RNA Genome Replication
importance of secondary RNA structure
First order information is confined in the SEQUENCE of RNA Second order information is confined in the STRUCTURE of RNA This structural diversity provides opportunities for specific interactions with other viral / cellular biomolecules
Initial Event Following NEG strand entry
Genome Replication into (+)ve strand RNA
How Retrovirus has evolved to cope with this ?
Has its own polymerase : - Reverse Transcriptase is packaged in the nucleocapsid
(+) RNA genome is converted to vDNA and integrated as proviral DNA
Viral Template used in HIV Transcription
incomplete circular DNA repaired - pregenomic RNA copied into ds DNA by viral enzyme
Viral Template used in hepatitis B Transcription
- transx soon in nucleus - associated with cellular histones
Viral Template used in herpes Transcription
- transx soon in nucleus - enter as mini-chromosomes
Viral Template used in papilloma (HPV)Transcription
converted to linear ds DNA
Viral Template used in parvo Transcription
Do cellular proteins participate in CoV genome replication and transcription?
Yes, # of cellular proteins that aids in Co V replication and Transcription
Are the exo and endonuclease part of CoV replicase complex ? and are they present in other RNA viruses ?
Yes, No Respectively
Action of brefeldin A; how it inhibits poliovirus RNA synthesis ?
fungal metabolite used in protein transport studies ; targets protein that regulates transport / secretory vesicles formation from ER and fusing to Golgi . - provides evidence for secretory vesicles requirement as platform
Identify some cellular and viral proteins associated with polymerase action (4)
host cellular protein(s): tubulin, actin, poly Rc binding protein, poly A pyrimidine tract binding protein
Reovirus replication uniqueness.
replication of genomic RNA occurs after RNA packaging inside viral capsids by v RNA polymerase - all unpackaged viral RNAs are mRNA by default.
What to learn from Reticulocyte lysate and Translation
ribosome binding to IRES sequence requires additional factors besides translation initiation proteins
General concept of poly (A) tract
secondary RNA structures, called cis-acting RNA elements allows RNADepRNAPolyase's to discriminate between self and non-self mRNAs 3' poly (A) tails are required for translation of (most) mRNAs Poly (A) is attached to the 3' ends of cellular mRNAs in the nucleus. RNA viruses replicate in cytoplasm
Influenza virus: role of nNS1 protein
selectively block Cellular RNA processing and translation inhibits both polyadenylation and splicing
Herpes virus : role of ICP 27 protein
selectively block Cellular RNA processing and translation inhibits splicing
Role of cytoskeleton proteins ( tubulin, actin) in virus replication (measles and RSV - respiratory syncytial virus only)
specific targeting probably ensures high local concentration of replication components -serves as anchoring site for polymerase Tubulin: stimulates measles virus replication and mRNA synthesis in vitro - Anti-tubulin Ab's inhibits replication of both these viruses Actin: ( forms microfilaments ), is needed by respiratory syncytial virus
identify if these structures are present in uninfected cells.
structures are absent in uninfected cells
Role of coronavirus induced cellular membrane structure
support their replication complexes;
Origin of membrane vesicles in SARS Co V infection
the membrane vesicles appears to originate from ER
coronavirus replication on membranous structures
to induce internal cellular membranous structures - support their replication complexes;
- synthesized by either viral / cellular enzymes - made either in nucleus or cytoplasm - must be compatible for host translation machinery
vmRNA
Do reovirus have an RNA polymerase packaged inside virions ? RNA pol is associated with inner / outer capsid
yes, The RNA-dependent RNA polymerase is part of the inner capsid
Advantages of transcription inhibition by viruses.
- cellular resources ( RNA building blocks, etc ) can be directed for vm- RNA production as well as RNA genome synthesis - competition for translational machinery is virtually eliminated amongst viral and cellular mRNA's
Why Replicate on Membrane Vesicles/ Advantage
- ensures localized concentration of replication components - optimizes packaging of progeny virions - provides physical support ( as platform)
Polio Viral Genome: Characteristics
- long with 5' and 3' UTR and an ORF A long poly protein is made by translation of the ORF - Various structural and nonstructural proteins are generated via alternate cleavage of the poly protein by virus encoded proteases
Negative strand viral RNA characteristics.
- template wrapped with nucleoprotein / nucleocapsid - RNA polymerase and accessory proteins are intimately associated with genome within nucleocapsid - provides stability as well as protection from cellular RNAses
Whats Canavanine also Fluorophenylalanine
Canavanine: arginine analog Fluorophenylalanine: blocks mature protein synthesis Added pre-cleavage no synthesis added post cleavage, P1 still made proteins
Uniqueness of CoV replicase coding sequence.
Comprised of 2 overlapping ORF's - The 5' end of the 2nd ORF overlaps the 3' end of the 1st ORF and is read in -1 reading frame (relative to the 1st ORF) - Replicase region is translated into 2 large polypeptides from 2 overlapping ORF1a and ORF1b
In Retrovirus, Are the input RNA is copied or translated?
Copied into cDNA then put into nucleus
RNA virus replication sites
Cytoplasm: 1) on membrane vesicles - ER derived Vesicle - Flu/Flavi - Late Endomsome Vesicle - Rubella - outer mitochondrial membrane - House Flock Virus 2) Cytoplasmic Inclusion - Reovirus 3) Cytoskeleton proteins - Measles/RSV
ER derived Vesicles
Cytoplasmic vesicles and examples of DenV/HepC/WnV/Flavi
- outer mitochondrial membrane - forms plant form where replication occurs
Cytoplasmic vesicles and examples of House Flock of Virus (HFV)
- Late Endomsome Vesicle
Cytoplasmic vesicles and examples of Rubella
- ER derived Vesicle - vesicles are formed from ER but they don't fuse with Golgi - disrupts Golgi but is NOT required for polio replication
Cytoplasmic vesicles and examples of polio
IRES and Translation Initiation Protein Requirements
Direct Binding: none/few initiation proteins are needed ( Hep C) Indirect Binding: all initiation proteins required (Polio V)
role of viral accessory proteins and helicase in genome replication
Role of Accessory Proteins: i. directs polymerase to correct intracellular replication site a. In nucleus: (Influenza - only RNA virus) b. In membrane vesicles of the cytoplasm: (Parvovirus) ii. targets polymerase to correct initiation site on RNA template Role of Helicase: encoded by viral genome - unwinds RNA sequences ( secondary and tertiary structures) - prevents pairing between template RNA bases and nascent strand
types of base pairings in RNA structure
Stem - complementary base pairs in sequence Pseudoknots - Loops
Phenol : Chloroform extraction / deproteinization of (-)ve RNA vs +ve RNA
(+) Strand RNA Virus : still infectious b/c the coding sequence is intact - can be translated by host ribosomes to make functional RNA polymerase (-) Strand RNA Virus : deproteinized genome is non-infectious since RNA polymerase protein is inactivated
Advantage of (+ ) RNA vs (-) RNA
(+) ve strand genome: (+) ve RNA genome is immediately translated inside host cell - exception : retrovirus genome, not readily translated inside cell (-) ve strand RNA genome: - provides stability as well as protection from cellular RNAses
. Nature of RNA template How (+) stranded genome differs from (-)ve strand RNA genome?
(-) ve strand RNA genome ( as in flu) - template wrapped with nucleoprotein / nucleocapsid - RNA polymerase and accessory proteins are intimately associated with genome within nucleocapsid (+) ve strand genome non - coated / covered ; basically naked genomes
Which one is translated as a large ORF ( 1a / 1ab?) and how it's processed ?
(1-ab) 803 kD fusion polypeptide The large polypeptide is cleaved***post/co translationally*** by VIRAL PROTEASE ( PART of the replicase )
Dynamics of Viral 5' end (Polio)
- 750 nucleotide sequence - Not capped but uses Viral protein genome link (VPg) as primer - is highly structured
Reticulocyte lysate (RRL) and Translation Characteristics
- Capped mRNA : causes efficient mRNA translation - Poliovirus translation was poor until extract added
Identify the generic enzymes associated with coronavirus replicase complex
- RNA dep RNA polymerase, helicase, protease
Why RNA viruses ( like polio ) inhibits host cellular transcription unlike influenza ?
- RNA viruses inhibits / blocks cellular transcription; they don't need cellular transcription machinery nor it's products - cellular resources ( RNA building blocks, etc ) can be directed for vm- RNA production as well as RNA genome synthesis - competition for translational machinery is virtually eliminated amongst viral and cellular mRNA's
How was influenza viruses PB1 identified ?
- amino acid sequence was aligned with known RNA dependent RNA polymerase motifs - analyzed for similiarity
Advantage of Poliovirus shutting down host Prot Synth
No competition Availablilty of ribosome for viral prot synthesis
Role of 1st 108 nucleotides vs rest of genome (Polio)
1st 108 nt's: forms a cloverleaf structure involved in Initiation of replication Of viral genome Rest of nt's: forms extensive stem loop structure involved in IRES mediated Translation Of viral protein.
End result of viral RNA processing
A. functional mRNA B. opportunities for post transitional regulation of gene expression
Importance of P1 and P3 in Polio viral genome
Essential to Function of Viral Genome - codes for capsid and polymerase
Where IRES 1st Identified ?
IRES 1st identified in picorna viruses and followed by others; Hepatitis C, pestivirus (?) and some cellular mRNA - little nucleotide conservation except pyrimidine tract of 24 nucleotides upstream of 3' end of viral IRES
List of mRNA that functions as proteins
Ig Binding BiP protein Ornithine Decarboxylase Vascular Endothelial Growth Factor Receptor (VEGFR) - Viral IRES contains extensive RNA secondary structures - important for RNA proteins binding
Where is cascade normally observed ?
Immediate early, early and late viral proteins herpesvirus transcription
RRL and Poliovirus Outcomes w/ or w/o HeLa celll extract
In HeLA cell extract Poliovirus 1) w/ uninfected cell extract: complete 100% transltion 2) w/o cell extract: Truncated Translation - translation stops after time - limited availability of cellular proteins
IRES cleavage in Poliovirus
Indirect -- - in Polio ELF4G cleaved on terminal end to provide binding site - also Cleavage of N term of ELF4g shuts down host prot synthesis - C terminal region has ribosome binding site ****- Cleavage mediated by viral 2 A protease - cleaves between G,Y AA's***
priming by capped RNA fragments (Ex)
Influenza-- steals 7Me-Gppp caps from cellular mRNAs by cleaving (cap snatching) - cleaved fragments are used to prime viral mRNA synthesis
RNA replication initiation in Bunya and Arena viruses
Initiation of Replication - begins at an internal C and follows "prime and realign" mechanism vs most de novo
Additional proteins invloved in IRES mediated Translation & Role
LA- Lupus auto antigen: - localized in nucleus; migrates to cytoplasm in PV infected cells - present in low concentration in RRL PTBP- Pyrimidine Binding Protein: nuclear protein - W/o this IRES mediated trans knocked off Poly RC BP: cellular poly rC binding protein Nucleolin : cellular nucleic acid binding protein ****They all maintain 3 dimensional structure of RNA ( possibly chaperone funct) facilitating binding of both ribosome as well as initiation proteins****
Features of CoV mRNA and the concept of nested mRNA's.
Nested set of 7 - 8 Co V mRNA's; with a common leader and 3' end - each mRNA has the same 3' terminus - each mRNA has the same 5' leader sequence of ~ 70 bases which is identical to the 5' end of genomic RNA
Is splicing associated with CoV mRNA synthesis ?
No - materials needed are in nucleus
Does it inhibit RNA polymerase ?
No : targets protein that regulates transport / secretory vesicles formation from ER and fusing to Golgi
Polymerase of Polio / Retroviruses / Hep C Virus
Polio: 3D pol HCV : NS5b (non-structural protein 5b) - most sought protein as drug target protein REtrovirus: reverse transcriptase
Protein priming vs priming (Ex)
Poliovirus has 22 aa covalently linked to 5' end of genome **Non nucleic Acid Primer - Viral Protein genome link** - (VPg) becomes polyuridylated (polyU); base pairs with poly A at 3' end of genome. - Interacts with RNA pol and targets it to primed 3' end of genome ( polio polymerase is primer dependent )
Why viral polymerase are a challenge to purify?
Polymerase Intimately associated with capsid , envelope , host cellular membrane
Types of Viral Translational Strategies (5)
Polyprotein synthesis Leaky scanning Reinitiation Supression of translation Ribosomal frameshifting
Difference between positive vs negative autoregulatory loops ?
Positive Loops: - alters rate of transcription initiation - viral proteins stimulate transx Negative Loops: - repress gene expression
Pox virus vs poliovirus general inhibition of transcription
Poxvirus induces rapid global inhibition of RNA synthesis by an identified viral protein Poliovirus induced transcription inhibition of RNA pol II ( best characterized) 3C pro cleaves TBP subunit of TF IIId; inhibits all cellular trsx activity - RNA viruses inhibits / blocks cellular transcription; they don't need cellular transcription machinery nor it's products
Whats Template Jumping?
Proposed Mechanism for CoV REplication - Leader sequence made then: - jumps to a new corresponding part of sequence and re-aligns
What's pseudoknots and loops in RNA structure?
Pseudoknots: Forms base pairs with nucleotide outside the structure Includes: LOOPs - hairpin loops, bulge loops, interior loops, multi-branched loops
Hall mark feature and resistance / sensitive to actinomycin D
RNA dependent RNA polymerase: unique in: - resistance to Actinomycin D, an inhibitor of DNA directed RNA pol - they inhibit cellular or viral RNA polymerase.
Explain and be clear of mechanism how poliovirus inhibits host cellular transcription ?
RNA pol II machinery transcribes viral DNA template resulting in viral transcription 3C pro cleaves (TATA Binding protein) TBP subunit of TF IIId; inhibits all cellular trsx activity
requirements and properties of viral RNA dependent RNA polymerase?
RNA polymerase must initiate and terminate at specific sites in the templatePolymerase may have requirement of: - host cellular protein(s) - viral accessory protein capsid and / or viral coded protein
How are they different from transcription cascade ?
Transcriptional cascade: - viral transcriptional units are activated in fixed sequence Allows viral genes transcription in a temporal controlled fashion - ensures viral proteins are made at different times of infectious cycle;
How is RNA polymerase activated inside reovirus particle ?
activated by proteolysis when REovirus outer capsid is degraded.
non structural proteins in Polio viral genome
are the functional proteins necessary for genome replication packaging and assembly etc
IRES Translation Steps
i. 40S subunit binds to IRES elements - in Polio ELF4G cleaved on terminal end to provide ribosome binding site - also Cleavage of N term of ELF4g shuts down host prot synthesis ii. scanning to initiation codon
How ribosomes bind mRNA for Translation
i. 5' dependent initiation -Most mRNA are translated by this mechanism - initiation complex binds to 5' cap structure, scans until it hits on AUG initiating codon ii. 5' independent initiation Internal ribosome entry site (IRES) - initiation complex binds at / just upstream of the initiation codon
(2) modifications for efficient translation in RNA processing
i. addition of m7GpppN to the 5' end ( capping ) ii. multiple (A) residues addition to the 3' end ( polyadenylation) Enzyme may be of viral or cellular origin
How Poly A tract acquired ?
i. encode a 3' poly A sequence on (+) strand and / or 5' poly-U on the (-) ve strand ii. Reiterative copying ("stuttering") on short 3' U-sequences on the (-) ve strand.
Recall 2 essential features of RNA virus replication
i. genome be copied faithfully from one end to the other, without skipping any nucleotides ii. RNA synthesized be effectively translated by host cellular machinery (i.e translation competent mRNA )
Extra Modifications for efficient mRNA Translation from pre-mRNA (4)
iii. splicing also accompanies when RNA is synthesized inside nucleus - no viral gene codes for splicing machinery; exclusive coded by host / cellular process iv. RNA editing ( internal chemical changes) in some viruses, single nucleotide is replaced or inserted at specific positions ( but NOT encoded by viral genome) v. RNA made in nucleus must be exported to cytoplasm for translation via cellular pathways - export mechanism can be altered during certain virus infection vi. RNA has a finite half life
Whats RRL?
immature rbc's with no Hb, but ribosomes are active for translation
structural Proteins in Polio Viral genome
includes capsid proteins coded by the P1 part
How do they do accomplish that role ? ( aka action of actnomycin D
intercalates between DNA turns denying the opportunity for Polymerase binding
Uniqueness of Co V genome and be clear why it's longest RNA ?
longest RNA genome and the polymerase / replicase is coded by ~70 % of the genomic RNA sequence Why? NO conclusive answer. Possibly: Co V replicase is a complex - of common RNA virus encoded enzymes includes: - RNA dep RNA polymerase, helicase, protease - a set of RNA processing enzymes not used by other RNA including: -sequence specific endoribonuclease , - 3' - 4' exoribonuclease, - ADP ribose 1- phosphatase, - cyclic phosphodiesterase - also, # of cellular proteins that aids in Co V replication and Transcription
Role of cytoplasmic inclusions as well as ( reovirus, ).
occurs on cytoplasmic inclusions - serves to provide platform and concentrate accessory proteins