Retroviruses: Oncogenesis, endogenous retroviruses, retroviral vectors

What was Rous Sarcoma Virus (RSV) first discovered in? What does it cause?

In birds and it causes sarcoma (connective tissue tumour) in chickens.

What did Vilhelm Ellermann and Olaf Bang show?

That chicken leukosis was caused by a virus later called avian leukosis virus (ALV)

What did Peyton Rous report?

The cell-free transmission of a chicken sarcoma which was the first time that a cancer could be transmitted (it is the virus transmitting the cancer). The virus is now called Rous sarcoma virus after Peyton Rous

Describe how Peyton Rous discovered Rous sarcoma virus

He homogenized the tumour from the chicken and then filtered the juice so it would not permit any bacterial cells to go through (only the smallest viral particles could go through) and used this innoculum to inject into a healthy chicken and it caused sarcoma

What are oncogenes? What were they first discovered in?

They are genes that if mutated will drive the formation of tumours. They were first discovered in viruses.

What was the cellular gene SRC first found in? What is the function?

First found in Rous sarcoma virus (v-SRC here). Function is non-receptor tyrosine kinase

Where is v-Src found in the Rous sarcoma virus? What does it do?

It is found downstream of Env and it drives DNA replication and expression so you have a lot of recipient cells.

How did v-Src get into the rous sarcoma virus genome? Is it present in avian leukosis virus?

Random recombinations of DNA and RNA happen sometimes and if beneficial the virus will keep it. In this case, v-Src was beneficial so it was kept. It is not present in ALV.

Where does v-Ras come from? How many versions of Ras do humans have? What percentage of different cancers have these Ras genes? What is the function of Ras?

Ras jumped out of the rat genome into the virus (Harvey rat sarcoma virus). 3 versions (H, K, and NRas). 30%. Small GTPase is the function.

What are two other cellular genes that are oncogenes? What is the corresponding viral gene? What is the function?

1. MYC. v-Myc (Avian myelocytomatosis virus). Function is transcription factor

2. FOS. v-Fos (FBJ murine osteosarcoma virus). Function is transcription factor

What did David Baltimore and Howard Teeming independently discover? What was discovered a year later?

Discovered an enzyme that could convert RSV viral RNA into DNA (RT). A year later RNase H activity was discovered.

What was the first human retrovirus to be discovered? What was discovered shortly after?

Human T-cell leukaemia virus 1 (HTLV-1). HIV was discovered shortly after

What genus does HIV belong to? Are they complex or simple? What genus does HTLV-1 belong to? Are they complex or simple? Are they found on different branches in the phylogenetic tree?

HIV- Lentiviruses and complex

HTLV-1- Delta retroviruses and complex

Yes as they belong to different genera

Is RSV and ALV similar to one another? Why or why not?

Yes they are and therefore found on the same branch (alpha-retroviruses) as they are both simple retroviruses. They are homologous except for v-Src in RSV.

What does HIV infect? What does alternative splicing produce? How many accessory proteins for HIV? What does infection with HIV destroy?

Infects CD4+ T cells

Produces 6 sgmRNAs

4 accessory proteins

Infection destroys CD4+ T cells leading to immunodeficiency

What does Tat and Rev do in HIV?

Tat- upregulated provirus transcription

Rev- regulates nuclear export of unspliced genomic RNA

What does HTLV-1 infect? How many sgmRNAs are produced from alternative splicing? How many accessory proteins?

Infects CD4+ T cells

Produces 9 sgmRNAs

6 accessory proteins

What does Tax and Rex do in HTLV-1? Are they functional analogs of Tat and Rev (are regulatory proteins)? Are Tax and Rex produced from the same sgmRNA?

Tax is a potent oncoprotein

Rex promotes nuclear export of genomic RNA

Yes they are. Yes they are through leaking scanning on the same sgmRNA

How is HTLV-1 basic zipper protein made (HBZ)?

It is produced from an inverted transcript. They have a different promotor at the 3’ LTR that makes a backwards mRNA (host RNA pol II will go in the opposite direction to the other transcripts)

What is HBZ important for?

It is an important oncogene

Does infection with HTLV-1 always lead to cancer? Do most adult T-cell leukaemia cells express HBZ or Tax?

No not always and most express HBZ and not tax

What are two human endogenous retroviruses that we have all the time? What genera do they belong to and are they simple or complex?

HERV-K (Beta-retroviruses and simple) and HERV-W (Gamma-retroviruses and simple)

What does the K and W mean for HERV-K and W?

It means lysine tRNA is associated with the genome (HERV-K)

It means tryptophan tRNA is associated with the genome (HERV-W)

What do retroviruses do?

They integrate into the chromosome and their genome becomes part of the chromosome of the host forever (or until the cell dies)

What do endogenous retroviruses arise from?

Integration of provirus into germline cells (egg and sperm)

Since the retrovirus is now in the germline cells, what happens?

Every time a sperm or egg is made the retrovirus genome is within because they integrated into this genome. All descendants of that infected organism will carry the same integrated provirus. Vertical transmission is occurring here.

What percentage of the human genome is retrovirus? How many nucleotides does our genome encode retrovirus?

8% retrovirus and every 1 in 10 nucleotide in our genome encodes for retrovirus

Are many copies of the endogenous retrovirus fully or partially inactivated? What does this mean?

Yes they are and this means that they don’t produce viral particles and can’t infect anything

Are some copies of endogenous retrovirus fully functional? What does this mean?

Yes and this means that they can be activated by different things and are in the blood (HERV-W and HERV-K). They can disrupt or activate host genes since they are integrating into host genome.

What is exaptation?

It is utilization of retroviral gene by the host

What is an example of exaptation?

ERVW-1 retroviral Env gene is used by the host as human syncytin-1 (has evolved to produce syncytin-1).

What is syncytin-1 used for? If we didn’t have retroviral Env from ERVW-1, would this be able to occur?

It drives fusion of trophoblasts to form the multi-nucleate syncytio-trophoblast layer in placenta. No placenta would not be able to perform this function without it. No trophoblast layer fusion would take place in the placenta which is important for human development

Why does it make sense that evolution exapated ERVW-1 retroviral Env function?

This is because glycoproteins of viral envelopes evolve to be very efficient at finding a membrane and fusing them together

Do different retroviruses in different mammals cause placenta fusion?

Yes they do

What are other functions of having endogenous retroviruses?

1. Regulates innate immunity because if a lot of them are being made then it sets off an alarm

2. Prevent other retroviruses from infecting us

Are mistakes that are made during replication selected against for endogenous retroviruses? What does this mean?

No they are not and this means that some have Gag, Pro-Pol, and no env. While some have just Env or for the most part they just have random LTR sequences and but also promotors that regulate gene expression.

Why can endogenous retroviruses make many mistakes during replication without it causing a problem?

This is because they are already integrated and don’t need to infect anything else and they aren’t making viral particles anymore

Describe what retroviral vectors are

You can gut retrovirus genomes to just their regulatory elements (LTR, PBS, packaging signal, and sometimes RRE) for packaging and expression but don’t have any retroviral genes. There is room to insert anything we want (trans gene) since Gag, Pol, and Env have been removed and now it is a vector

What happens in retrovirus transduction (when retroviral vector is used)

It will be able to transduce any actively dividing cell. Also instead of making structural proteins from translation, it will make proteins that we want to make. It also still makes a viral particle with with 2 pieces of RNA

How many packaging vectors are needed? Explain what they are

3 packaging vectors as one packaging vector will provide Gag and Pol with Pro attached to one. The second one will provide Env and the third will be our retroviral vector with the trans gene that we want to make. The first two vectors will be used to make mRNAs that are translated to make proteins for packaging. However, the mRNAs for Gag, Pol, and Env will not be packaged.

What vectors are most commonly used? Is this a type of retroviral vector?

Lentiviral vectors and yes it is

Do packaging constructs need to have LTR or packaging signal?

No they do not. Just need to have a promotor (usually CMV that drives expression)

Why is RRE sometimes used in a lentiviral vector?

It will boost production of that lentivirus if rev is also expressed

Why do we encode Env separately?

This is because it gives us flexibility to choose what type of envelope we want to put on it. It is also so we can make a pseudo virus.

What is the most popular Env that is used for packaging constructs?

The env from VSV because it has a broad tropism (can infect any cell) and we want this