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MICI 2100 > Lec 13-Genomes And Antiviral > Flashcards

Lec 13-Genomes And Antiviral Flashcards

1
Q

Speed of bacteriophage replication:

A

Fast

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2
Q

Steps of bacteriophage infection

A
  1. Viral DNA delivered into host E. coli
  2. Host gene expression is arrested, causes degradation and inhibits protein synthesis
  3. Enzyme synthesis after 5 mins
  4. DNA replication after 10 mins, where virus replicates its genome
  5. Formation of new virus particles after 12 mins
  6. Infection ends, bacteriophage triggers lysis of host and release of viral particles after 30 mins
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3
Q

Do bacteria have defence?

A

Yes

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4
Q

2 forms of antiviral defence:

A

Restriction endonucleases and CRISPR

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5
Q

What are restriction endonucleases?

A

Enzymes that bacteria have readily available to defend against viral infection. They cut dsDNA at specific nucleotide sequences called restriction sites (palindromic DNA), and cut up foreign viral DNA

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6
Q

Can DNA prevent endonuclease action?

A

Yes, host DNA is methylated by a host cell enzyme, which prevents cutting by host restriction endonuclease

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7
Q

How do restriction endonucleases cut DNA?

A

Makes 2 incisions at sugar phosphate backbone

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8
Q

What do names of nucleases reveal in restriction endonucleases?

A

Reveal source bacterium it came from (eg. Eco RI is from E. coli)

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9
Q

What is cut and paste gene cloning?

A

If gene of interest has restriction sites on both sides, get bacterial plasmid that has those sites too. Restriction enzymes cut gene of interest and open up bacterial plasmid. Ligation occurs using DNA ligase which combines the sticky ends the restriction enzymes have created. Creates one transformed plasmid

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10
Q

What does CRISPR stand for

A

Clustered regularly interspaced short palindromic repeats

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11
Q

What does CRISPR do?

A

Its a tool to edit genomes

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12
Q

Who made CRISPR

A

Doudna and Charpentier

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13
Q

Restriction endonucleases and CRISPR are ___

A

Complimentary

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14
Q

3 steps of RNA guided cleavage of foreign viral DNA

A
  1. Viral DNA is transcribed from CRISPR array on bacterial genome
  2. Loaded onto Cas9 nuclease (dsDNA cutting enzyme)
  3. RNA guides Cas9 enzyme to cut homologous incoming viral dsDNA
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15
Q

Do eukaryotes have restriction enzymes and CRISPR?

A

No

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16
Q

How do eukaryotes combat infection?

A
  • They have an immune sys, triggered by pattern recognition receptors (PRRs)
  • PRRs recognize foreign molec patterns
  • PRRs include molecs that recognize non-host nucelic acids, including viral dsRNA and 5’-triphosphate RNA
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17
Q

What are PAMPs?

A

There are multiple viral patterns (PAMPs) that eukaryotic PRRs can recognize, including nucleic acids and proteins

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18
Q

How do viruses make gene products (RNA and protein) and replicate their genomes?

A

Viruses depend on many parts of host machinery, so, they need to conform to machinery the host has (i.e. using host ribosomes for translation) or alter this machinery for their own use

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19
Q

What is the central dogma

A

DNA—>mRNA—>protein

20
Q

What does the work to make the dogma true?

A

Enzymes

21
Q

What is DNA dependent DNA polymerase?

A

Enzymes that replicate dsDNA into more dsDNA

22
Q

What is RNA polymerase?

A

Enzymes that replicate dsDNA to make (+)-sense RNA

23
Q

What does (+) or (-) sense mean?

A

(+) means ready to be translated, (-) means it is not

24
Q

What do ribosomes translate?

A

(+)-sense RNA into an AA chain

25
Q

Why do viruses often break the dogma?

A

Can copy RNA template into more RNA
Can copy RNA into DNA (reverse transcription eg. HIV does this)

26
Q

Viral genomes must make ____ that can be detected by ___

A

mRNA, host ribosomes

27
Q

All virus genomes are ___ or ____

A

DNA, RNA

28
Q

Baltimore classification of viral genomes

A

There are 7 types of viral genomes, each with a diverse composition. Many possible structures and tactics for encoding and decoding info in DNA/RNA

29
Q

What info is encoded in viral genomes?

A

Gene products (and regulatory signals required for rep of viral genome) and (+)-ssRNA

30
Q

Gene products are responsible for:

A
  • Assembly and packaging of genome into viral particles
  • Replication and timing of replication cycle
  • Defeating host defences
  • Spread virus to other cells and hosts
31
Q

Examples of gene products

A

RNA and protein

32
Q

What is (+)-ssRNA? Give an example

A
  • These viruses get directly translated in cytoplasm, are readily available
  • Generates polyproteins which get cleaved by either viral or host proteases into the viral proteins
  • In one movement, whole genome is turned into protein that gets chopped up into its parts
  • Eg. poliovirus: Express genes via direct translation in cytoplasm, replicate genomes by endorsing viral RNA-dep RNA pol (RdRp) that reads RNA and copies it into RNA product
33
Q

What would happen if you introduced (+)ssRNA polio in a lab and could introduce it into human cells?

A

You can make a virus!!:
Transcribe RNA in test tube, introduce it into cells, and create virus. You are experimentally bypassing PM to deliver intact (+)ssRNA and mimic uncoating. The whole thing needs to be translated and replication can begin.

34
Q

Steps of gene expression by a (+)-ssRNA genome in poliovirus:

A
  1. Genomic (+) RNA gets translated by host ribosomes to create long polyprotein
  2. Viral and host proteases create a cutting cascade that ultimately generates genome of all proteins that dictate polio infection
35
Q

Steps of gene expression from a (+)-ssRNA genome in Zika virus:

A
  1. Genomic (+) RNA gets translated by host ribosomes to create long polyprotein
  2. Polyprotein gets chopped up into capsid proteins by proteases
36
Q

Steps of gene expression from a (+)-ssRNA genome in Hepatitis C virus:

A
  1. Genomic (+) RNA gets translated by host ribosomes to create long polyprotein
  2. Polyprotein gets chopped up into capsid proteins by proteases
37
Q

Poliovirus gene expression from (+)-ssRNA outcome genome grouping:

A

Outcome genome is grouped by function, one else encodes structural/capsid proteins, the other encodes enzymes

38
Q

Autoproteolysis in poliovirus:

A

There are multiple viral proteases that cut the viral polyprotein as its being made (autoproteolysis). The protein folds as soon as it leaves ribosome, and folding leads to formation of protease sites that cleave the polyprotein

39
Q

Difference between polio and Zika gene expression:

A
  • Polio is icosahedral and Zika is enveloped, which means polio makes proteins in cytoplasm, Zika makes transmemb proteins in ER
  • Polyprotein is a bar shape in polio, but crosses ER membrane multiple times in Zika
  • A lot of folding and protease activity occurs in ER memb, bc a lot of these proteins end up in the envelope of the finished virus bc they were translated in the ER
40
Q

Difference between polio and hepatitis C:

A

Hepatitis C is an enveloped virus, translated in ER and spans its membrane many times like Zika does. Polio is icosahedral

41
Q

(+)RNA viruses are directly translated where? Processed by what into what?

A

Translated in cytoplasm, processed by proteases into their individual parts—enzymes and capsid proteins

42
Q

What is polyprotein processing?

A

It is a co-translational process that occurs from 5’ to 3’ end of genome. Folding happens as soon as any portion of protein exits ribosome, even as ribosome is still processing. Folding creates protease sites so it can also start cutting before ribosome is done

43
Q

What happens to (+)-ssRNA viruses that are translated in cytoplasm?

A

Translation generates polyproteins, which get cleaved by either viral or host proteases into the full complement of viral proteins

44
Q

Example of a strategy to interfere with virus replication?

A

Protease interference: Developing protease inhibitors, eg. paxlovin: viral protease inhibitor for SARS-CoV-2

45
Q

Why are proteases good drug targets?

A

The drugs against proteases have low toxicity because they are targeting viral enzymes and leaving host enzymes alone. We’re trying to develop a drug that will target only viral proteases and not host proteases

46
Q

Why is RdRp a good drug target?

A

We don’t have RdRp, only viruses do. Lots of room to develop RdRp inhibitor chemicals because we have nothing with the same specificity that would bind, so these would also be low toxicity drugs

47
Q

Can hepatitis C virus (HCV) be cured with antiviral drugs?

A

Yes

MICI 2100 (11 decks)

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