Title: HIV lifecycle with more accurate depiction of budding
1HIV 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
DSouza Summers, 2005, Nature Reviews 3 643-655
2Figure 9-15
Summary of HIV lifecycle
See previous slide for correction.
3Genomics and HIV
- Rapid sequencing of viral genomes
- Study transmission, mutation, drug resistance
- Genome wide association studies/genome
sequencing of elite controllers (long-term
non-progressors) - Elite controllers 1 in 3000
- Maintain 50 HIV/mL without anti-retroviral drugs
(typically 104-106 HIV/mL before drugs)
4Much of the human genome is non-coding
DNAResolves 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
5Endogenous 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 dont contain
functional genes.
Everybodys 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
6What is viral DNA doing in our genome?
- Producing viral particles
- Retaining transpositional activity
- Making proteins essential for host genome
function - Decaying into junk DNAEndogenous
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.
7Clicker question
- Is it likely that HIV could become an inherited
part of the human genome? - 1) YES
- 2) NO
8Clicker question
- What is inside the core of HIV?
- 1) A lipid bilayer
- 2) Receptors
- 3) Viral enzymes and RNA
- 4) Carbohydrates
- 5) Mitochondria
9Clicker question
- What is the typical course of an HIV infection?
- 1) AIDS--gtflu-like symptoms--gtsymptomatic--gtasympt
omatic - 2) Flu-like symptoms--gtsymptomatic--gtasymptomatic-
-gtAIDS - 3) Symptomatic--gtasymptomatic--gtflu-like
symptoms--gtAIDS - 4) Flu-like symptoms--gtasymptomatic--gtsymptomatic-
-gtAIDS
10Clicker question
- How many genes does HIV have?
- 1) 1
- 2) 10
- 3) 100
- 1000
- 10000
- 100000
11Clicker question
- HIV enters the host cell by
- 1) Budding
- 2) Receptor-mediated endocytosis
- 3) Fusion with the host membrane
- 4) Phagocytosis
- 5) Invitation
12Clicker question
- Where does HIV persist in a latent phase?
- 1) Hides embedded in the genome
- 2) Hides in the Golgi apparatus
- 3) Hides in the Endoplasmic reticulum
- 4) Hides on the Y chromosome
13Clicker question
- Where do the lipids on the HIV particles come
from? - 1) HIV has genes to make lipids for its particles
- 2) Lipids are acquired during processing in the
Golgi Apparatus - 3) A lipid bilayer is acquired from the host cell
during budding - 4) Lipids form spontaneously around the viral
core because the core is hydrophobic
14Clicker question
- HIV infects which of the following cell types
- 1) Sperm cells
- 2) Red blood cells
- 3) B cells
- 4) T cells
- Macrophages
- Liver cells
15Figure 11-23
HIV RNA is transcribed by viral reverse
transcriptase into DNA that integrates into the
host genome
- 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
Integrase enters cell along with viral genome.
RT enters cell along with viral genome.
16Consequences 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.
17Figure 9-15 part 3 of 4
HIV lies dormant in resting T cells and
replicates in activated T cells
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
cells normal defense mechanisms to transcribe
its genes. Another example of evolution at work!
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 cells RNA polymerase.
18Tat 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
19Figure 9-15 part 4 of 4
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. 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.
,
Note there are some inaccuracies in this
figure (maturation to a bullet-shaped capsid
occurs AFTER budding).
20HIV 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
DSouza Summers, 2005, Nature Reviews 3 643-655
21Figure 9-15
Summary of HIV lifecycle
Not clear how cDNA enters nucleus with integrase.
RT is primed by a host tRNA.
Capsid might uncoat at a later stage.
See previous slide for correction.
22How 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
23Genomics and HIV
- Rapid sequencing of viral genomes
- Study transmission, mutation, drug resistance
- Genome wide association studies/genome
sequencing of elite controllers (long-term
non-progressors) - Elite controllers 1 in 3000
- Maintain 50 HIV/mL without anti-retroviral drugs
(typically 104-106 HIV/mL before drugs)
24Much of the human genome is non-coding
DNAResolves 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
25Endogenous 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 dont contain
functional genes.
Everybodys 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
26What is viral DNA doing in our genome?
- Producing viral particles
- Retaining transpositional activity
- Making proteins essential for host genome
function - Decaying into junk DNAEndogenous
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.
27Clicker question
- Is it likely that HIV could become an inherited
part of the human genome? - 1) YES
- 2) NO
28Summary of HIV lifecycle
Bruce Walker, Harvard, HHMI holiday lectures
29What should you target to make an anti-viral drug?
Clicker question
- An activity that is critical for viral function
- An activity that is virally-encoded
- An activity that is not similar to host
activities - All of the above
30Potential anti-HIV drugs might
- Block attachment to host cell
- Prevent fusion of viral and host membranes
- Inhibit reverse transcriptase
- Inhibit integrase
- Inhibit HIV protease
31Attachment inhibition
32Recombinant soluble CD4 blocks gp120 binding to
cell surface CD4 on T cells
PRO 542 -- the first two domains of CD4 fused to
the constant (Fc) region of an antibody.
CD4 The T cell co-receptor and HIV receptor.
http//clinicaltrials.gov/ct2/show/NCT00055185 Th
e 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.
T cell plasma membrane
33(No Transcript)
34Could also block binding to CCR5 (HIV
co-receptor)
Progenics Pharmaceuticals
http//www.progenics.com/prod_pro140.cfm
35Pro 140
Pro 140 is designed to both block HIV and permit
normal chemokine binding.
36HIV fusion inhibition
37HIV binding and fusion
(movie from Dennis Burton, Scripps)
38The gp41 ectodomain is a 6-helix bundle
Structure thought to represent post-fusion state
of gp41
Chan et al (1997) Cell 89263
39Excellent animation of fusion mediated by HIV
gp41
- http//www.molecularmovies.com/movies/gp41_092707.
html
40(No Transcript)