Lec 14-Genomes And Antiviral II Flashcards
What does (+)-ssRNA virus genome do?
Mimics host mRNA and is directly translated into cytoplasm to make polyproteins. Viruses are always finding ways to mimic mRNA, just not always with a cap and poly-A tail
What do enveloped (+)-ssRNA viruses usually make? Where?
Make polyproteins at ER with many transmembrane domains
What do host and viral proteases do?
- Cut up long polyproteins into indiv proteins. Viral proteases typically cut themselves out
- Make RdRp’s
What do RdRp’s do?
Transcribe the gene to replicate it and make more RNA products such as (-)ssRNA intermediates and(+)ssRNA genomes. Viruses cannot have host enzymes do this, they have to make new genomes themselves
What does gene expression of (+)ssRNA on a zika virus translate?
The polyprotein translated is polymerase
What does (+)ssRNA make?
(-)ssRNA intermediate
What does (-)ssRNA intermediate make?
(+)ssRNA progeny genome
(+)ssRNA viruses will make ____ strands, used as template to make ___ strands
-, +
Polymerase reads ___ to make ___ or ___ to make ___
-, +, +, -
**will compliment any strand with its antiparallel strand
What is Ribavirin?
An antiviral drug
What are nucleoside analogs?
Many antiviral drugs are nucleoside analogs, meaning they resemble resemble a nucleoside
What virus does Ribavirin target?
Lots of them
Ribavirin is an inactive pro-drug. What does this mean?
Not activated until it is metabolized—is activated by phosphorylation
Incorporation of Ribavirin into RNA
Incorporated into RNA during genome replication:
- Ribavirin is chemically different enough that it loosens strict pairings of A-T and C-G
- Ribavirin is analogous enough to A and G both that it can bind to U or C
- Induces hypermutation in viruses that depend on RdRp
- This can be lethal to RNA viruses
Lethal hypermutation…what exactly happens?
Eg. (+)ssRNA original sequence
(-)ssRNA has R (ribavirin) replacing both A’s and G’s
(+)ssRNA doesnt know whether to bind C or U to R,
resulting in 2^n possible outcomes
How is hepatitis C transmitted?
Via blood
What does hepatitis C cause?
Hepatitis (liver disease) and liver cancer. People with chronic hepatitis C may need a liver transplant
Can hepatitis C be CURED?
Yes, with new anti-HCV drugs
How is HCV transmitted?
It is hard to transmit. Done through unprotected sex, blood/bodily fluid exposure, IV drug use
Can HCV be passed to animals?
No
What does Dr Lisa Barrett do?
Leads HCV elim in PEI and NS
What does Sofosbuvir do in HCV?
Sofosbuvir inhibits RdRp, resulting in no viral RNA synthesis. It is a prodrug for hep C, is metabolized into a nucleotide monophosphate to get phosphorylated and become an active triphosphate. This will be incorporated into the growing hep C molec, causing chain termination (stops the growing chain bc structure of ribose sugar in triphosphate inhibits elongation of new viral RNA)
Steps of coronavirus gene expression:
- Virus binds to host
- Attachment and endocytosis
- Release of viral genome into host cytoplasm. Has a (+)sense genome
- Engages with ribosome, starts making viral proteins through polyprotein synth
- Intermediate (-)sense genome is made
- Final (+)sense strand is made
What do polyproteins do in coronavirus gene expression?
Have 5’ cap. They dont go all the way to the end of the genome but they span most of it
Covid gene products:
Translation of 2 products:
1. ORF1A translates the shorter
2. ORF1A and ORF1B together translates the longer
- Covid makes their own proteases which are represented on both polyprotein products
- If it didnt make ORF1B, it couldn’t make polymerase as it is only encoded on 1B not 1A
Paxlovid on covid polyprotein:
Paxlovid targets a protease and prevents RdRp release. The protease should be cutting a lot of downstream things, but Paxlovid is encoded in the small, ORF1A produced polyprotein. This means that Paxlovid inhibits the entire second half of the protein
Why does processing with ORF1B result in longer polypeptide?
At stop codon on 1A, ribosome doesnt stop, instead slips into new reading frame to make another polypeptide
What causes an RNA pseudoknot?
Pseudoknot is a secondary structure just below reading site, makes ribosome pause to work through its complex structure
What does pausing at pseudoknot cause?
Ribosomal frameshifting: Ribosome slips while stalled, causing -1 slip that shifts reading frame
What viruses have ribosomal frameshifting?
Coronavirus, influenza, HIV
Why is ribosomal frameshifting beneficial to viruses?
Use this strategy to maximize potential of genome and make many types of proteins
Are ER-localized transmembrane coronavirus proteins in progeny?
No. They are nonstructural, meaning they aren’t in progeny
How many types of ER-localized transmembrane coronavirus proteins?
3
What enzyme do we need frameshifting to make?
RdRp, because it is only in the long polyprotein. So, we need to get past the stop codon using frameshifting in order to get to the 2nd half
What is Remdesivir?
Paxlovin is the first line of defence against coronavirus, remdesivir is the second. It inhibits RdRp causing chain termination and prevents viral RNA synthesis
What do ER-localized transmembrane coronavirus proteins do?
Sit in ER membrane, crossing it many times. Drive creation of viral factories in cytoplasm that are known as replication organelles. To do this, they steal ER membrane to make a compartment where viral RNA can be secretly replicated. We want this to be secret so immune system patter-recognition receptors dont recognize the foreign viral RNA products
What did the Dr Montserrat Barcena lab look at?
How do replication products made in secret get out? They discovered it is from a molecular pore around a replication organelle
Cryo-electron tomography
Cross-section of cell —> computer reconstruction to go to 3d. Many images combined can represent accurate structures. Was used to see the pore in which viral RNA escapes in Montesserat lab
What 2 things does RdRp do?
- Copies genome (+ genome to – intermediates to + product)
- Makes shorter sub-genomic +ssRNAs that encode structural proteins like spike
2 features of replication organelles:
- Hides viral RNA from PRRs
- Crown shaped pore allows ribonucleotides in, lets viral RNAs exit to be translated in cytoplasm or full-length genomes to be packaged into new viral particles
