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micb 202 > HIV - Reverse Transcriptase > Flashcards

HIV - Reverse Transcriptase Flashcards

1
Q

Chapter 10 - Retroviruses

A

f

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2
Q

HIV

A

HIV is the virus that causes AIDs the symptoms

It belongs to the subgroup of retrovirus known as lentiviruses or slow virus

Infections with lentivirus are characterized by a long interval between the initial infection and the onset of serious clinical symptoms

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3
Q

History of HIV:

A

Before HIV infection became widespread in the human population, AIDS-like syndrome were rare.

HIV has probably circulated in humans since the early 1900s
An increase in unusual infections

Infection cause of AIDS is caused after sexual contact, mother to infant transmission, transmission by blood and IV drugs use

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4
Q

History of HIV:

A

There are 2 types of HIV

  • HIV-1 and HIV-2
  • Both target/damage person’s immune cells by destroying CD4 T cells
  • TH cells are crucial in adaptive immune response

HIV1 is the form that is promarily circulating

HIV-2 was originally isolated from AIDs patient in west Africa

HIV 1 and 2 evolved in parallel, transmitted to humans by several zoonotic events

  • 1 came from chimpanzees and 2 came from sooty mangabeys
  • Both have the same mode of transmission similarly cause AIDS
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5
Q

HIV-1

A

There are 4 different groups

  • M, N, O, P

Each group is thought to be the result of an independent transmission of SIV into human

Group M can be further divided into subtypes - classes, based on genetic sequence

Some of the clades are known to be more virulent or are resistant to anti-retroviral drugs, or have faster disease progression

CRFs = circulating recombinant forms

Group M is the most commonly found

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6
Q

HIV Genome

A

Has 2 identical +ssRNS reverse transcriptase

Each RNA strand has a 5’cap, 3’poly A tail - looks like mRNA

Associated with it is tRNA acquired from the host cell in the previous infection

HIV genome is a polyprotein

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7
Q

Q1: After infecting the cell, the first step in replication cycle involves translation of RNA on a ribosome?

A
  • Not true!

it needs to be reverse transcriptase into dsDNA first before it can be translated

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8
Q

Q2: genome encodes for a polyprotein -
gag-pol-env protein

HIV would then cleave the individual proteins out of the polyprotein?

A

No, HIV does not need to cleave the individual proteins out of the poly protein
- RT not same as other viruses

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9
Q

HIV Genome structure

A

Enveloped

Roughly spherical in shape

Matrix layer under the envelope

The genome is packaged in a capsid like structure called the core

Core not capsid because it is a layer of protein that surrounds the nucleocapsid
- It does not completely dissociate /uncoat when the virus enters the cells cytoplasm (unlike other viruses)

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10
Q

Gag gene - group specific antigen gene

A

Polyprotein

Gag gene:

  • CA = capsids
  • NC = protein that coats genome to form nucleocapsid
  • MA = matrix protein

protease
- repeat sequences R, PBS

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11
Q

Pol Gene

A

Encodes for: polymerase gene

  • RT
  • IN = integrase
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12
Q

env Gene

A

Codes for viral envelope glycoproteins

  • Gp120 (anti receptor that binds to host’s virus receptor)
  • and Gp 41 (fusion peptide)
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13
Q

Reverse Transcriptase

A

tRNA acts as the primer

Uses the free 3’Oh on the tRNA as the primer to start reverse transcriptase process

There are 2 models on how the process of reverse transcription occur

Are both strands copied to DNA? - we dunno

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14
Q

Reverse Transcriptase

A
  1. RNA dependent DNA polymerase (RDDP) activity of the RT read the RNA template and synthesizes a complementary DNA strand
  2. RNase activity of the RT degrades the RNA strand so now left with ss DNA
  3. DNA dependent DNA polymerase (DDDP) activity of reverse transcriptase reads the DNA template and synthesizes a complementary DNA strand to make dsDNA
    - The resulting ends of the dsDNA is longer than original RNA due to RT process
    - This produces a promoter and transcription terminator sequence in the DNA sequence
    - LTR has thing important for transcription - promoter terminator etc
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15
Q

Q: Reverse transcriptase has 3 distinct enzymatic activities, each catalyses at a different site in the enzyme, which of the 3 RT enzymes is best target for antiviral therapy?

A

( activity 1: RDRP 2, RNase 3. DDDP)

best target for antiviral therapy is RDRP

  • because host cell do not have RNA dependent RNA/DNA polymerases
  • it is unique to virus only, host cell cannot read RNA template for transcription

Targeting RNase H or DDDP would be bad

  • Would also impact non infected cells and interfere with DNA synthesis
  • DNA pol 1 had RNase H activity to remove RNA primers from okazaki fragments (these are essentially DNA pol 1 and 2?)
  • Our cells only have DNA-dependent, no RNA dependent
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16
Q

Replication cycle

A

Core maintain it shape until step 4 - reverse transcriptase, production of dsDNA happen within the core in cytoplasm
(RT happens in 3, the step mentioned in chapter 10 picture above)

PICC

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17
Q

Retroviruses Replication

Challenge 1:

A

HIV must transcribe its RNA genome to DNA before integrating into the host cell genome

THe cell lacks a polymerase than can read RNA as a template and synthesize DNA and it lacks an enzyme that will integrate viral DAN into the host cell genome

Solution:

  • It encodes a gene for RT and IN and packages some of these enzymes in its capsid
  • RT has 3 activity site that can help synthesize dsDNA form +ssRNA
  • IN helps integrate
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18
Q

Retroviruses Replication

Challenge 2:

A

RT requires a primer to start the process of synthesizing DNA

Solution: virus packages tRNA molecule to use as a primer

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19
Q

Retroviruses Replication

Challenge 3

A

The eukaryotic ribosome can only translate the monocistronic mRNA,
but HIV has several proteins (and genes ), polycistronic

Solution: The virus uses both a polyprotein strategy - one reading frame multiple proteins

and alternative splicing pattern for viral mRNAs

20
Q

Retroviruses Replication

Challenge 4

A

The polyproteins needs to be cleaved to from the individual protein,
this requires a protease that recognizes the beginning and end of each protein

Solution:
- Virus genome encodes a protease that cleaves the polyprotein

  • It becomes active when the secondary and tertiary structures of the polypeptide are formed
  • The polyprotein undergoes self proteolytic cleavage as it forms/fold into ⅔ structure
  • One polyprotein is cleaved by a cellular enzyme
21
Q

Retroviruses Replication

Challenge 5

A

Eukaryotic ribosomes recognize mRNAs with 5’cap and poly A tail

Virus must compete with the cells mRNA for access to ribosomes

Solution

  • The viral genome also ready has these features (made in the replication cyle)
  • Viral mRNA is made using cellular RNA pol 2 and is processed with other cellular enzymes
  • resembles cellular mRNA
  • integration of virus’ genome into host cel genome made this possible

unlike previous virus’ we learnt - this is diff

22
Q

Retroviruses Replication

Challenge 6

A

Host cell RNases degrees RNA molecules

Solution:

  • The process of RT occurs within the virus’ core
  • This protects the RNA from RNaes and also keeps the enzymes needed for RT process and integration of DNA together
23
Q

HIV attachment and Genome entry

A

HIV binds to its receptor CD4 on host cell surface through the virus’ gp120 protein

  • This triggers a conformational change in gp120, allowing it to bind to its co-receptor CCR5 or CXCR4
  • This triggers conformational change in gp41, resulting in the fusion peptide inserting into the cell membrane

The envelope fuses with the cell membrane and the core of the virus particle enters into the cytoplasm
- not endocytosis of whole this - similar to polio?

24
Q

HIV attachment and Genome entry

- vid

A

First contact is through the viral envelope proteins and viral receptor CD4 to gp120

Result in confirmation change that allows envelope to insert gp41 into the cells membrane

Then envelope protein folds back and fuses with cell membrane

gp120/41 from the virus remain in the plasma membrane

  • the core of virus is released into cytoplasm
  • RNA coated with NP
25
Q

Chemokine receptor: CXCR4 and CCR5

A

The characteristic feature of all chemokine receptors is a serpentine 7 transmembrane spanning domain structure ( T cells)

Extracellular portions are involved in chemokine binding, while intracellular portions are involved in cell signaling

Some chemokine receptors have a role in infectious disease susceptibility or pathogenesis

In peripheral blood, T cells and monocytes express CXCR4 and CCR5

Some HIV strains prefer CXCR4 as co-receptors infecting mostly CD4 T cells

some prefer CCR5 - infecting both CD4 T cells and monocytes

26
Q

Chemokine receptor: CXCR4 and CCR5 mutation

A

Individuals that carry a mutation known as CCR5-delta32 in the CCR5 genes tend to be highly resistant to HIV infection (but not absolute)

This mutation is present in about 2% of caucasian population

If the person is homozygous for the CCR5-delta32, the resistance might result from both genetic loss of CCR5 on the cell surface as well as active down-regulation of CXCR 4 expression of the mutant by CCR5-delta32 protein

27
Q

Chemokine receptor: CXCR4 and CCR5 mutation

A

Protein shape and function are connected.

If a protein does not have the right shape, it likely won’t perform its function.

The altered shape caused by the deletion means that the chemokine receptor doesn’t bind chemokines and HIV can’t use it as a co-receptor to cause fusion peptides to be revealed

eg
- Has 4 transmembrane instead of 7, deletion of their allele the CCR5 gene

  • No C signaling protein inside
  • Does not function as a HIV receptor anymore
28
Q

HIV Genome

A

Even though HIV genome resembles mRNA, the first step isn’t translation

This is because when the virus infects the cell, the genome is only partially uncoated

The core remains mostly intact

  • It is porous so that nucleotides can diffuse in but not ribosomes
  • therefore it cannot be translated (no virus can do translation on its need need hosts ribosomes)

Inside the core with RNA genome are the RT and IN

Also inside the core are the tRNAs that the virus use as a primer for reverse transcriptions

These tRNA were packages with the viral genome during parcours replication cycle

Everything needed to convert ssRNA into dsRNA in the core

Basically stays inside till it form dsDNA then nucleus for integration

29
Q

HIV Genome Replication

A

Copying of RNA genome into a dsDNA genome - this is done by RT enzyme

The core delivers the virus dsDNA and integrase to the cell nucleus

The IN integrates the virus dsDNA genome into the host genome at particular target sequence

The transcription of the integrated virus dsDNA genome is done by the cell RNA polymerase
- cuz name it is pretty much same as mRNA (just with viral genome inserted in it )

30
Q

HIV Genome Integration

A

The process of integration is site specific

  • The target sequences needed for integration are located in many specific areas within the host cells DNA
  • can only insert itself in these areas on the DNA

The consequence to the host cell of this viral integration depends on whether it is integrated within a gene or its regulatory sequences

  • (how virus affects the DNA)
  • The consequence of the virus depends on if this site is transcribed frequently in the cell or not
  • One of the reasons why viruses go latent, if the region is not transcribed frequently

So not integrated in same place all the time but still specific

31
Q

The function of the core is important

A
  • Keeps the virus genome together with the RT so that dsDNA can be synthesized
  • Keeps the IN present so that it can be used when needed
  • Protects RNA from cells RNases
  • Prevents it from interacting with ribosomes
  • Delivers it to the nucleus

Lots of animations show the core falling apart and the RT and IN drifting off. If it did that, what would be the chances of the dsDNA being “re-united” with the integrase?

32
Q

HIV genome integration

A
  1. Recognition of target site by integrase
  2. Staggered cutting of host cell DNA at a target site resulting in short stretches of ssDNA
  3. Ligating viral dsDNA into host cell DNA
  4. Repair of ssDNA resulting in duplication of target

There are many copies of the target sites in the genome, but only one is used.

capping and polyadenylation done by host cell enzymes

33
Q

HIV Dormancy: After the dsDNA gets into the nucleus

A

HIV can remain dormant for long period of time

After cell activation, transcription of viral genome begins
-Changes in DNA that opens it up make it accessible for RNA pol2

34
Q

HIV genome replication

A

As the core is released into the cell

Virus core enters the cytoplasm and use microtubules motor to move to the nucleus

The RNA is coated with NP

Inside the core RT begins
The RNA is being coated with NP protein

RT copies RNA to DNA

Eventually genome is completely copied to dsDNA and is delivered to nucleus

The core has protected the RNA from RNases in the cytoplasm and ensured that integrase protein is still with the DNA

The DNA enters the cytoplasm with integrase attached

A target site is identified in the host cell DNA

  • Integrase cuts into the host cell DNA at target site and virus DNA is integrated
  • DNA repair is done
35
Q

HIV genome replication

A

Virus starts out with RNA strain as its genome

The RNA was reverse transcribed into DNA inside the virus’ core after the core entered the cytoplasm

This can’t happen before the virus is attached to host cell because
0 no source of nucleotides from the host cell
- and no ATP, need a host

Virus’ DNA permanently integrate into the host cell

Transcription of viral genome begins when changes in the DNA opens it up and make it accessible for RNA pol II

36
Q

HIV Translation

A

Limitation of the host cell ribosome: the genes are separated, need all of them

HIV needs to make many proteins - 3 strategies

  1. Spliced mRNA translated to make env poly protein
  2. Unspliced mRNA translated to make
    - Gag polyprotein
    - gag-pol polyprotein
    - the RNA transcript can be packaged into new virus particles to serve as genomic RNA
37
Q

HIV Translation

Strategy 1

A

Some viral mRNA are spliced - HIV can exploit the cells RNA splicing enzymes to make different mRNA

Splice at 5’ splice site then attach that spliced out 5’cap right before the env protein
- now instead of whole polycistronic DNA have only env gene

The spliced mRNA is translated by ER bound ribosome

The env polyprotein (aka gp160) is cut by CELLULAR ENZYMES in the Golgi to yield gp120 and gp41

38
Q

HIV Translation

Strategy 2

A

Some viral mRNA are unspliced

HIV can use polyprotein strategy to make several proteins

90% - Most of the time the ribosome start at the gag start codon and continues translating until the gag stop codon (aka just gag gene translated and made)

  • Need a lot of structural proteins per virus particle
  • Get the CA, NC, MA, PR
39
Q

HIV Translation

Strategy 3

A

Pol protein is never transcribed on its own, always gag-pol : 10% of the time
- Pol gene is in a different reading frame

In order to make the Pol polyprotein, the ribosome has to slip into a different reading frame
- Need only a few copies of pol enzymes per virus particle (but need lots of gag)

Ribosome takes a step back to shift reading frame, so now not a stop codon

40
Q

HIV Translation

Strategy 3: RIbosome Frameshifting

A

Strategy programmed into the genome

In the RNA there is a stretch of the nucleotides that forms a slippery sequence

There are also sequences that results in a secondary RNA structure like stem loops and pseudoknots

Pseudoknot causes the ribosome to stall at the slippery sequence

The ribosome will backtrack one nucleotide to resulting i in some mismatches between the tRNA and mRNA

Translation revolves on the backtracked ribosomes in the -1 frame

This allows it to skip over the gag codon and enter into the reading frame for the pol gene

Pseudoknot allows the ribosome to move back one nucleotide and shift the frame to avoid stop codon

(Codon = 3 nucleotide, shift back -1 changes the 2 nucleotides so doesnt read as a stop codon anymore)

41
Q

Assembly of New Particles and Budding

A

Env polyprotein gp160 is made by the ribosome on the ER and is cleaved to make gp120 and gp41 in the golgi and inserted into the plasma membrane

The gag and gag-pol polyproteins accumulate at the membrane in the area where gp120 and gp41 are found
(in entry, the gp120 and 41 are left behind on the plasma membrane)

The RNA genome and some tRNA accumulates at the membrane ready for assembly
Then the virus buds out
Ps the virus core is not made until the virus buds out (maturation)

42
Q

Movie key Points

A

Intact RNA that is part of the genome leaves the nucleus as it moves toward the plasma membrane, tRNA may bind to it

The gag and gag-pol proteins have accumulate just under the plasma membrane

The gp120 and gp41 have inserted in the plasma membrane

The RNA genome associates with the virus proteins and the virus starts to bud out of the cell

The virus exploits the cells ESCRT proteins to transport for release of virus particles
- Endosomal sorting complexes required for transport

43
Q

Virus Maturation:

A

After budding the virus needs to mature

Maturation happens after the virus particle has regressed from the cell

The RT and IN are not functional at this point (immature)
- they are still a part of gag-pol poly protein, needs to be cleaved

As the virus buds the protein undergo a conformational change
- The PROTEASE in the gag and gag-pol polyproteins become active and starts to cleave the into the individual proteins

The protein assembled within the final structure

Reassemble occurs within the envelope

NC binds to the RNA, MA coat the inside of the envelope, CA forms the core structure

44
Q

Anti-HIV strategies

A

Antiviral medications present a difficult challenge for the pharmaceutical industry

Antibiotics target structures of processes in the bacterial cells that are different or not present in eukaryotic cells
- Not suitable strategy

Viruses are exploiting the eukaryotic cell for their replication
-Something that inhibits the cellular RNA polymerase or ribosome would not be suitable

So need to find virus-specific enzymes that can be inhibited - this can be very difficult to find

HIV is very stealthy - it integrates into host cell: Hard to separate

Virus specific enzymes

  • RDRP - polio, influenza
  • RT (RDDP)
  • Integrase - HIC
  • Protease - HIV
  • Neuraminidase - HIC

Preventing viruses from entering the cells would be best, can be done with antibodies generated in vaccines or with drugs that block the virus binding site

45
Q

Nucleoside Analogs

A

Converted to nucleotide analogs by the cell by the addition of a phosphate group

Molecules that resembles a nucleotide in that it can be added to a growing nucleic acid chain, but it lacks the 3’OH end so no other nucleotide can be added
They act as a chain inhibitor and stop viral DNA pol

Different compounds may bind to the RT enzyme and stop it from working

(need Oh to attach to phosphate group_

46
Q

Reverse transcriptase inhibitors

A

RT inhibitors stops the virus from making DNA copy that is integrated into the host cell’s DNA after entry into the cell

micb 202 (19 decks)

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