Lecture 6

Question 1

coronavirus genome

Answer 1

• 1st gene from 5’ end occupies 2/3rd of the entire genome
• 1st gene is translated into a polyprotein and then cleaved to form mature functional protein
• Final 1/3rd encodes for virion structural and non-structural proteins (genes 2-7)

Question 2

coronavirus nucleocapsids

Answer 2

• have enveloped virions with helical nucleocapsids
• formed from N (nucleocapsid) protein bound to viral RNA in helical fashion, genome wrapped around the protein
• they resemble negative strand NA viruses

Question 3

coronavirus core

Answer 3

• may have spherical core structure formed via the M (membrane) protein

Question 4

what envelope proteins do coronavirus virions contain

Answer 4

• spike (S)
• membrane (M)
• envelope (E)
• nucleocapsid (N)
• some cases hemagglutinin-esterase (HE) proteins

Question 5

spike proteins (S)

Answer 5

• surface transmembrane glycoprotein protruding from surface
• responsible for viral entry and tropism
• generally forms trimers
• targeted by neutralizing antibodies and T-cells in infection, possible vaccine target

Question 6

Spike protein (S) structure

Question 7

Synthesis of spike proteins

Answer 7

• synthesized as a single polypeptide chain
• cleaved by a cellular proteinase to yield an N-terminal S1 domain and a C-terminal S2 domain

Question 8

spike protein composition

Answer 8

S1 Domain
- a globular receptor-binding domain
- recognizes specific cellular receptors and initiates attachment
- contains the receptor binding domain (RBD)
S2 Domain
- stalk fusion domain
- forms the stalk with a short C-terminal tail, hydrophobic transmembrane domain and exterior domain of interacting alpha-helices
- hydrophobic transmembrane domain allows for attachment and entry to the membrane

Question 9

Receptor binding domain (RBD)

Answer 9

• binds to host receptors to facilitate viral entry
• found in the S1 domain of spike proteins

Question 10

what receptors do alphacornaviruses bind to?

Answer 10

• bind to aminopeptidase-N
• family of zinc-binding metalloproteinases present on cell surface
• broadly distributed on epithelial and fibroblast cells in small intestine, kidneys, CNS
• species-specific

Question 11

what receptors do coronaviruses with HE bind to?

Answer 11

bind to sialic acid (9 carbon sugar) that is a common modification found on a variety of glycoproteins and glycolipids

Question 12

what receptors do betacoronaviruses bind to?

Answer 12

• SARS-CoV binds to metalloproteinase Angiotensin- converting enzyme (ACE2) and a co-receptor L-sign
• transmembrane serine protease 2 (TMPRSS2) that cleaves at S2 to activate viral fusion

Question 13

coronavirus entry by fusion

Answer 13

• mediated by fusion
• external S1 subunit mediates attachment through the RBD
• stalk subunit S2 (class 1 fusion protein) facilitates fusion
• conformational changes result in insertion of S2 into target cell membrane
• brings cell membrane and viral envelope into close contact
• can be pH dependent
• some can also form syncytia

Question 14

SARS-CoV 2 Entry

Answer 14

• can enter via endosomal or non-endosomal (fusion) pathway
• spike protein cleavage by TMPRSS2 (cell surface) or cathepsin L (endosomal route) required
• cleavage exposes hydrophobic amino acids in the spike that embed themselves into the host cell membrane

Question 15

endocytosis of SARS-CoV 2 for entry

Answer 15

• spike protein binds to host cell receptor (ACE2)
• triggers invagination of cell membrane
• forms the endosome
• the spike proteins fuse with the endosomal membrane to release the genome from the endosome

Question 16

membrane fusion of SARS-CoV 2 for entry

Answer 16

• binds ACE2 and TMPRSS2
• fuses at the membrane surface
• the genome is released into the cytoplasm and replicated directly

Question 17

pseudoknot

Answer 17

secondary structures formed in RNA via hydrogen bonds
- “fake knot”
- makes it difficult for the ribosome to go through
- causes it to pause and then a frameshift

Question 18

ribosomal frameshit

Answer 18

• used to deal with overlapping of reading frames
• ribosome ‘slipping’ by one nucleotide in 5’ (-1 nt) or 3’ (+1 nt)

Question 19

synthesis of viral proteins in coronaviruses

Answer 19

• replicase gene (Gene 1) is translated from genomic RNA into a polyprotein that is processed by viral proteinases
• gene 1 is comprise of ORF1a and ORF1b (partially overlapping reading frames)
• translation starts at start codon and goes 5’ to 3’
• ribosomes translating ORF1a pause at a pseudoknot and frameshift which allows for translation of ORF1b
• yields 2 polyproteins: pp1a and pp1ab

Question 20

what is the most conserved part of the coronavirus genome and what does it tell you?

Answer 20

• ORF1b
• it is critical to its function
• mutations in this region are not very well tolerated

Question 21

replication complexes

Answer 21

• site of viral RNA synthesis
• consists of viral and cellular proteins associated with membranes in the cytoplasm of the infected cell
• commonly observed on double membrane vesicles

Question 22

what is the advantage of the replication complexes being on the double membrane vesicles?

Answer 22

• vesicles will protect the viral genome