Brett's Flashcards
(33 cards)
1
Q
Influenza Virus genome
A
- RNA virus
- The genome consist of 7-8 RNA fragments, each coding for a viral protein
- 8 genes are responsible for the translation of 10-11 viral proteins.
2
Q
Influenza virus structure
A
- Nucleocapsid: RNA enclosed in a protein coat
- Surrounded by a lipid envelope
- Two glycoproteins present: HA and NA
3
Q
Influenza Virus Lifecycle in detail
A
- HA binds to cell GP at a Sialic Acid binding site
- Clathrin-coated pit endocytoses virion
- Conformational change: hydrophobic binding of HA to vesicle membrane - RNAs are released into cytoplasm for replication and transcription (vRNA and mRNA)
4
Q
Influenza Virus lifecycle
A
- The flu virus binds onto sugars on the surface of epithelial cells such as the nose, throat, lungs of mammals and intestines of birds
- Binding of the virus to the cell allows it to initiate its entry in the cell
- Two glycoproteins allow the binding and release of the virus from the surface of cells
- These proteins are integral membrane proteins (IMP) of the virus
5
Q
Neuraminidase
A
- Allows release of the newly formed viruses within the host
- Determinant of disease severity
6
Q
Hemagglutinin (HA)
A
- responsible for pathogenicity of the virus
- allows virus to adhere to endothelial cell sin the respiratory tract
- main determinant of immunity.
7
Q
Influenza:
Viral transcription of Translation
A
- accessory proteins and vRNA form a complex
- transported in the cell nucleus. RNA dependent RNA polymerase begins transcribing complementary positive sense vRNA
vRNA has two fates
- exported into the cytoplasm/translated (mRNA)
- remains in the nucleus (cRNA), replication of vRNA occurs in the nucleus.
8
Q
Influenza:
Production of viral mRNA
A
- The viral endonuclease (packaged in the influenza virus) snips off 13-15 bases from the 5’ end of the host mRNA
- Used as a primer for viral mRNA synthesis (all flu mRNA’s have a short stretch at the 5’ end which is derived from host mRNA
- The viral RNA replicase extends the primer and copies the template into complementary plus sense mRNA and adds a poly (A) tail
- Transcription results in 8 primary transcripts, one transcript per segment. So give rise to alternative transcripts
- 8 segments, last two have splice variance
9
Q
Influenza:
Replication of viral RNA
A
- RNA replication occurs in the nucleus using RNA replicase
- A full length, exact complementary copy of virion RNA is made (cDNA)
- cRNA is then used as a template for full length minus strand synthesis
- New minus strands can be used as a template for replication, mRNA synthesis or packaged.
10
Q
Influenza:
Release of newly formed influenza viruses
A
- Replicated vRNA, RNA replicases and other viral proteins are assembled into virions
- HA and NA cluster into a cell membrane bulge
- Virion leaves the nucleus and enters the membrane protrusion
- Mature virus buds from cell in host membrane containing HA and NA
- HA binding virus to cell surface via receptors containing sialic acid
- NA cleaves receptors allowing release of virus
11
Q
M-/T-tropic HIV
A
Two types of HIV strain in virus transmission
Early HIV transmission (virus in M-tropic)
- gp120 is able to bind to CD4 and chemokine receptors, CCR5
- found on macrophages
- occurs in 90% cases
Late phase HIV infections (virus is T-tropic_
- gp120 capable of binding to CD4 and chemokine receptor CXCR4
- found on T-lymphocytes
- phenotypic switch from M-tropic
12
Q
HIV:
Reverse transcription
A
- Viral reverse transcriptase (VRT) becomes active in cell cytoplasm
- Conversion of viral RNA into double stranded viral complementary DNA (cDNA) commences
- Reverse transcription is extremely error prone
- VRT also has ribonuclease activity and DNA polymerase activity
- -viral RNA degraded during the synthesis of cDNA
- -creates a sense DNA from the antisense cDNA
- cDNA and its complement form a double stranded viral DNA that is then transported into the cell nucleus
- vDNA integrates into the host cell genome (provirus). Carried out by viral enzyme called integrase
13
Q
HIV:
Viral replication
A
- Integrated DNA provirus transcribed into mRNA
- mRNA spliced into smaller pieces. Exported from the nucleus in the cytoplasm
- mRNA translated into the regulatory protein (tat, rev)
tat=encourages new virus production
rev-protein accumulates in the nucleus. Binds virus mRNA, allowing unspliced mRNA to leave the nucleus.
14
Q
HIV:
Retrovirus Replication Cycle
A
- Fusion of HIV to the host cell surface
- HIV RNA, reverse transcriptase, integrase and other viral proteins enter the host cell
- Viral DNA is formed by reverse transcription
- Viral DNA is transported across the nucleus and integrates into the host DNA
- New viral RNA is used as genomic RNA and to make viral proteins
- New viral RNA and proteins move to the cell surface and new immature HIV forms
- The virus matures by protease releasing individual HIV proteins
15
Q
HIV:
Retroviral genome
A
- Retrovirus contains two copies of the RNA genome held together by multiple regions of base pairing
- The RNA complex also includes two molecules of a specific celluar RNA (tRNA lys) that serves as a primer for the initiation of reverse transcription
- The primer tRNA is partially unwound and H-bonding near the 5’ end of each RNA genome in a region called the prier binding site.
16
Q
HIV Genome: Major Genes
A
gag
= “group specific antigen”. Encodes structural proteins, capsid, matrix, nucleoprotein (RNA binding)
pol =encodes enzyme -proteases cleaves viral polyprotein -RT/RNases for reverse transcription -Intergrase cuts cell DNA to insert proviral DNA
env= “envelope”
-encodes for envelope glycoproteins; surface, transmembrane
17
Q
Reverse transcription of HIV-RNA into dsDNA
A
- -During transport to the nucleus, the viral ssRNA genome is reverse transcribed into dsDNA by the viral RT.
- Reverse transcription goes in the 3’-5’ direction
- The tRNA which hybridises to the PB site provides a hydroxyl group for initiation of reverse transcription
- While ssDNA sequence is synthesised, the complementary ssRNA is degraded by RNase H function of RT - The DNA-tRNA hybrid molecule is then transferred to the 3’ end of the template and is used for first strand synthesis. Afterwards, the ssRNA is degraded except for the PP site, which serve as a new primer
- The initial second strand synthesis of ssDNA starts from the 3’-end of PP, which will be finally degraded.
The tRNA makes it possible to synthesise the complementary PB site. - After the tRNA is degraded, the first and second DNA strand hybridise at their PB site, which they harbour on their ends.
- Both strands will be completed by the DNA P function of RT. Compared to the ssRNA, both dsDNA ends now have a U3-R-U5 sequence that is also called a long terminal repeats (LTR)
18
Q
HIV:
Activation of viral transcription
A
- Integrated viral DNA may lie dormant
- –latent stage of HIV infection
- –last up to 10 years
- Viral replication is triggered
- –host cellular transcription factors are needed
- NF-kB is very important. Up regulated in activated macrophages
- Cells most likely to be killed by HIV are those currently fighting infection
19
Q
HIV:
T cell death
A
HIV infection leads to low levels of CD4+ T cells through:
- Direct viral killing of infected cells
- Increased rate of apoptosis in infected cells
- Killing of infected CD4+ T cells by CD8 cytotoxic lymphocytes that recognise infected cells
CD4+ T cells numbers decline below a critical level
- cell mediated immunity is lost
- body becomes susceptible to opportunistic infections.
20
Q
HIV:
Body’s response to loss of CD4 cells
A
- Body attempts to replace lost CD4 cells
- Over many years the body is unable to keep the count at a safe level
- Destruction of large numbers of CD4 cause symptoms of HIV to appear.
21
Q
HIV: Genetic variability- Mutations
A
- HIV has a very high genetic variability
- Fast replication cycle- generation of about 10^10 virions every day
- High mutation rates
- Generation of many variants of HIV in a single infected patient in the course of one day
22
Q
HIV: Genetic variability- Recombination
A
- Recombinogenic properties of vRT
- Single cell simultaneously infected by two or more different strains of HIV
- Genome of progeny virions maybe composed of RNA strands from two or more different strains
- This hybrid virions then infects a new cell where it undergoes replication
- vRT jumps back and fourth between the two different RNA templates
- Newly synthesised retroviral DNA sequence that combines the two parental genomes
23
Q
Life cycle of Lambda
A
- Virus enters cell
- PL and PR gets activated
- PL transcribes to make N protein
- PR transcribes to make cro protein
- Termination sites stop transcription but when enough N protein is made, transcription goes past these two stop sites (you can now make cIII and cII, replication proteins (O and P) and Q)
- There are also termination sites next to Q protein. Q protein will allow transcription past this site.
- If Cro protein blocks production of cI (goes lytic)
- If cII and cIII activates transcription to make cI (goes lysogenic)
24
Q
Bacteriophage Replication:
Lytic cycle
A
Lytic cycle= results in cell lyses and release of progeny phage
- Phage injects its DNA into bacterial cell
- Phage proteins are expressed and take over protein synthesis and DNA replication machinery of infected cell
- Phage DNA replication occurs
- Phage particles are assembled with phage DNA and protein
- Infected cell burst releasing 100-200 viral particles able to infect other cells
25
Bacteriophage Replication:
Lysogenic cycle
- Does not result in immediate lysing of the host cell
- Viral genome integrates with host DNA and replicates along with it fairly harmlessly (prophage)
- Virus remains dormant until host conditions deteriorate, (maybe due to nutrient depletion)
- Prophages activate and initiate the reproductive cycle resulting in lysis of the host cell
- Lysogenic cycle allows host cell to survive and reproduce, allowing the virus to be reproduced in all cell's offspring
26
Lytic or Lysogenic
- Cells with sufficient nutrients, protease activity is high which breaks down CII= Lytic lifestyle
- Cells with limited nutrients, protease activity is low making CII stable =Lysogenic lifestyle
- CIII appears to stabilise CII, both directly and by acting as a competitive inhibitor to the revelent proteases
- This means that a cell in trouble eg. lacking nutrients and in a more dormant state is more likely to lysogenis
- This would be selected for because the phage can now lie dormant in the bacterium until it falls on better times and so the phage can create more copies of itself with the additional resources available and with the more likely proximity of further infectable cells.
27
Genetic switch: Cellular proteases
- Activity of the cII protein plays a key role in directing lambda to the lysogenic or lytic cycle
- The cII protein is easily degraded by cellular proteases produced by E. coli
- Whether or not these proteases are produced depends on the environmental conditions
28
Cellular proteases choosing lytic cycle
Growth conditions are very favourable, the intracellular levels of the proteases are high
-The cII protein tends to be degraded
-Therefore, PRE cannot be activated and the lambda repressor is not made
-Instead, the cro protein slowly accumulates to high levels
-The binding of the cro protein to OR prevents transcription of the lambda repressor from PRM
-At the same time, the cro protein allows the lytic cycle to proceed
-Thus, environmental conditions that are favorable for growth promote the lytic cycle
This makes sense because a sufficient supply of nutrients is necessary to synthesize new bacteriophages
29
Cellular proteases choosing lysogenic cycle
-If the nutrients are limiting (starvation conditions), the cellular proteases are relatively inactive
-The cII protein builds up much more quickly than cro
-Therefore, the cII protein will turn on PRE
The lambda repressor is made
-Environmental conditions that are unfavorable for growth promote the lysogenic cycle
This makes sense because there may not be sufficient nutrients for the production of new bacteriophages
30
How do cells leave lysogenic cycle and go to the lytic cycle?
By stress
UV light
- recA (a cellular protein normally involved in DNA recombination) detects the DNA damage
- It is activated to become a protease
- It cleaves the lambda repressor and inactivates it
- This allows transcription from PR
- Cro protein accumulates favouring the lytic cycle
Nutrients
Anything that causes disruption of DNA induces lytic cycle
31
Lambda OR Region
The OR region provides a genetic switch between the two cycles
- The OR region contains 3 operator sites
- These operator sites control two promoters PR and PRM which transcribe in opposite directions
- The lambda repressor protein or the cro protein can bind to and or all of the 3 operator sites
- This binding governs the switch between the lysogenic and lytic cycle
Two critical issues influence this binding
1. The relative affinities that the regulatory proteins have for these operator sites
2. The concentrations of these regulatory proteins in the cell
32
Influenza
Mutation of vRNA
- absence of RNA proof reading enzymes
- RNA pol that copies the genome makes an error every 10 000 nucleotides
- Majority of newly manufactured viruses are mutants
- This enables the virus to alter surface anitgens slowly over time
Leads to antigenic drift.
33
Antigenic shift.
- More than one virus can infect a host cell at one time
- Allows mixing of the 8 separate segments of the vRNA and reassortment
- Rapid change in viral genetics and antigen expression
Leads to ANTIGENIC SHIFT
Both shift and drift allow the virus to evade host immune system
