RNA Genome Replication

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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


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