VERNON'S MODULE

Question 1

what is positive-sense?

Answer 1

viruses with a positive sense RNA genome means the genome is in the coding sense for protein synthesis and can act as mRNA upon entering the cell

so gets translated straight to protein

Question 2

what is negative-sense?

Answer 2

a virus with negative-sense genome means the genome is in the non-coding sense for protein synthesis and must be converted to positive-sense before protein translation can commence

so undergoes transcription by RdRp to become positive-sense RNA and then can undergo translation to protein

Question 3

outline replication of the positive-sense ssRNA virus poliovirus?

Answer 3

poliovirus replicates in the cytoplasm and its genome consists of one piece of +ssRNA with a polyA tail

the RNA is infectious if it was artificially put in a cell i.e. everything the virus needs to replicate is in that RNA

once virus has released genome into cell step 1 is translation to make protein (cause +ve sense)

after translation it has all the enzymes it needs to replicate genome to it replicates it (transcription) making complimentary sense copy called -ve sense replicative intermediate (antigenome)

antigenome is then a template which can undergo transcription to make more +ve sense so you can make more protein (via translation)

so you get build up of viral proteins and when concentrations high enough you get assembly

Question 4

why must RNA viruses encode their own RNA dependent RNA polymerase (RdRp) and what is it?

Answer 4

because we do not have RdRp in our cells and the virus needs it to replicate its RNA

RdRp is a transcriptase - RNA dependent cause RNA is the template and RNA polymerase cause RNA is the product

RdRp is usually part of the replicase complex

classic antiviral target since essential for viral replication and our cells don’t have it

Question 5

how does poliovirus get multiple proteins from a single gene?

Answer 5

polyprotein processing

Question 6

outline poliovirus polyprotein processing?

Answer 6

virus has to make multiple proteins from one piece of RNA - so it makes a polyprotein i.e. the RNA genome has only one gene with start and stop codon and one ORF

polyprotein has protease activity embedded within it so following translation it cleaves itself at cleavage sites to release multiple proteins

cleavage doesn’t occur simultaneously; proteins have different forms which perform different functions e.g. 3CD propol is a protease, 3Cpro is a protease and 3Dpol is a polymerase - protease activity different when attached and when not and the precursors of that process will have their own specific function

this is a mechanism by which poliovirus can maximise coding potential

Question 7

how does hepacivirus (HepC) of the family flaviviridae infect the host cell and replicate?

Answer 7

HepC associates with lipoproteins which associate with elements on host cell surface. E1 and E2 envelope glycoproteins interact with CD81 (host receptor) and then virus gets endocytosed via CLDNI and clathrin

endosome acidification changes viral glycoprotein and capsid (core protein unknown) structure causing fusion of viral and endoscope membrane –> genome release

genome is one piece of +ssRNA –> first step translation to protein then replicase complex makes -ve sense antigenome which is template for more +ve sense RNA

viral proteins assemble on ER and modify it to membranous web making replication factor

Question 8

what are the 5’ and 3’ non coding regions (NCR) of hepC genome?

Answer 8

HepC genome single RNA strand with one ORF and one start and stop codon and embedded protease activity for cleavage

but single RNA strands can undergo local basepairing to form double strands and complex 3D dynamic structures like hairpins and loops - this is what occurs at 5’ NCR and 3’ NCR at ends of hepC RNA genome

this is a critical way that so much info can be stored in a single stranded RNA genome

5’NCR - highly conserved, domain I is critical for RNA replication, domain II-IV are IRES, critical for initiating replication and establishing cap-independent translation

3’NCR - includes conserved 98bp X-tail, U(25) essential for replication, 5B structure essential

Question 9

how does canonical translation initiation occur (like normally in our cells w/o virus)?

Answer 9

met-tRNA forms pre-initiation complex with eIF3 and 40s ribosomal subunit which assembles on cap (methyl-guanosine group on end of mRNA) and scans till it finds a start codon. Once it does it brings in the 60s subunit and begins translating protein

Question 10

how does HepC initiate translation?

Answer 10

hepC doesn’t have methyl-guanosine cap so cannot use normal canonical translation

has IRES (internal ribosome entry site) in 5’NCR which allows direct interaction with 40s subunit and eIF3

it then brings in other components and scans to find start codon and initiates polyprotein translation

so IRES allows it to bypass cap binding complex components thus suppressing host cell translation and maximising its own production

Question 11

what kinds of translation initiation do +ssRNA viruses use?

Answer 11

some use IRES initiation e.g. hepC

however most +ssRNA viruses are capped and do not have IRES structures and so canonical translation initiation is normally used

Question 12

once hepC polyprotein has been translated, how does protein coding and processing occur?

Answer 12

same as poliovirus - polyprotein has proteases in it which cleave it

NS2 and NS3 are different proteases which release themselves and other proteins with different functions from each other to produce a series of protein

extra step (to poliovirus) is that core proteins and envelope glycoproteins are released from polyprotein by host cell proteases

so virus has adapted to also use host proteases

Question 13

where do HepC proteins go following translation and processing and what functions do they carry out?

Answer 13

they assemble on ER membranes which is where the replication complex assembles (hepC replicates on membranes)

many of these proteins carry out multiple functions e.g. NS4A and NS3 complex are proteases which don’t just cleave viral proteins but also host proteins to antagonise the innate immune response (TRIF and MAVS inactivation)

NS4B is responsible for membranous web formation which allows membrane area to be maximised for replication

Question 14

what are membrane associated replication complexes and what do they provide?

Answer 14

membrane bound replication sites - these are a hallmark of +ssRNA viruses

they provide compartmentalisation, localised concentration of viral proteins, RNA tethering to replication site, provides lipids for replication (hepC needs these) and also protects from innate dsRNA responses

hepC maturation occurs via budding into ER and cell secretion pathways completing the replication cycle

Question 15

what is the Sindbis virus?

Answer 15

family - togaviridae
genus - alphavirus

+ssRNA virus w more than one ORF (i.e. more than one gene on its genome)
30-42nm icosahedral virus in envelope
60-65nm diameter spherical virion
10-12,000 base genome
5’ cap and 3’ poly-A tail (canonically translated)
purified RNA is infectious

attachment via Lamina receptor, endocytosis entry then fusion with endosome, E1 and E2 type II fusion proteins on viral surface (in envelope derived from host membrane)

Question 16

outline expression and polyprotein processing of Sindbis non-structural (NS) proteins (first ORF)?

Answer 16

first ORF; things at 5’ end necessary for viral replication

40s ribosome starts scanning from methyl-guanosine cap and continues until it finds start codon and starts translating protein and continues until it finds stop codon

this polyprotein gets cleaved by viral proteases and produces a bunch of important proteins - NSP1/methyl-transferase (puts cap on mRNA, ours is in nucleus and virus replicates in cytoplasm hence why it must encode its own), NSP2 (protease for ppp and also helices for unwinding genome for replication), NSP3 (rep complex)

but stop codon doesn’t always work in which case ribosome just keeps going (read through) to next stop codon and when read through happens there are all four proteins for replication (incl. NSP4 - RdRp which can then synthesise full length antigenome from + strand)

BUT there is still the other ORF and we haven’t made any structural proteins yet

Question 17

outline expression of Sindbis virus structural (S) proteins

Answer 17

sub-genomic RNAs (sgRNA) are a common feature in +ssRNA viruses and can encode a polyprotein

-ve sense antigenome has very strong promoter for viral replicase for viral replicase and sometimes get (from an internal promoter part way through -ve sense) production of shorter part of +ve sense which will be the untranslated second ORF of the genome - the subgenome

this will get capped by methyl-transferase and seen as a piece of mRNA so translated into polyprotein until ribosome hits stop codon

protease cleaves it into structural proteins e.g. envelope, capsid proteins, E1 and E2 (surface glycoprotein dimers)

Question 18

what is polyprotein123 and polyprotein 1234?

Answer 18

P123 - sindbis virus NS proteins (before processing) without read through (Nsp1;methyl-transferase, Nsp2;Hel/Pro, Nsp3;rep complex)

P1234 - sindbis virus NS proteins (before processing) with read through (Nsp1;methyl-transferase, Nsp2;Hel/Pro, Nsp3;rep complex, Nsp4;RdRp)

they are the same polyprotein one is just bigger cause read through occurred

Question 19

when does RNA copying begin to occur in sindbis virus and what occurs following that?

Answer 19

when ORF1 (P1234) has been translated and processed cause that has all the NS replication proteins required for replication

first step is make antigenome from +ve sense template from which it can make more +ve sense and thus more protein

BUT sometimes instead of making full-length +ve sense it makes shorter one which gets capped and encodes the second ORF (containing structural proteins)

Question 20

why aren’t all proteins made at the same level (using sindbis virus as example)?

Answer 20

not all proteins needed in the same amount

one RdRp (Nsp4) can move along template many times but capsid needs lots of protein - so virus generally needs lots more structural proteins

RdRp recognises sgRNA promoter strongly so you tend to get more of this and the S proteins it encodes

Question 21

what are the two severe acute respiratory syndrome coronaviruses ?

Answer 21

SARS-CoV and SARS-CoV-2

SARS-CoV-2 has caused almost 800,000 confirmed cases and almost 7,000,000 deaths

Question 22

what does COVID-19 affect?

Answer 22

ACE2 receptor present in lots of tissues so lots of different symptoms e.g. neurological, pulmonary, cardiac, GI etc. because it causes systemic infection

Question 23

what underlying medical conditions make you more susceptible to severe COVID-19 and what does this indicate the importance of?

Answer 23

respiratory disease
heart conditions
immunocompromised
obesity
hypertension
diabetes
liver or kidney disease

hence importance of masks

Question 24

who else (other than those with underlying medical issues) are more susceptible to COVID-19?

Answer 24

disproportionately impacts ethnic minorities including Māori and Pasifika due to higher rate of chronic health conditions, crowded housing, difficulty accessing healthcare (so due to socioeconomic inequities)

Question 25

can COVID-19 be detected by PCR?

Answer 25

yes - viral load highest in nasophayrnx hence why you swab here

but once this decreases and can’t detect infection by PCR but symptoms may persist for as long as 12 months (long covid or post-acute symptoms)

this may be cause virus infection may remain systemic - tends to come right and less likely if vaxxed

Question 26

outline the coronavirus infection cycle?

Answer 26

bind ACE2 receptor via surface glycoproteins, cleavage, entry, endosome membrane fusion and +ssRNA genome release, two poly proteins translated, proteolysis, formation of replication complex which replicates genome and makes sgRNA, translation, structural proteins assemble on ER, assembly, budding

all occurs within cytoplasm

Question 27

what does the genome of coronavirus include?

Answer 27

includes viral proteases to process polyproteins

includes RNA polymerase to copy the genome which has limited proof-reading ability so makes some errors - ideal for identifying variants and tracking

big compared to other RNA virus; about 30,000bp

also makes a bunch of sgRNAs

Question 28

what kind of translation does coronavirus undergo?

Answer 28

canonical

Question 29

despite the replication module polyprotein of coronavirus consisting of ORF1a and ORF1b, why is it actually ORF1a and ORF1ab?

Answer 29

cap at start of ORF1a so ribosome reads along translating but when it gets to where it would usually terminate (between ORF1a and 1b) there is a pseudo knot which is a region of RNA with shitloads of base pairing complementarity and thus folded into a very stable complex structure

ribosome pauses here and either drops off (good cause needs more of the proteins from ORF1a) or shifts one base back and continues (different reading frame now)

so its always ORF1ab as it has a little bit of a at the start of it due to frameshift - produces 1ab protein

Question 30

how do SARS-CoV-2 polyproteins 1a and 1ab undergo polyprotein processing?

Answer 30

with virus encoded proteases PL2 and 3CL (encoded in each polyprotein)

they recognise and cut specific viral amino acid sequences called recognition sequences - PL2 cuts specifically between a glycine and alanine within its recognition sequences while 3CL (aka Mpro) cleaves after glutamine and before a range of other amino acids depending on what amino acids are flanking

so encoded within both polyproteins there are two proteases that specifically process polyproteins into proteins necessary for replication

Question 31

how does the subgenomic RNA get made for SARS-CoV-2 to produce its structural proteins?

Answer 31

SARS-CoV-2 makes a shitload of sgRNAs and they all have identical 3’ ends and same leader sequence at 5’ end and only the first ORF (gene) of each gets translated

so as RdRp synthesising antigenome it comes to transcription regulatory sequences (TRS) and it either carries on making -ve sense of next gene or shifts/skips to leader and then reinitiates RNA synthesis of neg sense of leader sequence

this results in a bunch of -ve sense sgRNA aka anti-mRNA and RdRp can use this as template to make +ve sense sgRNA

so coronavirus sgRNA are made by a series of antisense intermediates

and then that first ORF of each of these sgRNAs gets translated to protein

Question 32

when sgRNA is being made for SARS-CoV-2, what defines the skip point (where RdRp skips to leader)?

Answer 32

the transcription regulatory sequence (TRS) varies between each gene but all have same core sequence of 5’ACGAAC

the varying sequences determine how efficiently it either continues to make longer negative sense RNA or skips to leader

so virus regulates how much of each sgRNA it makes in accordance with how much it needs i.e. it makes lots of nucleocapsid protein

i.e. variation of the TRS regulates the skip

Question 33

how big is the coronavirus TRS?

Answer 33

ranges from 60-70bp for different coronaviruses

Question 34

what are some the families of -ssRNA viruses, how many segments in their genome and what similarities to they share?

Answer 34

orthomyxoviridae (8), bunyaviridae (3), arenaviridae (2), rhabdoviridae, paramyxoviridae, bornaviridae, filoviridae (all 1)

all of these hail from a common ancestor and share:
- one major capsid protein
- helical capsids
- enveloped with viral glycoprotein spikes
- 10-15kb genomes
- genome cannot serve for translation as no cap or polyA tail

Question 35

you need RdRp to make +ssRNA but you need +ssRNA to make RdRp, so how the fuck do -ssRNA viruses express their genome?

Answer 35

they carry RdRp in the virion so RNA alone isn’t infectious but nucleocapsid is cause it carries RdRp

so the first step for -ssRNA viruses is transcription to positive sense RNA

Question 36

what are the three things RdRp must do?

Answer 36

must synthesise mRNAs (necessary to make proteins from negative sense RNA)

must synthesise full length antigenome (otherwise cannot replicate genome)

must synthesise new full-length genomic RNA from antigenome (in order to replicate genome)

Question 37

what is vesicular stomatitis virus (VSV)?

Answer 37

of the family rhabdoviridae and a classic example of transcriptional control of gene expression (-ssRNA virus; transcription initiates replication NOT translation)

genome associated with many copies of nucleo(capsid)protein (N) which is wrapped up in a layer of matrix protein (M) which is surrounded by envelope which has trimer of viral glycoprotein (G) embedded in it. Associated with the polymerase (L) is phosphoprotein (P) both of which are critical to initiate replication

has terminal repeats (tr) on its genome

Question 38

outline the lifecycle of vesicular stomatitis virus (VSV)?

Answer 38

interaction between glycoprotein and receptor induces endocytosis of virus (receptor binding, endocytosis)

endosome acidifies and H ion influx causes VSV glycoprotein fusion protein to fuse w endosome membrane releasing genome into cytoplasm

-ve sense genome (template) so first step is transcription to +ve sense mRNAs (one for each of its proteins N, P, M, G, L) - these proteins have targeting sequences so things like Matrix protein and Glycoprotein get sent out to plasma membrane - also more production of P and L which are needed for genome replication

P and L allow for production of +ve sense antigenome which is template for more genome and more mRNAs

switch between making mRNAs and genome from +ve sense antigenome is determined by presence of Nucleocapsid protein (less N = more mRNAs and more N = more full length)

Question 39

how does VSV express multiple proteins when it has one piece of RNA?

Answer 39

transcription is sequential (N then P then M, G, L)

initiation at start of first gene (t) and it gets capped then polymerase makes copy till it gets to junction between N and P where it puts on polyA tail and releases mRNA

does the same for P, M etc. (each capped and tailed)

approx 2/3 of previous gene each time i.e. more N than P, more P than M etc.

amount of mRNA determines how much protein made so you get lots more N protein than L protein and this makes sense cause each genome copy needs lots more N than L

Question 40

what is controlling VSV protein expression?

Answer 40

PL complex (P and L) initiates at 3’ end at transcription initiation sequence and starts transcribing N mRNA and caps it so ribosome can assemble

reaches termination sequence between two genes and adds polyA tail and releases mRNA for translation

2/3 of the time PL complex then restarts transcription at initiation sequence (reinitiates) and starts transcribing the next gene

1/3 of the time is dissociates and reinitiates at the 3’ end of genome

Question 41

how does VSV add the polyA tail to its mRNA and what determines reinitiation?

Answer 41

termination of upstream gene occurs when RdRp reads through UUUUUU section of template and starts chattering and synthesising the polyA tail - this is called reiterative transcription

reinitiation for downstream gene transcription occurs if RdRp recognises sequence at 3’ end of downstream gene - this occurs 2/3 times and it then caps the 5’ end

1/3 of the time it releases RNP complex and goes back to start

Question 42

how does VSV replicate its genome if it terminates at junctions?

Answer 42

after making proteins it needs to make genome - to make genome it needs to make antigenome - how the fuck if it keeps terminating at junctions

L, P and N needed to make antigenome

as N (nucleocapsid protein) accumulates this improves replication processivity as N binds nascent RNA as its being synthesised stopping chattering and preventing transcript termination

this means gene junctions get ignored and you get full length antigenome

so as N increases it becomes more efficient at making full length

Question 43

what is sendai virus genome structure?

Answer 43

of the family paramyxoviridae

-ssRNA genome; similar five genes to those found in rhabdoviruses; (3’) NP (this is the N), P, M, HN, L (5’); also has nonstructural C gene and Fo between M and HN and also variants from P mRNA in diff ORF but not as important I think

3’ and 5’ ends are complimentary, makes new mRNAs the same as VSV

Question 44

how does sendai virus get alternate proteins from the P ORF - the C proteins?

Answer 44

after NP (nucleocapsid) turned into mRNA RdRp reinitiates, caps and makes mRNA for P, chattering –> puts polyA tail on

ribosome starts scanning through but sometimes doesn’t pick up and initiate translation at start codon at 104 (AUG), in which case it continues till it hits another which is at 114 (first alternate AUG) but if it misses that then 183 (2nd AUG) then 201 (3rd). Can also initiate upstream of 104 at 81 (ACG) - called leaky scanning

this shifts the reading frame so it no longer in same ORF as P and encodes a completely different protein

C protein is in different ORF to P and has a completely different amino acid sequence - C’ (initiation at 81), Y1 (183) and Y2 (201) are variants of C (114)

so through leaky scanning sendai virus can carry more than one coding sequence on one piece of RNA

Question 45

what is leaky scanning an example of?

Answer 45

TRANSLATIONAL control of protein expression

(even though it -ssRNA, occurs during translation step)

Question 46

how does sendai virus exhibit TRANSCRIPTIONAL control of protein expression through making alternate proteins from the P ORF?

Answer 46

30% of the time when RdRp is making P mRNA it sticks in a G at 1183 causing reading frame to shift by one frame

results in a protein that looks like P but with a C terminus

10% of the time it chucks in two Gs at 1183 meaning there’s a different frame shift to the other fucking frame shift making a P protein with a third C terminus

so there are three possible reading frames for P protein all resulting in proteins with different tails

Question 47

how many proteins can sendai virus make from P gene and why is this important ?

Answer 47

P gene makes P mRNA which can encode for P, C’, C, Y1, Y2 and sometimes V and W

so seven proteins from one sequence in the viral genome and all these proteins have a differing functions

this allows it to carry a lot of information in their genome

Question 48

what is sendai virus post-translational cleavage?

Answer 48

viral protein processing can include host cell proteases and viral proteins can also undergo a maturation processing event

Fo (fusion protein) is processed to F1 and F2

this involves two cleavage events carried out by host cell proteases; leader sequence removed during secretion and then cleavage into F1 and F2 in the Golgi

Question 49

what are the sendai virus protein coding strategies?

Answer 49

transcription initiation

non-templated editing

translation initiation (leaky scanning)

post-translational modification

Question 50

what is lassavirus?

Answer 50

of the family arenaviridae which have ambisense genome i.e two pieces of RNA with mixed sense on them

virions contain host ribosomes

Question 51

how do arena viruses code for multiple mRNAs?

Answer 51

genome has two ambisense genome segments

there’s no cap or polyA tail so these cannot act as mRNA even though there’s +ve sense gene at 5’ end - so because no way to initiate translation we call it -ve sense

but RdRp can recognise 3’ end and initiate transcription to make +ve sense copy and when it does this it will cap and tail it making sgRNA in +ve sense

genome can get replicated to full length - no cap or polyA tail on full length but RdRp can recognise UTR/NCR at 3’ end and make capped and tailed +ve sense sgRNA

S segment encodes two proteins: N and GPC
L segment encodes two proteins: replicase and Z

and each gene is transcribed as subgenomic mRNA

Question 52

what are some generic features of orthomyxoviridae?

Answer 52

enveloped

spikes of Haemagglutanin and Neuraminidase (H and N refer to antigenic type e.g. H1N1)

13,500 base, segmented negative-sense RNA

complementary 3’ and 5’ ends (so the genome ends base pair)

polymerase associated with homologous ends (i.e. carries polymerase in association w genome)

Influenza A (humans, birds, swine), Influenza B and C (both humans) and thogoto virus (mammals)

all these transmission by air except the last which is ticks

Question 53

what must the RNA segments of influenza virus do and what are the segments?

Answer 53

get into the nucleus (all 8) to initiate replication cycle

the segments are vRNAs with 5’ and 3’ UTRs which are transcribed to mRNAs

segments can be referred to as vRNPs - viral RNA wrapped up in nucleocapsid and replicase complex (PA, PB1 and PB2) associated with homologous 3’/5’ RNA ends

these 8 vRNPs get packaged in virus particle in 1 + 7 structure

Question 54

how does viral nucleoprotein get into the nucleus?

Answer 54

it is imported by our cells standard nuclear import machinery - importins and RAS

there’s a peptide sequence in nucleocapsid protein which targets the vRNP segments to nucleus

Question 55

what are the key proteins of influenza and their function?

Answer 55

polymerase/replicase (PB1, PB2 and PA complex) - replication of genome

haemagglutinin (H or HA) - viral attachment protein; bind sialic acid (host receptor)

nucleoprotein (NP) - binds RNA at 1 per 20 bases (genome packaging) and also facilitates full length RNA synthesis, part of vRNP complex

neuraminidase (N or NA) - release from host cell (cleaves receptor from cell to allow release after budding)

matrix protein (M1) - matrix protein (export assembly and packaging), targeting of vRNP complex to assembly site, bridges vRNP and envelope

Question 56

what are some other important proteins in influenza (but change between strains)?

Answer 56

M2 - tetramer in membrane of infected cells (present in low amounts), ion channel protein; essential for genome release

non-structural 1 (NS1) - range of activities

non-structural 2 (NS2)

PB1-F2 - apoptosis control and replication

PA-X - host shutoff activity

Question 57

what are the three types of RNA synthesised during replication of influenza?

Answer 57

negative genome

positive (anti)genome

positive mRNA

so three RNAs produced per segment - virion RNA (the genome), mRNA (for protein translation) and antigenome RNA (for replication)

Question 58

outline genome replication in influenza?

Answer 58

occurs in nucleus

viral RNP = template for PB1, PB2 and PA (replicase)

full length antigenome synthesis

RdRp promoter in 3’ region of template strand (remember polymerases synthesise 5’ to 3’)

Question 59

outline cap stealing and polyadenylation in influenza?

Answer 59

steals methyltransferase (cap) from host mRNAs as it leaves nucleus so it can interact w ribosome

uses these to prime mRNA synthesis (cap for mRNA)

poly-U tract for poly-A tail (needed for translation i.e. makes polyA tail via chattering)

8 segments encode >11 proteins

Question 60

what does the virion RNA (genome) of influenza look like?

Answer 60

sequences at 3’ and 5’ end will be identical between all segments - these flank the gene

negative sense (3’-5’)

Question 61

what does the mRNA (for protein translation) look like in influenza virus?

Answer 61

capped and truncated i.e. different at each end

cause 3’ end got polyA tail after RNApol chattered and dissociated 17-22 nucleotides before template actually finished

at 5’ end has got cap which was stolen from host along with hosts 10-13 first nucleotides

positive sense (5’-3’)

so this how influenza exhibits transcription control of mRNA production (cap stealing from host mRNA)

Question 62

what does influenza virus antigenome RNA (for replication) look like?

Answer 62

an exact complement of the genome/virion RNA in full length (unless there’s some genetic drift)

positive sense (5’-3’)

Question 63

outline the process of replicating the genome segments for influenza virus?

Answer 63

first must make antigenome in full length (from genome) and then gotta make genome again in neg sense

initiation, dinucleotide formation and then +cRNA transcription (antigenome; c meaning complimentary & + meaning sense)

the two nucleotides are primer for polymerase

does this and makes a copy of cRNA for every segment and this gets wrapped in nucleocapsid protein forming nucleocapsid (has replication complex associated with it too i.e. PA, PB1 and PB2) - this is cRNP complex

then it goes through next round of replication to make sense genomic (negative) sense vRNP from cRNP (which is the template)

does this in reverse with dinucleotide formation priming it - this works cause strands are complementary - makes vRNP (so this is the virion RNA)

Question 64

how does influenza virus cap stealing and mRNA synthesis occur?

Answer 64

(in nucleus) PB2 binds 5’ end of host cell mRNA, cleaves it 10-13 bases from end at an A nucleotide

this A is used to prime RNA synthesis and PB1 initiates transcription from 5’–>3’

primer gets extended by PA

transcription terminates at U track 17-22nt from 5’ end

reiterative transcription (chattering) on the U-tract generates polyA tail

polymerase complex dissociates

this is how influenza makes +sense mRNA

Question 65

why is influenza virus dependent on host DNA dependent RNA polymerase (DdRp) aka polII?

Answer 65

virus cannot replicate unless it can cap steal so uses host DdRp (polII)

no polII no mRNA

idk

Question 66

what are the mechanisms allowing flu segments to encode multiple proteins from limited genetic material?

Answer 66

post-translational cleavage

ribosomal choice between AUG codons (leaky scanning)

splicing of mRNA

couple stop-start translation of tandem cisterns (stop and start codons overlapping)

ribosomal frame shifting

Question 67

how does influenza make multiple proteins from its genome segments using NS segment as example?

Answer 67

NS transcribed to NS1 mRNA

but this protein sequence has acceptor and donor sites built into it and replication occurring in nucleus meaning it has access to host splicing machinery

so sometimes a region gets spliced out to make a different protein which is NS2

these all have the cap tho and get translated canonically

Question 68

how does influenza make multiple proteins from its genome segments using PA segment as an example?

Answer 68

PA (polymerase) makes PA mRNA via standard canonical translation

but can also undergo ribosomal frameshift making different protein (PA-X)

other protein variations can occur through leaky scanning

Question 69

how does influenza make multiple proteins from its genome segments using PB1 segment as example?

Answer 69

PB1 mRNA made by standard canonical translation but can also undergo some leaky scanning or pick up different reading frames giving shorter versions of the protein with different functions

Question 70

how does influenza make multiple proteins from its genome segments using M segment as example?

Answer 70

makes mRNA for M1 (matrix protein)

also makes M2 via alternate splicing (front of M1 gets fused onto another ORF further down to make the shorter spliced mRNA which can then be translated via canonical)

Question 71

explain what the fuck going on wit segment 7 of influenza A as it becomes protein?

Answer 71

segment 7 encodes M1 (matrix) and M2 (ion channel)

M2 mRNA derived from M1 mRNA by splicing

frame shift so only first 9 aa’s of M2 same as M1

this is possible because influenza replicates in nucleus

so altogether segment 7 (-ve) gets transcribed to M1 mRNA which has methyl guanosine cap stolen along w some host nt’s, also a polyA tail where the ribosome chattered. This can then be exported out of nucleus and translated to M1 protein by ribosome

OR sometimes as being transcribed it gets processed by host cell splicing machinery (there’s donor and acceptor sequences sitting in the mRNA) and so splicing brings different coding sequence forward to the front few M1 aa’s - this makes M2 mRNA and it then gets exported and translated

Question 72

why is it convenient for influenza A that it can only get M2 when the host cell splicing machinery gets involved while its making M1?

Answer 72

because virus needs lots more matrix protein (M1) than than M2 (ion channel)

so the whole splicing mechanism is how influenza A regulates how much of what protein it makes

Question 73

how does NS1 inhibit cellular mRNA processing and how does this benefit the virus?

Answer 73

NS1 binds host cell small ribonucleoprotein and CPSF which are important for splicing, cleavage and polyadenylation

this fucks up the host mRNA processing and export so it accumulates in nucleus

this is good for virus cause it now has a stash of 5’ caps to steal in the nucleus and it also stops host mRNAs going to ribosome leaving host cell machinery for viral proteins to exploit - over all makes viral replication more efficient

Question 74

what is PB1-F2?

Answer 74

encoded in segment 2 of influenza A - made by same RNA as PB1

leaky scanning by ribosome allows for it to be made sometimes depending on which start codon it picks

not in all influenza A strains and varies in length b between them - not in influenza B at all

Question 75

what is PA-X?

Answer 75

encoded by PA mRNA

occurs due to frame shifting when ribosome slips going over UUU - causes it to jump forward one so it moves into new frame making completely different aa sequence i.e. a completely different protein

variable between strains, is an RNA endonuclease, complements NS1 activity and affects innate immune response

Question 76

outline influenza B mechanisms of translation regulation of proteins?

Answer 76

NA and NB (influenza B version of NS1 and NS2) encoded on segment 6 - alternate start codon in different frame leads to leaky scanning –> NA

M1 encoded on segment 7 - reinitiation at overlapping start/stop codons leads to it making BM2 - this works cause needs more M1 than BM2 (ion channel)

Question 77

what are the defences our cells have against RNA viruses?

Answer 77

responses to non-normal RNA e.g. dsRNA (replication of RNA genomes generates dsRNA)

PKR/IFN pathway responds non-specifically

RNA interference responds specifically (sequence recognition)

Question 78

what mechanisms have RNA viruses evolved to avoid our intracellular defences?

Answer 78

inhibition of innate immunity

inhibition of RNA interference

anti-apoptotic factors

exploiting cellular RNA systems

Question 79

what are interferons (IFN)?

Answer 79

alpha, beta and lambda

commonly produced in response to dsRNA

IFN produced (mainly locally) by an infected cell - binds neighbouring cells and initiates the ‘antiviral state’

many isoforms of alpha-interferons - different responses to different viruses

IFNalpha, beta are type I interferons

IFNlambda is a type III interferon - largely expressed at epithelial surfaces

Question 80

give an overview of the antiviral state?

Answer 80

virus infects cells

RNA recognised and range of molecules including IFN produced

IFN is secreted and sets up protective state in nearby cells (even though they haven’t seen virus)

Question 81

what are the general effects of interferon?

Answer 81

stops replication by degrading RNA in an infected cell

stops replication any stopping translation in an infected cell

Question 82

how does interferon stop replication by degrading RNA in an infected cell?

Answer 82

dsRNA activates 2’,5’oligoA synthase

2’,5’oligoA synthase makes the oligonucleotide 2’,5’oligoA

2’,5’oligoA activates the latent mRNA endonuclease RNAseL (the enzyme that degrades RNA)

RNAseL degrades all mRNA in the cell (viral and cellular)

Question 83

how does interferon stop replication by stopping translation in an infected cell?

CHECK - INTERFERON OR PKR?

Answer 83

dsRNA activates dsRNA-dependent protein kinase (PKR)

PKR inactivates the translation initiation factor (eIF2)

eIF2 inactivation shuts down protein synthesis (both viral and cellular)

cells undergo apoptosis

Question 84

what is step 1 of the antiviral state?

Answer 84

IFN production

dsRNA induces interferon

IFN is secreted by infected cell

Question 85

how does the host cell recognise foreign RNA?

Answer 85

toll-like-receptors (in endosome) - specific for ssRNA, dsRNA

Protein Kinase R - recognise dsRNA

RIG1/MDA-5 helicases - recognise replicating viral RNA and 5’ phosphate on RNA

so TLRs in endosome as well as cytoplasmic molecules recognise viral RNAs

Question 86

what types of TLR recognise what type of viral RNA?

Answer 86

TLR3 recognises dsRNA

TLR7 and TLR8 recognise ssRNA

TLR9 recognises CpG DNA (incl viral DNA)

all these TLRs that respond to viruses are found in the endosome

Question 87

how does recognition of foreign nucleic acids by TLRs induce IFN production?

Answer 87

viral RNA in endosome gets recognised by TLR3 (dsRNA), TLR7 or 8 (ssRNA) or TLR9 (CpG DNA)

signal transduction

leads to translocation, transactivation and gene expression

Question 88

how does recognition of foreign nucleic acids by MDA/RIG induce IFN production?

Answer 88

cytoplasmic receptors MDA and RIGI recognise dsRNA (which ends up in cytoplasm through viral replication)

MDA or RIGI activate MAVS protein which associates with mitochondria

MAVS activation activates signal transduction pathway which leads to translocation, transactivation and gene expression (incl IFN production genes)

Question 89

what is step two of the antiviral state?

Answer 89

IFN activates antiviral state in adjacent cells

secreted IFN binds IFN receptors on neighbouring cells

triggers signal transduction and expression of anti-viral effector molecules (AVEMs)

neighbouring cells primed to produce AVEMs - most importantly 2’-5’oligoA/RNaseL and PKR

Question 90

when IFN(alpha/beta/lambda) binds IFN receptor on adjacent cells, how does the signal transduction pathway lead to gene expression?

Answer 90

IFN alpha and beta receptor got janus kinase (JAK) and tyrosine kinase (TYK)

binding incudes phosphorlyation event –> kinases phosphorylate STATS (signal transductor and activator of transcription)

STATS phosphorylate IRF (interferon regulatory factor)

IRF translocates to nucleus and activates ISRF leading to gene expression

same pathway but diff receptor for IFN lambda

Question 91

what are some of the things that get turned on by IFN signalling?

Answer 91

over 300 interferon stimulated genes

important ones:

OAS, RNASEL gene –> degrades viral and cell RNA, induces IFNa/b

PKR gene –> effects translation and blocks protein synthesis

also apoptosis factors, replicase chain terminators and more of the things needed to respond to incoming virus e.g. IRF, RIGI/MDA5, STATs

all these things turned on in adjacent cells which haven’t even seen virus yet

Question 92

what is the mechanism of the three key molecules in the establishment of the antiviral state?

Answer 92

PKR - a kinase of eIF2 is expressed but is inactive in the absence of dsRNA

2’-5’ oligoadenylate synthetase - enzyme to make 2’5’oligoA is expressed but is inactive in the absence of dsRNA

RNaseL - a non-specific RNase expressed but inactive in the absence of dsRNA

so the cell has turned on antiviral molecules but the are not active - if virus spreads to this cell it is primed to respond

Question 93

what is step 3 of the antiviral state?

Answer 93

cell has been activated by IFN and now gets infected

virus has replicated and made dsRNA

dsRNA activates AVEMs (anti-viral effector molecules)

if cell dies quick enough (before it finishes eclipse phase and starts making progeny) virus won’t spread

Question 94

how does dsRNA activated PKR block protein synthesis?

Answer 94

normal(canonical) translation process - eIF2 associated with GDP and needs to associate with GTP to initiate translation so binds eIF2B which phosphorylate GDP to GTP activating eIF2 which then links up w met-tRNA to form ternary complex –> initiate translation after which eIF2-GDP released to run it back

PKR phosphorylates eIF2 causing irreversible binding to eIF2B which reduces levels of eIF2-GTP thus preventing translation initiation

so as eIF2 and eIF2B begin depleting so does translation initiation and translation in the cell - this limits cellular protein production thus also virus replication

Question 95

how is PKR activated (it only functions in infected cells)?

Answer 95

when expressed PKR just sits there as a monomer (inactive) and must form dimer to become active

PKR contains dsRNA binding motifs (dsRBM1 and 2) and a kinase domain (which phosphorylates eIF2)

dsRBMs bind dsRNA allowing it to be used as a scaffold between two PKR molecules (dimer) and they can then phosphorylate each other -> active -> phosphorylate eIF2

Question 96

how does 2’-5’ OligoA synthetase and RNAseL cause an antiviral effect?

Answer 96

IFNa/b stimulates expression of 2’-5’OAS

subsequent virus infection produces dsRNA, 2’-5’OAS binds this and gets activated

2’-5’OAS synthesises 2’-5’ oligo adenylate –> these oligonucleotides bind inactive RNaseL monomers causing dimerisation of RNaseL to activate latent RNase leading to degradation of RNA within the cell (viral and cellular)

degrading RNA inhibits cell translation –> cell death

Question 97

how do some viruses block IFN at all stages?

Answer 97

influenza NS1 blocks IFN synthesis (production step)

pox viruses produce IFN receptor decoys (cell activation step)

adenovirus E1A blocks IFN signalling (signalling step)

Question 98

how do viruses block PKR?

Answer 98

influenza NS1 binds dsRNA and sequesters from PKR

adenovirus VA RNA I binds dsRBM of PKR (too short and so locks it in monomer form)

hepC NS5A binds PKR dimerisation domain preventing phosphorylation

vaccinia (DNA virus) K3L binds PKR catalytic site to stop substrate phosphorylation

HSV targets and dephosphorylates eIF2 to reverse PKR action

Question 99

why is virtually every step of the IFN response targeted by a virus?

Answer 99

cause if its important viruses will target it obviously

Question 100

what is RNAi?

Answer 100

RNA interference - the sequence-specific targeting of RNA molecules in a cell

conserved in mammals, plants, invertebrates

activated by dsRNA (i.e. from virus) and functions as an antiviral defence, anti-transposon defence and for fine tuning of gene expression (via short hairpin RNA) in our cells

Question 101

how does RNAi work?

Answer 101

dsRNA is recognised by DICER which cleaves it into 21 nucleotide fragments

strand forms RNAi silencing complex (RISC) with argonaut protein and unwinds strand - RISC can then go base pair with homologous RNA sequences

targeting of silencing complex to homologous RNA sequences in the cytoplasm

degradation of RNA and silencing of gene expression (if its a perfect match)

is a potential antiviral mechanism as if it does this on ssRNA genome its fucked

Question 102

how do viruses block RNAi and give examples?

Answer 102

they make proteins that block RNAi e.g.

flock house virus (+ssRNA, 2 segments, nodavirus) has RNA1 (RdRp) and RNA2 and on its genome is B2 made from sgRNA - has been experimentally shown in replicative system expressing GFP B2 is essential for virus to grow as blocks RNAi

NS1 has also been shown to block RNAi

Question 103

how do siRNAs cause RNAi?

Answer 103

long dsRNA recognised by DICER which cleaves them into 21nt siRNA duplex

this forms RISC complex with argonaut protein which removes passenger strand leaving only single targeting strand in complex

RISC complex can then base pair with mRNA with homologous sequence to targeting strand

if a perfect match that mRNA will be degraded

Question 104

how do microRNAs (miRNAs) cause RNAi?

Answer 104

pri(mary)-miRNA containing hairpin loop with ds regions made in nucleus and gets recognised by Drosha which clips of ends leaving hairpin loop and making it a pre-miRNA

pre-miRNA gets exported out of nucleus and its dsRNA recognised by DICER which removes passenger strand, loads other strand onto argonaut to make RISC complex and off it goes to bind mRNA

main difference to siRNAs is that they will cleave if perfect match but usually not so just get in the way by binding (and not degrading) usually at control regions at 5’ and 3’ UTRs causing TRANSLATIONAL REPRESSION (block ribosome from translating)

Question 105

do viruses make miRNAs and if so how?

Answer 105

yeah - often DNA viruses

virus can encode sequence for pri-miRNAs (or pri-viRNA) which will be produced in nucleus by polII along with other viral proteins

this gets recognised by drosha and processed leaving hairpin (pre-miRNA) and then exported via exportin to nucleus and so on

Question 106

what is an example of viruses regulating their own gene expression using miRNAs?

Answer 106

EBV has latent (low level productive infection) and lytic phases - transition to lytic requires upregulation of EBV DNA pol

EBV encodes many miRNAs including miR-BART-2 which targets and down regulates EBV DNA pol (BALF5) - so making this miRNA is how it suppresses itself in latent phase and turning it off sets off full blown replication cycle (lytic phase)

this is how it controls latent to lytic transition

Question 107

what is an example of viruses manipulating the host with miRNAs?

Answer 107

human CMV makes many miRNAs incl miR-US4-1 which targets host ERAP-1 which is important for MHC-I antigen presentation - leads to weaken cytotoxic T cell response and thus less clearance of HCMV infected cells

also makes miR-UL112 which inhibits MICB expression which decreases binding to NKG2D receptor –> less NK cell killing of HCMV infected cells

Question 108

what are some things in the host that DNA viruses can manipulate with miRNAs (viRNAs)?

Answer 108

innate and adaptive immune response

proliferation and cell survival

tumorogenesis

Question 109

what is an example of host miRNAs affecting a virus?

Answer 109

HepC replication requires host miR-122 which is specific to the liver

miR-22 binds two places in hepC 5’UTR/NCR which recruits argonaut protein to IRES

this stabilises IRES, promotes translation and protects the 5’ uncapped genome end (e.g. from our own innate immune sensors)

Question 110

miRNAs and siRNAs are examples of what?

Answer 110

non coding RNAs (ncRNAs) - RNA that doesn’t encode a protein

Question 111

what are some examples of viral modulation of host using other non-coding RNAs?

Answer 111

adenovirus VAI

flavivirus (dengue) sfRNA

Question 112

what does the adenovirus ncRNA VAI do?

Answer 112

folds into massively base paired and tight structure w ds regions - binds PKR and locks it in monomer preventing dimerisation and allowing protein synthesis to continue

also makes 10^8 copies per cell which is fuck loads so it allows it to saturate exportin5 and DICER which starts to effect host miRNA biogenesis and thus host gene expression of antiviral genes

Question 113

what does flavivirus sfRNA do?

Answer 113

sitting in 3’UTR of viral genome is three helix junction and RNA pseudonot

host XRN1 degrades viral RNA but gets stuck at this knot - so you get accumulation of these knots (which are the sfRNAs) and they mop up the XRN1 which leads to reduced mRNA decay and potentially cytokine storm

sfRNAs can also bind DICER supressing RNA silencing

sfRNAs also dampens IFN responses by sequestering transactivators for ISGs in stress granules and also by inhibiting RIGI activation which limits IFN induction

Question 114

what are some general principles of DNA viruses?

Answer 114

genome is essentially similar to host genome

most replicate in the nucleus

use host cell transcription machinery; RNApolII, processing/splicing, capping (have access to these cause in the nucleus)

some use host DNA pol

poxvirus does not of these tho

Question 115

what are some examples of viruses with ds DNA genomes?

Answer 115

parvoviruses - linear ssDNA - 5kb

papillomaviruses - circular dsDNA - 8kb

polyomavirus - circular dsDNA - 5kb

these are all small DNA viruses

Question 116

what are parvoviruses?

Answer 116

non-enveloped, icosahedron

18-26nm diameter virion

genome 5kb, linear single-stranded DNA

smallest DNA viruses

human parvovirus (PVB19) causes slapped cheek syndrome

many which infect animals e.g. feline panleukopenia virus (FPV), canine parvovirus (CPV-2a and 2b)

transmission in animals usually via faeces

Question 117

how do parvoviruses make protein?

Answer 117

virus binds receptor, internalised into endosome and following maturation releases genome which goes to nucleus and replicates (using our STAT5 proteins) and after making mRNA must make proteins

has 2 ORFs on its DNA genome - makes a whole bunch of proteins through splicing and leaky scanning

Question 118

how do parvoviruses replicate their genome?

Answer 118

once in nucleus host DNA pol and STAT5 initiate replication making DNA complimentary to neg sense template (genome)

DNA pol has strand displacement capability leading to quadruplex formation and complimentary regions base pair back on themselves leading to stem loop formation

linearisation and quadruplex formation continues along with displacement activity until you have a ds concatemer (many genome copies in one big ds molecule)

capsid comes along later in replication cycle and cleaves at recognition sequences in ds region to linearise it and begins packing it into capsid to produce new virus particle

Question 119

what is human papilloma virus (HPV)?

Answer 119

> 100 different HPV which infect epithelial cells and cutaneous or mucosal sites

different HPV cause different things from asymptomatic to benign warts to invasive cancer tumours

most are low risk types and don’t cause cancer

high risk type include HPV-16 and HPV-18; causes cervical mainly but also penile, tongue, head and neck cancers and cases of this worse in poor countries due to challenges accessing healthcare, diagnostics and treatment

Question 120

outline the commonality of HPV?

Answer 120

very common STD - 80% of sexually active people infected at some point

HPV causes 1 in 20 cancers, almost all cervical cancers and most anal, mouth and throat cancers

HPV16/18 responsible for 50% of cervical cancers and cervical cancer is third most common cause of cancer in NZ woman aged 25-44

1 in 5 men have high risk/oncogenic type HPV

HPV-related oropharyngeal cancer on the rise in NZ

> 99% cervical cancers are HPV+ however only <1 to 10% high risk HPV infections lead to cancer

Question 121

what are papillomaviruses?

Answer 121

non-enveloped, icosahedron, 55-60nm diameter virion

8kb linear circular dsDNA genomes

oncogenic virus but cancer not usual outcome of infection

replicate in nucleus of squamous epithelial cells

large and diverse group of viruses found in many animals

Question 122

what does the genes are in the genome of human papillomavirus (HPV)?

Answer 122

8 major ORFs

two classes of gene expression: late (L) and early (E)

E genes are functional genes (transcription, DNA replication) and come early in infection (e.g. E6 and E7 which are the oncogenes that get incorporated)

L genes are structural genes (capsid) and come on later (e.g. L1 and L2 which help with assembly)

there’s also the upstream regulatory region (URR) upstream of all these genes

Question 123

what are the promoters for the early and late genes of the HPV-16 genome and what are the polyadenylation signals?

Answer 123

p97 is the promoter for early genes and p670 the promoter for late genes

pAE and pAL provide the polyadenylation signals

multiple promoters and splicing key to making HPV proteins

Question 124

what are the roles of the E6 and E7 proteins in the HPV lifecycle?

Answer 124

E6 - oncoprotein: inhibits apoptosis and differentiation, regulates cell shape, polarity, mobility and signalling

E7 - cell cycle control: controls centrosome duplication

these are the two genes that cause cancer

Question 125

what are the steps of the HPV cellular replication cycle?

Answer 125

bind unidentified receptor

uptake

localised to early endosome

uncoating in late endosome

L2-genome complex released into cytoplasm, goes into nucleus establishes infection at PML bodies which are localised to areas of transcription in the nucleus

once infection established you get a burst of HPV genome replication which the E1 and E2 proteins are important for

Question 126

what are the HPV E1 and E2 proteins?

Answer 126

E1 is a helicase (enzyme the separates two annealed nucleic acid strands - it needs to do this to replicate ds circular DNA)

E2 is a specificity factor - binding specifically to DNA sequences in the URR (upstream regulatory region) - it defines the start of replication

Question 127

how does HPV genome replication happen (simply)?

Answer 127

cooperative binding of E1 and E2 dimer to origin at consensus sequence in the URR

recruitment of second E1 and release/displacement of E2

formation of E1 hexameric rings which are six copies of E1

then host DNA polymerase gets recruited to initiate replication of circular DNA

Question 128

how does HPV assembly take place?

Answer 128

L2 protein accumulates at nuclear structures (PML bodies)

L2 recruits L1 capsid protein to the PML bodies

PML bodies are also sites of HPV DNA replication

proposed that capsid proteins accumulate at these sites to facilitate packaging

Question 129

what are the stages of triphasic replication of HPV?

Answer 129

virus needs differentiating cells to replicate and initial infection as basal epithelial cells (so micro abrasion nessecary). These cells are gonna differentiate; moving up in layers to stratum corner so changing what they are as a cell throughout that differentiation process

so early stages you get maintenance of genome, later stages you get amplification of virus i.e. virus doing diff things at each stage of differentiation - its adapted to each phase of epithelial differentiation - this is called triphasic replication

stages of triphasic replication:

• initial amplification post infection
• stable maintenance of the genome
• vegetative amplification of viral DNA

Question 130

how is HPV is maintained as an episome (i.e. not integrated)?

Answer 130

initial amplification rapid and transient

DNA then stably maintained at an almost constant copy number during subsequent division of basal cells

viral genome number approximately doubles during S phase of the cell cycle

segregate equally into the nuclei of the daughter cells

4 to 6 E2-binding sites in the URR known as mini-chromosome maintenance element (MME)

E2 localises to the mitotic spindles and the MME to ensure partitioning of episomes during cell division

basically the E2 on the genome is targeting the progeny genome to the mitotic spindle so when the cell divides there is also a copy of the genome going into the daughter cells

Question 131

how does vegetative replication take place?

Answer 131

stratified squamous epithelial cells exit the cell cycle and stop dividing on differentiation - cellular polymerases and replication factors only produced in dividing cells so virus can’t grow in non-dividing cell

HPV’s do not encode any gene product able to replicate their DNA directly and the virus relies on cellular DNA replicative machinery

virus encoded (onco)genes (E6 and E7) re-establish a replication-competent state in infected cells (so cells that should be non-dividing reestablished to dividing)

Question 132

what are the two major oncogenes in HPV and what do they do?

Answer 132

E6 binds and degrades cell protein p53 which normally causes cells to arrest if DNA damage has occurred

E7 binds and inactivates cell protein Rb which normally prevents cell cycle progression

HPV E6 and E7 complement each other and mediate the HPV-associated epithelial cell transformation

high-risk HPV types have E6 and E7 of higher affinity for their cellular partners (p53/Rb)

Question 133

outline the differentiation dependent promoter activation of HPV?

Answer 133

initial infection at basal layer involves p97 promoter being turned on which leads to production of early genes e.g. E6 and E7, E1, E2

epithelial differentiation triggers differentiation dependent, E2 dependent viral promoter p670 which leads to increased expression of early genes E1 and E2, viral DNA, and later on you get late genes L1 and L2 (capsid proteins) resulting in increased copy number to thousands per cell

this is the process which happens over the course of years as initial infection leads to cervical cancer

Question 134

how does cervical cancer occur?

Answer 134

HPV infection at cervix in basal epithelial cells which keeps dividing and not differentiating as well as it should so it accumulates - most people this resolves and you lose infection

but unlucky people get integration of E6 and E7 which then take out p53 and Rb and cell goes into neoplasia which is the uncontrolled growth cycle

Question 135

how does expression of E6 and E7 oncoprotein cause deregulation of cell cycle?

Answer 135

normal cell cycle as G1 phase (cell growth) and then a major checkpoint for if anything wrong –> death before doing into S phase (DNA synthesis) where replication happens and then G2 phase with another checkpoint where if p53 recognises a mutation –> cell death

if no HPV present mutated cervix cells don’t divide cause mutation detected by p53 during G2 checkpoint and cervix remains healthy

but if HPV present p53 protein has been deactivated by E6 so cell continues to grow and E7 takes away regulating of cell division and youve got a tumour

Question 136

how do you see infected cells over time progressively adjust HPV?

Answer 136

if you end up with integration of oncogenes during infection the cells are not differentiated anymore and you don’t see any stratification you just see an outgrowth of cells

this shows as changes in expression patterns accompanying the progression to cervical cancer

doctors can rate the stage of cancer from low-grade squamous intraepithelial infection (LSIL) when there is still some productive virus being produced to HSIL to cervical cancer

Question 137

what are some examples of large DNA viruses with dsDNA genomes?

Answer 137

adenovirus

herpesvirus

poxvirus

all linear dsDNA

Question 138

what are some examples of poxvirus?

Answer 138

large mf linear dsDNA virus

a lot of genus but we will look at genus orthopoxvirus

this includes vaccinia virus, variola virus (smallpox) and monkeypox virus

Question 139

what is vaccinia virus?

Answer 139

large linear dsDNA genome with early, intermediate and later genes i.e. staged order of gene expression

80 proteins, one or two lipid envelopes

Question 140

outline the lifecycle of poxvirus?

Answer 140

two forms of the virus - intracellular mature virus (IMV) and extracellular enveloped virus (EEV)

both forms can infect cell and uncut on entry

temporal cascade of gene expression - early, intermediate, late

DNA transcribed to early mRNA (early genes) which gets made to protein and initiate DNA replication

then intermediate phase/genes which involves modulating cellular responses

then late stage genes involved in assembly

replication occurs entirely in cytoplasm (unusual for DNA virus) meaning it has to provide a lot of machinery itself, DNA not infectious and has many genes for host modulation

Question 141

what do the early genes of poxvirus do?

Answer 141

extracellular modulators - growth factors, cytokines, cytokine inhibitors

intracellular modulators - apoptosis inhibition, antiviral state inhibition, host range factors

enzymes to initiate viral DNA replication

it has to be able to replicate independent of our nucleus as a DNA virus so makes things like DNA pol, DNA helices, DNA ligase, holiday junction resolvase etc.

Question 142

outline genome replication in linear dsDNA genomes that do not form circles like poxvirus?

Answer 142

5’ and 3’ ends are covalently linked and the ends are terminal repeats - virtually entire genome ds base

uses viral proteins, nicks at specific sequence and unfolds out to linear sequence so now there is a primer and polymerase can copy around the covalent end back on itself

strands dissociate and self prime and then bidirectional synthesis yields two progeny genomes closed up by ligase

Question 143

what is the structure of vaccinia virus?

Answer 143

assembly occurs in replication factory and mostly involves late genes (assembly and release)

18 core associated non-enzymatic proteins

30 core enzymes and transcription factors

25 IMV membrane associated proteins and 8 EEV associated genes

Question 144

what is monkey pox virus (MPXV)?

Answer 144

most cases prior to the late 90’s restricted to central Africa

multiple small outbreaks around the world since then, current one began 2022 and spread to over 100 countries

largest animal reservoir is rodents which spread to primates and then people

mostly spread through contact and predominantly affecting gay males

cases on the rise in western pacific and SEA atm

causes monkey pox

Question 145

outline the lifecycle of money pox

Answer 145

similar to other pox virus

two forms - IMV and EEV

uncut upon entry of cell, early proteins (growth factors and immune defence), intermediate and then late (structural)

proteins localise to factory for assembly and EV buds out through Golgi or MV through lysis - the latter (MV, lysis) more stable so better at host to host transmission while former (EV, golgi budding) more cell to cell transmission

Question 146

what is herpesvirus?

Answer 146

enveloped dsDNA virus

genome consists of long and short fragments which may be oriented in either direction giving total of 4 isomers

three subfamilies:

• alphaherpesviruses: HSV-1, HSV-2, VZV
• betaherpesviruses: CMV
• gammaherpesviruses: EBV

sets up latent or persistent infection following primary infection, reactivation more likely during periods of immunosuppression, severity of these worsens if IC

Question 147

what is herpes simplex virus?

Answer 147

spread by contact - mainly secretions

primary infection usually trivial, disease mainly effects u5y/o’s

two peaks of incidence, 0-5 and the late teens (when sexual activity starts)

many individuals experience recurrences following primary infection

generally HSV1 = below the belt and HSV2 = above the belt

2/3 all people are HSV+ - very common

Question 148

outline primary infection and reactivation of herpes simplex virus?

Answer 148

primary - HSV spreads locally, causes short-lived viremia, virus disseminates through the body and establishes latency in craniospinal ganglia

reactivation - many triggers, common clinical manifestations include acute gingivostomatitis, cold sores and in extreme cases meningitis, encephalitis

Question 149

outline herpesvirus phases of gene expression?

Answer 149

once virus has entered cell there is staged infection process; alpha phase (immediate early), beta phase (early) and gamma phase (late)

replication in nucleus - VP16 comes in as transactivator turning transcription of alpha/immediate early genes - some of these turn on beta genes and some wind down alpha, some beta genes turn on gamma genes and some wind down beta etc.

so there is continual control of timing of gene expression across infection cycle using transactivators to turn on later genes and others to turn off the earlier ones

Question 150

outline the genome of herpes simplex virus?

Answer 150

dsDNA enveloped, 150kb genome

HSV1 and HSV2 share 50-70% genome homology

has terminal repeats and control regions on genome and produces some ncRNAs

Question 151

how does herpesvirus replicate its genome?

Answer 151

linear genome with complimentary single nucleotide 3’ extensions which can base pair to form circle

replicates by rolling circle replication which along with lagging strand synthesis produces concatemer

concatemer gets packed into virus head and cleaved to give a single genome

Question 152

how does herpesvirus move into our nerve cells to establish latency?

Answer 152

virus infection in epithelial cell

escapes cell and enters terminal end of nerves and protein pUL36-pUL37 interacts with dynein and kinesin to allow movement along nerve

dynein moves things retrograde (back to cell body) and kinesin anterograde (back out)

virus hitchhikes on these molecules to establish latency and if activated can hitch a ride on kinesin back out (this is how it can form a cold sore)

Question 153

how does herpesvirus establish latency?

Answer 153

infection is life-long due to latency in neurons which is allowed through host protein interaction

in latency viral genome circularises as an episome and produces miRNAs

miR-H2 and miR-H6 target the immediate early transactivators which drive virus lytic cycle - turning them off allows it to control latency stage

also makes miRNAs called LATS which block apoptosis of the infected neurons

reactivation can occur from various host factors e.g. HCF stress response protein which can turn off viral miRNAs