HIV - Reverse Transcriptase Flashcards

Question

Chemokine receptor: CXCR4 and CCR5

Answer 1

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

Answer 2

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

Answer 3

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

Answer 4

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

Answer 5

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 )

Answer 6

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

Answer 7

- 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?

Answer 8

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

Answer 9

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

Answer 10

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

Answer 11

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

Answer 12

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

Answer 13

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

Answer 14

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

Answer 15

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

Answer 16

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)

Answer 17

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)

Answer 18

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

Answer 19

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

Answer 20

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

Answer 21

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_

Answer 22

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