Thesis (PhD (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / Title page: Dept. of Genetics, Faculty of Science / ENGLISH ABSTRACT: Grapevine leafroll-associated virus 3 (GLRaV-3) is the type strain for the genus
Ampelovirus, family Closteroviridae. There has been only one report that claimed the
complete nucleotide sequence of GLRaV-3 (isolate NY-1, AF037268). Here we report the
complete sequence of the South African GLRaV-3, isolate GP18 (EU259806) and show a
significantly extended 5’ end. We used RLM-RACE to determine the 5’ end of GP18 and
found the 5’ UTR to be 737 nt compared to 158 nt in the NY-1 sequence. This extended
UTR was found in all other South African isolates of GLRaV-3 that were tested. In two
collaborative studies the existence of the extended 5’ UTR was confirmed and further
investigated. In the first study (Coetzee et al., 2010), metagenomic data generated by next
generation sequencing (Illumina Genome Analyzer II) was analysed for GLRaV-3 specific
sequences. Sequences similar to the GP18 isolate confirmed the sequence of the extended
5’ UTR. In the second study (Jooste et al., 2010), three genetic variants were identified and
their respective 5’ UTRs studied. Great diversity was observed between the 5’ UTRs of the
different genetic variants, however within a variant the 5’ UTR was found to be highly
conserved. Grapevine leafroll-associated virus 3 is a positive sense, single stranded RNA
virus that has been shown, like other closteroviruses, to produce subgenomic (sg) RNAs
during replication. These sgRNAs are deployed for the expression of the ORFs on the 3’
half of the genome. In this study a dsRNA blot confirmed the presence of three, 3’ coterminal
sgRNAs species [sgRNA(ORF3/4), sgRNA(ORF5) and sgRNA(ORF6)] in
GLRaV-3-infected plant material when using a probe directed at the coat protein gene. The
specific 5’ terminal nucleotides for these sgRNAs as well as four additional sgRNAs
[sgRNA(ORF7), sgRNA(ORF8), sgRNA(ORF9) and sgRNA(ORF10-12)] were
determined by RLM-RACE for GLRaV-3 isolate GP18. The construction of a GLRaV-3
mini-replicon, analogous to RNA1 of Lettuce infectious yellows virus, for the evaluation
of putative sg-promoters is also described. / AFRIKAANSE OPSOMMING: Grapevine leafroll-associated virus 3 (GLRaV-3) is ‘n lid van die Closteroviridae familie
en die hooflid vir die genus Ampelovirus. Tot dusver was daar net een studie wat die
volledige nukleïensuurvolgorde van GLRaV-3 gerapporteer het (isolaat NY-1, AF037268).
In hierdie studie rapporteer ons die volledige volgorde van ‘n Suid-Afrikaanse GLRaV-3,
isolaat nl. GP18 (EU259806) wat noemenswaardig langer is aan die 5’ kant. RLM-RACE
is gebruik om die 5’ eindpunt van GP18 te bepaal en daar is gevind dat die 5’
ongetransleerde streek (UTR) 737 nt lank is in vergelyking met die 158 nt van die NY-1
volgorde. Die verlengde 5’ UTR is gevind in alle Suid-Afrikaanse monsters wat getoets is.
Die verlengde 5’ UTR is bevestig en verder bestudeer tydens twee samewerkingsprojekte.
In die eerste studie (Coetzee et al., 2010), is metagenomiese data gegenereer deur
volgende-generasie volgordebepaling (Illumina Genome Analyzer II) en geanaliseer vir
GLRaV-3 spesifieke volgordes. Volgordes soortgelyk aan die GP18 isolaat het die
verlengde 5’ UTR volgorde bevestig. In die tweede studie (Jooste et al., 2010), is drie
genetiese variante van GLRaV-3 geidentifiseer en hulle onderskeie 5’ UTR volgordes
bepaal en bestudeer. Daar is groot diversiteit tussen die 5’ UTRs van die verskillende
genetiese variante gevind, maar tussen isolate van dieselfde variant is die volgordes
gekonserveerd. Grapevine leafroll-associated virus 3 is ‘n positiewe-sin, enkelstring RNA
virus wat al voorheen bewys is om, soos ander closterovirusse, subgenomiese (sg) RNAs te
produseer tydens replisering. Hierdie sgRNAs word ingespan vir die uitdrukking van die
ORFs op die 3’ helfte van die virusgenoom. In hierdie studie is ‘n dsRNA klad gebruik om
die voorkoms van 3’ ko-terminale sgRNAs [sgRNA(ORF3/4), sgRNA(ORF5) and
sgRNA(ORF6)] te bevestig in GLRaV-3 geinfekteerde plantmateriaal deur gebruik te
maak van ‘n peiler teen die kapsiedproteïengeen. Die spesifieke 5’ terminale nukleotiedes
vir hierdie sgRNAs sowel as vier additionele sgRNAs [sgRNA(ORF7), sgRNA(ORF8),
sgRNA(ORF9) and sgRNA(ORF10-12)] is bepaal deur gebruik te maak van RLM-RACE
op die GLRaV-3 isolaat GP18. Die konstruksie van ‘n GLRaV-3 mini-repliserings
konstruk, analoog aan die RNA1 van Lettuce infectious yellows virus, vir die evaluasie
van moontlike sg-promotors word ook beskryf.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/5417 |
| Date | 12 1900 |
| Creators | Maree, Hans Jacob |
| Contributors | Burger, J. T., Freeborough, M-J. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Format | 90 p. : ill. |
| Rights | University of Stellenbosch |
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