Virology term 1 - negative ssRNA viruses Flashcards
Genes in -ive ssRNA viruses
RNA synthesis machinery
Fusion entry machinery
Capsid assembly machinery
Innate immunity antagonism
Size of -ssRNA viruses
Usually small; 4-12 genes.
-ssRNA genome features
No need to have functional elements of mRNAs - no cap or poly A tail.
Always needs protection, always bound to nucleocapsid.
Replication occurs via a separate +ssRNA species.
Mononegavirales
rhabdo, paramyxo, filo and borna.
Best known rhabdovirus
VSV, a cattle pathogen.
Rhabdovirus proteins
N - nucleocapsid P M G – spike protein. L - polymerase in nucleocapsid.
Paramyxovirinae family
Sendai,
Mumps,
Measles
Nipah
Pneumovirinae
RSV
Segmented -ssRNA viruses
- Arenaviridae - Lymphocytic choriomenigitis virus
- Bunyaviridae
- Tenuiviridae
- Orthmyxoviridae
Orthomyxoviridae polymerase
Polymerase is like L protein split into 3 bits
PA endonuclease activity
PB1 polymerase module
PB2 cap-binding activity
Conserved motifs in an Rdrp
7, A-F. Conservation usually homomorphs, chemically similar, rather than sequence identity.
Core domains of Rdrp
Thumb
Fingers
Palm
Thumb domain Rdrp
Involved in RNA binding, and in some it helps stabilize initiating NTPs. Thumb domain contributes to formation of NTP channel.
Finger domains in Rdrp
Finger subdomain residues pack into major groove of RNA template. I.e. important in template binding.
Palm domain Rdrp
Palm subdomain motifs are A, C and D. N-terminal aspartates co-ordinate two divalent metal ions critical for polymerase function. These co-ordinate NTPs. Motif D is important in co-ordinating binding of the correct NTP.
Additional domains to Rdrp - attachment
Flexibly attached by linker domains. Includes endonuclease domain.
Additional domains to Rdrp. E.g. in VSV and flaviviruses.
o Viral mRNa 5’ cap sysnthesis
o Methyltransferase
o Guanine-M7-methyltransferase
o Polyribonucleotidyl-transferase/guanylyltransferase.
Rdrp template recognition in
IAV: association with a promoter leads to conformations change in promoter directing transcriptional/replicative activity.
Flaviviridae: circularisation of genome and RNA structures in 5’ UTR
Picornaviridae: 5’ UTR structures and circularisation
Brome mosaic virus (+ssRNA) uses another protein to cause association.
Rdrp initiation - de novo
initiating nucleotide serves as primer for a second nucleotide. These are base paired with positions +1 and +2 of the template. This interaction needs stabilizing - usually done by residues within the Rdrp.
Rdrp structure if using primer dependent initiation.
Use of primed initiation means that a template channel which can accommodate several base pairs of dsRNA is needed, so thes polymerases lack palm and thumb domain protrusions. E.g. Coronaviridae and Picornaviridae Rdrps
Types of primer initiation for Rdrp
Oligonucleotide primed (cap-snatching), protein primed, back-primed.
Motion after initiation - Rdrp
requires conformational rearrangements from apo structure to open form. Binding of the correct nucleotide causes conformational changes leading to the formation of the closed complex (these conformational changes alter between viral families). After catalysis, reverts to open complex, with translocation of template-nascent strand duplex and release of PP¬i.
Rdrps fidelity
o Depends on conserved motifs of polymerase domain. Incorporation of nucleotides is relatively robust.
o No proof-reading domains mutation rate several orders of magnitude higher than for DdDps.
Topics to cover if ‘viral RNA and polypeptide synthesis’ asked
Transcription
Translation
Replication
Under these, consider initiation and control.
Mononegavirales - transcription
mRNAs need to be monocistronic. Single promoter: at each gene end sequence terminate mRNA and release, and either dissociate or reinitiate at next GS sequence.
Mononegavirales txn units
Use conserved gene start (GS) and gene end (GE) to give these.
Mononegavirales. The first GS signal.
Important: in RSV if this is mutated downstream sequences only transcribed at 10% of rate of WT.
Mononegavirales. mRNA release at gene end.
GE sequence and polyA tract.
Processivity through these may be intrinsic to Rdrp - mutations in RSV increase it specifically through GE tracts.
Mononegavirales. Scanning between GE and GS.
Highly efficient - even if tract is increased in length, RSV reinitiation can occur.
Mononegavirales: control of protein proportions.
Transcriptional; single pol entry point followed by attenuation (shown by UV target size study of Ball and White). Rhabdo.
Possibly partly because RNA associated with N, so cis-acting structures difficult to see, so has to interact with 3’ end and then travel along. Engages within Le promoter
Mononegavirales: control of protein proportions, gene order.
- VSV; gene order exerts strong control as polymerase has 20-30% chance of termination as traverses gene, and long pauses between genes.
Paramyxoviridae: control of protein production when polycistronic RNAs.
Sendai and measles for P(C/V).
Leaky scanning – Kozak and etc. Polymerase stuttering complicates.
P AUG most often used.
Sendai has 4 different ORFs accessed by different start sites for C. One is an ACG start codon. Variant P proteins, variant V proteins.
RNA pol stutter accesses cys rich V insertion.
Pneumoviridae: control of protein production when polycistronic RNAs.
Reinitiation translation for different proteins.
Also filoviruses.
Filoviruses: control of protein production when polycistronic RNAs.
RNA editing, reinitiation translation.
Viruses using splicing in nucleus.
Bornaviruses, orthomyxoviruses, filoviruses.
Influenza virus: transcription initiation.
capsnatching – takes cellular RNAs, degrades RNA and uses small amount of undegraded + cap to prime. PB1 causes cap-binding, PB2 cap-snatching. In RSV (paramyxo, pneumo) capping probably done by L protein.
Segmented -ssRNA sites - accessing multiple ORFs
Alternative translation initiation – N and NS in bunya.
Frameshifting for overlapping ORF (bunya)
Splicing – orthomyxoviruses.
Influenza virus: control of protein proportions.
Increased levels of early proteins needed to transcribe late protesin successfully
Increasing protein synthesis by host manipulation: -ssRNA viruses.
Decrease mRNA nuclear export.
Destroy mRNAs non-specifically.
Influenzavirus: decrease mRNA nuclear export.
Prevent cleavage of pre-mRNA…
Decrease polyadenylation by binding polyA polymerase binding protein.
Influenza: protein responsible for preventing pre-mRNA cleavage.
NS1 by binding the cellular polyadenylation/cleavage complex
Mononegavirales: replication vs transcription.
Use different promoters from transcriptase; replicase and transcriptase different complexes.
RSV replication promoters.
trailer at 3’ of genome contains promoter for replication. Complement to trailer at 5’ end leads to formation of sense genome from antisense cRNA.
Encapsidation of RSV
Cis acting sequence in Le RNA appears to promote encapsidation.
Controlling the switch between transcription and replication. Rhabdoviruses
Controlled by phosphorylation of P.
Seems to depend on 2 promotors (can only take place if enough newly synthesised N available for encapsidation of replicated genome —> N trans-acting regulation based on quantity - needed to form replicase)
Controlling the switch between transcription and replication. Paramyxoviruses.
promoter control over transcription vs replication
Controlling the switch between transcription and replication. Pneumoviridae.
control using M2 proteins; M2-1 is a an anti-terminator (to stop intragenic termination, but encourages processivity, acts by binding P), while M2-2 downregulates transcription and upregulates replication.
Controlling the switch between transcription and replication. RSV.
Needs Le sequence.
Packaging the right RNA. Paramyxo.
Imbalance of synthesis. Packaging of anti-genomes does occur.
Bipartite promoter associating N with 6 nucleotides.
Rhabdovirus replication (mononegavirales)
Primary transcription
Replication via an intermediate.
Secondary transcription.
Order of genes in rhabdoviruses
N, P, M, G, L.
Rhabdovirus intergenic space
23 nt AUAC U7 G/CA Capping
Rhabdovirus intergenic space AUAC
C essential for termination, regulates slippage as well.
Rhabdovirus intergenic space U7
slippage: reduction in size abolishes termination.
Potential roles of leader RNA
Obligatory precursor to mRNA txn?
Abortive attempt at genome replication?
Le txn does not always proceed transcription (engineered U rich Le sequence and UV target size experiment).
Mononegavirales genome replication
Requires processivity.
Mononegavirales transcriptase complex.
L-Px3. P is phosphorylated.
Mononegavirales replicase complex.
L-(N-P). P is not phosphorylated.
Packaging the genome: rabies vs VSV
VSV: 5’ Tr sequence
Rabies: excess of -ive ssRNA.
-ive ssRNA plan lifecycle
Forms of RNA
Mechanisms
Control
-ive ssRNA plan lifecycle - mechanisms
translation, synthesis of viral RNA
-ive ssRNA plan lifecycle - controls
protein proportions (translation)
initiation (protein proportions transcription)
transcription/replication switch
Control of packaging of vRNA vs cRNA
-ive ssRNA plan lifecycle - forms of RNA
Monopartite vs segmented Genome mRNAs (incomplete of genome) Replicative intermediate panhandle structure Le RNA in some.
Le RNA present in
RSV. Check others.
-ive ssRNA plan lifecycle; mechanisms; synthesis of viral RNA.
Structure and action of Rdrp. Non-segmented transcription vs segmented transcription Control of protein proportions. Accessing different ORFs Transcription replication switch.
-ive ssRNA plan lifecycle; mechanisms; synthesis of viral RNA; non-segmented transcription.
Attenuation
Reinitiation
Transcription/replication switch monopartite
Rhabdo: different promoters used for replicase/transcriptase. This determined by levels of N and phosphorylation.
Paramyxoviridae: same promoter - probably determined by N or P phosphorylation.
Pneumoviridae: use M2 proteins.
P phosphorylation
Probably in domains II and III
vRNA packaging
Make more genome due to stronger promoter than antigenome. Rabies.
Use selective signal.
-ive ssRNA plan lifecycles; mechanisms; translation
Increasing coding capacity
Different types of initiation
-ive ssRNA plan lifecycles; mechanisms; transcription; increasing coding capacity.
RNA stuttering
RNA editing
Splicing
-ive ssRNA plan lifecycles; mechanisms; translation; increasing coding capacity
Reinitiation
Leaky scanning
Frameshifting
-ive ssRNA plan lifecycles; mechanisms; transcription; increasing coding capacity; RNA pol stuttering
Pneumovirinae - P(C/V).
-ive ssRNA plan lifecycles; mechanisms; transcription; increasing coding capacity; RNA editing
Pneumovirinae. o Insert 1 or 2 G residues at certain locations due to reiterative copying mechanism. Changes open reading frame for variant P proteins, or in some viruses cys rich V insertion. RNA pol stuttering.
-ive ssRNA plan lifecycles; mechanisms; transcription; increasing coding capacity; splicing
Borna and orthomyxo
Splicing mechanism
Uses cellular proteins. 2 trans-esterification reactions cutting out an intron. Requires a complex spliceosome for high degree of accuracy
Orthomyxo splicing
o M1 spliced to M2 and mRNA3
o NS1 spliced to NS2.
Roles of NS1 in influenza
Inhibiting nuclear export of mRNAs.
Increasing translation of vmRNAs.
Activating PI3K
IFN antagonist.
Bunya leaky scanning
N and NS,
Segmented virus.
Flu ribosomal frameshifting
At distinct signal in segment 3.
Segmented -ssRNA virus transcription.
cap-snatch/cap synthesise and transcribe.
mRNA may have multiple ORFs.
Ambisense coding strategy.
Accessing multiple ORFs in segmented -ssRNA virus.
Commonly: alternative translation initiation.
Rarely: ribosomal frameshifting.
Sometimes splicing: orthomyxo flu A segments 7 and 8.
Segmented viruses: transcription replication switch
cRNA stabilisation model: binding of NP to elongating strand enables pol to read all the way to the 5’ end of genomic RNA to give full length cRNA —> maintain integrity of viral genome.
If doesn’t also bind 5’ end, then shrinking loop does not occur, so no RNA pol stuttering at run of Us. No stuttering in 5% of cases –> replication. Differentiated by different structures (capped, associated with RNPs).
svRNAs
Cap-snatching in influenza
PB1 binds 5’ end of vRNA. Binds capped cellular mRNA.
PA endonuclease degrades most of mRNA. Remainder is used to prime Rdrp.
Influenza: polyadenylation.
Polyadenylation occurs in most cases on polyU tract. Shrinking loop hypothesis states that the snap-back hairpin structure of the RNA leads to Rdrp sitting on the polyU tract.
dsRNA families
Reoviridae
Reoviridae structure
Triple layered particle
Reoviridae genome structure
11 segments.
positive strand capped not polyA’d.
Antisense not capped
All monocistronic except seg. 11
Reoviridae segment 11
First AUG –> NSP5, second AUG –> NSP6.
TLP structure (dsRNA)
Core
DLP
TLP
Components of reoviridae core.
VP2, core protein 120 molecules/virion.
VP1, Rdrp also structural role.
VP3, capping enzyme.
VP3 (reoviridae)
Capping enzyme - methyltransferase, guanylyltransferase.
Components of DLP (reoviridae)
core + VP6 (inner capsid, required for transcription)
Components of TLP (reoviridae)
DLP + VP5, VP8 (before cleavage are VP4) and VP7.
Reoviridae - NSP2 role
NSP2 may regulate translation/replication switch. Binds both NSP5 and ssRNA.
Reoviridae - NSP4 role
is multifunctional, interacts with DLPs, releases Ca++ from stores (stabilises TLP), caps viroplasms, changes membrane permeability, acts as viral enterotoxin.
Reoviridae lifecycle
Attached, endocytosed, escapes from early endosome, transcription, translation, replication, assembly, budding into ER then losing membrane during exocytosis.
Reoviridae cleavage of VP4
Required for attachment.
Trypsin like proteases in the gut cleave VP4 to give VP5* and VP8* fragments.
Reoviridae attachment
VP8* has a galectin-like fold, which may or may not bind receptors contain sialic acid. Co receptors may include integrins and/or HSP70.
VP5* has lipophilic domains exposed in post-penetration umbrella conformation.
Reoviridae from escape from the endocytic vacuole.
Loss of the outer capsid is simultaneous with escape. ESCRT pathway may be involved.
DLP is transcriptionally active
Reoviridae transcription
Makes mRNA.
Replicative intermediate.
Channel specialisation model.
Reoviridae channel specialisation model.
now favoured model
each dsRNA associates with one polymerase complex and one channel. Other model is that one or two genome segments associate with several.
Reoviridae mRNA
not polyadenylated, but is circularised by NSP3, which recognises UGACC and binds eIF4G as well. Higher affinity than PABP, and PABP localised to nucleus (host shut off), so rotavirus mRNA preferentially translated.
mRNAs form panhandles.
Reoviridae VP1 structure in transcription - tunnels
• 4 tunnels lead to catalytic site of Rdrp
o Entry of NTPs
o Entry of template
o Exit of +ssRNA (product) – this tunnel is positioned so +ssRNA is guided towards class I channel in VP2 to go to cytoplasm.
o Exit of –ssRNA or dsRNA.
Reoviridae replication location
In viroplasms
Viroplasms
NSP2, NSP5 are enough to induce viroplasms by reoviridae.
Packaging
Panhandles may be important
2 different models - core-filling and concerted model.
Reoviridae - panhandles in packaging.
Association of UTRs with VP1 and VP3 may lead to selective packaging.
Core-filling model - reoviridae
RNA pumped into pre-formed core.
Concerted model - reoviridae
VP1 and VP3 associate with positive RNA, core assembles round it.
Reoviridae maturation and exit.
NSP4 acts as receptor for VP6 for budding into ER (transient envelopment). As it acquires VP4 and VP7 this is lost by an unknown mechanism.
Rhabdovirus replication as a drug target.
Closed form of the N-RNA may protect the viral genome from recognition by cellular TLRs such as RIG-1 (recognises the 5’ PPP) or TLR3 (dsRNA) —> drugs that mimic the factor that opens up the nucleoprotein would expose the viral RNA to innate immunity receptors, and drugs that stabilise the closed form of N would inhibit polymerase processivity
VSV: increasing translation of viral RNAs
VSV known to cause sequestration of eIF4E (viral proteins synthesis unaffected - suggests distinct mechanism) and M protein impedes export of mRNA from nucleus
Initiation paramyxoviruses
mRNA synthesis initiates at a nucleotide signal (gs1) within the genomic promoter at 3′ end
again have transcriptional gradient = dominant control for gene expression
RSV may use non-template initiation for genome replication —> method of error correction
polymerase activity modulated by free N levels
Leaky scanning - Kozak consensus
context of AUG codon affects 60S ribosome binding —> P gene AUG used most often but alternative start sites exist
C ORF in sendai has 4 possible products depending on start site e.g. one (C’) is an ACG (leaky scanning - good context but unusual start codon) —> some may have roles in blocking IFN response
Y1 and Y2 versions are probably translated by ribosome shunting as poor context —> ribosome directly translocated from upstream initiation complex to AUG without need for eIF4A to unwind secondary RNA structure
Gives control of amount of each protein produced
Borna RNA
Less known about them but = animal pathogens infecting CNS
Use nuclear replication presumably because of use of splicing machinery
Splicing generates subgenomic RNAs (in addition to usual control strategies)
Recent evidence for inserted copies into mammalian genomes via RT —> controversial link with psychiatric disease
Flu increasing coding capacity.
Ribosomal frameshifting to access an overlapping ORF e.g. in segment 3 of flu to produce PA and PA-X (endonuclease - cleaves host mRNAs) —> +1 frameshift thought to be stimulated by a slow to decode A-site codon with efficiency of 1-2%
Splicing to produce alternative mRNAs —> requires host machinery found in nucleus e.g. segments 7 and 8 (M1/M2 and NS1/NS2)
Alternative translation initiation on overlapping ORFs e.g. PB1 varients
svRNAs in flu
svRNAs correspond to 5’ end of vRNA —> expression correlates with vRNA accumulation and bias in RdRp towards replication —> may trigger viral switch from txn to rep through interactions with pol
depletion of svRNA has a minimal impact on mRNA and cRNA but results in a dramatic loss of vRNA in a segment-specific manner
Pneumovirinae translation
extra genes, long and varied intergenic regions and one overlap —> use start-stop translation (pol pauses until 2nd AUG recognised)
Flu leaky scanning
flu B NA and NB glycoproteins, fluA PB1, PB1-N40, PB1-F2
P
phosphoprotein. Cofactor for L.
Paramyxoviridae: initiation of synthesis using antigenome as template
Uses Tr promoter at 3’ end. In RSV this can happen by backpriming, otherwise de novo.
genome packaging in influenza
Export: cRNPs are not exported via Crm, wherease vRNPs are. Also U12 and U13 regions differentiate vRNA from cellular RNA.
vRNP interactions ensure that all segments are packaged; neither completely daisy chain nor completely master.
vRNP interactions in influenza
Slightly differ between different strains. 1 is at centre. Binds 2, 3, 7 and 8. 7 binds 4 and 6. 6 binds 3 and 7. 8 binds 5.
