Virology Flashcards
(177 cards)
1
Q
replication cycle
A
attachment entry uncoating biosynthesis (genome replication) assembly exit
2
Q
virus outcomes
A
cells: acute cytopathic infection, persistent, latent, cell transformation, abortive (shuts off replication), null (not infected even if virus get in)
organism: acute, subclinical, persistent and chronic, latent, slowly progressive, virus-induced tumour
3
Q
virulence vs pathogenicity
A
capacity to cause disease
ability to cause pathology
4
Q
iceberg principle
A
spectrum of outcomes from no infection to subclinical/silent to mild, sever, death
shape of iceberg differs for diff infectious agents (very pathogenic would be bigger top half out of water iceberg)
5
Q
interferons types
A
type 1 interferons alpha/beta - upregulate antiviral proteins and MHC I and build adaptive IR
type 2 interferon gamma - made by T cells, upregulate cytokines and antivirals and MHC I and II, part of adaptive IR more than innate
6
Q
IFN alpha and beta antiviral action
A
3 phases
1) induction - recognise non-specific pathogen PAMPs with host cell surface PRRs, initiates signal cascade and activates NFKB (TF) and IRF3/7 (if IFNalpha) which go to nucleus and bind IFNbeta promoter so interferons produced
2) priming antiviral state - interferon is cytokine which interacts with cells causing signal cascade and TFs bind to genes so ISGs transcribed
3) antiviral action of ISGs - virus specific, some activate PKR so disrupt protein synthesis, ISGs include pro-apoptotic factors so apoptosis (don’t use unless really have to)
7
Q
types of antibodies (+what have in common in how act)
A
neutralizing
non-neutralizing
both can activate complement and act as ligand for Fc receptors on phagocytic cells to activate complement
8
Q
neutralizing antibodies
A
bind virions and destroy infectivity and kill before infects cell
act on free particles so aggregate together
or act on early virus-cell to stop attachment to cell/endocytosis/uncoating/post-entry step
9
Q
non-neutralizing antibodies
A
bind virions
no effect or block neutralizing Abs so -ve effect
can activate complement
10
Q
cytotoxic T cells
A
recognise AG on MHC I which causes cell death by perforin/granzyme or inhibit virus growth by cytokines
11
Q
attachment
A
primary determinant of tropism (virus specific to cells)
12
Q
entry
A
enveloped viruses fuse with cell
non-enveloped (just protein shell) by endocytosis
13
Q
uncoating
A
structural proteins disassemble to release genome
14
Q
biosynthesis
A
key step
genome replicated and viral proteins synthesised
15
Q
assembly
A
proteins form particle structure, genome packaged, envelope acquired from cell
16
Q
exit
A
leave cell by budding/exocytosis/cell lysis
may have additional maturation step like reconfigure structure
17
Q
which parts of the host does the virus use for replication and what does it have for itself?
A
uses host cell ribosomes and machinery to make proteins but carries own polymerases
18
Q
positive sense
negative sense
ambisense
A
same sense as mRNA so directly translated
complementary to coding strand so copied to make mRNA for translation
part of RNA +ve so code for protein, some -ve so need to be copied to code protein
19
Q
influenza number of H and N types
A
17H
9N
20
Q
influenza structure
A
HA haemagglutinin - more of this than.. NA neuraminidase lipid envelope M2 ion channel matrix protein inside envelope RNP (ribonucleoprotein) - segments of RNA genome wrapped up in RNP 8 segments of flu genome - 8RNPs
21
Q
RNP (influenza)
A
RNA wrapped up in nuclear proteins to protect RNA
1 viral Pol on end (subunits PB1, PB2, PA) bind 2 ends of genome segment so 5’ and 3’
makes pan handle structure (strand round and back)
(picture on word document)
22
Q
what does PB1/2 and PA stand for in influenza?
A
polymerase basic protein
polymerase acidic
23
Q
influenza genome segments
A
8, encode diff proteins
largest Pol subunits, smallest non-structural (NS1/2))
all segments in order on word document
24
Q
entry of influenza into endosome
A
HA binds sialic acid on host
linked to galactose by alpha(2,6) in humans respiratory epithelium OR
alpha (2,3) in duck gut epithelium
SO diff strains prefer diff (tropism)
25
entry and uncoating of influenza
1) HA bind sialic acid so endocytosis
2) protons enter endosome - acid causes conformational change of HA
3) releases fusion peptides so covalent attached virus to endosome membrane
4) protons enter virus via M2 - conformational change so membranes fuse and vRNPs released from M1
5) release contents to cytoplasm
6) enter host nucleus with NLS (most RNA viruses replicated in cytoplasm, influenza in nucleus)
26
mRNA synthesis for influenza (happens for each 8 segments)
1) CLEAVAGE - cap-snatching, viral pol cleave to steal cap from host, PB1 bind 5' and 3' end to align host mRNA with viral, PB2 bind cap of host mRNA and PA cleaves
2) INITIATION - 1st 10 nucleotides of capped mRNA used as primer for viral mRNA, copy viral -ve sense RNA to capped primer
3) ELONGATION - PB1 elongates mRNA till poly-U at 5', adds poly-A tail
end up with viral mRNA copy of viral -ve sense RNA but with cap at 5' and poly-A at 3'
27
influenza cRNA purpose (and what is made first)
can't use copied mRNA with 5' cap 3' poly-A for replication so need to make +ve antigenome that is an exact complementary copy of viral RNA which is cRNA
mRNA is synthesised first to make viral proteins to help with replicating with cRNA
28
cRNA synthesis in influenza
cRNP: cRNA bound by NPs (nuclear proteins) so not degraded because no cap/poly-A, and prevents poly-A so exact copy of viral genome needed for replication
29
timing of mRNA/cRNA/vRNA synthesis in influenza
lots mRNA first and proteins made
cRNA stabilised as cRNP
lots vRNA made from each cRNP (so lots vRNA but little cRNP)
vRNA stabilised as vRNP to cytoplasm for assembly
30
leaky scanning of segment 2 of influenza genome
ribosomes don't always stop at 1st AUG when scanning for start codon - depends on sequence around AUG
strong initiation context: A/G (-3) ?? ATG G
(G at +4 from A)
31
ribosomal frameshifting of segment 3 of influenza genome
1% of the time, encounters frameshift signal CGU codon which is rare (not many tRNA for it) so ribosome pauses while waits for tRNA
UUU before CGU breaks H bonds and ribosome shifts forward by 1
32
accessory protein
not structural or enzyme
| interaction with host and manipulate cellular environment
33
3 ways NS1 works (multifunctional accessory protein) in influenza
1) Inhibits nuclear export of host cellular mRNA = prevent 3-processing
stop poly-A cleavage complex so no cleavage of pre-mRNA
bind polyA pol-binding protein so no polyadenylation
no polyA so no export and more ribosomes for viral mRNA (+steal caps)
2) increase translation of viral mRNAs = bind 5' viral mRNA, recruit initiation factors and ribosomes
3) IFN antagonist - bind RIG-I and PKR proteins so block IFN activation (no mRNA export also blocks IFN synthesis)
34
NS2 - influenza
nuclear export protein
| interacts with CRM1 (exportin 1) part of nuclear pore complex required for export
35
assembly of influenza virus
1) genome leaves nucleus and inhibits interferons
2) HA, NA, M2 line up in cell membrane
3) M1 bind C terminus of membrane proteins so trigger budding
4) NA gets virus out cell, cleaves sialic acid and prevents aggregation
5) need 8 diff vRNPs in each viral particle - 1 of each
36
how does influenza package the 8 segments of its genome?
not random because would need more than 8 to get all 8 types and can't fit
specific packaging - unique sequences and packaging signals so RNPs always arranged the same in particles
37
measles disease
lower respiratory tract infection so mistaken for other viruses
10-12 days incubation
rash
risk to unborn children - fatal brain disease (SSPE) - persistent, undetected till activated, long effect on IS after recovered, then more susceptible to secondary bacterial infections
38
measles structure
looks like flu but 50% bigger
envelope virus with glycoprotein spikes outside
protein shell of matrix under envelope
surface proteins haemagglutinin H and fusion protein F to bind to host
matrix protein M
large polymerase L with phosphoprotein P as cofactor
39
attachment and entry of measles
| binding, pathway in body
binds immune cells SLAM/CD150 or epithelial cells Nectin 4 receptor
vaccine measles binds CD46
lower respiratory tract to lymph nodes to systemic to epithelial and rash and upper respiratory for shedding
40
H monomer of measles
```
globular head domain
type II membrane protein (single transmembrane domain)
N-terminus cytoplasmic tail (CT) inside
dimerises with disulphide bonds on stalk
2 dimers form dimer so tetramer
```
41
attachment and entry of measles: roles of H and F
H for attachment, F for entry
H attaches - conformational change in H and F
F into host membrane - virus and cell membrane fuse, release RNP into cytoplasm
42
F monomer of measles
```
C-terminus cytoplasmic tail
TM
FP
translated as 1 polypeptide and cleaved to FP fusion peptide at N-terminus
forms trimer
```
43
measles transcription
1) pol recognise start and end sequence of each 6 genes and makes 6 mRNAs for each
2) cap and poly-A
3) pol lets go and keeps scanning
4) some pol go to beginning, some continue, and same at each gene so polar gradient model (more made of 3' genes)
44
measles replication
need antigenome RNA template for more viral RNA
mRNA made first so viral proteins like NP can wrap new RNA when made
converts pol so don't recognise start/stop so makes whole antigenome (not 6 mRNAs)
same time course graph as influenza -transcription then translation then antigenome and new genome
45
measles protein production
```
N
P
L
M
H
F
```
46
measles accessory proteins
encoded from P mRNA
C protein - leaky scanning
V protein - same N-terminus but diff C-terminus, from insert extra G nucleotide so change reading frame
virulence factors and inhibit interferons (inhibit IRF-3 so don't stimulate interferons) and inhibit JAK-STAT pathway
47
measles exit
H and F at cell membrane
matrix recognise their cytoplasmic tails so form shell with RNP
buds off
48
HIV origins
zoonotic transmission (trans-species)
HIV-1 = more widespread, chimpanzees, arose in region where they live, 4 transmission events, 4 groups (M main cause of epidemic, N, O, P)
HIV-2 = sooty mangabey, evidence form genetic homology and geography
49
how does HIV infect cells (mechanisms)
infect CD4+ T cells, so can't defend
bind cell, fusion of membranes, capsids deposited in cytoplasm, RNA reverse transcription, proviral DNA into nucleus and integrated to host chromatin (maturation specific to retroviruses), viral RNAs transcribed and translated
50
time course of HIV infection
1) primary infection: 1st few weeks, rapid rise in virus and rapid drop in T cells, mistaken for flu
2) fights back: virus drops, T cells rebound to set point level
3) clinical latency: years w/o symptoms, steady drop in T cells, symptoms when low enough
4) death from other diseases
51
how can you tell how long till AIDS if no treatment?
how high or low is the set point level that T cells rebound to
52
HIV genome and particle
1 strand +ve sense RNA, 2 copies so diploid
enveloped
LTRs on each end of genome
multiple genes: gag structural, pol enzymes, envelope glycoprotein (all retroviruses), all as 1 polyprotein then cleaved in maturation
tat, rev, vif, vpr, vpu, nef
53
gag HIV gene
structural
MA
CA (w/ 2 copies of genome)
NC wraps RNA
54
pol HIV gene
enzymes
reverse transcriptase
Int - integrate proviral DNA to host chromosomes
Pro
55
env HIV gene
envelope glycoproteins on outside
SU (surface) GP120
TM (transmembrane) GP41
forms trimer - 3 stalks and heads
56
tat and rev HIV genes
regulatory proteins for viral gene expression
57
vif and vpr
nuclear import
58
vpu and nef
accessory proteins modify host response to virus
59
HIV receptor binding, fusion and entry
1) GP120 (envelope glycoprotein) recognise CD4 and binds
2) conformational change in GP120 so expose binding site for co-receptors CCR5 and CXCR4 chemokine receptors on host
binding site hides under variable regions V1/V2/V3 so IS can't target (&HIV changes VR rapidly)
3) coreceptor binding so conformational change
4) insert fusion peptide GP41, 1 end in virus membrane, 2alpha helices, fusion peptide stuck in cell membrane so GP41 conformational change
5) alpha helices form hairpin so membranes close together and fuse
60
uncoating of HIV
capsid deposited in cytoplasm for RT
| cyclophilin A chaperone for protein folding and assembly needed in capsid or won't be infectious (because can't RT)
61
HIV reverse transcriptase
enzyme
enters and acts within partially uncoated viral core
2 activities: pol adds deoxyribonucleotides into DNA with RNA template
RNAse (ribonuclease) degrades RNA found in heteroduplex with DNA
62
reverse transcription of HIV (diagram on word page 3)
requires primer to make DNA
1) tRNA for lysine as primer to start making DNA 5' to 3'
2) RNAseH removes RNA just copied (R, U5)
3) DNA complementary to R jumps to other side and re-base pairs (R pairs to R on opposite strand)
4) synthesise back up to tb
5) RNAse H removes rest of RNA apart from polyP which is resistant but eventually removed, so acts as primer for other strand of DNA to make dsDNA
6) 2nd jump binding of 2 tbs
7) DNA synthesis in both directions so full dsDNA provirus slightly longer than RNA because extra U3 (U3 R U5) and U5 (U3 R U5)
LTR longer because contain control regions for gene expression
63
does DNA or RNA need a primer?
DNA pol needs a primer to work
| RNA does not!
64
nuclear import of HIV
DNA provirus w/ reverse transcriptase bound
Int bind 2 ends of DNA and then MA and Vpr bind
complex docks to nuclear pore complex and imports
Vpr allows infect non-dividing cells like resting T-cells, w/o it HIV would only be able to get into nucleus when cell division breaks down nuclear pore
65
HIV integration
1) processing - integrase breaks chromosome for viral DNA to slot in
2) joining - integrase joins viral to host DNA
3) repair - host DNA repair enzymes
66
proviruses infection
infection not cytolytic (cell don't burst)
replicate with host genome and propagate to every daughter cell (not HIV cause T cells don't replicate)
inherited if infect germ cell
may be latent
67
LTR importance in retroviruses
5' LTR contains promoter and enhancer (U3 with promoter with TF binding site so recruit RNA pol2, R start site)
3' LTR contains polyadenylation site (end of R beginning of U5)
68
HIV tat
viral transactivator impacts gene expression, enhances transcription starting from LTR
HIV dies without tat
w/o tat, HIV LTR makes poorly processive RNA pol so frequently drops off and makes truncated RNA
w/ tat, full RNA pol complex and full length transcription
69
HIV TAR element
tat-response element
downstream of transcription start site
can't move it around but position and orientation dependent so function as RNA not DNA
transactivator of viral promoter
70
loop binding factor (HIV)
cyclin T1 : Cdk9 complex (aka tat-associated kinase, TAK)
kinase activated by association with tat/TAR so phosphorylates C-terminus of RNA pol II and activates it
71
HIV LTR transcription: main points
tat (what happens w/o and w/)
TAR
loop binding factor
72
HIV nuclear export
rev-mediated
1) export competes with splicing mRNA: splice out gag/pol to get env, further splice to get 2kb with tat, rev, nef
2) Rev: regulator of virion protein expression
3) can't normal export of 9kb (gag, pol) and 4kb (vif,vpr,vpu,env) because contain introns
4) alternative nuclear export: RRE (rev-responsive element) mapped to env coding region, high affinity rev binding site, rev to activate intron-containing mRNA accumulation
73
what happens without HIV rev?
| and what is Rev's function?
mRNA for gag/pol/env is reduced
tat/nef unaffected
coat RNA in long fibrils
needs activation domain, nuclear export signals
binds exportin CRM1 and exits dependent on RAN-GTP
74
translation early proteins
translation late proteins
in HIV
Tat for transcription
Rev for gag/pol/env mRNA export
Gag, Pol, Env, Vif, Vpr, Vpu
75
translation of Gag/Pol (HIV)
overlapping reading frames
90% bypass frameshift signal so make Gag
10% makes most of Gag then frameshift -1 and make Gag-Pol fusion protein
76
HIV frameshifting process
stem-loop in RNA
ribosome pauses over poly-U slippery sequence
energy breaks bonds to cause slip in frame (from steric strain)
77
HIV Env
envelope transmembrane glycoprotein
needs ER bound ribosomes, co- translationally inserted to ER membrane
GP41 membrane, GP120 ER lumen (glycosylation of GP120)
follows secretory pathway to cell membrane
clusters in high cholesterol regions of membrane (lipid rafts)
78
HIV assembly
1) only unspliced full length RNA has packaging signal psi upstream of gag coding region downstream of splice donor
2) Gag binds psi, multimerises along RNA, dimerisation signal so base pairs with self and dimer of RNA coated in Gag
3) transport to cell membrane with Env - immature virus
require endosomal sorting complex for transport, ESCRT, for vesicle trafficking
79
what does HIV assembly require?
2 copies of full RNA
2000 Gag polyproteins
lipid envelope
env glycoprotein
80
HIV budding and maturation
protease self-cleaves from Pol so cleaves Gag and Pol separate
now mature
then capsid rearranges to icosahedral capsid and protects genome
now infectious virion
81
HIV accessory proteins
Vif - inhibit host APOBEC3G antiviral causes hypermutation of HIV
Vpu - inhibit host tetherin that prevents virus release
Nef - downregulate MHC I and CD4
82
HBV (hepatitis B) virus particle structure
quite small
Dane particles - infectious
non-infectious is empty and just surface AG in membrane or pleimorphic filaments (tube of membrane)
core icosahedral with rcDNA inside (not covalently closed like plasmid) attached to polymerase P
83
HBV genome structure
relaxed circular dsDNA = rcDNA
3.2kb partially double stranded
-ve strand slightly longer so overlaps +ve (overlapping 5' ends) maintains circular shape
5' of +ve strand attached to cap
DR1 and DR2 direct repeats (because RT)
strands not linked
-ve strand is 3.4kb and +ve is 2.4kb
4 genes: P-polymerase, C-core protein, S-surface AG (polypeptides L, M, S), X-transactivator of viral transcription
whole genome codes for protein
overlapping proteins in diff reading frames so very compact
3 sizes of surface antigen - small, medium pre-S2, large pre-S1
84
summary of HBV replication
attachment & entry - large version of sAG bind liver cell
penetration of nucleus - convert to closed circle
transcription - mRNA and pregenome
capsid assembly & pregenome RT - bud into ER
OR re-enter nucleus - copy closed form
a lot not known and just thought is true
85
HBV host
very narrow host range
| only hepatocytes
86
HBV entry & uncoating
1) L HBsAG (large surface antigen) attach + entry
2) endocytosis
3) travel to nucleus, release genome
4) genome completion: P on 5' of -ve removed, 5' cap on +ve removed, host repair extends +ve strand so ends join and covalently closed
87
HBV transcription
cccDNA (covalently closed circular) template for transcription by host RNA Pol II.
contain promoters & enhancers activate only in liver cells.
X transcriptional activator activates TFs and interacts with signalling pathways.
mRNA capped and polyadenylated and not spliced.
nuclear export w/ PRE (post-transcriptional regulatory element) uses CRM1/RanGTP pathway
```
4 types of mRNA made:
full length pregenome and core pol proteins, 3.5kb
2.4kb large sAG
2.1kb medium&small sAG
0.7kb X protein
start at diff places, end all same
```
88
HBV translation
1) P (polymerase) protein: RT domain and TP domain (terminal protein)
2) cellular chaperone complex induce conformational change in P to activate (X contributes)
3) P binds epsilon of pregenomic RNA
4) P:RNA signal for core particle assembly
89
HBV how is each mRNA translated?
3.5 kb encodes core protein and polymerase and makes 2 by leaky scanning (first AUG core, 2nd pol),
more core produced
2. 4kb monocistronic large sAG
0. 7kb monocistronic X protein
2. 1kb medium and small, 2 promoters so 2 diff mRNAs
90
HBV reverse transcription
1) after 5' transcription, AA primer of P protein jumps 5' to 3' end of pregenome RNA
2) -ve sense DNA extended
3) pregenome degrade apart from 5' cap - primer for +ve sense DNA
4) 2nd jump, primer pair with DR2 then adds nucleotides for +ve strand
5) switch to use DR1 and continue
6) runs out of DNTPs = incomplete genome
91
HIV vs HBV RT
1) ENZYME: similar motifs in pol of HBV and RNAse H domain in HIV.
HBV P protein has additional spacer and terminal protein, contains tyrosine 96 (primer for -ve sense DNA)
2) PROCESS: RNA in HIV, DNA in HBV,
genome RNA (mRNA for gag/pol) vs pregenome RNA (mRNA for C, P),
functions similar but HBV Pol also primer and encapsidation of pregenome,
RT complex: subviral core particles (capsid in cytoplasm) vs nascent subviral (just started assembly)
final dsDNA linear vs circular
DNA in nucleus integrated vs not integrated to host but separate and replicate when cell divides
92
HBV assembly
2 options: DNA back to nucleus or infectious particles bud out
depends on conc of large sAG, early infection low conc. so back to nucleus for more transcription, later higher L so into ER membrane, cytoplasmic tails out and core protein recognise tails so bud and released
93
Herpes particle structure
icosahedral core w/ nucleocapsid round it, w/ surrounding Tegument (important) made of virus coded proteins
envelope w/ lots glycoproteins (6) interact with host
massive genome
big coding capacity and variation so range of hosts
1 vertex of icosahedral capsid diff structure and function (portal vertex) - genome gets in/out particle
94
HSV (herpes simplex) interactions with body
2 interactions
in epithelial cells makes more virus and kills cells,
in neurones life long latency without killing so live with virus rest of life
95
HSV mechanisms
1) infects epithelium, replication and spreads to other individuals
2) infect sensory nerve endings - capsid with genome retrograde transport to cell body
don't replicate but latent in sensory root ganglia so persist for life because neuronal cells long lived
randomly reactivates and back through pathway to produce more virus
96
HSV infection types
oral herpes simplex virus type 1 - cold sore, reoccur same place
genital herpes simplex virus type 2
varicella zoster - 1st phase chicken pox, any part of body vulnerable to re-infection with shingles, reactivate group of nerves in particular area of body, re-infection not as frequent (only 1/2)
97
HSV1 (simplex 1) genome organisation
(152kbp long)
chunks of genome repeated (TRL TRs, IRL IRs)
partially diploid - 2 copies of several genes
intramolecular recombination - hairpin bend so recombine parts like TRL with IRL so turns around
4 diff genomes: standard, L inverted, S inverted, both inverted
98
herpes life cycle
ATTACHMENT: lots glycoproteins, B and gC essential for attachment to heparan sulphate proteoglycan on filopodia
ENTRY: 2 possible modes (probably mode 1)
1) attach to surface till find binding site for gD protein (and gM and gL), bind nectins on host and fusion
2) internalised by endocytosis. fuse endosomal membrane, no acidification
fusion of nucleocapsid to cytoplasm, cytoskeleton transports uncoated capsid to nucleus, genome out through nuclear pore to nucleus
99
HSV1 gene expression (transcription, temporal phasing)
transcription by host RNA Pol II in nucleus because double stranded
temporal phasing means gene expression is at diff times in phases and not all at once (all class 1 viruses)
100
temporal gene expression of HSV
1) VP16 from Tegument protein (transcriptional activator) turns on immediate-early genes (alpha) and makes complex with host cell proteins to help (HCF host cell factor, OCT1 (TF))
2) bind motif in promoters of early genes and turn on ICP0 and ICP4 early genes
3) early phase: provide enzymes for replication
4) trigger late: before/after replication begins, encode structural proteins to make particles
101
ICP4 (HSV)
turns on next phase of early gene expression (beta)
sequence specific DNA binding activity, not specific binding sites for ICP4 in early promoters
turns off own expression so not wasteful
102
ICP0 (HSV)
opposes host response by stopping host turning virus into closed chromatin so can transcribe
replace protamines with histones so host machinery can transcribe but this turns into silent compacted chromatin but open needed for transcription
103
HSV inhibition of host gene expression
shuts off early in process
UL41 from Tegument is a ribonuclease so shuts down host gene expression by degrading mRNA (own RNA subjected but made quickly so overcome)
bind VP16 inhibits in late infection
ICP22/ICP27 blocks transcription and splicing
large genome so significantly disrupt host and would kill so change in neurones so stay latent
104
concatemers (HSV)
linear genomes joined head to tail in long string (long straight lines)
produced by ongoing replication in rolling circle mechanism
but genome is not circular so make circular by 2 ends ligating covalently
105
HSV1 replication
1) single stranded nick in circle template
3 origins of replication
2) extends from 3' end and dispaces original strand so circle unrolls, goes round in circle, 1 strand made (2 not made at same time)
3) 2nd strand made normally in sections, linear double strand comes off circle, process carries on and doesn't stop at termination so lots joined together in concatemeric DNA
106
HSV replication proteins
targets for treatment
UL30 DNA Pol
UL23 accessory thymidine kinase - make substrate from which DNA synthesised
107
assembly of HSV particle
happens w/o DNA, requires scaffolding proteins not part of finished virus (degraded to leave empty space for linear DNA)
1) empty particle's portal vertex recognise end of concatemer
2) associates, enzymes in vertex cleave DNA
3) motor proteins of vertex use ATP to pump DNA into particle (need energy because charged DNA hard to compress)
4) cleavage to finished particle, concatemer package again (same process) - no free linear genome because packaged straight away
5) finish other layers, 2 possible mechanisms but 1st more likely
1) capsid bud through nuclear pore membrane to get envelope, then fuses back out w/o envelope (too big to pass through pore) then get proteins in cytoplasm and final envelope from Golgi then out in vesicle
2) all in nucleus, acquire tegument there
evidence: enveloped between inner and outer nuclear membranes
108
Picornaviruses characteristics (structure and genome)
```
single stranded +ve sense RNA.
7-8kb RNA.
single ORF.
non-enveloped icosahedral particles.
3 proteins make surface - VP1-3, VP4 inside, 60 copies.
genome with covalently attached VPg protein at 5' end.
cytoplasmic replication.
RNA-dependent RNA Pol
```
109
enteroviruses
gut
Polio, Coxsackie, Echo
4 species: Hev A/B/C/D
100 human ones
(group same with enteroviruses, genome wise)
110
rhinoviruses
common cold
A-C specie
then subdivided into serotypes (Ab response that virus elicits and how cross react with other viruses)
if Ab responds to 2 viruses then they're same serotype
200 human ones
111
poliovirus receptor
determines host - humans/primates
CD155 receptor on humans
transmembrane anchor, Ig super family
112
poliovirus structure (diagram lecture 9 page1)
5-fold axis (vertex) formed by VP1 pentamer
canyon is where receptor binds
binding initiates structural change to deliver genetic material so infection
113
poliovirus entry
attachment: receptor binding displaces sphingosine (pocket factor, natural host cell lipid molecule that fills pocket of polio)
increase flexibility of VP1 so VP4 interact with membrane and pull membrane around particle so RNA through to cytoplasm (probs need multiple CD155s)
114
VPg in poliovirus genome
instead of cap
115
poliovirus genome
diagram lecture 9
1 ORF so post-translational cleavage
P1 capsid protein, P2 and P3 enzymes
co-stranslational cis cleavage by 2A protease - releases P1
3C cleaves itself out and cleaves others like VP0 cleaved from VP3 which is cleaved from VP1 and 2A 2B 2C P3 cleaved from each other so all separate
VP4 with VP2 cleaved from VP0 after mature particle is assembled
116
functions of poliovirus genome parts
2A/3C proteases
2B/3A rearrange vesicles
3D RNA-dependent RNA Pol
3B (VPg) / 2C (helicase) accessory replication proteins
117
polio IRES element in replication (function, host translation difference)
internal ribosome entry site at 5' end of genome
where ribosome binds
the VPg cap protects but doesn't recruit ribosome like normal caps - no IRES recruits
so using IRES means translation is diff from host so 3C can cleave host cell cap recognition to stop host translation without affecting virus
118
polio replication
+ve sense RNA to -ve sense RNA to +ve to progeny
more +ve made than -ve and only +ve released from replication complex
amplification at each stage so lots RNA made
119
polio faecal-oral transmission
from contaminated water, replicates in gut
in follicle-associated epithelium in gut e..g M cells, Peyer's Patches
back to blood via lymph - transient viraemia
99% end here so minor and non-specific symptoms
120
polio other 1-2% pathogenesis
viraemia allows replication in CNS so destruction of neurones and deform
CD155 essential for pathogenesis but more widespread in body than tissue that PV infects so tropism is post-translational step
type 1 IFN response determines PV tropism: power on innate response differs between tissues so IFN alpha/beta prevents PV in non-neuronal tissues and 1-2% may have worse innate response
121
polio vaccine types
IPV - inactivated
1st vaccine, 3 serotypes, prevents disease from wPV but not replication in gut so doesn't block transmission
OPV (Sabin) - oral, live attenuated
prevent gut replication, 3 serotypes
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3 types of attenuation for polio vaccine
empirical process for serotype 3 Sabin: no guide, just worked, serial passage in diff hosts cause genetic changes because non-natural hosts so limits growth, still replicate but no disease
molecular basis: diff from parent at specific positions, reversion occurs (selecting in opposite direction) so changes again for succession in natural human host, see which part virulent by putting diff sequences in vaccine, difference in 5' UTR in live vs Sabin vaccine
attenuating mutations: reduce binding of polypyrimidine tract binding protein to IRES so can't recruit ribosomes and slow translation
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immune responses from OPV vs IPV vaccine
live OPV produce Abs at mucosal surface (delivered mucosally), only 1 dose needed but 2 given incase don't respond
killed IPV needs multiple doses
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IPV vs OPV advantages and disadvantages
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high amounts IPV, low OPV
injection IPV, oral OPV
IPV refrigerate, OPV requires more
IPV expensive
OPV could revert to virulence
IPV for immunocompromised
OPV interference with other gut enteroviruses
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vaccine works less in less developed world maybe altered IS from other pathogens
effective in western population - no polio for many years
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is polio eradicated in 2020?
no
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what makes polio eradication possible?
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no animal reservoir
effective cheap vaccine
lifelong immunity
no long term carriers
can't survive long outside body
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what makes polio eradication difficult?
inapparent infection
symptoms similar to other diseases
reverts to virulence
recombine with other viruses to make new
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VAPP
vaccine-associated paralytic poliomyelitis
problem with Sabin OPV
reverts to virulence within 2-3 days
vaccine 2 more likely than 3 or 1 (but not vaccinated now because eradicated)
circulate to cause small outbreaks (cvDPV)
mono/bivalent more effective against PV1/3
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ongoing polio issues
how long vaccinate after last case?
funding low so poor vaccine coverage
after eradication need isolate and contain well in labs before stop vaccines
but some people might still carry w/o disease
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what is the purpose of viral diagnosis?
clinical management
public health management
monitor and plan health care provision
blood screening before transfusions
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what are the features of an ideal diagnostic test?
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specificity
sensitivity
reproducible (same answer on same sample)
cheap (large no. and in poor places)
robust (w/o expensive training)
high-throughput (lots samples quickly)
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specificity of diagnostic test
how well focuses on particular virus
| how often false positive
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sensitivity of diagnostic test
how often miss correct diagnosis - false negative
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samples for diagnostic tests
saliva, nasal swab, urine, and faeces are easy and non-invasive
blood common and CSF, but invasive, require training and sterile equipment
biopsies - tissue samples, very difficult and invasive
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methods of viral diagnosis
direct detection: test for virus/components of it like AG or nucleic acids, limited by sample if not systemic
indirect detection: test for Abs for virus, better cause all systemic, sample likely to show true +ve, but memory persist after infection
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whole virus detection (historic diagnosis)
1) isolate, grow from sample, infect mouse/culture
2) observe cytopathic effect - appearance
3) electron microscopy
4) haemagglutination - virus bind RBCs and held in suspension in lattice so see colour, quantitative+quick but not specific to virus (so can add Abs)
5) Ab neutralisation test - if correct Ab to virus then destroy it, time consuming to test all Ab possibilities
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protein immunoblotting
immobilised protein bands on strip
add sample
if Ab in sample, will bind to bands
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ELISA
enzyme-linked immunosorbent assay
1) AG on base of well
2) test serum (sample) added
Ab binds to AG on base of well if present
3) add standard/2ndary Ab which binds patient's Ab,
linked to enzyme
4) enzyme convert substrate to colour to show +ve result
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specific antibody detection types
protein immunoblotting
ELISA
IgM capture ELISA
Antigen-capture ELISA
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IgM capture ELISA
shows current infection because IgM only initial reaction
1) Ab that will bind to human IgM put on base of well
2) add test serum and IgM binds Ab on base
3) add standard AG to detect IgM
4) add IgG specific to standard AG, with linked enzyme
5) substrate to colour so +ve result
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Antigen-capture ELISA
other way round to other ELISAs - test for AG not Ab
1) put Ab specific to AG on base of well
2) add test sample - AG binds if present
3) add Ab to detect AG
4) add IgG specific to this, with enzyme so detect colour
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nucleic acid detection
direct and specific
dot-blot nucleic acid hybridisation
qPCR
bDNA assay
DNA sequencing
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slot-blot (or dot-blot) nucleic acid hybridisation
base pairing between reagents you design and virus you know sequence of
PCR - depends on primer hybridisation for specificity so design primers to base pair with virus to amplify
then bright blot means virus present but don't know how much
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qPCR
quantitative so info about amount
continuously so real time PCR show how much during reaction
Light Cycler reaction - DNA-binding dye to quantify DNA made in PCR as it proceeds so plot product against number of PCR cycles (exponential) - dilutions shift curve right
Ct threshold value - see number of cycles required for product to cross threshold
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bDNA assay
like ELISA but base pairing
1) immobilise enzymes in well: microwell base, attach capture probe and capture extender
2) patient sample DNA base pair to capture molecules and immobilised
3) add another set of probes that detection molecules can bind to
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DNA sequencing for viral diagnosis
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determine subtypes
arrays on microchips
high-throughput sequencing
computer search
important for HIV changes from drug resistance
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Hepatitis C testing
more than 1 test to balance specificity and sensitivity
| anti-HCV Ab test with RT-PCR
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why are antivirals more specific than antibiotics?
viruses vary more
| also are in host cells so need to not target host processes
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what types of infection do antivirals target?
chronic/persistent/latent
| not acute because very narrow window to target and symptoms only occur later in replication so no time to target
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what do antivirals generally do to viruses?
slow/stop replication but don't eliminate
| just reduce level to which IS can eliminate so still need intact IS
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4 properties of antiviral therapies
specificity and potency in vitro
good selective index
good therapeutic index
good oral bioavailability
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specificity and potency of antivirals
specific so don't affect host
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good selective index of antivirals
in cell culture (in vitro)
lower conc that is toxic to cell that required to eliminate virus replication
50% toxic conc. divided by 50% virus inhibitory conc.
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good therapeutic index
in vivo
min toxic dose divided by therapeutic dose
pharmacokinetics - blood conc of drug under dose regime (ADME)
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difference between therapeutic and selective index of antivirals
therapeutic is in vivo and selective in vitro
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good oral bioavailability of antivirals
fraction that gets to circulation
| need stability, resistance to enzymes
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discovery of antivirals (luck or design)
a) high throughput screening of small molecules - see if any affect growth inhibition/enzyme inhibition but need know virus and structure
b) molecular modelling - use known 3D structure to design inhibitors
c) structure-activity relationships - modify leading compounds to enhance activity/pharmacokinetics/less toxic
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Northwick Park trials 2006
changed guidelines for drug development
| test 1 person at a time, small dose increases
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drug development of antivirals
first make sure good selective and therapeutic index and safe but active, before clinical trials
then... phases of clinical trials:
PHASE 1: 1st in man trial, small no. healthy, single small dose increasing, monitor adverse effects + pharmacokinetics, 40% fail
PHASE 2: small no. patients w/ virus, 30% fail, confirm same metabolism of patients and healthy, compare with placebo or other drug (double-blind)
PHASE 3: larger no., randomised double blind w/ placebo/other drug
broader spectrum of benefit:risk ratio
PHASE 4: after approval, monitor long term effectiveness/side effects, test on other groups
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why do 40% fail phase 1 of antiviral drug development?
40% fail this phase because pharmacokinetics not as good in humans as animals
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why do 30% fail phase 2 of antiviral drug development?
not effective enough
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how many phase 3 antiviral drug development trials do you need?
need 2 good trials in more than 1 country
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1st antiviral to be discovered
for herpesviruses - target DNA replication
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influenza antivirals (when given, how few of them work, interferons)
small window of opportunity - need to use prophylactically or immediately after symptoms
(specific examples on word but this is how they act):
block M2 proton channel OR
analogue of sialic acid to stop virus release OR
analogue of ribose nucleoside so active against RNA dependent RNA Pol
Type 1 interferons (alpha/beta) against Hep B & C, clear virus 20-30% cases but make feel like flu symptoms
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HIV resistance to antivirals
reverse transcriptase has high error rate so lots mutations
protease and integrase less likely mutations
cross-resistance among NNRTIs (resistance to 1 more likely also to other)
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HIV combination therapy of antiviral drugs
replication to minimum so less chance to mutate
takes longer to resist 3 diff drugs
resistant likely less virulent
target multiple cell types and mechanisms of distribution
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current treatment regime for HIV
HAART (highly active antiretrovirus therapy)
2NRTIs and 1NNRTI
OR 2NRTIs and 1PI
need to not stop taking them
therapy depends on country, age, illness, T cell count, viral RNA level, previous therapy, resistance profile, side effects, drug interactions
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new direction in antiviral therapy
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gene therapy
RNA-based drugs
protein-based drugs
blood stem cell therapy
T cell therapy
```
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gene therapy (antivirals)
modified genes interfere with viral replication in target cells
e.g. anti-HIV into CD4 T cells - Nef and rev switched around
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RNA-based drugs (antivirals)
occur naturally
ribozymes
antisense RNAs
RNA interference
RNA decoys
could cause mutations because rely on base pairing, alternatively can knock levels of cellular protein or combine RNA therapy so target multiple
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ribozymes (RNA-based drugs)
RNA enzymes bind and cleave viral RNA targets, cleavage to sequence of choice
[e.g. Tat mRNA, Rev mRNA, U5 of HIV]
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antisense RNAs (RNA-based drugs)
bind complementary mRNAs that want to target
nuclear retention/block translation/degradation of duplex
[e.g. Tat, Rev, U5 of HIV]
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RNA interference (RNA-based drugs)
short hairpin RNAs bind to themselves, degradation with exact same sequence
DICER in own antiviral machinery detect dsRNA infection and processed into short 21-23bps dsRNA (siRNA) interferes with RISC
unwinds siRNA and holds onto 1 strand and guides to bind to mRNA with same sequence
so target viral RNAs by expressing short hairpin (shRNA) recognised by DICER and cleave RNA with same sequence
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RNA decoys (RNA-based drugs)
small RNAs identical to protein-binding sequences (TAR) so sequester Tat/Rev viral protein so can't function
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protein-based drugs (antivirals)
modify viral proteins
[e.g. mutant Rev outcompetes wt Rev so no nuclear export]
or modify cellular proteins
[e.g. TRIM5alpha 1 AA change prevent HIV infecting]
intracellular Abs - intrabodies, bind target proteins to prevent function and degrade
ZFN (zinc finger nuclease) fusion proteins bind target and cleave and repair introduces deletions and mutations
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blood stem cell therapy (antivirals)
modified DNA in WBCs
autologous - own cells modified so no rejection
e.g. make T cells resistant to HIV
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T cell therapy (antivirals)
modify T cells to be resistant, proliferate in lab
| viral RNA levels unchanged but CD4 increased even year later so improved health
