Retroviruses andHuman Immunodeficiency Virus (HIV)

Lecturer: Bùi Thị Minh Diệu

Group members• Trần Hoàng Đệ• Lâm Tấn Hào• Huỳnh Lê Bảo Ngọc• Trần Hạnh Phước• Hoàng Nguyễn Phương Trinh• Lý Hoàng Tuấn

Outline• Part 1: Retroviruses:– Discovery & Classification– Methods to study retroviruses– Retrovirus structure– Retrovirus cycle

Outline• Part 2: HIV-1:– History– Signs & symptoms of AIDS– AIDS transmission and epidemiology– HIV-1 structure & replication– Treatment and prevention of HIV/AIDS

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

ClassificationGenus Examples HostAlpharetrovirus 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 1Simian immunodeficiency virusFeline immunodeficiency virus

Humans MonkeysCats

Spumavirus Simian foamy virus Monkeys

Table 1 7 genera of retroviruses

Methods• Apply many widely used methods in virology:– Purification of retroviral particles– Structural study with electron microscope

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

Electron microscope• Measure particle size• Define morphology• Study of retroviral structural proteins

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

Cryo-electron microscope• Observe virus directly as an unstained particle• Drawback: low-contrast image Apply computer-assisted program to generate 3D structure

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

Structure• Virion structure:

– Roughly spherical, 100nm in diameter

– Icosahedral or conical capsid

– Packaging 2 identical copies of (+)strand RNAand viral enzymes (RT, PR, and IN) in enveloped virion

Figure 2 A typical retrovirus virion

Structure• Genome structure:– The virus genome is a (+)strand RNA: 7–10kb

Figure 3 Structure of retrovirus RNA

Retrovirus life cycleEarly 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

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

Attachment and Entry

SU protein (virus) interacts with receptors (host)

TM protein changes conformation, allowingvirion 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

Reverse transcription• Reverse transcription: RNA DNA– Enzyme: reverse transcriptase 2 activities:

RNA/DNA dependent DNA polymerase, ribonuclease H

– No proofreading quasispecies– 9 steps

Figure 6 Reverse transcription. The first 6 steps

Figure 7 Reverse transcription. The last 3 steps

Integrase

Integration sites: random

(Cleave ligate)

Proviral DNA and host DNA can replicate together

Figure 8 Integration of proviral DNA into host cell

Integration

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

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

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

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

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

Ribosomal frameshifting

Figure 11 Ribosomal frameshifting

A heptamer

A 2nd structure

Figure 12 Retrovirus translation and post-translational modifications

Evn is glycosylated and cleaved SU & TM

Gag, Gag/Pol is myristylated

Assembly of the virionTwo 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

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

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)

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

AIDS Signs and SymptomsAcute

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

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)

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.

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

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

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

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)

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

HIV Replication• Entry and attachment of HIV-1• Functions of 6 additional proteins

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

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)

Tat• Transactivator of

transcription increases HIV-1 transcription by stimulating elongation by RNA pol II

Figure 20 Mechanism of Tat function

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

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

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

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

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

AIDS Treatment• Chemotherapy:– 5 classes of antiretrovirals, each with its own

mode of act (MOD) and drawbacks (D).• Immunotherapy• RNA interference

Chemotherapy• Nucleoside analog reverse-transcriptase

inhibitors (NRTIs)• Non-nucleoside RT inhibitors (NNRTIs)• Protease inhibitors (PIs)• Entry inhibitors• Integrase inhibitors

Nucleoside analog reverse-transcriptase inhibitors (NRTIs)

• MOD: Inhibit the viral reverse transcription• D: Has serious side-effects

Figure 22 Azidothymidine (AZT) – the first NRTI to be used

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

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

Entry inhibitors• MOD: Prevent membrane fusion by changing

gp41 conformation• D:– Not cost-effective– Ineffective against drug-resistance strains

Integrase Inhibitors• MOD: Inhibit strand transfer• D: Low barrier for virus to

easily overcome

Figure 25 Raltegravir: (left) Structure and (right) Marketable form

Immunotherapy• No effective vaccine is available Alternative treatment strategies are needed– Monoclonal antibodies are promising therapy– Using of immunogen need to be considerered

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

HIV/AIDS Prevention• Mother-to-Child Transmission• Sexually Transmitted Infections– Microbicides

• Post-exposure Prophylaxis• Pre-exposure Prophylaxis

Mother-to-Child Transmission• Treatment of the mother and infants with AZT– Combined with exclusive formula-feeding

Sexually Transmitted Infections• Safe sex message & practice– Important and cost-effective method

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

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

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

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

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