BCH 339M Exam 2

3 betacoronaviruses

MERS-Cov: 2012, 34% mortality rate

SARS-Cov1: 2002-2004, 10% mortality rate

SARS-Cov-2/COVID: 2019, 1% mortality rate

Function of the RBD

Receptor binding domain: binds to the receptor on host cells (ACE2) to enable viral entry. RBD sits in a down conformation but shifts into an up conformation to bind to the host cell receptor.

Structure of the Spike Protein

Trimer

S1 subunit: contains RBD, binds ACE2

S2 subunit: Anchors the S protein to the membrane, contains fusion peptides

3 Structural Mutation Strategies to Stabilize the Prefusion State

Proline residues: Add rigid proline residues at hinge points to prevent rotation of the protein backbone

Cavity Filling: Add bulky hydrophobic residues into cavities to create a more tightly packed core and stabilize the prefusion state

Cysteine residues: Add cysteine residues to sites that are close in the prefusion state but far in the post fusion state to create disulfide bonds and prevent conformation change.

Advancement in mRNA vaccines

Add modified base (e.g pseudouridine) to the RNA to prevent a dsRNA induced immune response (inflammation, mRNA degradation). Adding caps and poly-A-tails also slows degradation

Functions of the RSV-G and RSV-F Fusion proteins

G: Facilitates attachment to the host cell by binding to CX3CR1 and heparan sulfate proteoglycans

F: Mediates fusion of the virus to the host cell membrane. RSV-F may also be involved in attachment since it has been known to interact with receptors and deleting G does not eliminate infectivity

Influenza hemagglutinin (HA) function and different from RSV-G/F

HA mediates both attachment to host cells by binding to sialic acid and fusion of the host cell and viral membranes. HA completes both functions, is far more stable in the prefusion state, and is converted to the postfusion state by low pH.

Why did McLellan et al. (2013) wish to stabilize the prefusion conformation of RSV-F?

Neutralizing antibodies from people who recovered from RSV bound to the prefusion, not the postfusion state. He hoped that stabilizing the prefusion form would lead to the production of more neutralizing antibodies following immunization

What is palivizumab and why did it suggest that a successful RSV vaccine could be made despite decades of failed RSV vaccine clinical trials?

Palivizumab is a monoclonal neutralizing antibody that proved effective in treating RSV, leading to the belief a vaccine was possible. It bound to the antigenic site II that was present in both prefusion and postfusion conformations.

What are viral fusion peptides and why are they typically buried in prefusion conformations of viral fusion proteins?

They are highly hydrophobic amino acids that insert into the target cell membrane to begin fusion. Because they are hydrophobic, they must be buried so the prefusion structure remains soluble and doesn’t aggregate.

siRNAs: origin, function

siRNAs are small double stranded RNAs incorporated into the RISC complex to mediate degradation. They are originally long dsRNA fragments that are processed by Dicer into short fragments. One strand is incorporated into the RISC complex with Ago proteins and the other is degraded. They serve to direct the degradation of mRNA to which they are complementary.

microRNAs: origin, function

microRNAs are small, endogenous ssRNA that regulate gene expression by guiding RISC to the target mRNA and usually binding to the 3’ RNA. They originate from the organism’s genome, starting as pri-miRNA then being processed into pre-miRNA by Drosha. They are then exported to the cytoplasm where dicer cleaves then into mature miRNA and one strand is incorporated into the RISC complex. They serve to regulate translation of multiple different mRNA target.

What percentage (roughly) of bacteria and archaebacteria have CRISPR arrays? What is the range of the number of repeats typically found in a CRISPR array?

~40% of bacterial and ~85% of archebacterial strains have CRISPR loci. Most CRISPR loci contain ~50 repeats but as few as 2 and as many as 345 have been found. Up to 18 different CRISPR loci have been found in single bacterium.

3 stages of CRISPR Immunity

Adaption: cleavage of invading DNA and insertion into the CRISPR locus.

Expression: Transcription of the CRISPR locus and subsequent processing of the precrRNA into crRNA. Assembly of the RISC complex.

Interference: Cleavage of invading DNA.

pre-crRNA

RNA transcript from the CRISPR locus that contains most or all of the spacer repeats.

crRNA

cleaved product of the precrRNA, containing one spacer and variable adjacent sequences.

tracrRNA

A single RNA that binds to the cas nuclease and base pairs with the crRNA

sgRNA

single guide RNA, man-made combination of the tracrRNA and crRNA that simplifies the cas complex to only need 1 RNA instead of 2.

Function of the Palindromic repeats in CRISPR Arrays

Result in hairpin structures that can be recognized by CRISPR/Cas processing enzymes

Function of PAM sequences

Short nucleotide sequences next to the protospacer region of invading DNA. They are used by CRISPR as an initial recognition of DNA. They are not present in the bacterial CRISPR locus, so it serves as a mechanism for the bacteria to distinguish self from foreign DNA. However, it restricts the sequences that can be targeted by gene editors.

CRISPR-Cas9 enzymatic activity

Endonuclease activity by HNH and RuvC

How is Cas9 able to target a wide variety of DNA sequences for cleavage and is Cas9 able to target every possible sequence? Why or why not?

Target specificity is from the programmable crRNA/sgRNA to which cas9 binds

How does CRISPR/Cas9 unwind target DNA

The energy of cas9 binding to the PAM sequence bends the DNA and opens up the duplex so an RNA:DNA hybrid R-loop can form. The REC domain also binds and stabilizes the target DNA and crRNA. Once cleavage has occurred, the affinity is decreased and they can dissociate.

Why are CRISPR-Cas9 systems more tolerant of mismatches in regions distal from the PAM?

The crRNA binds to the PAM and moves distally. At regions further away from the PAM, more of the RNA:DNA hybrid has been formed so the complex is more stable and tolerant.

How is DNA cleavage prevented when using Cas9?

Mutate the endonuclease domain. Inactivating only 1 results in a nickase that cuts only 1 strand.

2 methods of DNA repair

1. Non-homologous end joining: broken ends are ligated together without a homologous template, resulting in nucleotide insertions or deletions, causing frame shifts that destroy protein function (knockouts).

2. Homology directed repair: Uses a template to repair the DNA. Can be made to match donor DNA so its used to modify/repair a gene.

Indel

an insertion or deletion of nucleotides within a DNA sequence, common in NHEJ and often results in a frameshift mutation that knockouts a gene

Base Editing

Base editing is a gene editing method using CRISPR-Cas9 fused with a base editor such as cytidine deaminase or adenine deaminase. A single stranded cut is made and the enzymes target single bases and can be used to create precise point mutations.

Prime Editing

Uses a nickase with reverse transcriptase fused to it and a RNA template containing a desired edit. A single stranded cut is made and reverse transcriptase then repairs the cut strand and incorporates the edit using the provided template. It was developed as a safer alternative to the ds breaks made by traditional CRISPR.