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Virology > Lecture 6 > Flashcards

Lecture 6 Flashcards

1
Q

coronavirus genome

A
  • 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)
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2
Q

coronavirus nucleocapsids

A
  • 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
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3
Q

coronavirus core

A
  • may have spherical core structure formed via the M (membrane) protein
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4
Q

what envelope proteins do coronavirus virions contain

A
  • spike (S)
  • membrane (M)
  • envelope (E)
  • nucleocapsid (N)
  • some cases hemagglutinin-esterase (HE) proteins
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5
Q

spike proteins (S)

A
  • 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
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6
Q

Spike protein (S) structure

A
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7
Q

Synthesis of spike proteins

A
  • synthesized as a single polypeptide chain
  • cleaved by a cellular proteinase to yield an N-terminal S1 domain and a C-terminal S2 domain
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8
Q

spike protein composition

A

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

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9
Q

Receptor binding domain (RBD)

A
  • binds to host receptors to facilitate viral entry
  • found in the S1 domain of spike proteins
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10
Q

what receptors do alphacornaviruses bind to?

A
  • 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
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11
Q

what receptors do coronaviruses with HE bind to?

A

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

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12
Q

what receptors do betacoronaviruses bind to?

A
  • 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
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13
Q

coronavirus entry by fusion

A
  • 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
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14
Q

SARS-CoV 2 Entry

A
  • 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
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15
Q

endocytosis of SARS-CoV 2 for entry

A
  • 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
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16
Q

membrane fusion of SARS-CoV 2 for entry

A
  • binds ACE2 and TMPRSS2
  • fuses at the membrane surface
  • the genome is released into the cytoplasm and replicated directly
17
Q

pseudoknot

A

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

18
Q

ribosomal frameshit

A
  • used to deal with overlapping of reading frames
  • ribosome ‘slipping’ by one nucleotide in 5’ (-1 nt) or 3’ (+1 nt)
19
Q

synthesis of viral proteins in coronaviruses

A
  • 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
20
Q

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

A
  • ORF1b
  • it is critical to its function
  • mutations in this region are not very well tolerated
21
Q

replication complexes

A
  • 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
22
Q

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

A
  • vesicles will protect the viral genome
  • has access to the ER
  • keeps everything in close proximity
  • do not need to diffuse long distances to carry out the process
23
Q

reticulovesicular network of coronaviruses

A
  • virus induced membrane alterations
    (ie. double-membrane vesicles, convoluted membranes, and double-membrane spherules
  • formed by a combination of cell stimuli to produce new membrane and modification of existing membranes by virus proteins (proposed to initiate formation of replication complexes)
  • nucleocapsid protein can be found in abundance at these sites, allows for encapsidation of genome as it is being synthesized
24
Q

coronavirus genome replication

A
  • proceeds using a ‘-‘strand intermediate (antigenome), directs synthesis of ‘+’ strand genome
  • most ‘-‘ strands associated with one or more growing ‘+’ strands in a replicative intermediate
  • ’-‘ strand RNAs only account for 1-2% of total viral RNA because each template can be used multiple times
25
Q

replicative intermediate

A

RNA molecule on which one or several growing RNA strands are being synthesized
- growing strand typically forms base-pairing to template RNA only near their growing 3’ end

26
Q

subgenomic mRNAs in coronaviruses

A
  • expression of genes downstream of replicase gene (gene 1) occurs from series of subgenomic mRNAs
  • all have identical untranslated leader (UTR) sequences at 5’ end, poly (A) tails
  • represent nested set (overlap, nested btwn othes) of mRNA
  • all contain overlapping sequences on their 3’ end
  • UTR attached to unique mRNA ‘body’ sequence with one or more open reading frames (ORFs)
  • transcribed from subgenomic negative strand mRNA templates
27
Q

discontinuous transcription in coronaviruses

A
  • negative strand mRNA templates for transcription of subgenomic mRNAs are made by discontinuous transcription
  • leader transcription-regulating sequence (TRS-L) at the 5’ end of the genome
  • multiple body transcription-regulating sequences (TRS-B) located upstream of genes encoding structural and accessory proteins
  • begins at the 3’ end of the genomic RNA and transcribes continuously until it encounters a TRS-B
  • pauses and dissociates the nascent RNA chain and jumps to the TRS-L (template switching)
  • each of the subgenomic ‘-‘ strands are used to make a ‘+” strand mRNA
  • enables coronaviruses to produce multiple proteins from a single RNA genome
28
Q

how can the discontinuous transcription model explain recombination between viral genomes?

A
  • viral RNA polymerase can switch between 2 different positive strand genome RNAs if they are both in the same cell
  • could be 2 different virus strains infecting the same cell, or mutations in the virus genome during replication
  • template switching may help with genome repair and/or generate new viral strains/variants
29
Q

potential consequences of a dissociation event during viral transcription (discontinuous transcription)

A
  • after disassociation, polymerase has to find a new template or transcription process will abort
    Possible outcomes following disassociation:
    1. aborted transcription
    2. re-association on same template and same genomic position
    3. re-association on same template at different genomic position
    4. re-association on homologous template at same genomic position
    5. re-association on homologs template at different genomic position
    6. re-association on non-homologous template (ie. cellular RNA)
30
Q

which re-association recombination event in discontinuous transcription is most likely to generate functional progeny?

A

same position on a homologous template

31
Q

where does assembly of coronavirus virions take place?

A
  • intracellular membrane structures
  • the endoplasmic reticulum-Golgi intermediate compartment (ERGIC)
32
Q

what is the endoplasmic reticulum-Golgi intermediate compartment (ERGIC)?

A
  • interconnected system of membranes that lies between ER and Golgi
  • involved in transport, processing and modification of proteins
  • generally located in the perinuclear (around or near the nucleus) region of the cell
33
Q

steps in assembly and release of cornavirus virions

A
  1. helical nucleocapsids containing genome RNA are delivered from site of synthesis to intracellular membrane structures fo packaging
  2. virus particles are formed by budding into the lumen of these membranes (virions acquire donut-shaped cores)
  3. progress to smaller and more uniformly dense cores as transit through golgi membranes, envelope proteins also undergo glycosylation
  4. secretory vesicles transport virions to cell surface for fusion with plasma membrane and release
34
Q

Membrane (M) and envelope (E) proteins in the formation of virus envelopes by budding

A
  • enveloped virus-like particles can be formed in ERGIC when only M and E are expressed
  • C terminal cytoplasmic tail of M thought to interact with packaging signals in nucleocapsid protein (N), ensures only full-length viral RNA gets packaged into virions
  • hemagglutinin-esterase protein (HE) if present and spike protein (S) get incorporated into the membrane through interactions with the M protein
35
Q

evolution of coronaviruses

A
  • bats are evolutionary pools (reservoirs) for new CoVs in mammals
  • subtle changes can alter tissue tropism, host range, and pathogenicity
  • changes can occur by: relatively high theoretical error rate of viral RNA polymerase and RNA-RNA recombination
  • some mutations may enhance virulence or cross species barriers, others may make virus less effective

Virology (16 decks)

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