Retroviruses and HIV

Question 1

descirbe the basic principle of how retroviruses work

Answer 1

RNA genome undergoes reverse transcription to form DNA copy

DNA copy integrates into host cell

integrated DNA is substrate for RNA transcription —> viral proteins made

Question 2

what genera of virus is HIV type 1

Answer 2

Lentivirus

(lenti = slow, lentiviruses cause slowly progressing diseases)

Question 3

what genera of virus is Human T-Cell Leukemia/Lymphoma Virus Type 1 (HTLV-1)?

Answer 3

deltaretrovirus, infects CD4+ T cells

in the US, almost exclusively in immigrants, via bodily fluids but much lower transmission than HIV

Adult T-Cell Leukemia/Lymphoma (ATL) cells look like flowers in smear

Question 4

what is the function of the following HIV-1 virion proteins?
a. p17/matrix
b. p24/capsid
c. p31/integrase
d. gp41
e. gp120
f. RT
g. p11/protease
h. p7/nucleocapsid

Answer 4

a. p17/matrix: holds core together with envelope
b. p24/capsid: forms core of virus
c. p31/integrase: integrates viral DNA into host DNA
d. gp41: transmembrane envelope protein, fusion of virus/cell
e. gp120: surface envelope protein, binds CD4 (receptor) (in complex with gp41) - the “spike”
f. RT: reverse transcriptase converts viral RNA to DNA
g. p11/protease: cleaves precursor polypeptides made by viral genome
h. p7/nucleocapsid: binds to nucleic acids to hold genome together

Question 5

which 2 HIV-1 virion proteins are involved with entering the host cell and how do they work together?

Answer 5

gp120: surface envelope protein, binds CD4 (receptor) (in complex with gp41) - the “spike”

gp41: transmembrane envelope protein, fusion of virus/cell

Question 6

what is the product of the following retrovirus genes:
a. gag
b. pol
c. env

Answer 6

a. gag: structural proteins
b. pol: RT, integrase, protease
c. env: viral surface and transmembrane proteins for viral attachment/fusion

Question 7

in retroviruses, the provirus is…

Answer 7

the DNA copy

Question 8

what about gp120 (surface) and gp41 (transmembrane) HIV envelope proteins help it to evade the immune system?

Answer 8

gp120 + gp41 form a trimer that is heavily glycosylated - acts as “shield” to protect viral proteins from recognition by host immune system

Question 9

what is the receptor for HIV, on what cells is it found, and what are the most prominent co-receptors?

Answer 9

HIV receptor is CD4, found on T cells and some macrophages

co-receptors: CCR5 and CXCR4 (chemokines)

Question 10

what are the 3 enzymatic steps of reverse transcriptase?

Answer 10

1. RNA-dependent DNA synthesis (1st strand)
2. RNAse H: degradation of RNA in RNA:DNA hybrid
3. DNA-dependent DNA synthesis (2nd strand)

*note that RT is associated with high error rate, insertions/deletions, recombinations —> high variability, no 2 HIV genomes are identical

Question 11

explain why there is such a high variability in HIV genome?

Answer 11

Reverse transcriptase is associated with high error rate (new mutation every 10 cycles), insertions/deletions/recombinations

no proofreading activity!

thus, no 2 HIV genomes are identical - makes it difficult to use targeted therapy

Question 12

what are the 2 enzymatic activities of retroviral integrase?

Answer 12

integrase cleaves host DNA and ligases proviral DNA into host genome

(host repair systems also play a small part in filling in gaps)

Question 13

explain the potential for latency of retroviruses (esp. HIV) and insertional mutagenesis

Answer 13

potential for latency - retroviral DNA (once integrated) is regulated like cellular genes and transcribed by RNA pol II, can be turned on/off

insertional mutagenesis - retroviral DNA contains enhancers/ promoters/ splicing sequences that can affect nearby cellular genes

*also note that retroviral integration can occur in germ cells, creating endogenous retroviruses

Question 14

what kind of vectors are used in CAR-T therapy?

Answer 14

retrovirus vectors! particularly lentivirus

[recall CAR-T = chimeric antigen receptor T cells]

Question 15

explain how treatment=prevention regarding the HIV epidemic

Answer 15

treatment decreases viral load = decreased ability to spread virus

undetectable = untransmittable

Question 16

how is HIV diagnosed?

Answer 16

1. screen with ELISA (window of insensitivity before antibodies are made, ~30 days post-infection)
   *can also use PCR - can test sooner but lower specificity
2. confirm with Western Blot (serum antibodies bind to viral antigens)

Question 17

what are the CDC recommendations regarding HIV screening/testing? specify for general population, high-risk individuals, and pregnant women/newborns

Answer 17

voluntary testing for ages 13-64 during health care encounters

annual repeated screening for high risk patients

all pregnant women and any newborn whose mother’s HIV status is unknown

Question 18

when should HIV treatment be initiated

Answer 18

at time of initial diagnosis !! ASAP

Question 19

what are the phases of HIV-1 infection

Answer 19

acute (nonspecific) —> asymptomatic (clinically latent) —> symptomatic (much worse opportunistic infections/malignancies if <200 CD4+ T cells/ml)

latent reservoirs are established within days!

Question 20

what are the following infections heavily associated with:
- toxoplasmosis, cryptosporidium
- pneumocystis carinii
- candida, cryptococcus, histoplasmosis
- M. avium, Mtb

Answer 20

these are all opportunistic infections commonly associated with AIDS patients

Question 21

what does HIV surface envelope protein gp120 bind to?

Answer 21

1. CD4 - receptor
   ALSO
2. CCR5 (binds CD4+ T cells and macrophages) and CXCR4 (CD4+ T cells) - co-receptors

*note that rare individuals have natural mutation in CCR5 that confers resistance to HIV

Question 22

how does gp14 transmembrane envelope protein of HIV interact with the host cell membrane?

Answer 22

after gp120 binds, envelope undergoes conformational change

hydrophobic N-terminus of gp41 uses harpoon-like mechanism to fuse viral and cell membrane

Question 23

where does transcription begin in the HIV genome? what is the viral transactivator?

Answer 23

5’LTR (long terminal repeat)

Tat: viral transactivator
[Rev is viral regulator]

Question 24

explain the relationship between NFkB and HIV viral transcription

Answer 24

HIV links its viral protein expression to NFkB so that viral expression is turned up with T cell activity (needs other T cells to be around to increase infection)

immune activation is a cofactor (enhances) AIDs development

[for ex, viral load increases following immunizations]

Question 25

what is the role of HIV Tat protein?

Answer 25

Tat is RNA binding protein, feeds back to its own promoter (positive feedback loop) to increase transcription 100x

Question 26

what is the role of HIV Rev protein?

Answer 26

HIV needs unspliced RNA to make a new virion - Rev promotes export of RNA with RRE (Rev response element) from nucleus which prevents it from being spliced to completion (so a new virion can be assembled) *required for HIV replication*

Question 27

how do HIV accessory proteins Nef and Vif counteract host anti-viral activities?

Answer 27

Nef: takes MHC I off the cell surface (some long-term non-progressers have Nef mutation) Vif: inhibits degradation of viral DNA

Question 28

monocytes, macrophages, and microglia can be infected by _____-tropic HIV

Answer 28

monocytes, macrophages, and microglia (also CD4+) can be infected by CCR5-tropic HIV (preferentially use CCR5 as co-receptor) - not killed, just dysfunctional CD4+ T cells are infected by both CCR5 and CXCR4 tropic HIV and killed *note that tropism is determined by gp120 (surface protein)

Question 29

how is tropism of HIV binding determined, and which form is present in early vs late infection?

Answer 29

tropism determined by hypervariable region of surface protein gp120 which binds CD4 and either CCR5 or CXCR4 co-receptor CCR5 (T cells, monocytes, macrophage, DC, microglia) preferential during initial infection - “R5 tropic virus” CXCR4 (activated T cells) preferential in later stages - “X4 tropic virus”, replicates faster

Question 30

what causes the decrease in CD4+ T cells in HIV infection?

Answer 30

DIRECT KILLING by cytopathic infection of HIV “exhaustion” of T cells also follows - chronic T cell activation induces senescence and apoptosis also immune destruction of infected cells, death of bystander cells (HIV toxic gene products attach)

Question 31

what is the pathogenesis of HANDs (HIV Associated Neuro Disease) thought to be caused by?

Answer 31

infection of monocytes/microglia (R5-tropic strain) —> pro-inflammatory cytokine release in CNS

Question 32

describe the progression of HIV to AIDS by what is happening to the CD4+ T cells

Answer 32

acute major loss of CD4+ T cells following initial infection + very high level of HIV replication before immune response begins… followed by dampening of HIV infection by CTL which is initially very effective - clinical latency… then viral levels begin to rise, opportunistic infections take hold, etc… full blown AIDS

Question 33

how does acute infection with HIV present?

Answer 33

mononucleosis-like symptoms, associated with high levels of HIV particles in peripheral blood and peripheral mononuclear cells (recall R5 tropism is seen in initial mucosal infection) acute CD4+ T cell loss

Question 34

describe the initial spread of HIV following infection

Answer 34

CCR5-tropic HIV infects founder population in mucosa and expands locally (1 wk) followed by explosive replication in lymph tissue (2-4 wk) followed by massive depletion of CD4+CCR5+ T cells in the gut (huge source of lymph tissue) via direct killing and bystander effect + punches holes in memory repertoire ultimate spread to lymph nodes with direct/indirect killing/death of CD4+ cells

Question 35

describe the effect of early HIV infection in the gut lymph tissue on the repertoire of memory T cells

Answer 35

when HIV spread to gut lymph it virtually destroys the T cell population there via direct killing and bystander effects this “punches holes” in the repertoire of Memory T cells there (specific for many opportunistic infections), which leads to early immune defects

Question 36

what is the initial immune response to HIV

Answer 36

NK cells, antibody production at 3-7 weeks, CTL initially quite effective but does not block establishment of life-long infection and cannot be sustained

Question 37

what is going on during the asymptomatic period of HIV infection? [on a cellular level]

Answer 37

low set point of HIV levels but persistence of replication in lymph tissues and CD4+ T cell destruction subtle immune defects develop due to targeting of memory T cells which decreases immune repertoire —> shift in T cell populations and cytokines

Question 38

what kind of mutations in HIV infection increase viral variability that leads to immune escape? 3

Answer 38

selective pressures for viral genomes that evade immune system lead to: - neutralizing antibody resistant mutants - mutations in CTL epitopes (conferring resistance) - mutation in MHC binding domains (not recognized by immune system)

Question 39

what kind of antibodies are initially made by the immune system in response to HIV infection, and why is this problematic?

Answer 39

important neutralizing domain (antibody target) is the hyper-variable region of HIV SU (surface envelope protein, binds CD4) - very sequence/patient specific, and HIV mutants frequently broadly neutralizing antibodies (to more conserved domains) develop later, after infection is already established

Question 40

regarding HIV progression, ____ levels indicate how far the disease has progressed, while ____ levels predict where the disease is headed (and if it is time to switch drugs)

Answer 40

regarding HIV progression, CD4 cell levels indicate how far the disease has progressed, while HIV RNA blood levels predict where the disease is headed (and if it is time to switch drugs) *when viral load goes up, CD4 cells are going to go down, and it is time to change drugs because resistance is emerging*

Question 41

what is the major roadblock to completely clearing HIV infection?

Answer 41

latent reservoirs!! cannot cure unless reservoirs are eliminated also co-factors such as immune stimulation and viral infections which may increase HIV replication

Question 42

what are some of the challenges hindering developing an HIV vaccine?

Answer 42

envelop variability (constant antigenic variation) + heavy glycosylation blocking Ab binding no examples of naturally acquired protecting immunity with viral clearance early establishment of latent reservoir means small window to block infection ethical issues for designing trials because PrEP is effective