Exam Two Flashcards
(59 cards)
Describe the structural features of a positive-strand RNA.
Model: polio
Non envelope. In a capsid. Icosahedreal.
Genome is in the sense form, upon entering the cell, the genome is translatable. Because of this feature, this can be termed as infectious in naked free form.
No need to package nonstructural components.
Cap dependent or independent.
One long polyprotein is formed. One start and stop codon.
- Explain why the genome of a positive-strand RNA virus is infectious.
- Explain why positive-strand RNA viruses do not need to package their RdRp enzyme in the virion structure:
- As soon as the genome enters the cell, host cell ribosomal proteins will begin synthesizing viral proteins
The virion amino acid sequence is translatable. In the positive naked free genome.
2.
The RdRp is a non-structural protein for these viruses.
Not available for host cell (only transcribes). All RNA viruses need to express RdRp in order to replicate RNA– evolved to complete this.
Describe the structure and function of the 5’ cap that is found on eukaryotic mRNAs:
Viruses follow the hosts’ rules.
The only way the host’s ribosome will assemble onto RNA– CAP Dependent translation.
Translation: assemble on RNA only with a CAP structure. Begins with recognition of the CAP.
- CAP is GTP linked to the first NT through a 5’ to 5’ triphosphate linkage
- CAP also has methylation: one at the GTP and two along the 2’ position
Transcription:
- CAP synthesizes here
Explain the general features of the cap-dependent translation process:
Again, the virus has to follow the rules of the host cell.
Translation will only be initiated after recognition of a CAP along the 5’ end
Explain the genome of poliovirus obtains its 3’ poly A tail.
HOST CELL
- cleave at CA region, poly A addition site
- poly A polymerase will add the poly A tail
PURPOSE
- prevents degradation at the 3’ end from other enzymes
- export from the nucleus to the cytoplasm for translation
- 3’ end bound to poly-A-binding-protein which has an affinity for CAP assembly protein (effect on translation)
POLIO
- genetically encode poly A tail produced during replication process
- poly U tract along the negative strand
- negative strand used to create a complementary positive strand
Describe the structure and function of the IRES
IRES: internal ribosomal entry site along the untranslated region
Location: upstream of start codon
Structure: NT positioned where RNA become folded into structure; intra molecular complementarity (G combined with C and A with U)
Function: ability to assmeble host cell ribosome just like a CAP
- Explain what a polyprotein is.
- Explain how the polyprotein of poliovirus is resolved into its individual proteins
- a large protein that is later chopped up; a very common protein strategy for positive RNA virions
- virus produces large protein with 1 start and stop region
proteases (provided by virion or host cell) cleave large poly protein to small proteins to mediate infection
Explain the general mechanism for replication of a positive-strand RNA.
Explain the location and replication functions of the non-translated regions of a positive-strand RNA.
3dPol last protein that is cleaved. 3dPol binds to 3’ end. Initiate replication from the negative strand 5’ to 3’ end.
All +RNA don’t package because they don’t need to, the sequence is translatable.
Need a negative strand to make more + strands.
- Positive to negative to positive to negative …
Explain why RNA viruses must synthesize their own RdRp enzyme,
not available in the host cell
needs to replicate RNA for infection
3dpolymerase (not structural not naturally brought in)
all positive do not package RNA polymerase, their genome is translatable
Explain how a region of an RNA molecule can fold into secondary structures such as a stem-loop or higher-order structure.
Understand intramolecular base pairing.
BP allow structure to fold up into utr sequences.
Create “markers”
Polymerase needs to know appropriate RNA (positive or negative viral RNA) to replicate.
Unique virus sequences, fold up and are recognize/physically bound to by polymerase.
Explain the fundamental steps in poliovirus assembly to form an infectious virion. Understand the concept of a packaging signal.
- protease cleavage produces VP0
- needs additional cleavage to produce subsequent structural proteins (need 4 in total) - formation of Proviron
- forming structural components into a pentamer (5 structural units) and later into an icosahedral (12 structural units) - RNA inside triggering a latent protease activity
- confirmational changes allow all 60 VP0 proteins to cleave themselves to produce actual virion structural products - right confirmation to bind to appropriate receptor and create a pore to leave cell for further infection
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Specificity issue during RNA selection for particular capsid, only + strand RNA should be placed into the capsid.
Sequence and structural component found in the RNA molecule that should be packaged. Not in negative strands or cellular mRNA.
HIGHLY CONSERVED
Explain the rationale for placing multiple different virus families into the mononegaviruses order.
What does placing multiple virus families into a single order imply about the evolutionary history of different viruses?
At one time, there was a common precursor for all these viruses.
Through evolution, these have specialized and branched enough to warren placement into their own family.
paramyxoviridae (measles), rhabdoviradae (rabies), filoriridae (Ebola)
Describe the general structural features of negative strand RNA viruses.
Why do these viruses incorporate their RdRp into the virus particle?
Why is the naked RNA genome of these viruses not infectious?
- no naked RNA; RNA coated with nucleocapsid structure (multiple NP or N proteins bound to genome)
- enveloped with a matrix sitting directly below the envelope, consisting of multiple M proteins
- RdRp need to package the individual proteins for the multicomplex (L and P; polymerase and phosphoprotein)
Describe the genetic organization of the viral genome.
Explain the general mechanism for transcribing the viral genes.
How do the mRNAs acquire their 5’ cap and poly A tails?
3’ to 5’
- leader sequence: polymerase access and binding site
- intergenic regions, separate linear sequence of individual genes
- # identical/highly conserved across the genome (change would cause detrimental effects for the virion)
- polymerase binding to leader sequence
- replication and mRNA synthesis that is complimentary in the 5’ to 3’ direction
- termination at the intergenic region (ig)
- slippage and recopying:
- repeated replication to create adenosine residues of poly A tails - mRNA released and next start region/next gene
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RNA polymerase creates poly A tail.
It also has the enzymatic capability to create the 5’ CAP.
- guanylyl transferase for GTP 5’- 5’ linkage.
- methylation
Explain the molecular basis of transcriptional attenuation.
How does this phenomenon occur?
What does the virus achieve by having the ability to regulate levels of its different mRNAs?
The order of the genome is relevant to the production necessary for the virus.
Genes closer to the 3’ end are needed in large amounts. Those closer to the 5’ end not so much.
The placement is important as the probability for reinitiating replication at the subsequent start regions decreases.
Explain the role of [N] in controlling the switch from transcription of viral mRNAs to replication of full-length genomes.
trigger for mRNAs or full-length molecules (replication) is determined by levels of nucleocapsid.
Early, [N] is very low. Only transcription.
Progression results in high [N] and meets a threshold that influences polymerase. Intergenic site ignored to create a full-length complement/genomes.
Explain the structure and function of signal sequence (glycoprotein synthesis).
Explain the function of the enzyme called signal peptidase.
Follow host’s cell rule!
Host cell ribosome translating mRNA for G protein docked on ER and translocating into lumen of ER.
Glycoprotein synthesis happens in the ER!
- Signal sequence: regions of proteins (N terminus) affinity for SRP. Given direction or order to a sequence.
- Induced stop in translation by SRP and signal sequence binding.
- SRP binds to SRPR.
- Translocan will extend and bring translated sequence into the lumen.
- Signal peptidase removes/cleaves signal sequence.
Explain the general process of glycoprotein synthesis, modification (carbohydrate addition), and transport from the RER to the plasma membrane.
glycoprotein dolichol: backbone carbohydrate structure
Asn will covalently bind to carbohydrate: N-linked glycosylation.
Asn-X-Ser/Thr
X = (any AA)
Sequence appears in lumen, enzyme transfer carbohydrate from dolichol to Asn.
Different compartments of Golgi Apparatus modifies the carbohydrate structures.
When finalized, the structure will begin to bud and take its envelope from the Golgi’s membrane. Glycoproteins will embed into the envelope.
Describe the fundamental structural characteristics of the influenza virus particle and genome.
- Matrix and envelope with glycoproteins HA and NA.
- 8 separate negative RNA strands with a nucleocapsid protein (NP).
- RdRp: consists of three proteins PB1, PB2, PA.
- M2 ion channel.
Explain why influenza A virus replicates in the nucleus, which is very odd for an RNA virus.
Explain the functional properties and the role of the M2 ion channel in the influenza virus replication cycle.
M2 transports the H+ to the inside the virion cell. Important for early entry process.
M proteins are naturally attached to the genome. H+ entry allows this complex to dissemble the group of 8 segmented genomes. Breaks the noncovalent bonds that hold M proteins and genome together.
Small RNAs are able to travel through the nuclear pore.
Explain the general process of how proteins are transported into the nucleus.
How the viral nucleocapsids of influenzas virus exploit this importation pathway to gain entry into the nucleus.
Could you explain what a nuclear localization signal is?
NLS AA sequence is embedded within the primary sequence protein which allows cellular importation.
NLS recognition allows the genome to get pulled into the nucleus.
Understand all key aspect of the cap-snatching mechanism as it relates to transcription of influenza mRNAs.
Cap snatching is utilizing (capturing) host cell CAP regions in order to transcribe viral mRNA. Will appear in the viral mRNA but not in the original microbial genome.
Stuttering is also involved to create poly A tails
Explain how gene segments 7 and 8 direct the synthesis of two different proteins each. This will require you to understand how alternative splicing works.
Immediate translation of mRNA results in one protein.
If spliceosomes encounter before translation the region encoded from the genome could differ producing a new/unique protein.
