MICR 3330 Lecture Notes - Lecture 6: Biopesticide, Gene Therapy, Endosome
27 Jun 2018
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Baculoviridae: Family
Why Study Insect viruses?
E.g. spruce budworm virus affects the loss of these trees in Canada’s forests.
Resulted in a 69 M ha loss of forest area due to insect viruses.
Baculoviruses, a Primer
Insect (not vertebrae) specific
Rod shaped
Enveloped virus
Two morphological forms: Biphasic replication = budded virus (cell to cell infection) and
occlusion derived virus (insect to insect)
DS circular DNA genome (80 kb to 180 kb)
Nuclear replication (hence some baculoviruses often referred to as “nucleopolyhedrovirus” or
NPVs)
Temporal cascade of virus gene expression
Species: Autographa Californica Nucleopolyhedrovirus (ACMNPV)
Genome: ds circular DNA genome, 133,894 bp (156 ORF)
Morphology: budded virus (cell to cell spread) and Occlusion derived virus (ODV) spread to
insects.
Budded virus (BV) envelope contains gp64 glycoprotein required for attachment
Genera: Alphabaculoviruus
Phylogeny
37 core proteins of 73 viruses
genome phylogeny reflects host phylogeny and virus taxonomy
Ancestor (Grammabaculovirus) in wasp hosts
Infection of host cell/insect ORF
ingestion of occlusion bodies (OBs) into alkaline gut
Liberation of ODVs in gut
Entry of midgut epithelial cells by membrane fusion
Nucleocapsid to nuclear pores via actin filament forces
Intranuclear vDNA replication and nucleocapsid assembly
Budded virus (Bvs) for cell to cell spread in caterpillar ( GP64 in membrane and VP39 in capsid)
Intranuclear assembly of polyhedral (occlusion bodies)
Liquefacation of larva and release of polyhedral 9viral chitnase and viral cathepsin protease)
Baculovirus Intracellular Replication Cycle
Virus attachment and penetration into cell
Movement of nucleocapsids to nucleus, uncoating of DNA genome
Temporal regulation of viral gene transcription
Viral DNA replication and protein synthesis
Assembly of nucleocapsids in nucleus
Nuclear egress and budding of nucleocapsids
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Embedding of nucleocapsids into polyhedral
Virus either ingested from contaminated leaf or from adjacent cell.
Polyhedra protects virus from external environment, but creates a problem when trying to uncoat
and render cell infectious.
Virus is smart and has learned that the gut of insect is alkaline (different form other environments
in this way). Thus, polyhedron is designed in a way that when virus enter alkaline gut of insect
envelope can break down and dissolve.
After dissolution, virus feeds its nucleocapsid inside cell.
Membrane fusion occurs with micro-villi (3), rods eventually make their way to nucleo-pore.
Actin polymerization allows to help push virus into nuclear pore.
Different routes if enters by budding (gp64) through endocytosis and change in pH by endosome,
will arrive at nucleo-pore as well.
Attachment/Entry and release in cytoplasm, transport to nuclear pore
1. Occlusion bodies (OBs) dissolve in alkaline pH (about pH 10) of gut
2. Occlusion derived virus (ODV) released into gut lumen.
3. ODV membrane fusion with microvilli membrane
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4. ODV nucleocapsids (NCs) released into cytoplasm following fusion
5. ODV NCs transported to nuclear pore via actin polymerization
6. Meanwhile, budded viruses (BV) bind to cell receptor (probably thru a glycoprotein) via viral
receptor binding protein GP64
7. Budded virus brought into cell by receptor-mediated endocytosis
8. Fusion of viral envelope and endosomal envelope following membrane fusion
9. NCs released into cytoplasm following membrane fusion
10. As per Step 5 above, BV nucleocapsids are transported to nuclear pore via actin mediated
polymerization
In nuclear pore, nucleocapsids enter
Viral dna gets uncoated, through the removal of proteins from nucleocapsid
Involves phosphorylation of p6.9 (a basic + charged protein), thereby allowing negatively
charged DNA to detach from proteins.
Both components need to be pull apart by making positive proteins into negative proteins =
repulsion and DNA will not be attracted to proteins.
Once DNA is uncoated, is now accessible to TFs
Immediately after uncoating, immediately early genes are transcribed and expressed, which are
TF that will take over cell machinery and enhance transcription of early viral genes (DNA pol,
helicase, DNA primase etc.).
Now DNA replication can begin followed by transcription of late genes using viral RNA pol =
structural proteins.
Nuclear Entry, uncoating and early expression
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
E. g. spruce budworm virus affects the loss of these trees in canada"s forests. Resulted in a 69 m ha loss of forest area due to insect viruses. Two morphological forms: biphasic replication = budded virus (cell to cell infection) and occlusion derived virus (insect to insect) Ds circular dna genome (80 kb to 180 kb) Nuclear replication (hence some baculoviruses often referred to as nucleopolyhedrovirus or. Genome: ds circular dna genome, 133,894 bp (156 orf) Morphology: budded virus (cell to cell spread) and occlusion derived virus (odv) spread to insects. Budded virus (bv) envelope contains gp64 glycoprotein required for attachment. 37 core proteins of 73 viruses genome phylogeny reflects host phylogeny and virus taxonomy. Infection of host cell/insect orf ingestion of occlusion bodies (obs) into alkaline gut. Entry of midgut epithelial cells by membrane fusion. Nucleocapsid to nuclear pores via actin filament forces.
