluted, but with loss of infective strength
- strength was increased by passage through intact leaves, but not by incubation in extracts of uninfected leaves
- agent is capable of replication, but only within living host cells
Name and describe two other early examples of “filterable” agents besides Beijernik
Loeffler and Froth: foot and mouth disease of cattle
Reed: yellow fever in humans
What is the differences between segmented and continuous viral genomes? Name an example for each.
Segmented: multiple nucleic acid segments that make up the entire genome (e.g. influenza A)
Continuous: one nucleic acid sequence (e.g. Sars-Cov2)
What is the purpose of the capsid?
- Encases and protects the genome
- mediated binding to the HC and entry of naked viruses
What are the symmetrical properties of viral capsids?
- capsid is symmetric
- comprised of multiple copies of more than one proteins
- the subunits themselves are asymmetric
How is helical symmetry defined? Name a virus that has helical symmetry.
- Length is defined by the length of the genome (not fixed by capsid)
- can be open ended , but some viruses have capping proteins on the end
- Virus: TMV, bacteriophade M13, rabiesvirus
What are the properties of icosahedral symmetry?
- 20 trianglular faces
- 12 vertices that have 5-fold symmetry
What are the properties of the simplest icosahedral capsids? Name an example of a virus.
- 60 subunits, 3/face
- e.g. canine parovirus
- each subunit contributes to both a face and vertex
What is the purpose of having more complex icosahedral capsid?
It uses more subunits which encloses a larger volume
How are bigger faces made in complex icosahedral capsid shapes?
Out of groups of smaller traingels, or other shapes that have a 3-fold axis symmetry (e.g hexagons)
What is the equation to determine the number of subunits/capsid (N), and what do the terms represent?
N = 60T, where T = triangulation number
What values can the triangulation number (T) be?
T = 1, 3, 4, 7, 9, 12
Name an example of a virus with a T= 3 capsid, how many subunits make up the capsid, and how are these subunits arranged?
Virus: nodavirus
Total subunits: 180 (60 x T -> 60 x 3 = 180)
Arrangements: 20 groups of 6 (hexons) and 12 groups of 5 (pentons)
How does a virus build a capsid if all the capsid proteins are the same?
the virus uses quasi-equivalent interactions to form pentons and hexons
How are capsids organized when T>1?
- 12 vertexes made of pentons (60 subunits)
- the remaining subunits (60T-60) are organized into hexons -> faces
- note for that T =4, some of the hexons straddle the boundaries between faces
Name a virus that contains combined symmetry
bacteriophage T4
Name a virus that contains complex capsid symmetry
Poxviruses -> small pox
What is the Baltimore classification system?
An unoffical classification system based of gene expression and replication strategy of viruses
Name a ssRNA (+) retrovirus, and does it pack any enzymes in viral particles and why?
HIV, yes it has to pre-package reverse transcriptase because the RNA is reversely transcribed to DNA and the DNA is transcribed to make mRNA, the RNA genome is not used for translation.
Name a ssDNA virus
canine parovirus
Name a dsDNA virus and does it pre-package it’s own ezymes and why or why not?
Adenoviruses, no it uses cellular DNA-dep RNAp
Name a ssRNA (+) viruses and does it pre-package its own enzymes, why or why not?
Sars-Cov2, no it only needs cellular machinery to translate it’s genome
Name a ssRNA (-) virus and does it pre-package its own enzymes, why or why not?
Rabies, yes it pre-packages RNA-dep RNAp because the genome cannot be translated without the positive sense RNA and thus cannot make it
Name a dsRNA virusus and does it pre-package any enzymes, why or why not?
Rotavirus, yes it pre-packages RNAdep RNAp because ribosomes cannot denature dsRNA, and thus no protein can be made until the dsRNA is denatured
What is the International Committee on Taxonomy of Viruses (ICTV)?
Offical classification system of viruses primarily based on genetic relationships
Name four methods that detect virus particles
- electron microscopy
- nucleic acid hybridization or PCR
- immunodetection of viral structural proteins
- hemagglutination
Name a viruses that leads to hemagglutination of eythrocytes?
influenza
How do you perform a plaque assay?
- prepare serial dilutions of virus stock
- inoculate aliquots of each dilution into susceptible cells
- overlay with agar to restrict virus diffusion (prevents viral progeny from spreading)
- count plaques
How are plaque assays quanitified?
plaque forming untis (PFU) per mL
Why is a viral titre usually less than the number of viral particles?
- viral particles are defective
- cells fight back
What are a few limitations of plaque assays?
- not every cell infected with a virus produced progency virions
- does not work with non adherent cells
- not all viruses kill cells
What are seven characteristics of viral receptors?
- protein or carbohydrate linked to a protein or lipid
- can be cell-type specific (HIV -> CD4 and chemokine R) or broadly distributed (e.g. EGFR)
- influence host-range of the viruse -> tropism
- serve normal cellular functions
- some serve as the receptor for more than 1 type of virus (e.g. SA: influenza, reovirus, rotavirus)
- some related viruses use different receptors (e.g major group rhinovirus -> ICAM1, minor group rhinovirus -> LDL)
- can play a major role in dictating cell-cell specificity of virus (e.g. CD4 and HIV -> macrophages express more CCR5 co-R)
Which co-receptors are used for HIV binding?
CCR5 or CXCR4 are used, with CCR5 required for efficient initial infection of a person
Where are G- viral receptors located?
on the outer membrane only
Is human CD4 necessary for infection by HIV?
Yes - CD4+ human fibroblasts were able to be infected by HIV
How do we know that human chemokine receptors are necessary for entry of HIV?
When human CD4+ mice were exposed to HIV, they were not infected
When human CD4+ and human chemokine receptors+ mice were exposed to HIV, they were infected
Note: most human cells inherently contain chemokine receptors
What mutation allows for resistance to HIV infection? Is this mutation recessive or dominant?
32bp del in the CCR5 gene, recessive gene
If someone is homozygous for D32 what is their phenotypic response to HIV
” heterozygous for D32 “
homozygous: highly resistant to infection
heterzygous: progress more slowly to AIDS
What caused selection for the D32 mutation in humans and why? Who contributed to the spread of the D32 mutation?
selection: Black Death (Y pestis uses CCR5) or smallpox (severity of disease is increased by CCR5 signalling)
spread: vikings?
What are the three steps to viral entry?
- getting the viral genome across the p.m OR get the phage genome across a membrane and a cell wall of bacteria
- uncoating
- capsids are stable
- must disassemble rapidly in a cell - delivery of the genome to the appropriate sub-cellular location
- e.g. nucleus or cytoplasm -> tend to be the most metabolically active
What is a common phage receptor located on G- bacteria?
LPS
How does bacteriophage T4 inject its DNA?
- legs bind to the recepor
- injects in viral genome (DNA)
What did the Hershey-Chase experiment demonstrate?
That the protein coat of phages remain at the cell surface while the nucleic acid is internalized; this allowed them to separate cells and the viral capsids to determine that DNA was the genetic material
What happens to viral proteins when enveloped viruses enter the HC?
viral proteins become integrated into the HC membrane
Describe HIV-1 entry
- the viral proteins that are involved are gp41 and gp120
- the viral receptors are CD4 and CCR5 or CXCR4
- gp120 binds to CD4 -> gp120 undergoes a conformational change that allows it to interact with CCR5 or CXCR4
- after binding to CCR5 or CXCR4, gp41 is exposed which can trigger fusion
How does respiratory syncytial virus enter HCs?
- viral F fusion protein binds insulin-like GF-1 R
- triggers a cellular signalling pathway that calls up an internal cellular protein (nucleolin) to the surface
- F then binds nucleolin
- fusion and cell entry
Which viruses undergo pore-mediated penetration?
picornaviruses - polio and major group rhinoviruses
Why do some viruses evolve to do endocytosis as a mode of entry?
To get their genome closer to its target site
Why do viruses use clathrin-mediated endocytosis?
it is fast
How do enveloped and naked viruses escape the early endosome?
enveloped: fusion
naked: lysis or permeabilization
What triggers enveloped viruses to escape the endosome
pH decreasing to 6
What are the steps to enveloped viruses escaping the endosome?
- viral fusion protein is bonded to the host receptor
- low pH causes unmasking of fusion-peptide
- host membrane penetration
- host membrane scission
- apposition
- hemifusion
- pore
How does influenza employ low pH of the endosome to uncoat its genome?
- protons enter virion by M2 ion channels
- low internal pH displaces M1 matrix protein from NP-RNA complexes
- nuclear localization signal on NP proteins targets RNP complexes to nucleus
How does adenovirus escape the lysosome?
lysis
How does reovirus escape the endosome?
local permeabilization of the endosomal membrane
How does the genome get delivered to its target?
on microtubules or actin filaments