7 & 8 - RNA Virus Replication Flashcards
1
Q
RNA viruses baltimore groups
A
3, 4, and 5
2
Q
How do RNA viruses replicate their genomes
A
- Using virally encoded RNA dependent RNA polymerase (RdRp)
- Some have methylase activities that synthesise mRNA caps
- Some may ‘stutter’ at poly U tracts to generate polyadenylated mRNAs
- Associated with other viral and cellular proteins to form replicase complex
3
Q
Three types of RNA synthesised during replication
A
- Genome
- Copy of the genome (anti-genome)
- mRNA
- Some viruses may also synthesise subgenomic mRNAs
4
Q
Three main processing steps of RNA
A
- Capping at 5’ end
- Addition of polyA tail at 3’ end
- Splicing to remove introns
5
Q
RNA viruse replication
A
- Usually in cytoplasm (besides influenza and HIV)
- plus strand RNA viruses have genomes that are functional mRNAs (may be capped and polyadenylated)
- Minus sense (-) RNA viruses must carry RdRp in capsid, to begin replication
6
Q
Earliest viral proteins synthesised
A
Those needed to synthesise new genomes, and RdRp
7
Q
Virus replication complexes (VRCs)
A
- Many viruses that replicate in the cytoplasm compartmentalise genome replication and transcription of proteins to virus replication complexes (VRCs)
- Escape recognition from host defences and recognition by toll-like receptors
- Within the VRC the + strand genome is used as a template to synthesize full-length anti-genomes (- sense), which remain hydrogen bonded to the + strand
8
Q
What are VRCs assembled by
A
- Non-structural viral proteins
- Viral genomes
- Host lipids
- Host proteins
9
Q
Class 4 (+) RNA viruses
A
- Express multiple proteins from a single genome (a capsomer and RdRp)
- Eukaryotic mRNA encodes just one protein
- Immediate translation is critical for viral replication because it results in synthesis of the viral RdRp
- RdRp subsequently synthesizes the replicative forms and viral mRNA
- The Class IV viruses encode a polyprotein that is proteolytically processed to release many individual proteins including the capsomers and an RdRp
10
Q
Viruses with genomes that lack 5’ cap
A
- Must compensate with structure that allows binding to ribosome (IRES)
- IRES-dependent translation initiation recruits translational machinery to an internal position in mRNA
- VPg is the protein primer for genome replication
11
Q
IRES
A
- Internal ribosome entry site
- Complex stem loop structure in the 5’ UTR
12
Q
initiation of franslation of mRNA
A
Occurs at elF4G initiation complex
13
Q
elF4G
A
- Eukaryotic initiation factor
- Serves as docking site for initiation factors and proteins involved in RNA translation
14
Q
Cap dependent initiation complex
A
- Normal initiation of host mRNA
involves the eIF4E protein binding to the 5ʹ cap and forming a complex with PABP - The small subunit of the ribosome (40S) is closest to the AUG start codon
15
Q
Poliovirus initiation complex
A
- The host ITAF protein binds to the IRES and substitutes for eIF4E
- The terminal VPg protein was removed from the 5ʹ end of the genome by a host enzyme.
- Poliovirus proteolytically degrades eIF4E, thus preventing cap-dependent translation of host mRNA.
16
Q
Events during gene expression and genome replications in some (+) strand RNA viruses
A
- After uncoating, the IRES enables the genome to be translated by making a polyprotein that is processed into individual proteins
- The proteins go on to form virus replication compartments in which double-stranded replicative forms are used to make mRNA and new genomes that are ultimately used to make new infectious virions
17
Q
Flaviviruses
A
- Enveloped, spherical virions, 10-12kb +ssRNA genomes
- Have 5’ cap similar to host mRNA
- Lack 3’ poly-A tail, instead 3’ URT folds into secondary structure with several stem loops
- Encodes a single polyprotein that forms 3 structural proteins (C, M, E) and 7 non structural proteins
18
Q
Translation of structural proteins through subgenomic RNA
A
- 2 distinct stages of gene expression that involves translation of proteins through subgenomic RNA
- e.g. SINV and CARS-Cov-2
- Genome divided into separate regions (encode non-structural and structural polyproteins, separated by non coding junction)
19
Q
Early stage gene expression
A
Expression of non structural proteins
20
Q
Later stage gene expression
A
Expression of structural proteins
21
Q
Multiple proteins can be encoded using overlapping mRNA sequences
A
- Via suppression of translation termination and ribosomal frameshifts
- Used to produce the less abundant polyprotein P1234 from sequences that overlap with those encoding more abundant P123
- E.g. RdRp is needed in smaller amounts than other NSPs
22
Q
Ribosomal frameshifts
A
- The ribosome is stalled on the slippery sequence by the pseudoknot structure in 3’
- in 10% of cases, the ribosome will backtrack one nucleotide, creating one or more mismatches between tRNAs and mRNAs
- Translation resolves on the backtracked ribosome in the -1 frame
23
Q
CoV subgenomic RNA transcription
A
- Each coronaviral RNA contains the common 5’ leader sequence of ~70nt fused to the body sequence from the downstream part of the genome
- During - strand synthesis, RdRp pauses when it crosses a TRS in the body (TRS-B) and switches the template to the TRS in the leader (TRS-L), resulting in discontinuous transcription leading to leader body fusion
- From the fused negative strand intermediates, positive strand mRNAs are transcribed
24
Q
TRS
A
Transcription regulatory sequence
25
SARS-CoV-2 genome organisation, subgenomic mRNAs, and the virion structure
- Each viral transcript has 5' cap structure and a 3' poly A tail
- Upon cell entry, the genomic RNA is translated to produce nonstructural proteins from two open reading frames
- Viral RdRp transcribes (-) sense RNA that serves as a template fro synthesis of (+) sense genomic RNA (gRNA), and subgenomic RNAs (sgRNA)
- gRNA is packaged with assembling virion
26
What do subgenomic (sgRNAs) encode
- Spike (s)
- Envelope (E)
- Membrane (M)
- Nucleocapsid (N)
- And several accessory proteins
27
All RNA genome viruses must encode what
- A RdRp
- No host derived enzyme is capable of synthesising complimentary RNA from an RNA remplate
28
Strategies used by (+)ssRNA viruses to encode multiple proteins within overlapping sequences
- Suppression of translation termination
- Programmed ribosome frameshift
29
Baltimore class 5 (-) ssRNA
- Genomes may be segmented or non segmented
- Genome of minus sense (-)strand RNA viruses cannot be used directly as mRNA
- Viruses package RdRp within the virion
30
Mononegavirales
- ss(-)RNA viruses with non segmented genomes
- Includes Rabies and Measles
- After uncoating, the (-) RNA remains associated with nucleocapsid proteins (NPs), other viral proteins (including RdRp) and associated factors needed for RNA synthesis
- This complex, including the genome, is called viral ribonucleoprotein complex (vRNP)
- RdRp becomes active as a transcriptase, using vRNP as a template to synthesise viral mRNAs
- When levels of NP reach a threshold, anti genomes are synthesised and new genomes which are packaged into new virions during maturation
31
Viral mRNAs
Have 5' caps and 3' poly A tails
32
Primary transcription
Uses the infecting genome as template
33
Replicase
Complex containing RdRp that produces antigenomes
34
Orthomyxoviridae (influenza virus)
- 8 genome segments, each bound to NP and to proteins PA, PB1, and PB2
- The 3' end and 5' end contain secondary structures
- The most abundant proteins in the influenza virion are encoded by dedicated mRNA, most are encoded by overlapping sequences
- Transcription takes place in nucleus, not cytoplasm
35
IVA virion structure
- Two surface glycoproteins, HA and NA, and the M2 ion channel protein embedded in the envelope, which is derived from host plasma membrane
- The ribonucleoprotein complex comprises a viral RNA segment associated with the NP and three polymerase proteins (PA, PB1, PB2)
- The M! protein is associated with both ribonucleoprotein and viral envelope
36
First step of influenza transcription
- IVA produces 10 seperate mRNAS with 5' methylated caps, however doesn't encode enzymes to synthesises these caps
- First step is cap snatching: the cap binding motif in PB2 protein (within vRNP) binds to host mRNA before it leaves the nucleus
- After PB2 binds to host mRNA, the PA protein cleaves the host mRNA between 9-15 nucleotides downstream of the cap
- Cleaved mRNA then serves as a primer for synthesis of mRNA that is complimentary to the RNA in the vRNP
- RdRp acts in cis to elongate the capped primer using vRNP as template whilst remaining attached to the 5' end of template genomic RNa
- mRNA is released and the vRNP returns to original config
37
Complementary RNP (cRNP)
- Genome replication occurs through a (+) strand intermediate (the antigenomes) called cRNP
- cRNP differs from mRNA in several ways, though they are both + strands
- The 5' end of mRNA is composed of host sequences (snatched caps), with a 3' poly-A tail.
- CRNP is a 'faithful' copy of the vRNP
- cRNPs are retained in the nucleus, vRNPs are targeted for export
38
cRNP synthesis
Occurs during early infection and for a short duration relative to the rest of the replication cycle
39
IVA Replication
- vRNP components enter nucleus after uncoating
- mRNA synthesis proceeds and is exported to cytoplasm
- Some proteins are translocated back into cytoplasm where genome is used to synthesize many antigenomes (antigenomes in a complex with NP are called
CRNP) and new genomes
- New genomes can be used to make more mRNA
- New vRNPs are exported to the cytoplasm for assembly and maturation, new virions exist host cell through budding
40
Two major groups of (-)ssRNA viruses that affect humans
- Mononegaviruses (rabies, measles)
- Orthomyxoviridae (Influenza)
41
Antigenomes and mRNA
Both (+) sense, but differ in both their structure and sequence
42
Baltimore class 3 dsRNA virus genomes
- Reoviridae
- Non enveloped, icosahedral virion with triple capsid structure
- Uncoating leaves a double layered particle (DLP)
- Segmented linear dsRNA genome with 11 segments coding for 12 proteins
- Each segment has 5' cap, no polyA tail
- Co-infection of cells with different rotavirus strains belonging to the same serogroup A, B or C undergo mixing of the genome segments (genetic reassortment)
43
Rotavirus viroplasm
- Cytoplasmic site of viral dsRNA synthesis
- During earlier phases of infection viroplasms are separate, older viroplasms fuse with one another
44
rotavirus genome replication
Early transcription of dsRNA genome by RdRp occurs inside DLPs
45
DLP
- VP6 is outermost protein, VP2 is underneath
- 11 dsRNA segments
- Every vertex of particle is fenestrated allowing for entry of NTPs and exit of newly synthesized mRNA
46
VP1/VP3 flower proteins
- VP1, VP3 are internal polymerase complex
- Transcribes capped (+) RNAs from each of the gene segments
- (+)RNAs serve as either mRNAs for synthesis of viral proteins on cellular ribosomes, or as templates for synthesis of (-)RNA
47
Rotavirus mRNA compared to host mRNA
- Each RNA segment has 5' cap, no polyA tail
- Viral NSP3 substitutes for PABP by binding to the conserved UGACC sequence near the 3' end of the viral mRNA
48
Gene expression and genome replication in rotaviruses
1. Cytoplasmic DLP catalyses primary transcription in which capped mRNA leaves each vertex of the virion
2. Translation of these proteins causes formation of viroplasm in which mRNA becomes enclosed by new DLPs
3. Inside the new DLPs copying of (+) strand templates results in formation of DLPs containing dRNA genomes
4. The new DLPs cause an exponential increase in viral mRNA and protein
5. After a few hours the virus switches from gene expression and genome replication to the assembly phase of the replication cycle
49
Example of a rotavirus protein that is structural and has enzymatic acivity
VP1
