Transcript Overview of your immune system

HIV lifecycle with more accurate depiction of budding
Translation of unspliced
mRNA gives viral Gag, and
occasionally, Gag-Pol
proteins.
These assemble to form
immature particles.
Proteolytic cleavage of
Gag (by HIV protease)
produces matrix (MA),
capsid (CA), and
nucleocapsid (NC), which
rearrange to form the
mature virus.
A Gag-Pol protein
D’Souza & Summers, 2005, Nature Reviews 3: 643-655
Summary of HIV lifecycle
Figure 9-15
See previous slide
for correction.
Genomics and HIV
• Rapid sequencing of viral genomes
– Study transmission, mutation, drug resistance
• Genome wide association studies/
genome sequencing of “elite controllers” (“long-term nonprogressors”)
– Elite controllers: ~1 in 3000
– Maintain 50 HIV/mL without anti-retroviral drugs (typically
104-106 HIV/mL before drugs)
Much of the human genome is “non-coding” DNA;
Resolves the “C-value paradox” -- observation that genome
size does not reflect complexity
• Human genome: 1.5% protein-coding genes;
98.5% non-coding DNA.
– Much of the non-coding DNA are transposable elements
(transposons), sequences of DNA that can move to different
positions with the genome; i.e., mobile genetic elements.
• Discovered by Barbara McClintock in corn (1948).
– Retrotransposons are one class of transposable
element. They paste copies of themselves into
genome in multiple places.
• Retrotransposon DNA is first transcribed into RNA.
• RNA copied into DNA by a reverse transcriptase (often encoded by
the transposon itself).
This should sound familiar…
Endogenous retroviruses in the human genome
•
8-10% of human genome codes for retroviruses that are inherited
along with other genes.
Endogenous retroviruses are copied from a viral RNA
genome and inserted as proviruses into the host genome.
Intact retroviruses have LTRs (long terminal repeats) and
coding sequences. Other retroelements can transpose but
don’t contain functional genes.
Everybody’s genome is littered with the remnants of ancient retroviral
infections. The total burden of retroviral infection on the human genome is
total to about one and a half average-sized chromosomes. There are more
retroviruses in your DNA than there are genes.
Paul Bieniasz, 2008
What is viral DNA doing in our genome?
• Producing viral particles
• Retaining transpositional activity
• Making proteins essential for host genome function
• Decaying into “junk” DNA
Endogenous retroviruses can move between species.
Are xenotransplants safe?
Need complete genomic sequences in many species to determine
the number and location of endogenous retroviruses, their role
in genome evolution, and their contributions to human disease.
Clicker question
Is it likely that HIV could become an
inherited part of the human genome?
1) YES
2) NO
Clicker question
What is inside the core of
HIV?
1) A lipid bilayer
2) Receptors
3) Viral enzymes and RNA
4) Carbohydrates
5) Mitochondria
Clicker question
What is the typical course of an HIV infection?
1)
2)
3)
4)
AIDS-->flu-like symptoms-->symptomatic-->asymptomatic
Flu-like symptoms-->symptomatic-->asymptomatic-->AIDS
Symptomatic-->asymptomatic-->flu-like symptoms-->AIDS
Flu-like symptoms-->asymptomatic-->symptomatic-->AIDS
Clicker question
How many genes does HIV have?
1) 1
2) ~10
3) ~100
4) ~1000
5) ~10000
6) ~100000
Clicker question
HIV enters the host cell by
1)
2)
3)
4)
5)
Budding
Receptor-mediated endocytosis
Fusion with the host membrane
Phagocytosis
Invitation
Clicker question
Where does HIV persist in a latent phase?
1)
2)
3)
4)
Hides embedded in the genome
Hides in the Golgi apparatus
Hides in the Endoplasmic reticulum
Hides on the Y chromosome
Clicker question
Where do the lipids on the HIV particles
come from?
1)
2)
3)
4)
HIV has genes to make lipids for its particles
Lipids are acquired during processing in the Golgi
Apparatus
A lipid bilayer is acquired from the host cell
during budding
Lipids form spontaneously around the viral core
because the core is hydrophobic
Clicker question
HIV infects which of the following cell
types:
1)
2)
3)
4)
5)
6)
Sperm cells
Red blood cells
B cells
T cells
Macrophages
Liver cells
HIV RNA is transcribed by viral reverse transcriptase into DNA
that integrates into the host genome
Figure 11-23
• Viral genome enters cell after fusion of viral and host cell membranes.
• Viral reverse transcriptase protein packaged together with viral genome
transcribes viral RNA into viral cDNA (complementary DNA).
• Viral cDNA integrated into host cell genome by viral integrase.
• Integrated cDNA is called the provirus.
• Analogous to integrated prophage in a phage  lysogen
RT enters cell along
with viral genome.
Integrase enters
cell along with viral
genome.
Consequences of integration of HIV genome
into a host cell chromosome
• HIV DNA that is integrated into a chromosome is
duplicated when cell divides, therefore all progeny
of the infected cell will contain HIV DNA.
• Individuals with HIV in its latent, proviral form
are healthy and show no signs of AIDS.
• HIV can be passed among healthy individuals
because HIV can residue as a provirus in T cells.
Healthy individuals whose T cells contain
integrated HIV DNA can transfer HIV in blood or
semen, both of which contain T cells.
HIV lies dormant in resting T cells and replicates in activated T cells
Figure 9-15 part 3 of 4
Tat and Rev promote viral
replication in activated T cells.
NFkB is a host cell transcription
factor that binds to the viral
LTRs to initiate transcription by
the host cell’s RNA polymerase.
NFkB is normally
supposed to be used by
the cell to transcribe
genes related to host
defense -- it enters the
nucleus to transcribe
genes only under
conditions of stress (e.g.,
when T cells are
activated).
Note that HIV is using
the host cell’s normal
defense mechanisms to
transcribe its genes.
Another example of
evolution at work!
Tat is required for HIV-1 replication
• Tat = transactivator
protein
• Tat protein binds to start
of a new HIV RNA strand
• Tat binds to TAR:
Transactivator Active
Region
• Tat binds to TAR and
activates transcription of
HIV genes
Final steps in HIV assembly and budding from host cell
HIV needs to export
unspliced, singly spliced,
and multiply spliced mRNAs
from the nucleus to the
cytoplasm in order to make
all of its proteins.
Figure 9-15 part 4 of 4
,
Note there are some
inaccuracies in this figure
(maturation to a bullet-shaped
capsid occurs AFTER budding).
Eukaryotic cells normally
prevent export of
incompletely spliced
mRNAs.
Fully spliced Rev mRNA
leaves the nucleus and gets
translated in cytoplasm.
Rev protein then enters the
nucleus and binds to a
specific site on the viral
RNA and to a host
transport protein to force
export of unspliced viral
mRNA.
HIV lifecycle with more accurate depiction of budding
Translation of unspliced
mRNA gives viral Gag, and
occasionally, Gag-Pol
proteins.
These assemble to form
immature particles.
Proteolytic cleavage of
Gag (by HIV protease)
produces matrix (MA),
capsid (CA), and
nucleocapsid (NC), which
rearrange to form the
mature virus.
A Gag-Pol protein
D’Souza & Summers, 2005, Nature Reviews 3: 643-655
Summary of HIV lifecycle
Figure 9-15
RT is primed by
a host tRNA.
Not clear how
cDNA enters
nucleus with
integrase.
Capsid might uncoat
at a later stage.
See previous slide
for correction.
How does RT reverse transcribe only its own RNA?
Host tRNALys3 packaged into virions along with
HIV RNA.
• 3’ terminal 18 nucleotides of tRNALys3 anneals
with 18 nucleotides at the 5’ of the HIV RNA (the
primer binding site)
• tRNALys3 serves as a primer for RT.
• RT priming for in vitro reverse transcriptions
done with oligo-dT or random primers
Genomics and HIV
•
Rapid sequencing of viral genomes
– Study transmission, mutation, drug resistance
• Genome wide association studies/
genome sequencing of “elite controllers” (“longterm non-progressors”)
– Elite controllers: ~1 in 3000
– Maintain 50 HIV/mL without anti-retroviral drugs (typically
104-106 HIV/mL before drugs)
Much of the human genome is “non-coding” DNA;
Resolves the “C-value paradox” -- observation that genome
size does not reflect complexity
• Human genome: 1.5% protein-coding genes;
98.5% non-coding DNA.
– Much of the non-coding DNA are transposable elements
(transposons), sequences of DNA that can move to
different positions with the genome; i.e., mobile genetic
elements.
• Discovered by Barbara McClintock in corn (1948).
– Retrotransposons are one class of transposable
element. They paste copies of themselves into
genome in multiple places.
• Retrotransposon DNA is first transcribed into RNA.
• RNA copied into DNA by a reverse transcriptase (often encoded by
the transposon itself).
This should sound familiar…
Endogenous retroviruses in the human genome
•
8-10% of human genome codes for retroviruses that are inherited
along with other genes.
Endogenous retroviruses are copied from a viral RNA
genome and inserted as proviruses into the host genome.
Intact retroviruses have LTRs (long terminal repeats) and
coding sequences. Other retroelements can transpose but
don’t contain functional genes.
Everybody’s genome is littered with the remnants of ancient retroviral
infections. The total burden of retroviral infection on the human genome is
total to about one and a half average-sized chromosomes. There are more
retroviruses in your DNA than there are genes.
Paul Bieniasz, 2008
What is viral DNA doing in our genome?
• Producing viral particles
• Retaining transpositional activity
• Making proteins essential for host genome function
• Decaying into “junk” DNA
Endogenous retroviruses can move between species.
Are xenotransplants safe?
Need complete genomic sequences in many species to determine
the number and location of endogenous retroviruses, their role
in genome evolution, and their contributions to human disease.
Clicker question
Is it likely that HIV could become an
inherited part of the human genome?
1) YES
2) NO
Summary of HIV lifecycle
Bruce Walker, Harvard, HHMI holiday lectures
Clicker question
What should you target to
make an anti-viral drug?
1) An activity that is critical for viral function
2) An activity that is virally-encoded
3) An activity that is not similar to host
activities
4) All of the above
Potential anti-HIV drugs might
• Block attachment to host cell
• Prevent fusion of viral and host membranes
• Inhibit reverse transcriptase
• Inhibit integrase
• Inhibit HIV protease
Attachment inhibition
Recombinant soluble CD4 blocks gp120
binding to cell surface CD4 on T cells
CD4
The T cell co-receptor
and HIV receptor.
PRO 542 -- the first two domains
of CD4 fused to the constant (Fc)
region of an antibody.
D1
D2
D3
D4
T cell plasma membrane
http://clinicaltrials.gov/ct2/show/NCT00055185
“The purpose of this study is to determine any adverse
effects of PRO 542 after administration and to
determine the anti-HIV effects of PRO 542 in the
patient.”
Could also block binding to CCR5
(HIV co-receptor)
Progenics Pharmaceuticals
http://www.progenics.com/prod_pro140.cfm
Pro 140
Pro 140 is designed to both block HIV and permit normal chemokine binding.
HIV fusion inhibition
HIV binding and fusion
(movie from Dennis Burton, Scripps)
The gp41 ectodomain is a 6-helix bundle
N-peptide
C-peptide
Heptad repeats form a-helices with one hydrophobic face; e.g., leucine zipper
Trimer of coiled coils
Structure thought to
represent post-fusion
state of gp41
Chan et al (1997) Cell 89:263
Excellent animation of fusion
mediated by HIV gp41
http://www.molecularmovies.com/movies/gp41_092707.html