Iahi: 3.5 - Retroviruses

Front 1

Who do retroviruses infect?

Back 1

Insects --> Fish --> Man

Front 2

What are the potential infection consequences of retroviruses?

Back 2

- No ill effects
- Tumors - rapid onset or long latency
- Wasting diseases, neurological disorders
- Immune deficiencies (HIV)

Front 3

What are the genetic properties of retroviruses?

Back 3

- Acquire host cell sequences - oncogenes
- Insert into host cells chromosome - can activate or inactivate genes --> cancer
- Rapid genome evolution - acquire mutation through replication and recombination

Front 4

How are viruses classified now?

Back 4

Based on genomic sequence

Front 5

Enveloped viruses have what extra proteins?

Back 5

- Env protein embedded in membrane
- Matrix protein under lipid bilayer

Front 6

The capsid (core) of a virus is made of what structural proteins?

Back 6

Group specific AntiGens (GAG proteins):
- Matrix proteins (MA)
- Capsid proteins (CA)
- NucleoCapsid proteins (NC)
- Protease (PR)

Front 7

What is the genome of retroviruses?

Back 7

2 copies of a (+) ssRNA genome (only virus that is "diploid" and accounts for recombination potential)

Front 8

What is the significance of retroviruses having 2 copies of the +ssRNA?

Back 8

- Only virus that is "diploid"
- Accounts for recombination potential

Front 9

What is the simple retrovirus genomic organization?

Back 9

5' end cap
- R (repeat)
- U5 (unique to 5' end)
- gag gene: MA, CA, NC, PR*
- polymerase (pol) gene: RT, IN
- envelope (env) gene: SU, TM
- U3 (unique to 3' end)
- R (repeat)
3' end poly-A tail

*PR is in pol reading frame in HIV

Front 10

How do the retroviral genomic RNA get capped (5' end) and polyadenylated (3' end)?

Back 10

Made by host's Polymerase II - so gets the same modifications as host mRNAs

Front 11

What proteins does the "gag" gene encode (5'-3')? How are they synthesized?

Back 11

- MA - matrix
- CA - capsid
- NC - nucleocapsid
- PR - protease*
- Made as a polyprotein that then gets clipped (*note - PR is in pol gene reading frame in HIV)

Front 12

What proteins does the "pol" gene encode (5'-3')? How are they synthesized?

Back 12

- RT - reverse transcriptase
- IN - integrase
- Made as an extended polyprotein
* PR is in the reading from of pol gene in HIV (5' end)

Front 13

What proteins does the "env" gene encode (5'-3')? How are they synthesized?

Back 13

- SU - surface domains
- TM - transmembrane domains
- Made as a precursor and gets cleaved into SU an dTM domains

Front 14

What is the complex retrovirus genomic organization (e.g., HIV)?

Back 14

- Organized similarly to similar retroviruses (w/ gag, pol, env genes)
- Except numerous additional genes/proteins
- mRNAs for additional genes ("accessory proteins") are generated by complex alternative splicing

Front 15

What is the point of the accessory proteins of complex retroviruses (HIV)?

Back 15

Endow HIV with its unique properties, various functions

Front 16

What are the two phases of the replication cycle in retroviruses?

Back 16

A-D - precedes integration
E - integration into host DNA
F-J - post-integration

Front 17

What are the steps of replication of retroviruses?

Back 17

A - adsorption
B - penetration and uncoating
C - reverse transcription
D - transit into nucleus
E - integration into host DNA
F - viral RNA synthesis, host pol II
G - RNA processing
H - virion protein synthesis
I - assembly and budding
J - capsid maturation (proteolysis)

Front 18

What happens during the first step of the replication cycle of retroviruses?

Back 18

A - adsorption
- Virus binds cell via env protein and a host cell receptor
- HIV receptor is CD4 / CCR5
- Other viruses use amino acid transporters, LDL-like receptor, etc.

Front 19

What happens during the second step (after adsorption) of the replication cycle of retroviruses?

Back 19

B - penetration
- Viral envelope fuses with cell membrane either at cell surface or in endosomes after endocytosis

Front 20

What happens during the second step (after penetration) of the replication cycle of retroviruses?

Back 20

B - uncoating
- Genomic RNA is only partially uncoated (remains in protein particle in cytoplasm - prevents translation)
- RT, IN and some of the gag proteins remain associated with incoming genomic RNA
- Proteins are needed to convert ssRNA genome to dsDNA, nuclear import, and integration

Front 21

What is the purpose of the genomic RNA of retroviruses only be partially uncoated after penetration into the host cell?

Back 21

Do not want translation at this point - protein particle prevents association with ribosomes - goal is to convert ssRNA to dsDNA

Front 22

What happens during the third step (after partial uncoating) of the replication cycle of retroviruses?

Back 22

C - reverse transcription
- Conversion of ssRNA to dsDNA w/ RT
- Integrated DNA ("Provirus") is longer than the template RNA w/ U3 and U5 duplicated at ends to form Long Terminal Repeat (LTR)

Front 23

What are the functions of Reverse Transcriptase (RT)?

Back 23

- RNA-dependent DNA polymerase (copies RNA --> DNA)
- DNA-dependent DNA polymerase (copies a 2nd strand of DNA from first strand)
- Error-prone polymerase, ~5 errors made per genome, leads to rapid evolution and drug resistance

Front 24

What happens during the fourth and fifth steps (after reverse transcription) of the replication cycle of retroviruses?

Back 24

D/E - Transit to nucleus & Integration
- Integration requires dsDNA have access to host DNA
- Integrase recognizes and is specific for sequences at ends of dsDNA (i.e., ends of U3 and U5)
- Integration reaction is at random part of host sequence via IN protein
- Now, virus is a permanent resident of host cell's DNA

Front 25

How do retrovirus' dsDNA gain access to host DNA for integration?

Back 25

- Many retroviruses can't cross nuclear membrane and need cell division to integrate
- HIV CAN cross nuclear membrane, important for infection of non-dividing cells, and for gene therapy vectors

Front 26

Where does integration of retrovirus' dsDNA into host DNA occur?

Back 26

Essentially random - integration reaction is NOT specific for host sequences

Front 27

What happens during the sixth step (after integration of dsDNA into host) of the replication cycle of retroviruses?

Back 27

F - proviral transcription
- LTR directs synthesis of viral RNA
- 5' U3 binds transcription factors
- Beging transcription at R region

Front 28

How do the LTR (Long Terminal Repeats) direct synthesis of viral RNA?

Back 28

- 5' U3 contains binding sites for cellular transcription factors required for high level RNA synthesis
- U3 has signals recognized by the cell's transcription machinery, which directs transcription at beginning of R region

Front 29

How does U3 on the LTR influence which tissue/cells a retrovirus is active in (e.g., tropism)?

Back 29

- Spectrum of proteins that bind the U3 determines which tissues/cells it is active in
- E.g., HIV LTR requires transcription factor NFκB (only expressed in activated T cells)

Front 30

How does HIV RNA regulate which types of cells it is transcribed in?

Back 30

- HIV LTR requires transcription factor NFκB, which is only expressed in activated T cells
- Not transcribed in infected memory T cells because memory cells do not express NFκB

Front 31

What happens during the seventh step (after proviral transcription) of the replication cycle of retroviruses?

Back 31

G - RNA processing
- All viral RNAs are polyadenylated, some must be spliced to generate the env mRNA
- A large portion must remain full length to serve as gag-pol mRNA and as genome for progeny virions
- Complex retroviruses (HIV) do much more alternative splicing

Front 32

What are the three fates of retroviral RNA:

Back 32

1. Full length RNA --> genomic RNA
2. Full length RNA --> gag-pol mRNA
3. RNA splicing --> env mRNA (and others)

Front 33

Why is retroviral RNA splicing incomplete?

Back 33

To preserve full-length RNA for genomic RNA and gag-pol mRNA

Front 34

What happens during the eighth step (after RNA processing) of the replication cycle of retroviruses?

Back 34

H - Translation
- Most abundant protein is gag and gag-pol
- Gag initiates at an AUG start codon, and ends at a stop codon at end of gag
- Gag-pol is made from same AUG start coon, but ribosomes ignore or circumvent the gag stop codon and continue to end of pol (only happens about 5% of time, so RT and IN proteins are less abundant, also not needed in surplus, than gag)
- Both gag and gag-pol proteins are eventually cleaved by protease (PR) domain to release individual proteins
- Env protein is made from spliced mRNA on rER --> golgi --> inserted into plasma membrane

Front 35

How are the gag and gag-pol proteins related?

Back 35

- Both start at the same AUG start codon
- gag protein stops synthesis at stop codon
- gag-pol protein ignores gag stop codon and continues to end of pol (only happens 5% of time)
- Eventually cleaved by protease (PR)

Front 36

What are the env proteins?

Back 36

- Env gp160 precursor protein
- Cleaved into gp120 and gp41 by cellular protease
- Cleavage must happen because gp160 cannot support membrane fusion (virus would be made but could not fuse w/ target cell)

Front 37

What happens during the ninth/tenth steps (after translation) of the replication cycle of retroviruses?

Back 37

I/J - virion assembly and budding
- Packaging requires signal Psi (ψ) contained in unspliced RNA; splicing removes ψ signal
- Budding - viral gag and gag-pol polyproteins recruit RNA and assemble under cell surface; gag protein interacts w/ env and budding occurs as particle forms
- Maturation - proteolysis of gag and gag-pol by PR occurs after budding, causing protein rearrangements and core to become more dense
- Viral particles still form and bud if proteolysis is inhibited, but viruses are not infectious

Front 38

What is the action of PR inhibitors?

Back 38

- Inhibit proteolysis of gag and gag-pol by PR (which occurs after budding)
- Viral particles still form and bud if proteolysis is inhibited but they are NOT INFECTIOUS

Front 39

How were retroviruses discovered?

Back 39

As agents isolated from naturally occurring tumors in animals, that, when inoculated into naïve animals, would again cause tumors

Front 40

What are the types of retroviruses?

Back 40

- Non-transforming = non-acute or slow tumor viruses
- Transforming = acute tumor viruses

Front 41

What are the characteristics of non-transforming retroviruses?

Back 41

- Non-acute or slow tumor viruses
- Tumors take 6 mo to 1 yr to appear
- Do not transform cells in culture (low freq., i.e., no cancer phenotype)
- Viruses do not contain oncogenes
- Tumors are caused by activation or inactivation of host genes

Front 42

What are the characteristics of transforming retroviruses?

Back 42

- Acute tumor viruses
- Tumors occur within weeks
- Causes tissue culture cells to become "transformed" or cancer-like
- Viruses harbor a mutated copy of a cellular gene involved in growth control, "oncogene" --> rapid tumor onset upon introduction via infection

Front 43

What was the first oncogene to be identified?

Back 43

Src - in Rous sarcoma virus (avian virus)

Front 44

Which groups of people are more commonly infected by HIV?

Back 44

- Homosexual men
- Heroin addicts
- Hemophiliacs
- Haitians

Front 45

HIV patients have a decline in what cells?

Back 45

CD4 T cells

Front 46

When was the death rate from AIDS highest for males under 40 years old? What caused a decline?

Back 46

- Peaked in 1994-1995
- Declined w/ use of HAART (Highly Active Antri-Retroviral Therapy)

Front 47

In 2011, how many adults and children are living with HIV?

Back 47

34 million

Front 48

What are the known transmission routes of HIV infection?

Back 48

* Sexual transmission (vaginal and anal)
- Perinatal transmission (intrauterine, breast milk) - can be reduced w/ antiviral treatment
- Inoculation in blood (transfusion, needling sharing, needle-stick)

Front 49

What are some of the additional genes/proteins in the complex retrovirus, HIV?

Back 49

- 6 Accessory proteins (Tat, Rev, Vif, Vpu, Vpr, Nef)
- 2 Regulatory proteins (Tat and Rev)
- 2 Restriction factors (Vif and Vpu)

Front 50

What are the 6 additional accessory proteins of HIV required for?

Back 50

Replication and / or pathogenesis

Front 51

What is the function of Tat and Rev? Type of proteins?

Back 51

- Essential, crucial for viral replication (attractive targets for therapy)
- Regulatory proteins

Front 52

What kind of protein is Tat? Function?

Back 52

- Regulatory protein / accessory protein of HIV
- Tat = Transactivator of Transcription
- Absolutely required for transcription (mRNA synthesis)

Front 53

Which protein is absolutely required for transcription?

Back 53

Tat

Front 54

What kind of protein is Rev? Function?

Back 54

- Regulatory protein / accessory protein of HIV
- Rev = Regulator of Virion Expression
- Allows structural gene expression by promoting transport of unspliced RNA from nucleus to cytoplasm (export)

Front 55

Which protein allows structural gene expression by promoting transport of unspliced RNA from nucleus to cytoplasm?

Back 55

Rev

Front 56

What is the function of Vif and Vpu? Type of proteins?

Back 56

- Viral proteins that overcome cellular defenses, or "restrictions"
- Restriction factors

Front 57

What kind of protein is Vif? Function?

Back 57

- Restriction factor / accessory protein
- Vif = Virion Infectivity Factor
- Causes a cellular antiviral protein (deoxycytidine deaminase) to ge degraded; otherwise is incorporated into new virions where block RT in next cell by inducing massive mutations in viral dsDNA

Front 58

What protein causes deoxycytidine deaminase to be degraded?

Back 58

Vif

Front 59

What kind of protein is Vpu? Function?

Back 59

- Restriction Factor / Accessory protein
- Promotes virion release from cells by inhibiting a host protein "tetherin" which otherwise blocks release of virus from the cell
- Works on other enveloped viruses

Front 60

What protein promotes virion release from cells by inhibiting "tetherin" (red linkages)

Back 60

Vpu

Front 61

Are Tat, Rev, Vif, and Vpu good anti-viral targets?

Back 61

Hypothetically yes, but so far nothing has been developed that targets them effectively

Front 62

What receptor/cells does HIV bind to?

Back 62

CD4 on immune cells:
- CD4 Helper T-cells
- Dendritic Cells
- Macrophages
- Microglia

Front 63

How does HIV bind to / affect CD4 Helper T-cells?

Back 63

Binds; this is the main cell population that is depleted in AIDS

Front 64

How does HIV bind to / affect Dendritic Cells?

Back 64

Can bind HIV at CD4 receptor, but can not be productively infected; can assist in dissemination

Front 65

How does HIV bind to / affect Macrophages?

Back 65

Infected via binding to CD4 receptors, but not efficiently killed - reservoir of virus production

Front 66

How does HIV bind to / affect Microglia?

Back 66

Binds via CD4 receptors; leads to brain infection - contributes to AIDS dementia

Front 67

For HIV what is necessary for membrane fusion?

Back 67

CD4 binding AND co-receptor

Front 68

What are the HIV tropisms / co-receptors? Types of cells that are infected?

Back 68

- M-tropic (R5-tropic) & CCR5 co-receptor - infects primary T-cells and macrophages (NOT T-cell lines)
- T-tropic (X4-tropic) & CXCR4 co-receptor - infects primary T-cells and T-cell lines (NOT macrophages)

Front 69

What is M-tropic binding responsible for? What kind of people have this kind of infection?

Back 69

Initial infection and transmission, and predominates in asymptomatic persons

Front 70

What is T-tropic binding responsible for? What kind of people have this kind of infection?

Back 70

Associated with disease progression, arises at AIDS stage of infection

Front 71

What chemokines can specifically inhibit M-tropic HIV? How?

Back 71

- RANTES, MIP-1α, MIP-1β
- Occupy receptor (CCR5 co-receptor)

Front 72

What chemokines can specifically inhibit T-tropic HIV? How?

Back 72

- Cytokine Stromal Derived Factor 1 (SDF-1)
- Natural ligand for CXCR4

Front 73

What do HIV co-receptor expression levels correlate with?

Back 73

Cell permissiveness

Front 74

What is the basis for strain tropisms?

Back 74

Env sequence of different HIV types have preference for different co-rereceptors (most concern is for M-tropic virus, which is source of person-to-person transmission)

Front 75

What is the explanation for why some rare individuals remain seronegative despite high-risk behavior and presumable repeated viral exposure?

Back 75

- For some, explanation is a 32bp-deletion in CCR5 gene (co-receptor for M-tropic HIV)
- Causes non-functional CCR5
- WT:Δ32 heterozygotes (~10%) get infected but progress to disease more slowly - express about half as mucha s normal
- Δ32:Δ32 homozygotes (~1%) are highly resistant to infection - normal despite lack of CCR5 expression on surface

Front 76

What are the steps of the fusion process of HIV?

Back 76

1/2: Env initially contacts CD4 and induces a conformation change in Env to expose co-receptor binding site
3/4: gp41 "fusion domains" are exposed, and fusion domain enters cell membrane
5: co-receptor (CCR5) engagement triggers a "snapback" of N- and C- terminal helical regions of gp41 (yellow and red cylinders), which brings membranes together and fuses them

Front 77

What happens in the first two steps of the the fusion process of HIV?

Back 77

1/2: Env initially contacts CD4 and induces a conformation change in Env to expose co-receptor binding site

Front 78

What happens in the third and fourth steps (after initial contact of Env w/ CD4) of the the fusion process of HIV?

Back 78

3/4: gp41 "fusion domains" are exposed, and fusion domain enters cell membrane

Front 79

What happens in the fifth step (after gp41 fusion domain is exposed) of the the fusion process of HIV?

Back 79

5: co-receptor (CCR5) engagement triggers a "snapback" of N- and C- terminal helical regions of gp41 (yellow and red cylinders), which brings membranes together and fuses them

Front 80

What can block snapback in the 5th step of HIV fusion process?

Back 80

T20 "C" peptides (Fuzeon antiviral) can bind the N-terminal helical region and block snapback

Front 81

What are the steps of pathogenesis to HIV infection?

Back 81

1. Initial HIV infection
2. Spread to lymph nodes
3. Virus infects T cells, replicates, and gets into circulation (viremia)
4. Asymptomatic phase

Front 82

Where/how does HIV infection usually occur?

Back 82

At mucosal surfaces or by blood products

Front 83

After an initial HIV infection, what happens?

Back 83

Spreads to lymph nodes - DC cells can bind and carry HIV to nodes, where T cells reside and are infected

Front 84

What happens after HIV spreads to the lymph nodes?

Back 84

Virus infects T cells, replicates to high levels, and spills into circulation (viremia)

Front 85

What happens during the asymptomatic phase of HIV?

Back 85

- FDC traps virus, keeps viremia low, but nodes (especially GALT - gut associated lymphoid tissue) are major sites of replication (1 billion/day)
- GALT deteriorates late in infection (destruction of lymph tissue)

Front 86

What is the most important site of infection of HIV?

Back 86

GALT - gut associated lymphoid tissue

Front 87

What are the disease mechanisms of HIV?

Back 87

- Direct killing of CD4 T-cells by HIV
- Indirect effects on infected CD4 T cells
- Impairment of immune system function

Front 88

How does HIV directly kill CD4 T cells?

Back 88

- Massive virus production leads to membrane linkage and death
- Syncytia (fused cells) induced by fusion of infected cell with uninfected cells (via Env on infected cell interacting w/ CD4/CCR5 on uninfected cell) - cells eventually die (could kill uninfected cells via by-stander effect)
- Apoptosis induced by infection, some evidence that cells undergo apoptosis even if unproductive infection

Front 89

How does HIV indirectly kill CD4 T cells?

Back 89

- Immune response kills infected cells, important for clearing initial viremia
- Soluble gp120 may bind uninfected cells, now susceptible to ADCC (antibody dependent cellular cytotoxicity)

Front 90

How does HIV impair immune system function?

Back 90

- CD4 T-cell function altered, and loss of CD4 T cell help --> leads to severely compromised immune system
- Infected macrophages are dysfunctional --> aberrant immune system

Front 91

What are the clinical features of the acute phase of HIV infection and seroconversion?

Back 91

- Initial burst of virus production coincides w/ decreased CD4 T-cells
- Early vigorous CTL, subsequent humoral response, w/ FDC help, clears viremia
- Immune response only "appears" to control infection
- High level virus production persists in lymph nodes / GALT

Front 92

What are the clinical features of the asymptomatic phase of HIV infection?

Back 92

- Continued strong immune response, but gradual decline in CD4 counts
- Progression measured by CD4 counts, CD4:CD8 ratio, "viral load" by measuring RNA by PCR; (patients w/ low "set-point" have better prognosis)

Front 93

What are the clinical features of the symptomatic/AIDS phase of HIV infection?

Back 93

- Late in infection, CD4 cells depleted (< 200 / µL)
- Immune system begins to fail
- Viremia increases
- Patients susceptible to many opportunistic infections

Front 94

How does the amount of virus change throughout the three stages of an HIV infection (acute / asymptomatic / symptomatic-AIDS)?

Back 94

- Stage 1 - acute - Initial high amount of virus
- Decline during clinical latency through Stage 2 - asymptomatic
- Stage 3 - symptomatic/AIDS - amount of virus rises
(YELLOW)

Front 95

How does the CD4 and T-cell count change throughout the three stages of an HIV infection (acute / asymptomatic / symptomatic-AIDS)?

Back 95

Continually gets lower through all three stages
(PURPLE)

Front 96

How does the amount of anti-HIV-1 antibody change throughout the three stages of an HIV infection (acute / asymptomatic / symptomatic-AIDS)?

Back 96

- Stage 1 - increases
- Stage 2 - plateaus and declines at end (beginning of AIDS)
- Stage 3 - very low
(BLUE)

Front 97

When are tests done for HIV?

Back 97

- Identify infected persons to initiate treatment
- Identify "carriers" who may transmit HIV to others
- Follow course of disease
- Evaluate efficacy of treatment

Front 98

What serological laboratory tests are done to diagnose HIV?

Back 98

- Ab ELISA - initial screening, detect Ab to virus (rapid oral test - inexpensive w/ fast results)
- Ag ELISA - detect p24 antigen earlier than Ab
- Western Blot - confirmation test, uses patients Ab to detect HIV protein

Front 99

What laboratory tests are used to diagnose HIV?

Back 99

- Serological (Ab ELISA, Ag ELISA, Western Blot)
- RNA RT-PCR

Front 100

How does RT-PCR diagnose HIV? Uses?

Back 100

- Detects virus in blood
- Quantitate virus in blood
- Very sensitive (≤ 50 HIV RNA copies / mL)
- Detects virus before seroconversion (high risk groups, newborns)
- Gauges viral load in asymptomatic patients w/ low titers (important for prognosis)

Front 101

What are the therapeutic targets for HIV?

Back 101

- Any step in replication cycle, especially virus specific proteins
- RT inhibitor
- Protease inhibitors
- Fusion inhibitor (T-20)
- Entry inhibitors (CCR5 co-receptor antagonist)
- Integrase inhibitor
- HAART - Highly Active Anti-Retroviral Therapy

Front 102

Is it necessary to completely block HIV replication to remain healthy?

Back 102

No - progression is related to viral load

Front 103

Which kind of HIV therapy can reduce viral load by 30-100x alone?

Back 103

Protease inhibitors

Front 104

What is HAART? Outcomes?

Back 104

- Highly Active Anti-Retroviral Therapy
- Triple therapy ("cocktails") of different inhibitors
- Virtually eliminate virus production in some individuals for many years
- Virus undetectable in plasma, increased CD4 cell counts and obvious clinical benefit
- Long-term patients experience toxicity

Front 105

What did HAART allow determination of?

Back 105

HIV replication dynamics, 1/2 life of HIV

Front 106

How long does it take to clear free virus and infected T cells w/ HAART?

Back 106

~2 months

Front 107

How long does it take to clear other "compartments w/ longer half-lives (macrophages and FDCs) w/ HAART?

Back 107

~1-2 years

Front 108

How long does it take to clear infected memory T cells w/ HAART?

Back 108

Long-lived, can detect virus from those on HAART for > 5 years (resting T cells); would need > 75 years to clear this compartment

Front 109

Why is the enthusiasm for HAART and a "functional cure" for HIV tempered?

Back 109

- Not all patients respond to HAART
- Drug regimen is difficult to follow (getting easier w/ 1x/day pills)
- Toxic effects seen in long term HAART users
- Inaccessible pool of virus

Front 110

What kind of drugs are being developed to treat HIV?

Back 110

- New attachment inhibitors (anti-CD4 antibody)
- New CXCR4 and "dual receptor" inhibitors, and anti-CCR5 antibody
- RT inhibitors to common drug-resistant RT viruses
- New integrase inhibitors
- Maturation inhibitors that work on gag and gag-pol proteins