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CSB351Y1 Study Guide - Rna Virus, Reverse Transcriptase, Plant Virus


Department
Cell and Systems Biology
Course Code
CSB351Y1
Professor
Mounir Abou Haidar

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The 35S RNA is particularly complex, containing highly structured 600 nt long leader
sequence with 6-8 short ORF (encode proteins 2-35 aa in length)
This leader is followed by 7 tightly arranged longer ORFs that encode all the viral proteins
The mechanism of expression of these proteins is very special
ORF VI protein (TAV, encoded by the 19S RNA) controls translation reinitiation of major
ORF on the polycistronic 35S RNA
Ribosomal shunt translational strategy

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Replication cycle of CaMV
1. Entry of the virus into the host cell
2. The virion is transported to the nuclear pore
3. Genome imported into the nucleus
4. Repair of DNA sequence discontinuities and association of the genome with histones to form
a minichromosome
o The viral DNA is transcribed by cellular RNA polymerase II into the pregenomic 35S
and subgenomic 19S RNA
5. Translation of the 19S RNA results in production of P6
6. P6 condenses into cytoplasmic virus factories and transactivates the translation of all other viral proteins from 35S RNA
7. Among these, P5 replicates the genome by reverse transcriptase using 35S RNA as a template
8. The newly transcribed viral DNA is encapsidated, resulting in generation of virus particles in the virus factories
9. With this last step, the minimal time required for one round of replication is over, symbolised by the clock
The replication is similar to that of other retroviruses
The 35S RNA contains at the 5’ end region 13 nt complementary to the
3’ end of tRNA Met and 190 nt of repeated sequences at the two ends
of the RNA
The 3’ OH end of the tRNA Met is used as a primer for the reverse
transcriptase to start the synthesis of a complementary DNA of the virus
The ribonuclease H activity of the RT degrades the RNA hybridised to
the cDNA
However, small pieces of RNA are resistant to the RNase H activity
They serve for synthesis of a 2nd strand DNA
These small RNA pieces are located at 3 different positions on the viral
DNA and at their position the DNA is interrupted
Assembly
A specific interaction takes place between the CP of CaMV and the conserved
purine-rich sequence in the central part of the CaMV pgRNA leader (35S RNA)
35S RNAs are RT into dsDNAs in the form of immature virions which are likely
assembled in inclusion bodies in the cytoplasm
Summary of CaMV Replication cycle
Once introduced within a host cell, virions migrate to the nuclear envelope,
where they presumably decapsidate (uncoating)
The viral genomes then enter the nucleus where they form minichromosomes
that are transcribed by the host RNA polymerase II to generate 2 mRNAs, the
polycistronic 35S RNA comprising the entire genome encoding a single protein,
P6 (TAV or transactivator protein)
In the cytoplasm, P6 is translated from the 19S RNA and aggregates in small
inclusion bodies, where it transactivates translation of all other viral proteins
from the 35S RNA
The 35S RNA is also the template for the production of viral genomic dsDNA,
which is probably encapsidated into virions during or shortly after its synthesis
The progeny virions are sequestered mostly into the P6 inclusions that grow over time and form viral factories
The 35S promoter of CaMV is widely used in biotechnology
The promoter of the 35S RNA is a very strong constitutive promoter responsible for the transcription of the whole CaMV
genome
It is well known for its use in plant transformation
It causes high levels of gene expression in dicot plants
However, it is less effective in monocots, especially in cereals
The differences in behavior are probably due to differences in quality and/or quantity of regulatory factors

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The promoter was named CaMV 35S promoter because the coefficient of sedimentation of the viral transcript, whose
expression is naturally driven by this promoter, is 35S
One of the most widely used, general-purpose constitutive promoters
The CaMV promoter is used in most transgenic crops to activate foreign genes which have been artificially inserted into the
host plant
It is inserted into transgenic plants in a form which is different to its naturally occurring state arising in its natural Brassica
plant hosts
This enables it to operate in a wide range of host-organism environments which would otherwise not be possible
35S promoter is active in all plant tissues: leaves, roots
Recombination
Cloned CaMV DNA is infectious
2 overlapping pieces of the genome are also infectious, suggesting that recombination of CaMV DNA exists
2 pieces of CaMV DNA from 2 different strains of the virus were used to infect plants
The results indicate that a portion of the virus particles obtained have the characteristics of the 2 strains
Can Caulimoviruses integrate into the host genome like retroviruses?
Do not integrate obligatorily into the host genome
But ‘illegitimately’ integrated sequences have been found for several genera to date
These have been named commonly ‘endogenour plant pararetroviruses region of chromosomes, are passively replicated
together with the host DNA, and are inherited from generation to generation
Lecture 14 Viroids, virusoids and ribozymes
Identification of viroids “like a virus”
Eg. Potato spindle tuber disease (PSTV)
Reported in potato in the 1920’s
Potato tuber looks like a spindle
Symptoms slowly spread through fields (an infectious agent of sorts)
Eg. Cadang-Cadang disease
Killed a large number of coconut palms
Great economic loss
Geographical distribution
Viroids are found over many parts of the world infecting a large number of plant species
Transmission of PSTV
No arthropods, seed, or soil transmission
By contact in the field and mechanical transmission in the lab
Viroids
Needs CP to be transmissible
Don’t code for anything except replication
dsRNA into nucleus
RNA polymerase of the cell (DNA dependent)
1 replicated in the nucleus; a few not replicate in the chloroplast these have ribozymes
Viroids are infectious agents capable of self-replication in plants
They consist of a few hundred nt of highly complementary, circular, ssRNA, but are not encapsidated by a CP
The smallest discovered is a 220 nt RNA associated with the rice yellow mottle virus (RYMV)
In comparison, the genome of the smallest known viruses capable of causing an infection by themselves are around 2k nt
Viroid RNA does not code for any protein
The first viroid to be identified was potato spindle tuber viroid (PSTVd)
Some 33 species of viroid have been identified
Classification
Viroids are common plant pathogen which are a serious economic problem
25 different viroid sequences have been determined and numerous variants identified
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