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MICR337 - Virology > MATLOOB'S MODULE > Flashcards

MATLOOB'S MODULE Flashcards

1
Q

what are the factors driving viral emergence?

A

ecological changes

human demographics

international travel and trade

virus change and adaption

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2
Q

what drives virus change adaption?

A

a molecular change in virus-host interactions

this can be caused by genetic changes in virus (genetic drift), a virus circulating in multiple hosts or a virus adapting to multiply in a new host

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3
Q

what is genetic drift?

A

changes in the genetic structure of a virus

caused by mutations and happen during replication

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4
Q

what are the factors causing genetic drift?

A

lack of 3’-5’ proofreading capability in RNA-dependent RNA polymerase (RdRp) and reverse transcriptase

pressure from host immune system

rapid replication for many progeny; viral RNA polymerases exchange accuracy for efficiency and on average each progeny ends up with at least one point mutation

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5
Q

when does genetic drift occur?

A

all the time in the virus lifecycle

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6
Q

what is antigenic Drift?

A

a genetic drift that decreases the antigenicity of a viral protein

allows it to escape the existing antibody-mediated immunity thus problematic for host (no antibodies for it)

maintains the viral fitness (ability to infect and multiply in new cell as good as previous generation)

can increase viral fitness via epistatic mutations, if one aspect of viral fitness compromised this will improve another aspect to maintain it (I think)

examples of this = HA or NA of influenza virus or spike protein of SARS-CoV-2

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7
Q

how does a virus circulating in multiple hosts contribute to virus change and adaption?

A

generation of a new virus through antigenic Shift where genetic material exchanged between two viruses resulting in shift in antigenicity (common in viruses with segmented RNA genome e.g. pigs infected with avian and human influenza A viruses)

generation of new virus through genetic recombination where an exchange of genetic material occurs between two viruses (common in positive-sense RNA viruses e.g. bats harbouring multiple coronaviruses)

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8
Q

what is zoonosis in viruses?

A

a virus adapting to multiply in a new host

caused by point mutations in viral proteins (RBPs) and RdRp (RBP has to mutate as must be able to bind receptor in new host)

examples include avian influenza A viruses H5N1, H7N3, H7N7, H7N9

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9
Q

describe influenza virus?

A

enveloped 80-120nm diameter vision

segmented, -ssRNA genome, ~14kb

8 RNA genes segments encoding 17 proteins

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10
Q

what are the major proteins in influenza virus and their function?

A

HA - receptor binding

NA - release

M1 - virion shape

M2 - entry

NP - RNA binding

PA/PB1/PB2 - RdRp

NS1 - host antagonism

influenza virus utilises all components of the host cell during life cycle

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11
Q

what are the four types of influenza virus, what do they infect, and how do they transmit among their hosts?

A

influenza A viruses infect variety of mammal and avian hosts, awaiting bird as reservoir host

influenza B and C mainly infect humans

influenza D infects cattle and pigs

transmission via:

aerosols for humans
faecal-oral route for birds
contact for cattle

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12
Q

how is influenza A subtyped and which subtypes cause human infection?

A

based on HA and NA proteins

16 HA and 9 NA subtypes known - all found in aquatic birds (reservoir)

some found in bats

H1, H2, H3, H5, H7 and H9 subtypes can cause infection in humans

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13
Q

what are the drivers of emergence of influenza virus variants?

A

antigenic drift

antigenic shift

zoonosis

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14
Q

outline antigenic Drift in influenza virus?

A

occurs in both HA and NA genes of type A and B influenza viruses

main reason for recurring seasonal flu epidemics

main reason for the requirement of annual flu vaccination (gives you updated HA and NA antibodies which neutralises updated virus)

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15
Q

outline antigenic Shift in influenza viruses?

A

also known as genetic reassortment and only seen in type A influenza viruses

occurs when two influenza A subtypes (e.g. avian and human subtype) infect same cell in something like a pig; segmented genome means novel subtype can have combination of those segments; may lead to new HA etc which then will probably cause a pandemic

this is how the influenza pandemic was caused

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16
Q

what are the three properties a virus must acquire to cause a pandemic?

A

bear a receptor-binding protein to which there is little or no pre-existing immunity in humans

able to cause disease in humans

capable of sustained human-to-human transmission (critical)

many get one or two cause genetic drift happens all the time but third much more rare (thank fuck)

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17
Q

what influenza subtypes have acquired the three essential properties for pandemic formation?

A

H1N1, H2N2 and H3N2

have all caused pandemics in the past and are all type A

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18
Q

what was the 1918 Spanish flu pandemic?

A

originated in USA, approx 50 million deaths

caused by H1N1 subtype

two theories of origin:
- avian H1N1 virus adapted to humans through mutations
- a human H1Nx virus reasserted with avian HxN1 virus

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19
Q

what was the 1957 asian flu pandemic?

A

originated in China, approx 2 million deaths

caused by H2N2 subtype, a lineal descendant of 1918 H1N1 subtype reasserted with an avian subtype

acquired three novel genes HA, NA and PB1

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20
Q

what was the 1968 Hong Kong flu pandemic?

A

originated in Hong Kong, approx 1 million deaths

caused by H3N2 subtype which was was an H2N2 subtype reassorted with an avian subtype

acquired two novel gene segments HA and PB1

thought to be milder than 1957, possibly cause N2 antibodies remain in population

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21
Q

what was the 2009 swine flu pandemic?

A

originated in Mexico, approx 200,000 deaths

caused by H1N1 subtype, generated by two-step genetic assortment between two unrelated swine subtypes

80% deaths younger than 65 (prob cause older people were around when OG H1N1 pandemic happened)

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22
Q

what are the zoonotic influenza viruses?

A

avian influenza A viruses H5N1, H7N7, H7N9 and H9N2

swine influenza A virus H3N2

case fatality rate 30-60%!

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23
Q

what is avian influenza A virus H5N1?

A

originated in HK 1997

first time an avian influenza A virus subtype was discovered to infect humans

remains endemic in poultry and causing sporadic infections in humans (mortality rate >50%)

sparked lots of fear of an impending bird flu pandemic

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24
Q

what is avian influenza A virus H7N9?

A

originated in China 2013

a novel avian influenza A virus

generated by a two-step reassortment between H7Nx, HxN9 and H9N2 avian influenza virus

caused infections in several waves due to closing/re-opening of live bird markets

mortality rate >35%

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25
Q

what is coronavirus?

A

enveloped, 100-160nm diameter virion

linear, +ssRNA genome, ~30kb

4 structural proteins: S (pike), E, M, N

16 non-structural proteins (nsp 1-16)

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26
Q

what are the functions of the proteins in the coronavirus?

A

Spike (S) - receptor binding

E - assembly, release

M - virion shape

N - RNA binding

Nsp 1-16 (RdRp, viral protease, host antagonism)

purely cytoplasmic (all its processes occur in cytoplasm)

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27
Q

what are some coronavirus hosts?

A

people

bats

cattle

cats and dogs

pigs

rodents

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28
Q

what are the driving factors of the emergence of coronaviruses?

A

genetic recombination

zoonosis

drift (genetic & antigenic)

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29
Q

what are the two proposed mechanisms of coronavirus genetic recombination?

A

replicative

non-replicative

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30
Q

what is replicative genetic recombination in coronavirus?

A

linear RNA genome so one cell being infected by two or more coronavirus which replicate at same time and RNA polymerase can then SWITCH TEMPLATE so replicates genome from other virus

forms hybrid RNA cause two sets recombine to form progeny genome

called template switching and is common

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31
Q

what is non-replicative genetic recombination in coronavirus?

A

during replication one genome breaks into two and recombines and ligates with the other one

called breaking and joining and not common

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32
Q

what is SARS coronavirus?

A

emerged in 2003, Hanoi, man died from it

emerged through sequential recombination events between coronaviruses in bats and then jumped from bats to civet cats

approx 8000 cases 770 deaths

wasn’t very transmissible so easy to control

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33
Q

what is MERS coronavirus?

A

emerged in 2012 in man in Saudia Arabia who died

emerged through sequential recombination events in bats then from bats to camels

chilled in camels for at least 30 years before jumping to humans in 2012

approx 2,500 cases, 900 deaths, 27 countries

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34
Q

what is SARS coronavirus 2?

A

emerged Dec 2019 in China in a woman and two men

one man died, others recovered

> 85% genome identity with 2003 SARS coronavirus

emerged from recombination event between a bat coronavirus and a pangolin coronavirus

> 768 million cases and >6.9 million deaths currently

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35
Q

outline drift in SARS-CoV-2?

A

numerous mutations in the original virus

several antigenic variants (delta, omicron) (this caused by antigenic drift)

antigenic drift is prolific in this one and current covid vax is bivalent

potential to become endemic in nature

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36
Q

what are the three key determinants/factors of viral pathogenesis?

A
  • epidemiological factors e.g. population status (certain populations more susceptible e.g. old cunts, people without immunity)
  • host factors e.g. receptor, proteases, antiviral proteins, genetic polymorphisms (in some antiviral proteins hence why some people more susceptible than others)
  • viral factors e.g. RBP, polymerase, virulence proteins (some viruses have more effective/stronger binding ones of these)
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37
Q

what does viral antagonism refer to?

A

ability of virus to subvert host innate viral defence

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38
Q

viral pathogenesis is multi-stage and multi-genic; what does this mean?

A

it involves multiple stages and multiple genes

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39
Q

what does infectivity mean?

A

ability to infect host cell

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40
Q

outline the infectivity of influenza virus?

A

HA::receptor (HA is the receptor binding protein)

influenza A virus HA exhibits receptor tropism

influenza A virus exhibits tissue tropism

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41
Q

what is receptor tropism in influenza A?

A

HA of different influenza A subtypes binds different receptors

receptor in humans is alpha-2, 6 sialic acids

receptor in avian hosts is alpha-2, 3 sialic acids

receptor in bats is MHC class II

note that humans still have all three receptors

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42
Q

how does influenza A exhibit tissue tropism in human hosts?

A

tissue tropism refers to what tissues the virus infects

alpha-2, 6 sialic acid (SA) is predominantly found in the upper respiratory tract

alpha-2, 3 sialic acid (SA) is more abundant in lower respiratory tract

and why avian flu fucks up that part of your lungs

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43
Q

why are pigs a mixing vessel for influenza A virus and what does this mean for the virus?

A

both a-2, 6 SA and a-2, 3 SA are present in upper respiratory tract of swine hosts

this means antigenic shift is easier in pig cells cause both viruses infect the same area

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44
Q

why is temperature important for influenza A infections?

A

can determine how efficient RNA polymerase (viral replication) functions

different sites of infection in different hosts are different temperatures

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45
Q

outline the site of infection for influenza A in avian hosts?

A

a-2, 3 SA is present in both respiratory tract and gastrointestinal tract

this means transmission can occur via feral oral route as well

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46
Q

outline the structure of influenza virus HA?

A

globular head connected to fibrous stem by hinge domain

within globular head is receptor binding domain which binds SA (so has to be exposed on surface)

within fibrous stem is fusion domain/peptide which is located close to hinge domain

also a stop domain buried inside envelope

all these structures are important for HA function

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47
Q

how do specific amino acids in the HA receptor binding domain determine receptor tropism?

A

HA receptor binding domain is where all the action happens like antigenic shift

certain amino acids in the RBD effect the strength of the interaction between HA and SA - which amino acids these are changes between influenza subtypes

these amino acids are especially important as most pandemic viruses had L226 amino acid (which entails strong binding to alpha-2, 6 SA)

main point is single amino acids can determine the strength of binding between RBD and receptor

(IS THIS DUE TO CLEAVAGE MOTIF)

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48
Q

outline influenza A membrane fusion?

A

HA binds receptor for entry but has to cleave (mediated by a protease) to bind and enter as this exposes fusion peptide

M2 protein creates proton channel for proteins to enter viral envelope

proton shakes shit up causing virus ribo nuclear proteins (VRNPs) (these are the gene segments) to come out of cytoplasm and uncoating takes place

gene segments go in host nucleus and start replication process

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49
Q

what is the HA cleavage motif in human and low-pathogenic avian influenza A subtypes?

A

human and low-pathogenic avian influenza A subtypes HA contain single basic amino acid at cleavage site

trypsin-like proteases localised mainly to respiratory or intestinal tract recognise this motif/sequence

localisation of trypsin-like proteases restricts virus infection locally

cleavage motif in HA is critical for influenza A pathogenesis

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50
Q

what is the HA cleavage motif in high-pathogenic avian influenza A subtypes like H5N1 and H7N7?

A

HA in high-pathogenic avian subtypes contains multiple basic amino acids at cleavage site

proteases which recognise these (such as furin) are ubiquitously present in host body which can allow systematic infection and increased pathogenicity

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51
Q

what is the main difference between the HA cleavage motif in human/low-pathogenic avian subtypes of influenza A and high-pathogenic avian subtypes?

A

only a single basic amino acid in low-pathogenic cleavage site recognised by localised trypsin-like proteases keeping infection local

multiple basic amino acids in high-pathogenic cleavage site recognised by ubiquitously-present proteases allowing systematic infection

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52
Q

outline replication by influenza A?

A

replication determined by RNA dependant RNA polymerase which is a complex made up of the proteins PA, PB1 and PB2

this controls the level of viral RNA replication and transcription

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53
Q

what does the E627K mutation in PB2 of the influenza A RNA pol complex do?

A

is critical for adaption and virulence of avian influenza to transfer to mammals as it changes PB2 structure so it can function efficiently at lower temperature in the human upper respiratory tract (33 degs)

all pandemic, seasonal and zoonotic avian influenza A (H5N1, H7N7 and H7N9) subtypes contain this mutation

H1N1 2009 pandemic virus was only one without this mutation and this prob why it wasn’t so virulent

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54
Q

what does the D701N mutation in PB2 of influenza A RNA pol complex do?

A

critical for pathogenesis in zoonotic avian influenza A subtypes

associated with increased virulence in mammals

promotes nuclear localisation of PB2 by increasing its binding to nuclear import protein importin-alpha (RNA pol has to go in nucleus for transcription and replication so this mutation makes that import in efficient as)

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55
Q

what are the two mutations in PB2 that are critical for zoonotic avian influenza A pathogenesis?

A

E627K

D701N

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56
Q

outline how NA effects transmissibility of influenza A?

A

NA is a sialidase which cleaves surface sialic acid leading to release of viral progeny

if NA is low activity there is virion aggregation on surface meaning less release and less spread

NA is high activity in pandemic viruses

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57
Q

outline the infectivity of coronavirus (CoV)?

A

spike::receptor

coronaviruses exhibit some receptor tropism

the receptor for SARS-CoV-2 is ACE2

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58
Q

what is ACE2?

A

angiotensin-converting enzyme 2

a transmembrane protease which is important in regulating blood pressure and fluid balance

processes angiotensin II into angiotensin 1-7 resulting in vasodilation

is also an interferon-stimulated gene so there is increased ACE2 expression after viral infection (that’s fucked)

ACE2 is highly conserved in mammals and exhibits no tissue tropism. It is expressed in multiple organs including lungs, heart and kidney and so SARS-CoV-2 can infect all of these

part of the reason cunts can get real severe covid

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59
Q

outline the structure of the CoV spike?

A

two parts S1 (receptor-binding domain) and S2 (membrane-binding domain)

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60
Q

what component of the CoV spike is responsible for most of the evolution and adaption of the virus between hosts?

A

S1 (receptor binding domain)

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61
Q

outline the evolution and adaption of the SARS-CoV-1 spike?

A

two critical positions in S1 RBD (479 and 487) where evolution of amino acids occurred

began as bat virus (479Arg and 487Ala) then to civet virus (479Lys and 487Ser) then to human virus (479Asn and 487Thr)

so civet was the intermediate host

basically changed strength of spike binding ACE2 in respective hosts

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62
Q

outline the evolution and adaption of the SARS-CoV-2 spike?

A

S1 RBP positions 479 and 487 changed to 493 and 501

bat virus evolved to pangolin virus which then adapted to human virus with 493Gln and 501Asn

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63
Q

outline the evolution and adaptation of the MERS-CoV spike (in terms of host)?

A

bat virus evolved to camel virus which then adapted to infect humans

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64
Q

outline CoV spike cleavage?

A

two cleavage sites in the spike - one in S1 and one in S2 (not like influenza A HA)

cleavage at these sites exposes fusion peptide and occurs by host proteases (like influenza A HA)

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65
Q

how has the SARS-CoV-2 spike evolved to allow for more cleavage and what does this mean for the disease it causes?

A

it has evolved to contain multiple basic amino acids in its motif (so motif getting bigger) at the cleavage site allowing it to be recognised by multiple proteases e.g. Furin, cathepsin etc.

since ACE2 present in multiple organs and cleavage site accessible to proteases in multiple organs it means SARS-CoV-2 can cause quite severe disease

cleavage site the same as in MERS-CoV and SARS-CoV but motif is bigger in SARS-CoV-2

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66
Q

what are the mutations in SARS-CoV-2 spike affecting binding affinity to ACE2?

A

Asn501Tyr in S1 receptor domain (Omicron)

Pro681Arg in S1 cleavage site (Delta)

fuck rewatch this

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67
Q

how does replication of viral RNA occur for SARS-CoV-2?

A

Nsp1 degrades host mRNA and then binds viral +ssRNA and takes it to the ribosome to be preferentially translated and blocking mRNA entry channel in ribosome for host mRNA

this means antiviral proteins from host might not get translated (e.g. IFN)

68
Q

outline SARS-CoV-2 transmissibility?

A

SARS-CoV-2 release by exocytosis (not budding like influenza A)

ORF3a promotes viral progeny release by lysosomal exocytosis

69
Q

how do host immune systems recognise viruses?

A

PRRs looking for PAMPs on virus

they bind the PAMP and induce signalling cascade

70
Q

what are the three main categories of PRRs recognising viral PAMPs?

A

TLR

RIG-1

NLR

71
Q

what are the three main categories of PRRs recognising viral PAMPs?

A

TLR

RIG-1

NLR

72
Q

outline the host innate antiviral response?

A

production of a shit load of cytokines (e.g. interferon and interleukin), chemokines and interferon-stimulated genes

these may act on the cell that produced them (autocrine) or on other cells (paracrine)

73
Q

outline the three main mechanism of viral antagonism of the host innate antiviral response?

A

sequestration of proteins

inhibition of expression

mimics/decoys

74
Q

outline the sequestration of proteins mechanism of viral antagonism?

A

interfere with formation of a function complex that host cell is trying to make

arrest the proteins in an intracellular compartment (stopping activated proteins from moving)

75
Q

what is the inhibition of expression mechanism of viral antagonism?

A

down regulation of transcription, nuclear export and degradation of host mRNA

down regulation of translation, degradation and cleavage of host polypeptides

76
Q

what is the mimics/decoys mechanism of viral antagonism?

A

uses analogous and homologous viral proteins

host cell thinks its interacting with host proteins but they viral

77
Q

how does influenza virus antagonise the host innate antiviral response?

A

influenza virus has NS1 which is highly expressed and binds host molecules to sequester and interfere stopping them from performing their function

NS1 usually in nucleus but if TRIM25 (host molecule involved in activating RIG-I pathways) being expressed in cytoplasm it is exported out of nucleus to bind and sequester TRIM25 stopping it from activating viral recognition pathways

scientists use NS1 attenuated vaccine

78
Q

how does SARS-CoV-2 use sequestration to antagonise host innate antiviral response?

A

uses Nsps and ORFs to sequester and interfere

79
Q

how does Ebola virus protein VP35 antagonise host antiviral response?

A

VP35 sequesters host IRF3 into specialised compartments where it can’t perform its function

these compartments are called inclusion bodies and they are sites of Ebola replication

80
Q

how does adenovirus protein VII sequester HMGBI?

A

HMGBI is host nuclear protein (alarmin) which is a host immune activator and danger signal against DNA viruses

Protein VII sequesters it in the nucleosome

81
Q

how does influenza A NS1 antagonise host antiviral responses via inhibition of expression?

A

interferes with mRNA processing and export (already processed mRNA)

so NS1 does multiple functions

NS1 stops protein complex that usually exports mRNA from nucleus to cytoplasm thus stopping mRNA trafficking

this has been proven by removing complex domain NS1 binds and then mRNA trafficking occurred as normal

82
Q

how does degradation of mRNA by viral endonuclease occur?

A

viral endonuclease target host mRNA and cleave it by chopping in the middle of the sequence (exonuclease chop from the end)

they can distinguish between host and viral mRNA cause of the different structure

83
Q

how does influenza virus PA antagonise the host innate immune response through mRNA and polypeptide degradation?

A

PA is a viral endonuclease that degrades HDAC mRNA and activates host caspases (e.g. caspase3) to degrade HDAC polypeptides

HDACs are deacetylases that are involved in inducing immune response - they are anti-influenza virus factors

this has been proven through knockdown of viral PA expression which rescued HDAC mRNA and polypeptide levels as well as inhibition of caspase3 activity rescuing HDAC at polypeptide level

84
Q

what is the pro and anti-inflammatory response?

A

inflammation at the site of infection takes form as redness, pain, heat, swelling and is caused by immune cells congregating at the infection site (pro-inflammatory response)

anti-inflammatory response involves inflammation resolution and its purpose is clearance of virus-infected cells

both are mediated by cytokines and chemokines

85
Q

what do pro and anti-inflammatory cytokines do?

A

chemokines attract immune cells (e.g. to site of infection)

cytokines activate immune cells

86
Q

what are some examples of pro and anti-inflammatory cytokines and chemokines and what do they do?

A

pro-inflammatory cytokines - IL-1, IL-6, IL-12 and TNF (promote leukocyte activation)

anti-inflammatory cytokines - IL-4, IL-10 and TGF-beta (suppress pro-inflam cytokines)

chemokines - IL-8 (recruits immune cells)

87
Q

how do viruses antagonise the host innate antiviral response using mimics or decoys?

A

using viral proteins which are analogous/homologous to immune molecules

common in large DNA viruses like poxviruses and herpesviruses; large genome can accomodate many genes

these genes encode proteins called:

virokines - mimic cytokines and chemokines to block the host receptors/pathways

viroceptors - decoy cytokines and chemokines which capture host molecules (i.e. make them bind the wrong thing)

88
Q

how do herpesviruses use mimics and decoys?

A

has a large dsDNA genome of 120-230kb and 100 ORFs

secrete mimics of cytokines like vIL-10, vIL-17 and mimics of chemokines like vCXC1

also secretes decoy soluble cytokine receptors or binding proteins e.g. vCKBP3

also has decoy membrane bound chemokine receptors e.g. vCKR

89
Q

how do poxviruses use mimics and decoys?

A

they make a shitload of soluble decoy cytokine receptors and binding proteins, membrane bound decoy cytokine receptors, and also make a bunch of cytokine and chemokine mimics

an example is vIFN-a/b BP (binding protein) as a decoy for host IFN-alpha/beta receptor

the infected cell with secrete the decoy receptor which goes and binds uninfected cells and pretends to be IFN receptors thus stopping further signalling

90
Q

how did the evolution of pathogen mimics occur?

A

they evolved to be able to express the same sequence due to coevolution

DNA viruses are better because the DNA is common between humans and the virus

some bacteria can also do the mimic/decoy shit too

91
Q

outline virus assembly?

A

is an organised and multi-step process

may occur in one or more than one compartments of the host cell (e.g. cytoplasm or nucleus or both or more)

all components required to make virus particle must be transported to that compartment and so the transport machinery of the host cell plays a critical role

92
Q

what is the replication site?

A

where replication of viral genome takes place

93
Q

what is the assembly point?

A

where everything comes together to assemble viral progeny

94
Q

what is the overarching process common to all viruses in order to replicate?

A

entry to host cell

replication site

assembly point

release from host cell

95
Q

where do DNA viruses such as Adenovirus and Polymovirus assemble?

A

exclusively in the nucleus

most DNA viruses assemble in the nucleus

96
Q

where do most DNA viruses replicate their genome?

A

the nucleus

prefer nucleus of host cell because because they utilise host components (e.g. transcription factors) for DNA replication

97
Q

what viruses exclusively replicate in the cytoplasm?

A

coronavirus like SARS, MERS

picornavirus like Polio, Hepatitis A

poxvirus like Smallpox, Vaccinia

(first two RNA virus third is DNA virus)

98
Q

what are cytoplasmic sites of virus assembly (give examples of virus that assemble there)?

A

endoplasmic reticulum (e.g. coronavirus)

golgi complex (e.g. herpes virus)

viral factors or inclusion bodies - specialised compartments that only exist in infected cells (vaccinia virus, ebola)

99
Q

where does MERS-CoV assemble?

A

in the cytoplasm; specifically between tubules in the endoplasmic reticulum

100
Q

where does vaccinia virus assemble?

A

in the cytoplasm; specifically in viral factories

101
Q

what viruses assemble on plasma membrane but replicate elsewhere?

A

retrovirus (HIV)

rhabdovirus (VSV, Rabies)

102
Q

what viruses undergo assembly in multiple compartments of the host cell?

A

influenza virus

herpesvirus

they replicate their mRNA in nucleus, then go to cytoplasm to synthesise some proteins which get imported back into nucleus to assemble a core structure which is exported to assemble complete virus particle at plasma membrane

103
Q

where does herpesvirus assembly occur?

A

both the nucleus and cytoplasm; specifically the Golgi

104
Q

what are the two key components of all viruses and what is the third optional one?

A

protein - involved in viral capsid, receptor-binding, polymerases

nucleic acid - involved in viral genome

all viruses have those two components

if a virus is enveloped it also has lipid which is involved in viral envelope

105
Q

why must viral components be targeted to the assembly site?

A

proteins only synthesised in cytoplasm

genome can be synthesised in cytoplasm or nucleus

but then these have to be targeted to site of assembly so they can be transported there

106
Q

how does targeting of viral proteins occur?

A

no different than the way host proteins targeted as virus just using host machinery

proteins get synthesised two ways in cytoplasm:

  • on free ribosomes (post-translationally targeted)
  • on membrane-bound ribosomes (rER) (co-translationally targeted)

so viral proteins use these processes

107
Q

outline post-translational targeting for host cells?

A

occurs on free ribosomes in cytosol when polypeptide is synthesised (after translation) - these kinds of proteins get targeted to mitochondria, nucleus etc and are soluble proteins/polypeptides

108
Q

outline co-translational targeting for host cells?

A

occurs for proteins synthesised by membrane bound ribosomes in rER, proteins get targeted while being translated and get targeted to membranous compartments e.g. lysosomes, plasma membrane, secretory vesicles (which courier these proteins)

109
Q

how do viral proteins and host cell machinery know the destination

A

through molecular signals i.e. specific amino acid sequences in the polypeptide

these amino acid sequences/signals are recognised by host cell transport machinery (e.g. secretory vesicles)

110
Q

all proteins synthesised in cytoplasm, so how do they get into nucleus if that’s where they’re needed for assembly?

A

by simple diffusion (passive process) (rare)

or

by specific nuclear signals and receptors (active process) (common)

111
Q

what does passive transport into the nucleus involve?

A

occurs for smaller proteins (<40kDa)

nucleus has pores so if the proteins can fit through these you just slide them through (passive diffusion)

112
Q

what does active transport into the nucleus involve?

A

occurs for larger proteins

facilitated by host proteins importin alpha and importin beta

importing recognise nuclear localisation signals (NLS) on viral proteins - these are specific amino acid sequences

113
Q

what are nuclear localisation signals (NLS)?

A

what host proteins importin alpha and beta recognise on viral proteins to transport them into nucleus for assembly

NLS are less than 20aa long and rich in basic amino acids

114
Q

what are some examples of NLS?

A

KGTKR in nucleoprotein (NP) of influenza virus

RKLKR in M1 protein of influenza virus

GPNKKKRKL in VP2 protein of Simian virus 40

all the K’s and R’s are basic amino acids hence all NLS are rich in basic amino acids

115
Q

outline the nuclear import pathway?

A

protein containing NLS binds importin a

importin a binds importin b

importin b binds to components of nuclear pore complex (NPC)

active transport (ATP involved) of complex through NPC involving GTPase Ran

dissociation of protein containing NLS from importins

importins cycled back to do the same to other proteins

116
Q

how does trafficking of influenza virus nucleoprotein (NP) occur?

A

NP synthesised in cytoplasm

goes to nucleus and binds vRNP (RNA and polymerase complex)

vRNP gets out of nucleus with help of nuclear protein signals as well as nuclear export protein (facilitates export)

complex travel through cytoplasm on microtubules as the transport medium

then localises on plasma membrane

117
Q

what is the experimental basis for trafficking of influenza virus NP?

A

culture virus and eventually NP protein starts getting exported and eventually nucleus will be clear and most NP found in cytoplasm or plasma membrane (where it needed for assembly)

mutate NP amino acids to alanine to remove polarity thus interfering with protein structure and subsequently its interaction with importins

so when mutated the protein just stayed in cytoplasm and nucleus stayed empty indicating signals they mutated were NLS

mutation shows that region is a limiting factor for virus and so could be effective antiviral target

118
Q

how does targeting of viral proteins to the ER occur?

A

some viral proteins don’t need to go nucleus cause they are membrane proteins so not in nucleus

ER targeting signals is how system knows these are membrane proteins

two signals:

  • signal peptide (a sequence of 16-22 hydrophobic aa’s, at N-terminus of a polypeptide)
  • trans-membrane domain (a sequence of 20ish hydrophobic aa’s, can be anywhere on the polypeptide, provides signal of where to stop targeting so protein doesn’t get out)

there are also ER retention signals for insertion of viral proteins into ER membrane

so the specific amino acid sequence is determining the fate of the polypeptide

119
Q

what occurs during processing of viral proteins in the ER?

A

multiple modifications e.g. glycosylation, di-sulfide bond formation, folding, oligomerisation

onece protein processed in ER it is targeted to the Golgi where more sugars are added

120
Q

what occurs during processing of viral proteins in the Golgi complex?

A

oligosaccharide processing and maturation e.g. removal of mannose, addition of N-acetylglucosamine, galactose and sialic acid

mature proteins are then targeted to the plasma membrane or other cellular compartments

121
Q

how does trafficking between organelles occur?

A

occurs through vesicles which have specific coatomer proteins (COP) on their outside which initiate vesicle formation

vesicles contain a docking protein called v-SNARE allowing them to dock on target compartments containing matching receptor (t-SNARE)

when this docking occurs vesicle will fuse with target membrane and release its cargo inside

122
Q

what are the five types of membrane proteins?

A

type I (N terminus luminal and C terminus cytoplasmic)

type II (N terminus cytoplasmic and C terminus luminal)

type III (multiple transmembrane domains; I and II have just one)

anchored proteins (stay on luminal side but anchored to membrane by lipid or GPI)

peripheral proteins (just bind a protein already inserted such as type I-III)

123
Q

how does targeting of viral proteins from the ER to the Golgi occur?

A

proteins that want to exit have motifs/amino acid sequences called ER exiting or export signals on cytoplasmic tail

cytoplasmic tail exposed toward outside so COP can recognise them and they are trafficked to Golgi in vesicles - this is an active process catalysed by Sar I GTPase

v-SNARE docks with t-SNARE to release cargo - catalysed by Rab GTPases

this process already exists in host cell so viruses just exploit it and don’t encode their own COP or v/t-SNARE - but they do have specific signals on their cytoplasmic tail

124
Q

give an example of how a viral protein is trafficked from ER to Golgi?

A

VSV G protein has di-acidic amino acid (DXE) motif in its cytoplasmic tail

COP will recognise this DXE sequence and take the protein into the vesicle

125
Q

how was it shown that exit motifs (export signals) exist on viral membrane proteins?

A

stain the cell with different dyes for different compartments and to visualise that when you mutate the motif the viral proteins no longer localise at the Golgi

this has been shown for M protein of MERS-CoV

can also be shown that ER-localised proteins like VACV localise to Golgi when appended with VSV G cytoplasmic tail

126
Q

how does signal-sequence independent targeting of viral proteins occur?

A

these proteins are synthesised on free ribosomes and don’t have transmembrane domain or signal peptide so are anchored to membranes through lipid modifications

but need to be targeted to a membranous compartment such as mitochondria and so one of the ways they get to it is getting modified by lipids

two kinds of lipid modification - myristolation and palmitoylation

these lipid modifications facilitate the association of the protein to the membrane through anchoring

127
Q

what is myristoylation?

A

modification of a 14-carbon fatty acid chain

modification occurs at the N terminal glycine and motif for that is MGXXXS/T (X meaning could be anything and S/T meaning its always one of those two)

examples include HIV Gag protein and Lassa virus Z protein

128
Q

what is palmitoylation?

A

modification of a 16-carbon fatty acid chain

modification occurs on cysteine

examples include vaccinia virus F13 protein

129
Q

how has Lassa virus Z protein membrane association w/o signal sequence of transmembrane domain been shown through experimentation?

A

align Z protein sequence in lab to see no transmembrane domain or signal sequence but then gotta confirm this in lab so…

you can centrifuge the membrane at high speed to seperate out the components and the non-membrane bound proteins are heavier cause more compact and so will sediment

you can then run these on gel to see which is heavier and then distinguish if membrane bound or not

they did this with wild type Z protein and Z protein with glycine at myristoylation site mutated to alanine and you could see the mutant Z protein was heavier cause not membrane bound

this is called membrane flotation

130
Q

how does targeting of viral proteins without (or with weak) targeting signals occur?

A

weak meaning to weak to interact with host cell machinery

these proteins interact with other viral proteins which do contain targeting signals and basically piggy back off them

examples include bunyavirus G2 protein using G1 protein for targeting to Golgi

or SARS-CoV-1 spike protein using M protein for targeting to Golgi

131
Q

how was it shown that SARS-CoV-1 spike protein can target to Golgi despite having no targeting signals?

A

visualise with microscopy

when spike protein expressed alone it localised at ER but when co-expressed with M protein it localised to Golgi

shows that M protein helping spike exit ER and target to Golgi

132
Q

how does transport of the viral genome occur?

A

no need if virus assembly occurs at site of genome replication

otherwise it occurs in complex with the DNA or RNA-binding viral proteins

e.g. influenza (genome replicates in nucleus but assembly at plasma membrane) so viral RNA binds nucleoprotein (NP) and takes it there OR retroviral RNA taken to assembly site by Gag protein

133
Q

what is the medium of transport for viral proteins?

A

medium is the cytoskeleton which is made up of three components:

  • microfilaments
  • microtubules
  • intermediate filaments

the first two are dynamic in that they polymerise at one end and depolymerise at the other

134
Q

how is influenza A vRNP transported through the cell?

A

has to come out of nucleus and go through the cytoplasm

does so via microtubules (medium of transport)

135
Q

how does transport of the whole virion on microtubules occur?

A

microtubules have motor proteins called kinesin and dynein

virions interact with these via viral proteins

examples include Gag of HIV, A36 of vaccinia virus, capsid proteins of herpesvirus

136
Q

how does transport of lipid for virus assembly occur?

A

is derived from membranes of host cell compartment where final step of virus assembly and/or release occurs

e.g. Golgi membrane for vaccinia virus and herpes virus, ER membrane for coronavirus, plasma membrane for influenza virus

137
Q

what are the three ways virus release from infected cells occurs?

A

by budding (HIV and influenza) or exocytosis (herpesvirus) at plasma membrane - enveloped viruses

by cell-to-cell spread (e.g. vaccinia virus and plant viruses) - enveloped viruses

by cell lysis - mostly non-enveloped viruses (adenovirus)

138
Q

outline how viral release occurs by budding?

A

e.g. influenza virus

occurs at lipid rafts (lipid/cholesterol rich area)

all the influenza virus components assemble under the raft where there are lots of actin filaments

M2 and dynamic actin filaments help the bud start to form and once complete NA nicks the bud and virus released

139
Q

outline how virus release by cell-to-cell occurs?

A

e.g. vaccinia virus

mature virus particle travels along microtubules

goes under plasma membrane and actin filaments start polymerising behind virus particle forming actin tail which propels it through plasma membrane and into neighbouring cell

140
Q

how does viral release by cell lysis occur?

A

e.g. adenovirus

it expresses ADP (adenovirus death protein) which bursts the cell membrane allowing viral release via cell lysis

141
Q

how do you visualise virus particles live?

A

fuse viral proteins with a fluorescent protein e.g. GFP, RFP, CFP

fuse to cytoplasmic tail cause protein incorporates into envelope with the GFP on outside cause cytoplasmic tail on outside

then you can visualise the movement of the virus particles

142
Q

what is the WHO Global Alert and Response (GAR) system?

A

for public health emergencies

under the International Health Regulations agreed to by WHO-member countries; they must notify WHO of any outbreaks happening

143
Q

what are steps taken by WHO GAR system after being notified of outbreak?

A

disease surveillance and notification of public health events to WHO

verification of those public health events

risk assessment

determination if those events constitute a public health emergency of international concern

coordination of international response, containment and mitigation

goal: break inter and intra species virus transmission

144
Q

what are some key viral/host factor targets?

A

infectivity: block receptor-RBP interaction and virus entry e.g. vaccines, monoclonal antibodies, drugs

replication: block virus replication and assembly e.g. drugs targeting polymerases, poly protein processing

antagonism: strengthen host innate antiviral defence e.g. interferons, anti-inflammatory drugs

transmissibility: block virus release from host cells e.g. drugs targeting factors involved in virus release

145
Q

what are the four types of preparations for viral vaccines?

A

whole virus - inactivated (killed) by heat (60C) or chemicals (formalin)

whole virus - attenuated (live but non-virulent i.e. can replicate but can’t cause disease) by routine culture in unfavourable conditions or mutations

virus-like-particle - made when a viral protein with inherent ability to form a viral structure is expressed alone i.e. no genome

viral mRNA - purified and encapsulated in lipid nanoparticle or expressed from a vector (e.g. non-replicating virus or plasmid)

146
Q

how are virus-like-particles (VLPs) made?

A

produced at large-scale in bacterial, yeast, insect, mammalian and plant cells

gene of interest is cloned in a plasmid (bacterial/yeast cells) or viral vector (insect/mammalian/plant cells)

highly immunogenic cause structurally very similar (just no nucleic acid) to virus

147
Q

what is a drawback of making VLPs in insect cells?

A

glycosylation v important for immunogenicity of the protein

drawback of making VLPs in insect cells is that their glycosylation system slightly different so VLPs produced in insect cells slightly less immunogenic

148
Q

how do you make viral mRNA in purified formulation?

A

clone gene of interest into plasmid

linearise the plasmid

transcribe the gene into mRNA

chemically add 5’-guanine (G) cap

purify the mRNA

encapsulate in lipid nanoparticles (LNPs)

relatively simple process but requires a lot of infrastructure and has some logistical issues

149
Q

how do you express viral mRNA from an adenoviral vector?

A

adenovirus used as vector to make covid vax; have to remove virulence

E1 locus critical for virulence but not for replication - delete E1 locus by homologous recombination

this allows it to be used as a vector cause its replicating but not causing disease

then you clone in your gene of interest and grow recombinant virus in mammalian cells

150
Q

what are antiviral drugs?

A

two kinds - virus-directed (direct-acting) or host-directed (directed at component of host involved in viral life cycle) viral therapeutics

they are chemical analogues of crucial components and either compete with that component or sequester it

big challenge with these is drug resistance

151
Q

what some examples of virus-directed viral therapeutics?

A

entry inhibitors (e.g. fuzeon, amantadine)

genome replication inhibitors (e.g. acyclovir, azidothymidine, xofluza)

synthesis/assembly inhibitors (e.g. paxlovid, maviret - both inhibit viral proteases)

release inhibitors (e.g. tamiflu)

152
Q

discuss the mechanism of action of fuzeon (enfuvirtide)?

A

is a synthetic 36-amino acid peptide entry inhibitor for HIV

HIV enters through fusion with plasma membrane - its RBP is called Env and has two domains (gp41 and gp120)

gp120 binds host CD40 receptor causing conformational change, then gp120-CD40 complex bind CCR5 co-receptor causing gp41 to disassemble and expose fusion peptide allowing viral envelope to fuse and genome to enter host cell

fuzeon binds gp41 domain preventing it exposing fusion peptide and consequently preventing HIV entry

drug less effective nowadays due to resistance

153
Q

discuss the mechanism of action for amantadine (symmetrel)?

A

once influenza in endosome the M2 protein forms an ion channel in envelope allowing influx of protons and subsequently release of vRNPs

amantadine is a synthetic tricyclic amine which blocks the M2 ion channel inhibiting vRNP release and consequently influenza virus entry

Ser31Asn mutation in M2 widens the channel and decreases amantadine binding so amantadine doesn’t really work anymore cause mutation widespread in circulating influenza

154
Q

what is the mechanism of action for xofluza (baloxavir)?

A

oral drug for influenza virus - inhibits genome replication; specifically inhibits RNA polymerase subunit PA

influenza RNA polymerase three subunits - PB2 captures host pre-mRNA, PA cleaves the G cap and uses it as a primer and then PB1 elongates it - this is how viral transcription occurs

baloxavir targets and binds the PA inactivating it so no cleavage meaning viral mRNA cannot be primed

thus viral transcription cannot occur

155
Q

how do acyclovir and azidothymidine work?

A

both are analogues of nucleotides used in replication process

acyclovir is a synthetic guanasine analogue used for herpesvirus

azidothymidine is a synthetic thymidine analogue used for HIV

156
Q

what is the mechanism of action for paxlovid?

A

synthesis/assembly inhibitor (specifically viral protease inhibitor) for SARS-CoV-2

SARS-CoV-2 positive sense RNA virus so genome acts as mRNA so as soon as viral genome enters host cell it starts synthesising polypeptide which has to be cleaved into individual proteins by viral proteases - in the case of SARS-CoV-2 this is Mpro

so Mpro gets inhibited and viral replication lifecycle cannot proceed

157
Q

outline the mechanism of action for tamiflu and the molecular basis for resistance to it?

A

binds neuraminidase (NA) and stops it acting on HA and SA so virus stays attached to infected cell

His275Tyr mutation repositions Glu277 making the binding pocket a bit bigger and reducing tamiflu binding to NA

158
Q

what might host-directed viral therapeutics target?

A

host factors which are required by virus to infect/multiply (e.g. selzentry)

components of the host immune system (e.g. interferon)

compounds which dampen the hyper-immune response (e.g. immune modulatory or regulatory molecules)

159
Q

how does selzentry (mariviroc) work?

A

is a host-directed therapy for HIV which blocks co-receptor CCR5

160
Q

how are interferons used as a therapeutic?

A

large scale production using recombinant DNA technology

can then be purified and packaged and administered to patients to supplement the host immune response

161
Q

why might we provide compounds which dampen the immune response and give some examples of these compounds?

A

production of pro-inflammatory cytokine during immune response can overreact and cause cytokine storm and tissue damage

need to dampen the expression of pro-inflammatory genes

e.g. glucocorticoid or corticosteroid which are types of steroid hormones or dexamethasone for covid-19

162
Q

what might be some next-generation viral therapeutics?

A

gene editing tolls e.g. CRISPR and RNA interference

163
Q
A
163
Q
A
164
Q
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MICR337 - Virology (2 decks)

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