Replication of RNA viruses II

2/07

(-) strand RNA viruses

• not infectious by self
• RdRp is INSIDE the viron, bc needed for transcription to be done upon entering cell
• transcription/translation
  • RdRp used to make mRNA from (-) strand viral RNA
  • once transcribed, can translate mRNA -> single proteins (NOT polyproteins)
• replication
  • makes fullsized (+) RNA (NOT mRNA) intermediate, but is NOT translated
  • instead, use (+) RNA as template to make more (-) RNA
    
    

notes

(-) rna viruses

- their genomes are opposite polarity of mRNA

- NOT directly infective

– use RNA dependent RNA polymerase that they carry in their virion

- enables them to make mRNA from their genome ( - RNA -> mRNA )

Monocystronic -> made into single proteins

Replication of negative strand requires full size (+) RNA intermediate to create more negative strand

- (+) intermediate used to make (-) RNA, so makes a strand of the original polarity

what kind of proteins do (-) sense RNA viruses make?

monocystronic proteins (make single proteins, not polyproteins)

diff btw mRNA and antigenome (+) RNA

mRNA

• shorter than genome template + RNA
• contains 3' poly A tail
• contains 5' cap

antigenomic (+) RNA

• exact copy of template genome
• no 3' poly A tail
• no 5' cap

what viruses are (-) strand RNA viruses?

single (nonsegmented) genome

• measles
• mumps
• rapies
• VSV
• RSV

segmented genome

• influenza

replication / trans.+transl. of (-) strand RNA virus (nonsegmented genome)

transc/trans

- strand RNA -> transcription -> monocystic mRNAs -> translation = proteins

replication

• (-) strand RNA vgenome -> make (+) strand RNA template -> make (-) strand progeny RNA

proteins + (-) progeny RNA = progeny virus

replication principle is same in non-segmented/segmented viruses

for either:

• virus (-) RNA bind receptor
• -> viral membrane fuse cell membrane
• then viral polymerase is in capsid
  • transcription or replication

newly made nucleocapsids associated w/ matrix and glycoprotein - modified plasma membrane = enveloped virus

(-) RNA virus replication occurs in

cytoplasm (except flu)

(-) RNA virus leave cell by

budding

bc the proteins assoiciate w/ matrix and glycoprotein modified plasma

nonsegmented (-) RNA viruses

(-) RNA

• transcription
  • virion RNA pol respect initiation-termination signals
  • = generate individual mRNAs for each protein
• replication
  • virion RNA pol will not stop in transcription recognition signals
  • = generate full-length (+) RNA indermediate template = NOT mRNA

can swtch between transcription/replication mode

replication of paramyxoviruses

(-) genome released in cell

• Transcription mode
• Then either go down replication or translation route
  • Either made template (+) RNA or mRNA depending which it is doing

segmented (-) RNA virus

• allows for reassortment to occur
  • = good for viral pathogenicity (can only do reassortment if segmented)
• diff gene segments encode diff proteins
• transcription
  • viral RNA polymerase generate mRNAs from each genomic segment
  • each segment contains its own signals to initiate transcription
• replication
  • viral RNA pol will generate (+) RNA intermediates from each genome segment which will be copied into (-) RNA genomes

pretty much same thing as nonsegmented

flu virus

segmented (-) strand RNA virus

• replicates in host cell nucleus (this virus is the EXCEPTION)
• replicate in nucleus bc flu transcriptase use host cell 5' cap mRNA as a PRIMER for viral mRNA synthesis

consequence: viral endonuclease PB2 cuts cell's mRNA 5' cap structure as well as 10-13 nucleotides from cellular mRNA

• = capped RNA oligonucleotides function as primer for transcription initiation
• = disabling host cell protein synthesis

what is unique about the flu virus?

it replicates in the host nucleus because the mRNA's 5' cap structure is required to work as the virus' primer for transcription

= host cell cannot make any more proteins

ds RNA viruses

• naked - double capsid
• most - segmented genomes
• mRNA made only from one template strand of each segment
  • uses (-) strand template!!! (-> so make (+) RNA = mRNA)
• transcription
  • similar to ds DNA genome -> (-) strand template
• necessary enzymes must be encoded, packaged in virion + carried to next cell
• after infection initiation -> several proteins in virion particle removed by protease in intestinal tract -> form intermediate/infectious subviral particle
  • this is the functional particle
  • virus must become activated by GI tract - if not, gets deactivated
  • virus ingested, activated by protease in GI tract
• DS GENOME - in ISVP
  • but viral mRNA is in cytoplasm outside the ISVP

method of DS RNA replication

conservative replication

ISVP binds receptor (sialic acid) to ___

and whole virions are taken up by ___

penetrate the cell

receptor mediated endocytosis

ISVP

• The viral particle that has been activated by the GI tract protease which allows it to become functional and do its work in the cell upon entering
• releases core into cytoplasm
• enzymes in the core initiate mRNA production
• each (-) strand use as template by virion core enzymes
  • individual mRNAs made and mRNAs leave core to be translated
• later, virion proteins and (+) RNA segmentes form cores
  • (+) RNA copied to make (-) RNA in cores = ds genome

retroviruses

• enveloped SS genome
• capsid w/ 2 identical copies of (+) strand mRNA genome
• copies of reverse transcriptase (RT) and integrase
• has 2 tRNAs - used as primers
• mRNA genome is NOT infectious bc does not encode RdRp
• encode RNA dependent DNA polymerase and replicate thru dna intermediate
• contradicted central dogma bc shows RNA can -> produce DNA (backwards)
• important in humans bc responsible for
  • HIV
  • HTLV-I
• important for studied/technology
  • gave rise to enzyme used for PCR

retrovirus replication cycle

2 (+) strand RNAs

1 RNA -> give rise to cDNA strand (reverse transcriptase)

-> remaining RNA strand degraded

-> use cDNA to create complementary cDNA using DNA-depdendent DNA polymerase from host cell

-> integrate into cellular host genome

(make sure these steps are correct)

notes

Starts w/ 2 (+) strands

One + strand RNA -> make ss DNA strand using RT

Then next RNA -> ss DNA using RT

So now have ds DNA

-> nucleus

why do retroviruses have 2 copies of RNA?

not sure

maybe it increases probability of successful DNA synthesis bc if one is broken, RT can switch templated + copy the RNA = DNA synthesis

genome of retrovirus

3 major genes

• gag -> capsid proteins
• pol -> polymerase, protease, integrase
• env (envelope) -> glycoproteins

end of each RT genome = long terminal repeat sequence (LTR)

LTRs -> contain promoters, enhancers and other gene sequences used for binding of diff transcription factors

oncogenic retroviruses -> contain oncogenes

complex retroviruses (HTLV) -> encode other regulatory proteins

HIV

viral attachment to receptor/coreceptors (CCR5 and CXCR4) necessary for infection

attach CD4 (Helper T) cells exclusively

if someone does not have receptor -> they are resistance to infection

once retroviruses released into cytoplasm

• RT use virion tRNA as primer + make complementary (-) strand DNA (cDNA)
• RT act as ribonuclease H
  • degrade RNA genome left + make (+) strand DNA
• during synth of provirus, sequences from each end of genome are duplicated = attach the LTRs to both ends
• process creates sequences necessary for integration + makes enhancer and promoter regions in the LTRs for transcription regulation
• ds DNA delivered to nucleus + integrated into host chromosome

once integrated into host chromosome

• viral DNA transcribed (in the cell's genome) by the host's RNA pol II
• = full length RNA
  • can either:
    • proceed to make
      • mRNA
      • translation = proteins
    • assemble into new virions

RT OCCURS WITHOUT FULL UNCOATING OF THE GENOME

RT OCCURS WITHOUT FULL UNCOATING OF THE GENOME

Nucleocapsid partially dissolved

WHICH EXPLAINS WHY THE GENOME (mRNA) IS NEVER TRANSLATED

(thus not full genome is used)

virus acts as cellular gene so its replication depends on:

• extent of methylation of viral DNA
• cell's growth rate
• ability of cell to activate enhancers + promoters encoded in the lTR

stimulation of the cell by mitogens, certain lymphokines, or infection of the cell w/ exogenous viruses produces transcription factors which bind LTR + activate viral transcription

viral oncogenes promote cell proliferation = stimulate transcription and viral replication

comparison of (+) strand RNA viruses and retroviruses