This document summarizes retroviruses and HIV. It discusses the discovery and classification of retroviruses. The structure of retroviruses includes a spherical capsid containing two copies of RNA and viral enzymes. The retrovirus lifecycle includes early reverse transcription of RNA to DNA and later integration into the host genome and assembly of new virions. HIV causes AIDS and was isolated in 1983. It is a global pandemic transmitted sexually or through blood. Treatment includes antiretroviral drugs that target reverse transcriptase, protease, entry, and integration. Prevention focuses on safe sex practices, mother-to-child transmission prevention, and pre-exposure prophylaxis.
2. Group members
• Trần Hoàng Đệ
• Lâm Tấn Hào
• Huỳnh Lê Bảo Ngọc
• Trần Hạnh Phước
• Hoàng Nguyễn Phương Trinh
• Lý Hoàng Tuấn
3. Outline
• Part 1: Retroviruses:
– Discovery & Classification
– Methods to study retroviruses
– Retrovirus structure
– Retrovirus cycle
4. Outline
• Part 2: HIV-1:
– History
– Signs & symptoms of AIDS
– AIDS transmission and epidemiology
– HIV-1 structure & replication
– Treatment and prevention of HIV/AIDS
5. Discovery
• The finding of retrovirus (Rous, 1910) & reverse
transcriptase (Temin & Baltimore, 1971)
revolution
Figure 1 (from left to right) Peyton Rous, Howard Temin & David Baltimore
6. Classification
Genus Examples Host
Alpharetrovirus Rous sarcoma virus Chickens
Betaretovirus Mouse mammary virus Mice
Gammaretrovirus Murine leukemia virus Mice
Deltaretrovirus Human T-cell leukemia virus type 1 Humans
Epsilonretrovirus Walleye dermal sarcoma virus Fish
Lentivirus Human immunodeficiency virus type 1
Simian immunodeficiency virus
Feline immunodeficiency virus
Humans
Monkeys
Cats
Spumavirus Simian foamy virus Monkeys
Table 1 7 genera of retroviruses
7. Methods
• Apply many widely used methods in virology:
– Purification of retroviral particles
– Structural study with electron microscope
8. Purification of retroviral particles
• On the basis of size and density
• Achieved by centrifugation
– Retroviral density ~ 1.16 g/ml 35% w/w sucrose
• In retroviruses, physical/infectious particles
> 100:1 flaws in properties measurement
• Most purified retrovirus: AMV early
biochemical study of viral proteins
10. Electron microscope
• Use 2 techniques, each with its own drawbacks:
– Negative staining: deformations in particles
– Thin-section:
• Require harsh fixation
• Final appearance depends on plane of sectioning
• Particle size can also be measure by rate zonal
sedimentation, but usually result in doubling in
actual size
11. Cryo-electron microscope
• Observe virus directly as an unstained particle
• Drawback: low-contrast image
Apply computer-assisted program to
generate 3D structure
12. Methods to study retroviruses
• To sum up:
– The exact arrangement of retroviral components
remains uncertain
– Models are necessary to represent the virion structure
with suitable modification and prediction
– A complete understanding of retrovirus may have to
wait for the development of new techniques
13. Structure
• Virion structure:
– Roughly spherical,
100nm in diameter
– Icosahedral or conical
capsid
– Packaging 2 identical
copies of (+)strand RNA
and viral enzymes (RT,
PR, and IN) in enveloped
virion
Figure 2 A typical retrovirus virion
15. Retrovirus life cycle
Early phase
• Virus enters the
cell copies
RNA genome
inserts the copy
into the host cell
genome.
Late phase
• Expression of
viral RNA
• Synthesis of viral
proteins
• Assembly of
virions
16. Early phase
• Retrovirus enters cell by the
fusion pathways.
• Viral RNA is converted into a
double-stranded DNA copy
by reverse transcription.
proviral DNA
• A copy of proviral DNA is
integrated into the cellular
genome at a random site.
Figure 4 Early phase of retrovirus life cycle
17. Attachment and Entry
SU protein (virus)
interacts with
receptors (host)
TM protein changes
conformation, allowing
virion membrane to fuse with
host plasma membrane
Figure 5 Retrovirus attachment and entry. Fusion of virion membrane and plasma
membrane, the virion contents is modified to be the transcription complex
18. Reverse transcription
• Reverse transcription: RNA DNA
– Enzyme: reverse transcriptase 2 activities:
RNA/DNA dependent DNA polymerase,
ribonuclease H
– No proofreading quasispecies
– 9 steps
22. Late phase
• Expression of viral RNA through transcription
of proviral DNA
• Synthesis of viral proteins through translation
and post-translational modification
• Assembly and budding of virions
23. Expression of viral RNA
• 2 identical long terminal
repeats LTRs:
– Left LTRs: signaling the
transcription initiation
precisely at U3/R junction
– Right LTRs: signaling
cleavage and
polyadenylation of the
transcript
Figure 9 Transcription of proviral DNA
24. Expression of viral RNA
• Differential splicing
generates multiple mRNAs
• All retrovirus make at least
2 mRNAs:
– Unspliced form Gag &
Gag/Pol proteins
– Singly spliced form Evn
proteins
25. Synthesis of viral proteins
• Gag: many structural proteins
• Pol: fewer enzyme molecules
• 2 mechanisms to ensure the Gag and
Gag/Pol in a proper ratio (~ 95%:5%):
– Suppression of translation termination
– Ribosomal frameshifting
26. Suppression of translation termination
• Correct recognition of stop codon
UAG separating gag and pol
reading frame gag protein
only
• Gln-tRNAGln misreading UAG as
CAG 1/20 time translational
readthrough Gag/Pol
polyprotein
– Stimulated by pseudoknot
Figure 10 Suppression of translation
termination
28. Figure 12 Retrovirus translation and post-
translational modifications
Evn is glycosylated
and cleaved SU
& TM
Gag, Gag/Pol is
myristylated
29. Assembly of the virion
Two different assembly
pathways:
Core assembly and then
budding
“B-type”, “D-type” viruses
Core assembly and budding
simultaneously
“C-type” virus
Figure 13 2 assembly pathways in
retroviruses
30. Assembly of the virion
• Only full-length RNA is encapsidated, thanks
to psi () signal
• As virion assembled and extrudes, protease
cleaves Gag, Gag/Pol mature & functional
form
Virion become infectious
31. History
• 1959: 1st case of HIV infection in human from
Democratic Republic of the Congo (Africa)
• 1981: A group of healthy young male in Los
Angeles/San Francisco showed significant depletion of
their immune system suffered opportunistic
infections (pneumonia)
The term acquire immunodeficiency syndrome (AIDS)
32. History
• 1983: A retrovirus isolated from the blood of
individuals with AIDS was characterized
human immunodeficiency virus type 1
(HIV-1)
Figure 14 (from left to right) Luc Montagnier, Barré-Sinoussi, and Robert Gallo successfully
isolated and characterized HIV-1 at the same time
33. AIDS Signs and Symptoms
Acute
infection
• Last several
weeks
• Mononucleosis
or influenza-like
syndromes
Clinical
latency
• Two weeks to
>20 years
depends on
several factors
• Few or no
symptoms
AIDS
• Low CD4+ T cell
level
• Various
opportunistic
infections,
cancers
Figure 15 3 stages of HIV infection
34. HIV Transmissions
• HIV-1 was probably
transmitted to humans
from chimpanzees
infected with SIVcpz.
• HIV can be transmitted
from an infected person
to another through:
Blood, semen, vagina
secretions, breast milk.
• Activities that allow HIV
transmission:
– Unprotected sexual contact
– Direct blood contact
(injection drug needles,
blood transfusions…)
– Mother-to-Child
transmission (Vertical
transmission)
35. AIDS Epidemiology
• HIV/AIDS is a global pandemic
– approximately 35.3 million people living with HIV
globally (2010), of which: 3.4 million children <15
• Sub-Saharan Africa and South East Asia are 2
regions most affected.
36. Sub-Saharan Africa
• 12% world population <> contribute
to 2/3 people infected with HIV
• More women are infected than men
• Reasons:
• Widespread of sexually transmitted
diseases
• Unsafe blood transfusions
• Poor state of hygiene and nutrition
• Poor economic conditions
• Lack of sex education
Figure 16 Map of HIV prevalence in
Africa in 2007
37. South & South East Asia
• 4.2 – 4.7 M adults and children infected.
– Largely concentrated in: injecting drug users, men
who have sex with men (MSM), sex workers, and
clients of sex workers and their immediate sexual
partners
38. Vietnam
• 220k people living with HIV (0.47% population)
(2007):
– 65% are injecting drug users (IDU): sharing needles
• Women are more exposed to risk of contracting HIV
– 90k (2007)
– Reasons: from their partners – undisclosed IDU, from
men having pre-marital or extra-marital sexual
relationships
39. Structure
• Specific features:
– Cone-shaped capsid:
Wide end: 40–60nm,
narrow end: 20nm
Figure 17 Diagram of HIV-1 virion
structure (Only 1 mRNA molecule is
shown covered with CA for clarity)
40. Structure
• Genome structure is
very complex
• Splicing of HIV-1
primary transcript
>25 mRNAs, coding for
Gag, Gag/Pol, Env and
6 additional proteins Figure 18 Genome structure and RNA splicing
pattern of HIV-1
42. gp120 (SU) (HIV-1)
binds to CD4
receptor (cell
surface)
gp120 changes conformation
interact with chemokine
receptors (CCR5/CXCR4) gp41
(TM) change conformation Fusion and
release of
nucleocapsid
HIV-1 Attachment
and Entry
Figure 19 Model of HIV-1 entry
43. 6 addition proteins
• Virion protein R (Vpr)
• Viral infectivity factor (Vif)
• Virion protein unique to HIV-1 (Vpu)
• Transactivator of transcription (Tat)
• Regulator of expression of virion protein (Rev)
• Negative effector (Nef)
45. Rev
• Regulator of expression of virion
protein mediates cytoplasmic
transport of viral 9-kb (full-
length) and 4-kb (singly spliced)
mRNA
Figure 21 Mechanism of Rev function
46. Virion protein R (Vpr)
• Confer HIV-1 the special ability to infect non-dividing cell
• Facilitate the packing of enzyme uracil DNA, glycolase in
the virion remove deoxyuridine, which blocks
transcription
• Arrest infected cell at G2 stage, at which transcription of
HIV-1 is the most active
Enhance HIV-1 replication at multiple levels
47. Viral infectivity factor (Vif)
• Structure: 139-amino acid protein
• Found in cytoplasm of infected cells
• Function: Vif prevents action of host protein
APOBEC3G viral DNA is not mutated by
APOBEC3G increases virion infectivity
48. Virion protein unique to HIV-1 (Vpu)
• Structure: 81-amino acid protein
• Found in Golgi apparatus, endosome
compartment of infected cells
• Function: enhance the release of progeny virions
– Degradation of CD4
– Enhancement of virus release from the plasma
membrane
49. Negative effector (Nef)
• Structure: 210-amino acid protein
• Found in the inner face of plasma membrane
• Function:
– Decrease in the expression of CD4 and MHC1
– Enhancement of virus infectivity
– Modification of cell signaling
enhance release of progeny virion
50. AIDS Treatment
• Chemotherapy:
– 5 classes of antiretrovirals, each with its own
mode of act (MOD) and drawbacks (D).
• Immunotherapy
• RNA interference
53. Non-nucleoside RT inhibitors (NNRTIs)
• Very varied chemical structure
• MOD: Cause allosteric
inhibition
• D:
– Side effects
– Ineffective against HIV-1 and
drug-resistant mutants
Figure 23 Nevirapine (NVP) (left) and
Efavirenz (EFV) (right) – 2 common
NNRTIs
54. Protease inhibitors (PIs)
• MOD: Compete with
protease enzymes
– Efficacy in triple therapy (used
in combine with 2 NRTI) -
Highly active antiretroviral
therapy – HAART
• D: Frequent and severe side-
effects Figure 24 Ritonavir – the PI
usually used in HAART
55. Entry inhibitors
• MOD: Prevent membrane fusion by changing
gp41 conformation
• D:
– Not cost-effective
– Ineffective against drug-resistance strains
56. Integrase Inhibitors
• MOD: Inhibit strand transfer
• D: Low barrier for virus to
easily overcome
Figure 25 Raltegravir: (left) Structure and (right) Marketable form
57. Immunotherapy
• No effective vaccine is available
Alternative treatment strategies are needed
– Monoclonal antibodies are promising therapy
– Using of immunogen need to be considerered
58. RNA Interference
• Response to the double stranded RNA
• Target both HIV genes and host cell receptors
Reduce the chance of synthesis of HIV
proteins
• Face numerous challenges for clinical
application
62. Microbicides
• Microbicides: cellulose sulfate and PRO-2000,
and non-ionic surfactants applied topically
to vagina/rectum disrupt virus – cell
interaction
appropriate for women
• D: May cause inflammatory effect
63. Post-exposure Prophylaxis
• Use antiretroviral drugs (ZDV, truvada & kaletra)
immediately after possible exposure to HIV:
– Occupational exposure
– Sexual exposure
– Pre/Post-natal treatment
Reduce the risk of HIV infection
64. Pre-exposure Prophylaxis (PREP)
• The use of antiretroviral drugs (i.e. truvada)
prior to exposure
• Concerns:
– Rapid development of resistance in cases of
transmission
– People undergo PREP may have some risky
behavior
65. Conclusion
• The discovery of retroviruses and reverse transcriptase is
phenomenal
• No available method provides a complete understanding of
retroviruses
• Retrovirus cycle consists of 2 phases
• HIV-1 is a typical member of retroviruses
• HIV has caused global pandemic – AIDS
• Currently, there is no effective treatment or vaccine to
completely eliminate HIV
66. Thank you for your attention
Figure 26 The red ribbon is a
symbol for solidarity with HIV-
positive people and those living
with AIDS.
World AIDS day: December 1st