VERNON'S MODULE Flashcards
what is positive-sense?
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
what is negative-sense?
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
outline replication of the positive-sense ssRNA virus poliovirus?
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
why must RNA viruses encode their own RNA dependent RNA polymerase (RdRp) and what is it?
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
how does poliovirus get multiple proteins from a single gene?
polyprotein processing
outline poliovirus polyprotein processing?
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
how does hepacivirus (HepC) of the family flaviviridae infect the host cell and replicate?
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
what are the 5’ and 3’ non coding regions (NCR) of hepC genome?
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
how does canonical translation initiation occur (like normally in our cells w/o virus)?
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
how does HepC initiate translation?
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
what kinds of translation initiation do +ssRNA viruses use?
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
once hepC polyprotein has been translated, how does protein coding and processing occur?
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
where do HepC proteins go following translation and processing and what functions do they carry out?
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
what are membrane associated replication complexes and what do they provide?
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
what is the Sindbis virus?
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)
outline expression and polyprotein processing of Sindbis non-structural (NS) proteins (first ORF)?
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
outline expression of Sindbis virus structural (S) proteins
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)
what is polyprotein123 and polyprotein 1234?
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
when does RNA copying begin to occur in sindbis virus and what occurs following that?
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)
why aren’t all proteins made at the same level (using sindbis virus as example)?
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
what are the two severe acute respiratory syndrome coronaviruses ?
SARS-CoV and SARS-CoV-2
SARS-CoV-2 has caused almost 800,000 confirmed cases and almost 7,000,000 deaths
what does COVID-19 affect?
ACE2 receptor present in lots of tissues so lots of different symptoms e.g. neurological, pulmonary, cardiac, GI etc. because it causes systemic infection
what underlying medical conditions make you more susceptible to severe COVID-19 and what does this indicate the importance of?
respiratory disease
heart conditions
immunocompromised
obesity
hypertension
diabetes
liver or kidney disease
hence importance of masks
who else (other than those with underlying medical issues) are more susceptible to COVID-19?
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)
can COVID-19 be detected by PCR?
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
outline the coronavirus infection cycle?
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
what does the genome of coronavirus include?
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
what kind of translation does coronavirus undergo?
canonical
despite the replication module polyprotein of coronavirus consisting of ORF1a and ORF1b, why is it actually ORF1a and ORF1ab?
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
how do SARS-CoV-2 polyproteins 1a and 1ab undergo polyprotein processing?
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
how does the subgenomic RNA get made for SARS-CoV-2 to produce its structural proteins?
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
when sgRNA is being made for SARS-CoV-2, what defines the skip point (where RdRp skips to leader)?
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
how big is the coronavirus TRS?
ranges from 60-70bp for different coronaviruses
what are some the families of -ssRNA viruses, how many segments in their genome and what similarities to they share?
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
you need RdRp to make +ssRNA but you need +ssRNA to make RdRp, so how the fuck do -ssRNA viruses express their genome?
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
what are the three things RdRp must do?
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)
what is vesicular stomatitis virus (VSV)?
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
outline the lifecycle of vesicular stomatitis virus (VSV)?
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)
how does VSV express multiple proteins when it has one piece of RNA?
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
what is controlling VSV protein expression?
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
how does VSV add the polyA tail to its mRNA and what determines reinitiation?
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
how does VSV replicate its genome if it terminates at junctions?
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
what is sendai virus genome structure?
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
how does sendai virus get alternate proteins from the P ORF - the C proteins?
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
what is leaky scanning an example of?
TRANSLATIONAL control of protein expression
(even though it -ssRNA, occurs during translation step)
how does sendai virus exhibit TRANSCRIPTIONAL control of protein expression through making alternate proteins from the P ORF?
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
how many proteins can sendai virus make from P gene and why is this important ?
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
what is sendai virus post-translational cleavage?
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
what are the sendai virus protein coding strategies?
transcription initiation
non-templated editing
translation initiation (leaky scanning)
post-translational modification
what is lassavirus?
of the family arenaviridae which have ambisense genome i.e two pieces of RNA with mixed sense on them
virions contain host ribosomes
how do arena viruses code for multiple mRNAs?
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
what are some generic features of orthomyxoviridae?
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
what must the RNA segments of influenza virus do and what are the segments?
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
how does viral nucleoprotein get into the nucleus?
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
what are the key proteins of influenza and their function?
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
what are some other important proteins in influenza (but change between strains)?
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
what are the three types of RNA synthesised during replication of influenza?
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)
outline genome replication in influenza?
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’)
outline cap stealing and polyadenylation in influenza?
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
what does the virion RNA (genome) of influenza look like?
sequences at 3’ and 5’ end will be identical between all segments - these flank the gene
negative sense (3’-5’)
what does the mRNA (for protein translation) look like in influenza virus?
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)
what does influenza virus antigenome RNA (for replication) look like?
an exact complement of the genome/virion RNA in full length (unless there’s some genetic drift)
positive sense (5’-3’)
outline the process of replicating the genome segments for influenza virus?
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)
how does influenza virus cap stealing and mRNA synthesis occur?
(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
why is influenza virus dependent on host DNA dependent RNA polymerase (DdRp) aka polII?
virus cannot replicate unless it can cap steal so uses host DdRp (polII)
no polII no mRNA
idk
what are the mechanisms allowing flu segments to encode multiple proteins from limited genetic material?
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
how does influenza make multiple proteins from its genome segments using NS segment as example?
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
how does influenza make multiple proteins from its genome segments using PA segment as an example?
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
how does influenza make multiple proteins from its genome segments using PB1 segment as example?
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
how does influenza make multiple proteins from its genome segments using M segment as example?
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)
explain what the fuck going on wit segment 7 of influenza A as it becomes protein?
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
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?
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
how does NS1 inhibit cellular mRNA processing and how does this benefit the virus?
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
what is PB1-F2?
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
what is PA-X?
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
outline influenza B mechanisms of translation regulation of proteins?
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)
what are the defences our cells have against RNA viruses?
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)
what mechanisms have RNA viruses evolved to avoid our intracellular defences?
inhibition of innate immunity
inhibition of RNA interference
anti-apoptotic factors
exploiting cellular RNA systems
what are interferons (IFN)?
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
give an overview of the antiviral state?
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)
what are the general effects of interferon?
stops replication by degrading RNA in an infected cell
stops replication any stopping translation in an infected cell
how does interferon stop replication by degrading RNA in an infected cell?
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)
how does interferon stop replication by stopping translation in an infected cell?
CHECK - INTERFERON OR PKR?
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
what is step 1 of the antiviral state?
IFN production
dsRNA induces interferon
IFN is secreted by infected cell
how does the host cell recognise foreign RNA?
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
what types of TLR recognise what type of viral RNA?
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
how does recognition of foreign nucleic acids by TLRs induce IFN production?
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
how does recognition of foreign nucleic acids by MDA/RIG induce IFN production?
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)
what is step two of the antiviral state?
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
when IFN(alpha/beta/lambda) binds IFN receptor on adjacent cells, how does the signal transduction pathway lead to gene expression?
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
what are some of the things that get turned on by IFN signalling?
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
what is the mechanism of the three key molecules in the establishment of the antiviral state?
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
what is step 3 of the antiviral state?
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
how does dsRNA activated PKR block protein synthesis?
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
how is PKR activated (it only functions in infected cells)?
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
how does 2’-5’ OligoA synthetase and RNAseL cause an antiviral effect?
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
how do some viruses block IFN at all stages?
influenza NS1 blocks IFN synthesis (production step)
pox viruses produce IFN receptor decoys (cell activation step)
adenovirus E1A blocks IFN signalling (signalling step)
how do viruses block PKR?
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
why is virtually every step of the IFN response targeted by a virus?
cause if its important viruses will target it obviously
what is RNAi?
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
how does RNAi work?
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
how do viruses block RNAi and give examples?
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
how do siRNAs cause RNAi?
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
how do microRNAs (miRNAs) cause RNAi?
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)
do viruses make miRNAs and if so how?
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
what is an example of viruses regulating their own gene expression using miRNAs?
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
what is an example of viruses manipulating the host with miRNAs?
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
what are some things in the host that DNA viruses can manipulate with miRNAs (viRNAs)?
innate and adaptive immune response
proliferation and cell survival
tumorogenesis
what is an example of host miRNAs affecting a virus?
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)
miRNAs and siRNAs are examples of what?
non coding RNAs (ncRNAs) - RNA that doesn’t encode a protein
what are some examples of viral modulation of host using other non-coding RNAs?
adenovirus VAI
flavivirus (dengue) sfRNA
what does the adenovirus ncRNA VAI do?
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
what does flavivirus sfRNA do?
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
what are some general principles of DNA viruses?
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
what are some examples of viruses with ds DNA genomes?
parvoviruses - linear ssDNA - 5kb
papillomaviruses - circular dsDNA - 8kb
polyomavirus - circular dsDNA - 5kb
these are all small DNA viruses
what are parvoviruses?
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
how do parvoviruses make protein?
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
how do parvoviruses replicate their genome?
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
what is human papilloma virus (HPV)?
> 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
outline the commonality of HPV?
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
what are papillomaviruses?
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
what does the genes are in the genome of human papillomavirus (HPV)?
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
what are the promoters for the early and late genes of the HPV-16 genome and what are the polyadenylation signals?
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
what are the roles of the E6 and E7 proteins in the HPV lifecycle?
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
what are the steps of the HPV cellular replication cycle?
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
what are the HPV E1 and E2 proteins?
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
how does HPV genome replication happen (simply)?
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
how does HPV assembly take place?
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
what are the stages of triphasic replication of HPV?
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
how is HPV is maintained as an episome (i.e. not integrated)?
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
how does vegetative replication take place?
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)
what are the two major oncogenes in HPV and what do they do?
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)
outline the differentiation dependent promoter activation of HPV?
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
how does cervical cancer occur?
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
how does expression of E6 and E7 oncoprotein cause deregulation of cell cycle?
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
how do you see infected cells over time progressively adjust HPV?
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
what are some examples of large DNA viruses with dsDNA genomes?
adenovirus
herpesvirus
poxvirus
all linear dsDNA
what are some examples of poxvirus?
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
what is vaccinia virus?
large linear dsDNA genome with early, intermediate and later genes i.e. staged order of gene expression
80 proteins, one or two lipid envelopes
outline the lifecycle of poxvirus?
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
what do the early genes of poxvirus do?
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.
outline genome replication in linear dsDNA genomes that do not form circles like poxvirus?
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
what is the structure of vaccinia virus?
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
what is monkey pox virus (MPXV)?
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
outline the lifecycle of money pox
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
what is herpesvirus?
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
what is herpes simplex virus?
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
outline primary infection and reactivation of herpes simplex virus?
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
outline herpesvirus phases of gene expression?
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
outline the genome of herpes simplex virus?
dsDNA enveloped, 150kb genome
HSV1 and HSV2 share 50-70% genome homology
has terminal repeats and control regions on genome and produces some ncRNAs
how does herpesvirus replicate its genome?
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
how does herpesvirus move into our nerve cells to establish latency?
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)
how does herpesvirus establish latency?
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
