Lecture 16

There are three major genes produced by the retroviruses - what are they are how are the translated?

Each is each translated as a polyprotein and cleaved into mature proteins. gag: nucleocapsid proteins pol: reverse transcriptase, protease, and integrase env: envelope proteins

RT can use what templates?

Either RNA OR DNA.

What can RT use as a template?

Essentially, this enzyme can use either the RNA or the DNA and you have to get the dsDNA.

How are the mRNAs of theretrovirus transcribed?

mRNAs transcribed from integrated provirus DNA by host RNA polymerase II.

Do our bodies use reverse transcription?

No. Our body doesn't have an enzyme that can take RNA and make DNA. We have always believed that we go from DNA to RNA to protein. But viruses have introduced this RNA reversing back to DNA. So that is what makes this so unique.

What are some of the retroviral pol proteins? (2)

Pol protein encodes enzymes: PR (protease) RT (Reverse Transcriptase which has both DNA polymerase and RNase H activities) IN (Integrase)

What do most HIV drugs target at this time? (2)

Protease and integrase. * Anywhere in the life cycle, you want to think about drugs being developed but there are a lot of problems with T cells being the main target.

How is the integrated viral RNA transcribed?

RNA polymerase II of the host cell transcribes the integrated viral DNA genome.

HIV-1 has a huge amount of genetic variants. What are the main factors governing HIV-1 genetic diversity? (4)

Very high replication rate (~10^10 new viruses/day). Very high mutation rate, low fidelity of RT (1:2000-1:5000). Very high recombination frequency (2-30 events/replication),~10% of circulating strains are the result of inter-subtype recombination. Strong and persistent selective pressure from the immune system. * This supports to why we don't have a vaccination yet - there are just so many variants.

Describe the integration process of HIV. (3)

Viral integrase binds to the 2 ends of linear viral DNA close to host DNA Integrase removes the 2 3' terminal nucleotides Cleavage of 2 host phosphodiester linkages for ligation to 3' viral ends. * Now that you have that dsDNA, it can go thorugh the nuclear pore in the host cell and essentially what happens is the integrase protein that the virion carried into the cell is in the cytoplasm and is associated with the dsDNA and keeps it in a loop. Then it goes through the nuclear pore and the integrase allows it to essentially integrate into the chromosome through this double stranding nicking and recombination event.

What region of the host genome controls transcription initiation?

U3 region contains transcriptional enhancers that interact with cellular transcription factors and determine transcription initiation

What happens directly upon HIV entry into the host cell?

Upon entry into the cell, the RNA genome is reverse transcribed to ds proviral DNA within the viral nucleocapsid.

Is the replication cycle of the retrovirus similar to other viruses?

Yes - it is actually really normal, apart from the reverse transcription that happens and the incorporation into the host chromosome.

Why does the retrovirus have to make the ds DNA?

You have to because that is what can integrate into the chromosome.

After integration: Synthesis of viral mRNA happens by...

cellular RNA polymerase II uses the integrated provirus as a template to make genomic mRNA.

The issue that you will find when we start looking at all the HIV viruses is there is a ton of variation if you sequence them all. The reason for that is because...

the enzyme RT leads to really high mutation rates.

Gag and Pol proteins made on ___________ mRNA.

unspliced

HIV has only 10 kb with ~ 9-15 gene products but can affect ~1500 different normal cellular functions. This supports just how much _______________ there is in the HIV.

variation * Remember there is just so much mutation that goes on here. The genome was actually really small! So there are 1500 different cellular functions that are changed just because you are infected with HIV. It changes who you are.

Acute infection you still have CD4 T cells. As the CD4 T cells start to decrease this is called....

clinical latency. * Viral RNA is pretty low here because it is just hiding out in latency. Once it establishes this latency - the virus isn't actively replicated - but it is integrated into the cell. So the viral load is low. Some of it might be replicating but it depends what T cells are active in your body. Note: Acutely, the viral RNA is high at the beginning but you may not know that you are infected because you don't have any symptoms because you have high T cells. But you can test positive.

Pol protein encodes all of the...

enzymes.

Does the RT have good proofreading?

No - it makes a lot of mistakes and leads to high mutation rates.

KNOW THAT THE ENZYME CAN USE BOTH RNA AND DNA AS A TEMPLATE AND IN GET DS DNA FROM THAT ONE ORIGINAL RNA STRAND.

GOT IT? GOOD.

What is a major example of a retrovirus?

HIV.

Describe the integrase function.

Integrates retroviral DNA into host genome via endonuclease activity. * There are drugs being developed to this. If we can stop the integration into the host genome, that would be an important tool.

What is the genome of the retroviruses?

Linear ss RNA, positive sense.

Once the conformational change has occurred and the seoncdary binding of chemokein receptors has occurred, what happens next?

The HIV virion fuses with the hot cell.

What are the two major challenges with HIV-1 therapy? (2)

Viral latency - we don't know how to target the latent T cell population. The latency is a problem because when we think about our immune cells that are being targeted, the memory cells that are so important are harboring the HIV for us and not allowing it to be targeted by the drugs. SO this is a really big problem. Genetic variation and resistance - how can we possibly create a vaccine to that - it is impossible. Think about the flu .The flu doesn't have this many variants even! It only has HA and NA. So can we really create a vaccine to HIV?

How is reverse transcription now manipulated by us?

We use it on our labs to study the RNA of things! So we isolate the RNA, we use reverse transcriptase, make cDNA and then we can do PCR to look at various levels of the transcript that we are interested in - mostly everyone that does molecular biology uses reverse transcriptase. Before we ran these southern blots - this is a lot more quantitative.

When is it considered AIDS?

When you don't have any CD4T cells left. * The viral RNA spikes at this point.

Is the retrovirus enveloped?

Yes - remember because it envelopes out with all the proteins and RNA it uses before even developing the virion. * This initiates the initial binding to T cells.

Binding and entry: Retroviruses enter cells by the _________ pathway.

fusion

Even if you study anything to do with HIV literature - you'll always come across these coding areas - gag, pol and env. Each of these encodes for ______________ products.

multiple

The activity of RT results in the production of _____________ ______ which is found in a _____________________ complex.

proviral DNA preintegration

What are the main reasons for why we don't have an HIV vaccine yet? (9)

1. Extensive viral calde and sequnec diversity. 2. Early esabtlishment of latent viral reservoirs. 3. There are holes in our understanding of immunology - if we don't understand the memory totally then how are we going to work with these? 4. Viral evasion of humoral and cellular immune responses. 5. Ab responses typically type special and we don't know how to make a vaccine to all these different clades. 6. No method exists to elicit broadly reactive neutralizing Abs. 7. Attenuated viruses unsafe for human use. And if it's always subunit then we always have that problem of genetic mutation. 8. Lack of small animal model. We have monkeys but that isn't even that great because some monkeys survive HIV and we don't know why. 9. Little pharmaceutical interest. There is already HAART and it is bringing in money. It takes someone like Bill and Melinda Gates because it's not all about the money - they want to spend all their money before they die. No one was studying HIV before Bill and Melinda Gates.

RT has has efficient extension from ___' ___________ primers so it can just keep on going forward allowing it be mutate a lot.

3' mispaired

Reverse transcriptase produces what?

A DNA copy of the RNA genome.

What allows binding to specific chemokine receptors?

A conformational change.

The first time RT makes the first strand from RNA, it makes what?

A ss DNA.

What are quasispecies?

A viral quasispecies is a group of viruses related by a similar mutation or mutations, competing within a highly mutagenic environment.

In humans, what disease are associated with retroviruses? (2)

Acquired immune deficiency syndrome (AIDS) (a major global pandemic today (more than 33 million people infected). A variety of cancers in humans, monkeys, mice, cats, sheep, birds, etc.

Step by step, what is the complete retrovirus replication cycle?

Attachment of the virion to a specific cell surface receptor Penetration of the virion core into the cell SPECIFIC TO HIV: Reverse transcription within the core structure to copy the genome RNA into DNA Transit of the DNA to the nucleus Integration of the viral DNA into random sites in cellular DNA to form the provirus Synthesis of viral RNA by cellular RNA polymerase II using the integrated provirus as a template. This is read like any other gene would be read - this is a random integration so you don't know where it is going to integrate. Processing of the transcripts to genome and mRNAs Synthesis of virion proteins Proteolytic processing of capsid proteins and assembly and budding of virions.

What is interesting about SIV that makes us really curious to study it?

But SIV doesn't cause disease in all monkeys - we really want to understand this because what can their immune system do to survive this?

Once the DNA that was produced by the reverse transcriptase is integrated into the host chromosome, then how is the mRNA produced?

By the host RNA polymerase. * That is done like any of the host genetic material.

HIV-1 infects and kills primarily what cells? What is the result of this infection?

CD4+ T cells resulting in severe immune deficiency for >90% of people infected.

What is the use of current HIV therapy?

Current drug therapies prevent disease progression but do not eliminate latently infected cells.

Describe protease function.

Cuts Gag/Pol polyprotein into mature proteins after assembly. * Major class of anti-HIV drugs are Protease Inhibitors.

What are some of the retroviral env proteins? (3)

Env protein encodes: SU surface glycoprotein TM transmembrane protein

How does AIDS develop?

From HIV which replicates in and kills T lymphocytes and macrophages. Note: There are some AIDS denialism people that think that HIV does not cause AIDS. There is no evidence for that - all the evidence says that HIV depletes CD4 T cells and leads to aids.

What are the two mRNAs that are made by the viral genome in transcription?

Full length mRNA serves as mRNA for GAG and POL A fraction is spliced removing GAG/POL reading frames and serves as mRNA for ENV. * The gag and pol get made into a polyprotein. A splicing event splices out the gag and pol so that you get env by itself. So the differential splicing gives rise to different polyproteins.

What are some of the retroviral gag proteins? (3)

Gag protein proteolytically processed into: MA (matrix) CA (capsid) NC (nucleocapsid)

What is the current therapeutic strategy we have for HIV infected individuals and what is it's goal?

HAART (Highly-Active Anti-Retroviral Therapy). Goal: To keep a low viral load by controlling replication. It doesn't erradicate the virus since can't eliminate the latent virus reservoirs.

What is the typical course of an untreated HIV-1 infection?

HIV-1 infection results in depletion of CD4+ T cells, rendering the host immune-incompetent. As a result, opportunistic infections by other pathogens are often fatal. * Entry into the host, localized replication at the site of infection and release of the virus into circulation. The immune clear the virus from peripheral circulation but it remains in the lymph nodes and continues to replicate and destroy the lymph. When it gets rereleased you have no immune response and will die from infections.

Understanding the HIV replication and life cycle, why does it make sense that it could lead to cancer?

If it is inserted by a promoter or enhancer, it'll be transcribed just like any other cellular gene would depending on what is regulating it. So this has the potential to cause cancer because what if it integrates into something that is lethal or right before the cell cycle.

What is the significance of proteases in research?

If we can block these then you can't form the mature visions - so then you wouldn't be shedding anymore virions.

Inside even any one individual, there is the intra-pateient evoluation. Explain. (6) There are etreme diversification of viral sequences (high mutation and recombination rates) from the time of infection to the development in AIDS. Give examples.

Immune escape from Ab or CTL. Changes in replication capacity. Resistance to anti-retroviral drugs. Changes in viral tropism (different types of T cells) Changes in cytopathicity. Deterioration of immune competence.

What does the integration of proviral DNA allow?

It becomes a part of the host spreading through cell division and to offspring and can not be "cured".

Why can't the HAART therapy cure HIV?

It can't cure because we don't know how to target the latent pool of viruses that is because we have memory T cells that hide in your body until they need to be activated so all those memory T cells that might have HIV integrated into their chromosome, drugs aren't going to target them because they are not activated. Only your T cells that are activated with the virus in their chromosome are actively replicating HIV. HAART targets those pools of virus and will keep it low.

What is unique to the retrovirus virion? (3)

It contains the reverse transcriptase, this enzyme that we haven't seen before. Two identical genomes packaged in virions. 5' cap, 3 poly(A) tail (that capping and polyA tail doesn't' usually happen in prokaryotes, so they make themselves look like the host and so they can avoid host defense).

As soon as the nucleocapsid is released into the host cell by fusion, what happens?

It is broken down by cellular proteases and the ss RNA is released. Then the RT activity on the ssRNA is totally independent of the nucleocapsid. The dsDNA is then bound to integrase and transported to the nucleus where the dsDNA is integrated into the host chromosome.

What is the major disadvantage to RT's high mutation rate and cause of mutations?

It makes it really challenging to produce a vaccine.

What two things make curing HIV infection really difficult?

It's high mutation rates and the latency of infection.

The RT mutations rates are due to the lacking of what enzyme activity?

Lack of 3'-5' exonuclease proofreading activity. Normally we have this proofreading system and it make a mistake and goes back and repairs it. This enzyme doesn't have that activity.

There are a number of different retrovirus genera. Give two examples.

Lentiviruses and delaretroviruses.

RT is a dimer that has what two activities?

RNA-dependent DNA polymerase activity and ribonuclease H activity (Nuclease specific for RNA in RNA:DNA hybrids)

Before integration: Virus uses __________ to produce the DNA from RNA.

RT

What are the problems with HAART (Highly-Active Anti-Retroviral Therapy)? (4)

Resistance Cost (it's really expensive). Side effects (it's really toxic). Availability

What does the retrovirus carry within it's virion?

Retroviruses package two identical copies of a positive-strand RNA genome along with the viral enzymes reverse transcriptase, integrase, and protease in an enveloped virion.

Retroviruses have a unique replication cycle based on what two major things?

Reverse transcription and integration of their genomes.

What are some of the important proteins that the HIV virion is carrying with it?

So env is the transmembrane proteins and the surface proteins, there is the polymerase that is the RT. There are other enzymes like protease - really critical for the life cycle virus - an active target to control HIV by drugs. Integrase allows it to integrate into our chromosomes. There's others like the capsid protein and matrix protein.

Describe the overall life cycle of HIV as an example of retroviruses.

So you get fusion, you have have the CD4 receptor. Once that binds, it facilitates the second receptors (a chemokine receptor) that allows for fusion and release of the nucleocapsid and then you get disintegration of the capsid proteins to release the viral RNA. The RNA connects with RT and the RT makes the dsDNA. The dsDNA integrates into the host DNA facilitated by integrase. This can be transcribed, all the products are translated, and new viral RNA then associates with the envelope proteins that is within the plasma membrane. So they are produced, exported and then they form these cluster. When the virus is replicating, it can create its virions and that required the protease that the virus carries. Basically, the clusters will have all the polypeptides then the proteases cleave the polypeptide to release the proteins needed to form the capsid.

What do we know of how monkeys are able to survive the HIV infection and not die from AIDS?

Somehow the monkeys control the T cell responses, they always have a reservoir of T cells. They carry it with them but they are not going to die - like a cold sore. They can control heir T cells - the T cells never get depleted. We don't know how that works yet. We just know that this is an active area of research to try to figure this out.

What are deltaretroviruses known to cause?

T cell leukaemia.

Describe the translation of the Gag and Gag/Pol polyproteins. (4)

Termination codon separates Gag and Pol Ribosome occasionally shifts reading frame one nucleotide and resumes translation in a new frame Termination UAA stop codon is no longer recognized and ribosome reads through Result is the generation of Gag/Pol.

What is the baltimore classification of retroviruses?

The are sRNA positive sense. They have a viral RNA-dependent DNA polymerase (reverse transcriptase) that makes ss DNA, then ss/dsDNA and finally dsDNA.

Which of the major products are made together and which are not?

The gag and pol are made together and the env protein is made in the splicing event.

The second time RT makes the second strand, it makes what?

The other ss DNA (called complimentary DNA) to make double stranded DNA. * It uses it's first ss DNA as a template. It starts using RNA then uses the single copy DNA to make the cDNA strand to get dsDNA.

What is protease required for?

The release of the mature virus.

What is the purpose of having the alternative reading frames?

This allows gag to be produced alone sometimes - and ensures that there are more gag structural proteins and less pol enzymes. * The termination codon separates the gag and pol. So you can get gag by itself or you can get the gag/pol. If it reads through the stop codon, you'll no longer recognize it because it skipped over. The idea is that this would ensure more gag structural proteins. You only needs a few enzymes so this way you get more gag than gag/pol.

What part of HIV activity makes it so dangerous?

This also allows the integration to happen - that is why it happens to be so dangerous because cells that they infect, T cells, they are not always actively proliferating - there are a whole bunch of them that are memory -so they are latent and so that is why it is really important for how IV stays around for so long and because they are just integrated into those cells.

What is HAART made of?

This is a cocktail of drugs - the fusion inhibitors, protease inhibitors and an inhibitor of the RT.

HIV is highly related to Lentivirus from non-human primates. What is unique in studying this evolution?

This is interesting because they don't get AIDS! Can we figure this out? Will this help us understand how we could not get sick as well? Because they do get infected.

Describe retroviral entry into the cells.

The retroviruses enter by fusion. Receptors include: CD4 and the chemokine receptors CCR5 (which defines the T helper subset of cells) and CXCR4 (for HIV-1). So first the hIV binds the CD4. When that interaction happens, you get a conformational change and that change pulls the virus and the cell membrane together which then enables a second receptor to bind - that is CCR5 or CXCR4. Essentially then, that pulls the virus and membrane completely together for fusion of the viral envelope and the host cell. So then what happens is that the nucleocapsid gets put inside the cytoplasm and all the proteins disintegrate through proteases and such.

From translation of HIV, what are the three MAJOR products that you get?

The three major products that you get are gag, pol and env.

Genetic diversity has killed every aspect of therapeutic strategy. Thus what is the effect that therapy has?

Therapy can keep viral load lower but it cannot cure the disease.

Explain the recombination frequency of the retroviruses.

There are two genomes in each virus. So we don't consider this like segments because they are identical. It's not like the orthomyxoviruses where the segments are unique and can be reassorted. But you can still get heterodimeric progeny - so that is another factor as to why there is so much variation.

What are the lentivurses known for?

These are retrovirses that lead to the examples of HIV (type 1) that causes and infects humans leading to AIDS. There is something really similar in monkeys - it is an immunodeficiency and we think it that HIV probably developed from it (it is called simian immunodeficiency virus).

When researchers observed the distribution of HIV-1 genetic clades what did they see?

They noticed that there are different clades in different parts of the world so the environment might play a role.

In the transcription of the viral genome, how many mRNAs are made and how do they arise?

differential splicing generates 2 mRNAs

Viral RNA is converted into what by reverse transcription?

double-stranded DNA copy

Retroviruses express three sets of genes. They are...

gag (capsid proteins), pol (viral enzymes), and env (envelope proteins).

HIV-1 RT has a really _______ incorporation error rate.

high * This depicts that you get a really high error rate because of this enzyme - that is the fidelity - it makes mistakes. There are 5-10 mutations per genome per replication cycle.

Proviral DNA is imported into the nucleus and ____________ into the host cell chromosome at __________ sites.

integrated random

Lack of proofreading capability leads to high mutation rate and generation of ____________.

quasispecies

A copy of proviral DNA is integrated into the cellular genome at a ____________ site.

random

Viral RNA is converted into a double-stranded DNA copy by ____________ ______________.

reverse transcription

Retroviruses have a unique replication cycle based on two things...

reverse transcription and integration of their genome * This discovery of the RT enzyme shattered the central dogma of molecular biology which stated the flow of genetic information was from DNA to RNA.

RNA converted to DNA by...

reverse transcription.

Env proteins made on _________ mRNA

spliced

There are additional regulatory genes expressed on _____________ mRNAs.

spliced

Gag and Gag/Pol polyproteins are made by... (2)

suppression of termination and use of alternative reading frames.

Retrovirus infect certain species and cells based on the ___________ ___________.

surface protein *it needs that CD4 receptor and that is why it really affects the T cell population. * HIV Gp120 binds to the CD4 molecule present on T cells.

Transcription of the viral integrated DNA depends on...

the appropriate set of transcription factors as normal cellular genes do. * It is regulated with all the other host material, so transcription is really complicated and we don't fully understand but it does use the TFs just like the cellular genes do.

Viral proteins derived from the gag (group-specific antigen), pol (polymerase), and env (envelope proteins) genes are incorporated in ___________.

virions * The virus carries all of these with it in the virion.