Module 6 - Magor (assembly + exit of viruses; Polio, RT virus + Herpes)

*****Recap the general infection → exit life cycle of POLIO

Polio = non-enveloped picornavirus (not RT); (+) RNA strand genome; 4 proteins x 60 = capsid

1. Virus attaches to PVR

2. Uncoating of the virus at the cell membrane

3. + Strand RNA virus = directly begin synthesis of polyprotein

4. Initial cleavage is autocatalyzed = releases proteases for further cleavage

5. When [capsid proteins] = high enough → auto assemble in a concerted assembly with RNA (based on chance)

*****Elaborate on Polio protein synthesis: when is the a.a. folded into proteins, Does cleavage occur BEFORE or AFTER folding?

Proteins are folded while still in the polypeptide

• Proteins are cleaved AFTER FOLDING by proteases (aka. cleavage occurs after all the steps shown in the image)

******Why does Folding occur BEFORE cleavage?

INTRAMOLECULAR INTERACTIONS (key element of protein folding) are favored in the intact polyprotein

********How does the polio virus make sure that an RNA genome gets in every virion? (red question)

Cleavage of VP0 → V2 + V4 requires the RNA to be inside

• V4 = needed on the inside of capsid to provide stability

• Only in the presence of RNA in the capsid, Cleavage of the VP4 off the polypeptide occurs so that it can stabilize the capsid

What is the idea behind this Polio vaccine

• what challenges does this type of vaccine present in terms of Assembly of the virus

Idea = synthetic vaccine → no genomic material = Virus free

• made in a way to look like the virus but has no way of ever replicating

Challenge: Polio vaccine requires the presence of the RNA genome inside the capsid for the cleavage of V0 → v2 + V4 and for V4 to be cleaved off in order for V4 to be able to stabilize the capsid + complete assembly

Need a way for the capsid to still package in the absence of RNA

****How was this done?

• What aspects of the virus did it change?

Preserve the immunogenic + antigenic characteristics while:

• Stabilization the determinants of the attenuation of the virus = editing the 5’ UTR

• Reduce evolvability of the virus = prevent reversion ← done by reducing evolvability

• Ultimately increase the genetic stability

****Moving on from Polio → RETROVIRUSES

• What is an example of a RT virus that we have already covered?

HIV

*****HIV (as a RT virus)

• is it enveloped or not

• What is packaged within the capsid

• What type of Genetic material does it have?

ENVELOPED + spike proteins

Within capsid = RTase, Integrase, Protease

Genetic material = + strand RNA

****Why does HIV package integrase + RTase in the virion despite being a + strand RNA virus? (red question)

Packaging it means that it doesn’t need to synth any proteins immediately after entering the cell = helps evade T-cell immunity? (something to be covered in the future?)

*****What does HIV use integrase + RTase for?

• Briefly outline how it replicates + transcribes itself (what machinery does it use; host or self)

HIV uses RTase to turn it’s +strand RNA into dsDNA → then uses integrase to integrate the dsDNA into the host genome

• It then uses HOST transcriptional machinery to make more copies

• it also locates to the nucleus thus can use the Alternate splicing of transcript mechanism of the host = increase variation + evolution

******Where is HIV + RNA strands made in the host?

Nucleus

****Where is HIV Proteins translated in the host?

1. Secreted or Transmembrane proteins = made on the rough ER

2. Other viral proteins eg. RTase, Protease + Integrase = translated on ribosomes in the Cytoplasm

True or false: HIV also assembles retroviral particles from a polyprotein precursor? like polio

TRUE

******What is the order in which proteins are made by HIV viruses in the host?

Made in order that they want to assemble them (genes are also assembled in this order)

• Early genes = Capsid proteins first (MA, CA + NC)

• Late genes last (PR, RT, IN)

*******What is the ratio of Late Gene proteins to early gene capsid proteins?

• WHY is this the case?

Only 1/10 transcripts encode RT → only 1/10 transcripts make it to the pol gene + get transcribed (processivity?)

• gag = gene region for early proteins (capsid)

• pol = gene region for late proteins (RT, IN + PR)

WHY? Virus needs 10x more capsid proteins to facilitate assembly than it needs RT, IN + PR protein/enzymes when packaging

• only need ½ of each late protein

******What are gp120 + gp41 again?

HIV surface proteins (Spike proteins)/ envelope glycoproteins that work together to mediate viral attachment, fusion, and entry into host cells

*****Where do the membrane proteins gp120 + gp41 get made?

On the rough ER b/c that they need to be transported to the host cell surface to become surface proteins for the Enveloped membrane of HIV

********What is the process of localization of viral Proteins to the cellular membrane? (like gp120 + gp41 of HIV)

1. Made on Rough ER → secreted into the ER lumen(secreted) or lumen(transmembrane protein)

2. Rough ER membrane + lumen containing these proteins bud off into the Golgi

3. Golgi buds off into → rough vesicles that fuse with the cell membrane

******Where does the assembly of enveloped viruses (Like HIV) frequently take place?

At the plasma membrane

• in order to facilitate budding off + taking host membrane (containing virus surface proteins) with it

****Moving on from RT virus (HIV) → DNA viruses

• What is an example of a DNA virus introduced in this lecture?

Herpesvirus

*****What type of Genetic material does Herpes virus have?

dsDNA

******What enzymes is requires for the Transcription of the Herpes virus genetic material into mRNA to code for viral proteins?

DNA-dependent RNA polymerase

******Do DNA viruses NEED to package a DNA-dependent RNA poly for transcription → mRNA?

NO = can use the HOST machinery BUT some DNA viruses synthesize their own

*******What is the general trend seen in DNA viruses that encode their own enzymes for replication + transcription instead of using host?

LARGER DNA viruses encode their own enzymes

******Does the HERPES virus encode it’s own Enzymes?

YES = ONLY DNA POLY → still uses host RNA poly

******Structure + genome organization of Herpes virus

• is it an enveloped or non enveloped virus

• What are the contents within the capsid

• Size of genome

ENVELOPED protein = contains Spike proteins

• Only packages LINEAR dsDNA within capsid

LARGE GENOME SIZE with large # of genes

*****Why does the Herpes virus have such a large genome?

It has 2 life cycles = need to encode genes that code for proteins that promote latency

• Active + latent (think phages in Genet 270)

****What are the 3 common characteristic of exponential viral DNA replication mechanism?

1. Template directed

2. Each strand is copied beginning at origins

3. uses a DNA dependent DNA poly

Same as in mammal DNA replication = semi-discontinuous (okazaki fragments) bi-directional replication (2 rep. forks)

******True or false in DNA viruses the replication of the DNA genome must occur until the end

TRUE

• The entire DNA genome must be replicated to the ends

****How many copies of DNA can be replicated per cell in a day (Herpes virus)

50 000 copies from one DNA template

• New DNA’s serve as template

******What condition must be met for the DNA virus to be able to replicate its DNA in the host?

• how does it address this issue?

Need generation of dNTPs

• solution = Tricks host to start cell replication process

******In what stage of the CELL CYCLE does generation of dNTPs for DNA synthesis only occur?

S phase

• thus virus needs to trick host cell into starting cell rep + begin the S phase

Primase?

Enzyme that synthesizes the RNA primer needed to provide the 3’OH for DNA replication

*******How does the Herpes virus address the end Replication problem?

ROLLING CIRCLE mechanism of DNA replication

***Give a simple break down of RCR + What is the end result?

end result = CONCATOMER

****Which method of packaging does the HERPES virus use to package the genome into the capsid?

HEADFUL packaging (think genet 270)

******What is the mechanism of Headful packaging in the case of the Herpes virus?

Direct repeats = mark the end of a genome + help package only one genome into the capsid

• proteins bind to the repeats

• Empty capsid binds to these proteins

• DNA is stuffed in

• Stuffing occurs until proteins on the other end are reached

• Cleavage of remaining concatemer of DNA

*******WHERE does viral DNA synthesis occur for HERPES?

In specialized compartments

• DNA templates + enzymes = sequestered into discrete sites in the NUCLEUS

• Product of one replication cycle becomes templates for the next? (many RCR at a time)

*****What is the difference in the DNA polymerases between host + viral?

• which one does herpes use again?

Host DNA poly has Proof reading during synthesis

Viral DNA poly may NOT proofread

• herpes uses VIRAL DNA poly (uses host RNA poly)

*****What is Acyclovir

Antiviral medicine that is converted to a product that competitively inhibits Herpes virus DNA poly

******HOW does Acyclovir Work?

Looks a lot like guanosine = incorporated into the DNA of virus during replication BUT it has no 3’ OH to continue DNA synth = Chain terminator

****There are 2 life cycles of the Herpes virus: What is the REPRODUCTIVE cycle?

• What are the first/early proteins vs. the late proteins

Early/first = transcriptional activator of genes

• these trancripts make proteins needed later in synthesis

Late = capsid assembly + membrane proteins

****There are 2 life cycles of the Herpes virus: What is the LATENT cycle?

• Where does this lifecycle of the Herpes virus occur

Transcripts are produced from parts of the genome to produce proteins required to maintain latency

• WHERE = primarily in the NEURONS (not normally attacked by immune system)

• Also think genome may be hiding in TELOMERES (not transcribed = easy to hide)