emma (L23)

Question 1

HIV and HBV statistics

Answer 1

HIV:
34 million infected worldwide; 2.5 million new infections in 2011
1.7 million deaths from AIDS (15% were children) in 2011

HBV:
400 million people chronically infected worldwide
15-25% will die prematurely: hepatocellular carcinoma, cirrhosis

Question 2

Human immunodeficiency virus

Answer 2

Causes destruction of T cells of immune system.
T cells normally defend body against bacteria, fungi, viruses. When blood contains less than 200 T cells/μl, AIDS develops.

HIV infects cd4+ T cells
causes their destruction - T cells have a shorter half life when infected
so get shortage of T cells (which would protect us otherwise)

Question 3

origins of HIV

Answer 3

Zoonotic transmission (trans-species). Simian to human:

HIV-1: from SIVcpz. Evidence: Viral genetic homology; geography of viruses. At least 4 transmission events gave rise to HIV-1 groups M, N, O and P. Group M viruses are estimated to have entered humans in the 1930s.

HIV-2: from SIVsm. Evidence: Viral genetic homology; geography.

Keys to cross-species transmission: (1) initial infection and (2) simple transmission within the new host (includes urbanization, trucking/trade, air travel)

Question 4

Retroviral Replication Cycle

Answer 4

Virus - cell surface interaction
Membrane fusion and capsid release
Reverse transcription of genome RNA into DNA (called proviral dna)
Transfer of proviral DNA to nucleus
Integration of provirus into host chromatin
Viral gene expression and progeny genome synthesis
Assembly of particles and maturation
Release from cell by budding

Question 5

genes in HIV genome

Answer 5

there are 2 copies in a viral particle (diploid)
ssrna is 10kb long, has LTRs on either ends (long terminal repeats)
along the genome we have a nmbr of genes that have a:
- gag gene encodes the structural genes of the virus
- pol gene encodes the enzymes of virus
- env gene encodes the envelope glycoproteins of virus
gag pol and env are common between all retroviruses

Question 6

proteins in HIV genome

Answer 6

TAT and rev are regulatory proteins involved in gene expression
VPR, VPU and nef are accessory proteins used
vpr involved in nuclear import
other proteins involved in modifying the host response to the virus (important role)

hiv is an enveloped virus
under membrane - layer of protein called the matrix protein (similar to flu and measles)
within viral particle there is another protein capsid made up of capsid protein - inside the capsid is the 2 copies of viral genome (+ve sense rna) wrapped up in nuclear capsid protein

R2 - reverse transcriptase - will catalyse the reverse transcription process of rna to dna
integrase - responsible for integrating the proviral dna into the host cell chromosomes
protease - during maturation process, cleaves the polyprotein into individual segments, to rearrange themselves into the correct structures seen in infectious particle

(gag and pol are single genes - because gag is translated as a polyprotein, others as one protein. polymerase translated as polyprotein too)
envelope gene - also a polyprotein, has 2 parts to it - surface and transmembrane (forms a trimer)

Question 7

HIV Receptor Binding

Answer 7

DIAGRAM IN L23 S14

Question 8

HIV fusion and entry

Answer 8

DIAGRAM IN L23 S15

Question 9

what does productive uncoating require?

Answer 9

Productive uncoating requires a cellular chaperone protein (cyclophilin A)

Chaperones
facilitate protein folding and assembly

Virus particles lacking cyclophilin A fuse and enter but cannot reverse transcribe

• Point mutations in capsid that cannot bind cyclophilin A
• Viruses produced in the presence of Cyclosporine A

Efficient RT may require expansion or partial disassembly of the viral capsid core

RT mechanism essentially as for ALV etc

Question 10

Reverse Transcriptase

Answer 10

Enters cell in particle; incoming genome has no chance to be translated to give new enzyme.
Enzyme acts within partially uncoated viral core

RT protein includes a domain with a distinct activity (RNaseH)
RNaseH degrades RNA specifically when in a heteroduplex with a DNA strand

Question 11

Reverse Transcription Process

Answer 11

(-) strand primed by tRNA bound at tb
RNase H exposes the DNA copy of R and U5
Base pairing between R’ and the second copy of R (1st template jump)
Continued (-) strand synthesis and template RNA degradation
Poly purine oligo (polyP) is specially spared degradation to prime (+) strand DNA synthesis
(+) synthesis uses new (-) DNA as template and continues until a modified base in tRNA blocks extension; tRNA degraded by RNaseH
tb and tb’ at 3’ ends of (+) and (-) strands base pair (2nd template jump)
extension of both strands to completion

DIAGRAM IN L23 S19-20

Question 12

proviral genome has to get to nucleus

Answer 12

proviral genome has to get to nucleus
matrix protein, integrase and vpr are needed proteins
red lines - represent dna provirus
reverse transcriptase molecule still bound to dna
then have integrase enzyme - present in virus particle, comes in with the virus and binds to the 2 ends of the viral dna
then have binding of matrix protein and vpr
those 3 proteins are involved in the docking of that preintegration complex with the nuclear pore complex
allows import into nucleus

this virus has vpr → simple retroviruses dont have additional proteins like vpr. can only get into nucleus during cell replication when nuclear membrane breaks down
hiv has vpr so can infect non dividing cells (like resting T cells)

Question 13

integration process steps

Answer 13

1) Processing (Integrase)
2) Joining (Integrase)
3) Repair (Host cell)

Question 14

integrated proviruses

Answer 14

Infection is not cytolytic

Provirus will replicate with the host genome at every round of division and propagate to every daughter cell

Provirus can be inherited between generations if virus infects a germ cell

Genomes of humans and other species contain large amounts of vestigial retroviral sequences

Question 15

Basic Retrovirus Gene Expression

LTRs on both 3’ and 5’ ends

Answer 15

if the cell is dormant/non-activated/resting, the provirus might sit latent in the genome for a long time
once gene expression is activated, then productive viral replication can happen (provirus has to be transcribed)
provirus acts like a cellular gene and is transcribed by the host using polymerase 2, which generates mRNAs that are capped and polyadenylated

this is the integrated provirus with LTR on each end
simple retroviruses - genes encoded are gag pol and env genes
reverse transcriptase produces these LTRs that are a bit longer than the viral rna
5’ LTR consists of the U3,R,U5 region (contains promoter for gene expression and the enhancer)

5’ end contains the transcription factor binding site, to recruit rna pol 2, to transcribe the viral rna
transcription start site is at the beginning of the R region

3’ end has the LTR that has the polyadenylation site, signal to make polyadenylated mrnas
using signals the host rna pol 2 will start transcribing on left, and make the full length genomic mrna (this mrna encodes gag and pol, can become new viral particles)
to produce M it needs to be spliced (splice donor just upstream of gag, and the splice acceptor at the end of pol → so you splice out gag and pol regions and produce the mrna that contains the M gene)

HIV is a complex retrovirus and has additional factors that aid it in its life cycle
TAT is a viral transactivator (so impacts the gene expression process)

Question 16

how is the TAT gene essential for HIV infection?

Answer 16

Tat is a Transactivator of Transcription
HIV LTR linked to a reporter gene showed up to 1000x increase in expression when Tat supplied
In studies to map the promoter elements targeted by Tat, found TAR (Tat-response) element, immediately downstream of txn start site
TAR function is absolutely position and orientation-dependent

Suggests TAR might function as RNA, not DNA

Question 17

mechanisms of TAT action - in the presence and absence of TAT

Answer 17

IN ABSENCE OF TAT
The HIV LTR assembles a poorly processive RNA polymerase complex
Once clear of the promoter, RNA pol frequently drops off the template, producing a truncated RNA
Short RNA comprising TAR can often be detected

IN PRESENCE OF TAT
The RNA polymerase complex is converted to a fully processive mode
A high proportion of initiation events lead to full- length transcription

Question 18

Tat and the Loop Binding Factor

Answer 18

Loop factor is cyclin T1 : cdk9 complex (a.k.a. Tak – Tat- associated kinase)
– Activated by association with Tat/TAR and phosphorylates Cter of RNA pol II

Question 19

action of Tat

Answer 19

1. pol 2 and associated TFs (with ptefB). to be functional, we need phosphorylation of C terminal domain. this is bound to the RNA. part of the dna that encodes the Tar element is shown.
2. as rna pol 2 transcribes this rna, it folds into the stem loop that forms Tar, and it recruits the TAT protein and cyclin t1 and cdk9 also bind.
3. this activates the kinase activity in cdk9 and causes hyperphosphorylation of the C terminal domain.
4. this converts the rna pol2 complex into a highly processive enzyme which makes very efficient and full length RNAs.

→ tat is needed for full length and efficient transcription of HIV RNAs

Question 20

rna species of 9kb, 4kb and 2kb

Answer 20

9kb - unspliced - gag + pol
4kb - singly spliced - vif + vpr + vpu + env
2kb - multiply spliced - tat + rev + nef

Question 21

The Rev-minus Phenotype

Answer 21

Rev – regulator of virion protein expression

Compare gene expression from Rev-plus and Rev- minus viral DNA constructs:

In the absence of Rev, cytoplasmic mRNA for Gag, Pol, Env are reduced
In the absence of Rev, mRNA for Tat, Nef are unaffected
I.e. post-transcriptional effect

Question 22

Sequences Necessary for Rev Function

Answer 22

Used a Rev-dependent reporter construct to map sequences required for Rev to activate gene expression.

Sequences mapped to the Env coding region:

– Rev-responsive element (RRE) necessary for Rev to activate mRNA accumulation of an intron- containing construct

Question 23

function of RRE

Answer 23

RRE confers Rev-dependent export on mRNAs that contain it and that would otherwise be retained
RRE has a complex secondary structure

Rev

RRE binds Rev at a high-affinity site in RRE and Rev then multimerises on the RNA

Question 24

function of REV

Answer 24

In vitro, Rev coats RNA to form long fibrils
Does it work by occluding splice sites?
No, because of existence of transdominant negative mutants in the Activation domain
I.e. RNA binding and multimerization is not enough
Rev Activation domain is a nuclear export signal
Binds the exportin, Crm1, and exits dependent on RAN
Pathway of ribosomal 5S RNA export
Export competes with splicing, but splicing inhibition is not the mechanism of Rev action

Question 25

translation of HIV proteins

Answer 25

Early proteins include Tat (required for efficient transcription) and Rev (required for export of the late mRNAs for Gag, Pol, Env etc.).
Tat and Rev translated in the cytoplasm and then transported into the nucleus, and allow the nuclear export of nuclear mrnas for the late proteins:
Gag, Pol, Env and accessory proteins Vif, Vpr, Vpu are
late proteins.

Question 26

shift translation of ga and pol

Answer 26

Gag and Pol are translated from the full length HIV RNA.
The two proteins are in different, overlapping reading frames.
9 out of 10 ribosomes translate Gag and terminate at the Gag Stop codon.
Pol is produced by ribosomal frameshifting:
1 in 10 ribosomes translate most of Gag and then undergo a -1 frameshift: the ribosome slips back by 1 nucleotide and continues translating in a different reading frame to produce a Gag-Pol fusion protein

Question 27

How does frameshifting occur?

Answer 27

A stem-loop structure forms in the HIV RNA which causes the ribosome to pause during translation.

The ribosome pauses with its A and P sites containing a UUUUUUA slippery sequence .

10% of the time, the ribosome slips back 1 nucleotide, and then continues to translate = Gag-Pol fusion protein

Question 28

env protein production

Answer 28

Env is a transmembrane glycoprotein.
Co-translationally inserted into ER membrane.
Gp41 region of Env traverses the membrane, gp120 region of Env in the lumen of the ER.
Glycosylation of gp120 region occurs in the ER.
Env follows the route of membrane and secretory proteins in vesicles via the Golgi to the cell membrane.
Env clusters in regions of the membrane that are high in cholesterol, called lipid rafts.

Question 29

particle assembly requires:

Answer 29

2 copies of full length HIV RNA
Approx. 2000 molecules of Gag protein
Lipid envelope containing Env glycoproteins

Question 30

how does the HIV particle ensure that it only packages full length 9kb RNAs?

Answer 30

packaging signals just upstream of the gag coding region and just downstream of the splice donor
gag polyprotein binds to the packaging signal through its nucleocapsid domain and it multimerises along the rna

end up with rna coated in gag polyprotein
the rna contains a dimerisation signal (that is a palindromic sequence, pairs with itself)
so you end up with a dimer molecule, coated in gag
this occurs in cytoplasm and then it is taken to plasma membrane in the region where the lipid Raf contain the envelope glycoproteins

Question 31

the Gag-RNA complex

Answer 31

The Gag-RNA complex forms in the cytoplasm. This is transported to a region of the cell membrane containing Env proteins to form an immature virus particle.

This process requires cellular proteins called ESCRT (endosomal sorting complex required for transport) complexes, which usually function in vesicle trafficking.

Question 32

during/after the budding of immature viral particles from the cell

Answer 32

During or after budding of the immature virus particle from the cell, viral Protease self-cleaves from Pol, and cleaves the Gag and Gag-Pol polyproteins.

structural rearrangement = infectious virion

LONG NOTES IN L23 S55

Question 33

What are the roles of Vif, Vpu and Nef?

Answer 33

Accessory proteins involved in counteracting host antiviral and immune responses.

Vif – inhibits host cell APOBEC3G, an antiviral enzyme that causes hypermutation of the HIV genome.
–> protein that inhibits APOBEC3G (which is a cytosine deaminase), will cause hypermutation in the hiv genome so that it cant encode its normal protein, virus dies (vif gene counters APOBEC3G)

Vpu – inhibits host cell tetherin, a cell surface molecule preventing the release of viral particles.
–> counteracts antiviral protein called tetherin (which evolved to encounter any envelope virus and protect our cells against many enveloped viruses, because it tethers cell particles to the surface and doesnt allow their release) – VPU degrades tetherin to allow its particles to be released from the cell surface

Nef – causes the downregulation of MHC Class 1 and CD4 from the cell surface.
–> prevents superinfection of the cell with further hiv particles

Question 34

2 types of hepadnaviruses

Answer 34

• orthohepadnaviruses for mammals
  ( human hepatitis B virus
  woodchuck hepatitis virus*
  ground squirrel hepatitis virus)
• avihepadnaviruses for ducks
  (duck hepatitis B virus*
  heron hepatitis B virus)

each has a specific host animal, and specific for some hepatocyte cells in liver

Question 35

hepatitis B virus statistics

Answer 35

Parenterally transmitted jaundice (serum hepatitis) first documented in 1885.

Outbreaks often linked to improperly sterilised syringes and needles.

Surface antigen from HBV identified in the serum of patients with serum hepatitis (= hepatitis B) in 1967.
HBV particles (infectious particles) identified by Dane et al. in 1970. HBV DNA isolated in 1974.

Purified surface antigen licensed as a vaccine in 1981. (surface antigen used to vaccinate and stimulate the immune system to neutralise hep B particles)

Question 36

hep B virus particles

Answer 36

. 45nm dane particle - infectious particle, virulent, smaller than most viruses like flu and hiv (100nm in diameter). has an outside envelope with proteins on surface, core particle with genome.

. 22nm HBsAg - found in blood too, non infectious, empty, only has surface antigens.

. pleomorphic - long tube of membrane with surface antigen

(( a lot more empty surface particles than there are infectious particles. so they flood the system and soak up the immune response so that the virus can maintain a chronic infection with the individual ))

Question 37

dane particle structure

Answer 37

membrane shows surface antigens
black line is core icosahedral particle, has hep B genome
double stranded dna molecule - it is a relaxed circular form (not covalently closed circle like a plasmid) is attached to the p protein

DIAGRAM IN L23 S63

Question 38

HBV genome

Answer 38

Relaxed circular DNA (rcDNA).
3.2 kb partially double stranded.
Circularity maintained by overlapping 5 ends.
DR1 and DR2 are direct repeats.
(-) strand slightly longer than unit length; (+) strand less than unit length.

(( it is attached to the polymerase at the 5 prime end and slightly overlaps with itself. it is 3.4 kb and is slightly longer than the unit length
the positive strand is attached to an RNA 5 prime cap but it is 2.4 kb so is a bit shorter than the unit length

they are not conveniently joined into a circular shape so the shape is only maintained by the overlapping region of both 5 prime ends of the strand ))

Question 39

genes on HBV genome

Answer 39

HBV has 4 genes:
P – polymerase 
C – core protein
S – surface antigen
(3 polypeptides – L, M, S)
X – transactivator of viral transcription

Genes are overlapping – the whole genome codes for protein (very compact).
3 sizes of S protein
- S, only has S region of protein
- M, medium has a little bit more of the N terminus
- L, large has a bigger extension on N terminus of protein

Question 40

Key Events in the HBV Life Cycle

Answer 40

DIAGRAM IN L23 S66-67

1. Attachment and entry
2. Penetration of the nucleus
3. Genome conversion to covalently closed circle
4. Transcription producing mRNA and pregenome
5. Capsid assembly and pregenome reverse transcription
   6a. Virus envelopment by budding into ER, then exocytosis
   6b. OR re-entry to nucleus, amplifying DNA copy number

Question 41

1. HBV entry and uncoating

Answer 41

Hepadnaviruses have very narrow host range and only infect hepatocytes.

The large (L) HBsAg is required for attachment and entry. The cell surface receptor is unknown.

HBV particles are though to be taken up by endocytosis and membrane fusion occurs between viral and cellular membrane. However, the mechanism is unknown.

The core particle travels to the nucleus and releases the genomic DNA, but the mechanism of uncoating is unknown.

Question 42

3. HBV Genome Completion

Answer 42

5’ cap is seen on -ve sense DNA strand (black circle)
5’ of +ve sense DNA strand is attached to a cap associated bit of RNA
the overlapping of repeat regions is also seen

within the nucleus it is converted to the covalently closed circular DNA form
for that to happen the P protein and the 5’ cap have to be removed. the positive sense strand then needs to be extended and then joined
this is all carried out by the host cell DNA repair enzyme

Question 43

4a. HBV transcription - process

Answer 43

cccDNA is a template for transcription by host RNA Polymerase II.

HBV DNA contains a transcription enhancer that is only active in liver cells.

HBV X protein is a transcriptional activator;
doesn t bind DNA itself
activates transcription factors
interacts with cell signalling pathways

mRNAs are capped and polyadenylated, but not spliced.

Nuclear export of mRNAs to the cytoplasm depends on a post- transcriptional regulatory element (PRE) that uses the CRM1/RanGTP pathway (like Rev/RRE). BUT no viral protein is required.

Question 44

4b. HBV transcription - rna sizes produced

Answer 44

3. 5 kb – pregenomic RNA, encodes P and C proteins.
4. 4 kb – encodes large (L) surface antigen.
5. 1 kb – encodes medium (M) and small (S) surface antigens.
6. 7 kb – encodes X protein.

Different transcription start sites but all end at a common polyadenylation site

Question 45

5. Encapsidation of pregenomic RNA into core particle

Answer 45

reverse transcription process
the P protein is composed of the reverse transcriptase domain and a terminal protein domain. the reverse transcriptase domain is similar to the hiv one (it has both polymerase activity and RNA H activity). but different because if the TP domain

it is activated by why the binding of cellular chaperone proteins. the viral protein also contributes
please change the confirmation of the P protein and it becomes active so that I can bind to to the pregenome RNA

once they are bound, it is a signal for the core particle to start assembling
C protein start forming that complex and within the core particle, the reverse transcription starts taking place

Question 46

REVERSE TRANSCRIPTION PROCESS

Answer 46

DIAGRAM IN L23 S75-76

Question 47

6. viral assembly

Answer 47

viral assembly is the last step and not much is known of it

RT is going on in the assembling core particle
the particle has to complete with its viral genome
the choice (between budding or going back to the nucleus) depends on the concentration of the large surface antigen on the cell

at the initial infection there aren’t many viral proteins made yet so there is a local sensation of large surface antigens which means that the core will go back to the nucleus
later on in the infection when the the infected cell has translated more viral particles, there is a high concentration of large proteins which are translated on membrane-bound ribosomes and inserted into the er.
if there is a high concentration of L proteins on the er, the core protein can recognise the cytoplasmic tail of the the large surface antigen, so it will bind to it and and but into these cellular compartments
then we end up with a viral particle that will follow a secretory pathway out of the cell

we know there is interaction between the core particle and the large proteins but otherwise we don’t know much more of this process

Question 48

comparison of HIV and HBV reverse transcriptases

Answer 48

Terminal protein of HBV P protein is the primer for (-) strand DNA synthesis – first nucleotide is added to tyrosine 96.
HIV RT uses tRNAlys as the primer for (-) strand DNA synthesis.

Both enzymes have a reverse transcriptase (RNA-dependent DNA polymerase) domain and an RNase H domain.

Question 49

how does the new genome decide which route to go to once it is assembled?

Answer 49

The concentration of L (large) surface antigen determines the fate of core particles.

L, M, S surface antigens accumulate in ER-Golgi membranes. C (core) protein interacts with L.

Low concentration of L leads to the core particle/viral DNA entering the nucleus, where it is converted to cccDNA.

There can be up to 10-20 copies of cccDNA in one cell nucleus. Each can be a template for transcription.

High concentration of L leads to the core particle budding into the ER. Virions exit the cell by exocytosis.

Empty HBsAg particles (containing mostly S) also bud into the ER and undergo exocytosis.

Question 50

the difference in timing of RT in retrovirus and hepadnavirus

Answer 50

this shows the difference in timing where the RT process occurs between these 2 viruses
RETRO - +ve sense rna genome that as soon as it gets into the cell, it is reverse transcribed into the dna
that is then the template for transcription by host rna pol II to make more rna which is the genome rna come out of the cell in virus particle

HEPA - double stranded dna is our viral genome which is transcribed by host cell rna pol II, so we have double stranded dna goes to rna. that is the template to RT to make more ds dna to leave the cell in a viral particle

the order of events is different
retrovirus - RT is an early event (as soon as capsid gets into cytoplasm)
hepadnavirus - RT is one of the last events (just before budding out of cell)