HIV Virology/Replication

Question 1

HIV is surrounded by a lipid membrane with two different membrane associated proteins. What are these two proteins?

Answer 1

1. gp41: transmembrane glycoprotein
2. gp120: A docking protein.

take the 1’s away from these proteins and you get 420 remember?

Question 2

HIV has 3 enzymes that it uses to take over. What are they?

Answer 2

1. Protease
2. Integrase
3. Reverse transcriptase

Question 3

What kind of virus is HIV? DNA? RNA?

Answer 3

RNA retrovirus. It goes from RNA to DNA using its magically reverse transcriptase protein.

Question 4

There are two kinds of HIV, HIV-1 and HIV-2. What is their distribution.

Answer 4

HIV-1: EVERYWHERE

HIV-2: Western Africa

Question 5

Within HIV-1 we divide infection types into groups. There is O group, N group and M group. Which one is the most common and describe its classification.

Answer 5

M group is the most common (95% of all infections). This group is split into different CLADES (A, B, C, D etc) that we use to classify what strain of HIV is where in the world.

O group: outlier group

N group: not M or O group. I did not make this up.

Question 6

What clade is most common in North America (and Europe, Australia and South America too)

Answer 6

Clade B.

Because we’re the BEST!

A,C,D in Africa.
C in Asia.
E in the Asian Pacific

Question 7

What host receptors does HIV bind to?

Answer 7

On the CD4 cell, the CD 4 receptor binds to gp120, and the CCR5 receptor binds to gp41. CCR5 is predominantly affected in the PRIMARY infection.

CXCR4 is the CD4 receptor that is predominantly affected in the progression to AIDS.

Question 8

Which receptor is more ubiquitously expressed on the CD4 cell, CXCR4 or CCR5?

Answer 8

CXCR4

But remember that the first receptor HIV goes for is CCR5

Question 9

We always talk about CD4 cells being affected by HIV. What other immune cells are HIV targets?

Answer 9

1. Monocytes/macrophages

2. Dendritic cells (aka Langerhans cells in epithelium)

Question 10

Where does HIV find all the CD4 T cell to infect? What are the main target tissues?

Answer 10

Mucosal tissue: lots of activated CD4+

1. Cervix
2. Vagina
3. Anus
4. Rectum
5. Foreskin

Question 11

Once HIV infects the CD4/Macrophage/Dendritic cells in the genital mucosa, what happens next?

Answer 11

These infected cells go to the genital lymph nodes. Here they replicate more and then travel either free or associated with cells through vessels leading to systemic lymphoid replication.

Question 12

Let’s talk about the genes of HIV and what they code for. Name the 3 most important genes and their products

Answer 12

Gag: codes for nucleocapsid core protein
Pol: codes for reverse transcriptase protease, and integrase
Env: codes for the envelope glycoprotein

Question 13

Walk me through HIV replication, from first site to entry into the cell. General terms first.

Answer 13

1. HIV spots a CD4 cell from across the room
2. HIV gp120 binds to CD4 receptor
3. gp41 then binds to CCR5
4. They binding leads to conformational changes that expose the p41 fusion protein that helps the membranes of HIV and CD4 cell fuse.
5. Viral capsid is released into the CD4 cell
6. HIV has gained access to the cell. Shit’s bout to go down.

Question 14

Now HIV is in the cell. WHAT HAPPENS NEXT??

Answer 14

The viral RNA is released and reverse transcriptase is used to make some good old DNA. Viral DNA duplicated to make double stranded DNA. This goes to the nucleus and is integrated into the host DNA by integrase enzyme. Transcription occurs, so now all the viral proteins have been made. You make a new virus and now you’re royally fucked.

Question 15

Describe attachment and entry of HIV in detail.

Answer 15

1. CD4 binds to gp120
2. This causes conformational change in gp120 so that it also binds CCR5, and also releases gp41’s fusion protein.
3. Gp41’s fusion protein integrates into the host membrane and then folds in on itself. This allows viral entry to occur.

Question 16

DNA formation. Discuss in detail.

Answer 16

Viral mRNA is made into DNA by reserve transcriptase enzyme. The enzyme starts working at the LTR (long terminal repeat) site. Reverse transcriptase also destroys the RNA after DNA is made. DNA is then duplicated so you’ve got a double stranded helix.

Question 17

Why does HIV mutate so much?

Answer 17

1. Can use either strand of RNA to make DNA

2. Reverse transcriptase lack self-correcting function that our DNA polymerases have.

Question 18

What protein helps the double stranded viral DNA into the nucleus?

Answer 18

Viral protein R (Vpr) puts the dsDNA in the PIC (preintegration complex) which contains the integrase enzyme and other transport proteins

Question 19

What exactly does integrase do?

Answer 19

It nicks the viral DNA at the LTR site making “sticky ends” and nicks the host DNA at complimentary sites. Cellular ligase enzyme seals the viral DNA into the host. And now you’re infected with HIV for the rest of your life.

Question 20

Where on the LTR does TRANSCRIPTION occur? Not reverse transcription! Making mRNA after the HIV is already in the host genome.

Answer 20

The TAR site (tac activated response element). On the viral DNA.

Question 21

What accessory protein initiates transcription of the host genome?

Answer 21

TAT protein (trans-activator of transcription). This protein has a TAR binding domain which makes sense since that’s where we are starting transcription!

Question 22

Ok so TAT binds to TAR and then a bunch of other cell cycle proteins join the party so you have this big protein complex on the to-be transcribed DNA. What’s the point?

Answer 22

This complex activates RNA polymerase II, which does the transcribing. Now you’ve got mRNA.

Question 23

You have to get the transcribed mRNA out of the nucleus so that it can be translated into proteins in the cytoplasm. Remember that this mRNA transcript is made in spliced pieces. What HIV protein helps to transport our HIV transcripts to the cytoplasm.

Answer 23

HIV rev protein binds to RRE (rev response element), which is on many (but not all) HIV transcripts. Rev helps get these suckers out so that HIV can unleash its rath.

Question 24

So we translate our RNA transcripts into proteins in the cytoplasm. Do the 3 protein we care most about: gag, pol and env, just start doing their job or are they cleaved first?

Answer 24

Env: is translated as gp160 and then cleaved into gp120 and gp41
Pol: translated as a precursor protein with gag and cleaved into its active parts
*Gag: translated as a precursor protein but is not cleaved until it leaves the cell. Since it is cleaved by proteases, this is the site of action for protease inhibitor antiretrovirals.

Question 25

What does HIV Nef protein do?

Answer 25

Enhances infection and replication

Question 26

What does HIV protein Vpu do?

Answer 26

It degrades CD4 receptor and tetherin on the infected cell, allowing for release of HIV from the cell and prevents it from just bind to the CD4 receptor of the same cell.

Question 27

What does HIV protein Vif do?

Answer 27

When reserve transcriptase is doing its job making DNA from RNA, APOBEC3G (a host protein) tries to introduce hypermutations into the viral DNA. Vif unfortunately degrades APOBEC3G. Nice try host.

Question 28

What do all the little viral transcripts do now that they’ve been translated and are ready to infect

Answer 28

They assemble at the host cell membrane, self assemble, and bud off into the world. They are not infectious cells until the viral proteases cleave the gag protein, though.

Question 29

Review: what are the major steps in HIV replication

Answer 29

1. Attachment and entry
2. Reserve transcription (cytoplasm)
3. Integration (nucleus)
4. Transcription (nucleus)
5. Translation (cytoplasm)
6. Assembly
7. Release and maturation

Question 30

What is a super infection of CD4 T cells.

Answer 30

CD4 cell get infected with more than one HIV virus type. These different viruses can form recombinant forms of HIV! Wahhhhh!!!!!

Question 31

What protein antibodies does the p24 ELISA look for?

Answer 31

Gag protein antibodies.

Question 32

What is seroconversion?

Answer 32

First appearance of HIV antibodies in serum. Can take between 4-12 wks.

Question 33

What is the eclipse phase?

Answer 33

Time after infection where we have no way to detect HIV infection. Viral RNA is the quickest method of detection and that doesn’t becoming positive until about day 10.

Question 34

What method of HIV detection is relied on the most?

Answer 34

HIV RNA PCR is a more sensitive test than p24. It is positive earlier and stays positive longer. Combine with a HIV ELISA to detect chronic disease.