Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2010 Aug 3:7:180.
doi: 10.1186/1743-422X-7-180.

3'-coterminal subgenomic RNAs and putative cis-acting elements of Grapevine leafroll-associated virus 3 reveals 'unique' features of gene expression strategy in the genus Ampelovirus

Sridhar Jarugula  1 , Siddarame Gowda, William O Dawson, Rayapati A Naidu
Affiliations
Comparative Study

3'-coterminal subgenomic RNAs and putative cis-acting elements of Grapevine leafroll-associated virus 3 reveals 'unique' features of gene expression strategy in the genus Ampelovirus

Sridhar Jarugula et al. Virol J. .

Abstract

Background: The family Closteroviridae comprises genera with monopartite genomes, Closterovirus and Ampelovirus, and with bipartite and tripartite genomes, Crinivirus. By contrast to closteroviruses in the genera Closterovirus and Crinivirus, much less is known about the molecular biology of viruses in the genus Ampelovirus, although they cause serious diseases in agriculturally important perennial crops like grapevines, pineapple, cherries and plums.

Results: The gene expression and cis-acting elements of Grapevine leafroll-associated virus 3 (GLRaV-3; genus Ampelovirus) was examined and compared to that of other members of the family Closteroviridae. Six putative 3'-coterminal subgenomic (sg) RNAs were abundantly present in grapevine (Vitis vinifera) infected with GLRaV-3. The sgRNAs for coat protein (CP), p21, p20A and p20B were confirmed using gene-specific riboprobes in Northern blot analysis. The 5'-termini of sgRNAs specific to CP, p21, p20A and p20B were mapped in the 18,498 nucleotide (nt) virus genome and their leader sequences determined to be 48, 23, 95 and 125 nt, respectively. No conserved motifs were found around the transcription start site or in the leader sequence of these sgRNAs. The predicted secondary structure analysis of sequences around the start site failed to reveal any conserved motifs among the four sgRNAs. The GLRaV-3 isolate from Washington had a 737 nt long 5' nontranslated region (NTR) with a tandem repeat of 65 nt sequence and differed in sequence and predicted secondary structure with a South Africa isolate. Comparison of the dissimilar sequences of the 5'NTRs did not reveal any common predicted structures. The 3'NTR was shorter and more conserved. The lack of similarity among the cis-acting elements of the diverse viruses in the family Closteroviridae is another measure of the complexity of their evolution.

Conclusions: The results indicate that transcription regulation of GLRaV-3 sgRNAs appears to be different from members of the genus Closterovirus. An analysis of the genome sequence confirmed that GLRaV-3 has an unusually long 5'NTR of 737 nt compared to other monopartite members of the family Closteroviridae, with distinct differences in the sequence and predicted secondary structure when compared to the corresponding region of the GLRaV-3 isolate from South Africa.

PubMed Disclaimer

Figures

Figure 1
Figure 1
A schematic diagram of the GLRaV-3 genome. The ORFs, numbered as 1 to 12 above the diagram, are shown as boxes with associated protein designations. L-Pro, leader proteinase; AlkB, AlkB domain; MET, HEL, and POL, methyltransferase, RNA helicase, and RNA-dependent RNA polymerase domains of the replicase, respectively; p6, a 6-kDa protein; p5, a 5-kDa protein; HSP70 h, a HSP70-homologue; p55, a 55-kDa protein; CP, the major capsid protein; CPm, the minor capsid protein; and p21, p20A, p20B, p4 and p7 are the 21-, 19.6-, 19.7-, 4- and 7-kDa proteins, respectively. Below the genome map is a representation of (right) the 11 putative subgenomic messenger (m) RNAs for the 3' genes and (left) the polyproteins from ORFs 1a and 1b. The subgenomic mRNAs and their transcription start sites identified in this study are shown with an asterisk. Arrow head indicates site of +1 ribosomal frameshift.
Figure 2
Figure 2
Northern blot analysis of total RNA extracted from grapevine (cv. Merlot) infected with GLRaV-3. Northern blot hybridizations were carried out using a positive-stranded gene-specific riboprobes containing 3'terminus, p20A, p21, CPm, and CP sequences. Position of subgenomic (sg) RNAs is indicated by arrows on the left. Location of sgRNAs for CPm, p55 and HSP70h were tentative and indicated with an asterisk. The non-specific band present in all lanes is indicated by an arrow head.
Figure 3
Figure 3
RACE analysis of 5'NTR of GLRaV-3. (a) The schematic diagram showing the locations of primers used and expected size of amplicons and (b) agarose gel showing virus-specific DNA fragments (shown by arrow head on the right) amplified from cDNA made using primer AR. Lane 1 shows 883 bp fragment amplified with primers AAP and M1012 and lane 2 shows 1059 bp fragment amplified with primers AAP and M1013 primers. Lane M shows 1kb plus DNA marker (Invitrogen) for estimating the size of amplified DNA fragment. The size of marker DNA bands is indicated to the left. See Materials and methods for primer details.
Figure 4
Figure 4
Pairwise comparison of 5'NTR sequences of GLRaV-3 isolates from Washington (WA) and South Africa (SA). (a) The alignment was adjusted manually and residues that are unique to each isolate are shown by an asterisk beneath them. The 65 nt tandem repeat in WA isolate is represented in bold and as underline (red and blue colored text), and extra sequence present only in South Africa isolate is in bold italics (green colored text), (b) pairwise alignment of 65 nt tandem repeat in Washington isolate showing differences at four nucleotide positions.
Figure 5
Figure 5
The computer-predicted secondary structure of the NTRs of GLRaV-3. (a) 5'NTR of Washington (WA) and South Africa (SA) isolates and (b) 3'NTR of WA isolate (complement).
Figure 6
Figure 6
Transcription start site (TSS) of four subgenomic (sg) RNAs. (a) Nucleotide sequence of the portion of the genomic RNA showing the TSS of sgRNAs specific to CP, p21, p20A and p20B and (b) the predicted secondary structure of the minus-strand sequences around the TSS of the sgRNAs. Numbers indicate nucleotide coordinates with the genomic RNA. The TSS is indicated by a bent arrow (with +1 adenylate underlined) in (a) and by an arrow in (b), termination codon of the preceding ORF is underlined and marked with an asterisk and the translation initiation codon is in bold and marked with an arrow.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Karasev AV. Genetic diversity and evolution of closteroviruses. Annu Rev Phytopathol. 2000;38:293–324. doi: 10.1146/annurev.phyto.38.1.293. - DOI - PubMed
    1. Martelli GP, Agranovsky AA, Bar-Joseph M, Boscia D, Candresse T, Coutts RHA, Dolja VV, Falk BW, Gonsalves D, Jelkmann W, Karasev AV, Minafra A, Namba S, Vetten HJ, Wisler GC, Yoshikawa H. The family Closteroviridae revised. Arch Virol. 2002;147:2039–2044. doi: 10.1007/s007050200048. - DOI - PubMed
    1. Dolja VV. Beet yellows virus: the importance of being different. Molecular Plant Pathology. 2003;4:91–98. doi: 10.1046/j.1364-3703.2003.00154.x. - DOI - PubMed
    1. Dolja VV, Kreuze JF, Valkonen JPT. Comparative and functional genomics of closteroviruses. Virus Res. 2006;117:38–51. doi: 10.1016/j.virusres.2006.02.002. - DOI - PMC - PubMed
    1. Salem NM, Chen AY, Tzanetakis IE, Mongkolsiriwattana C, Ng JC. Further complexity of the genus Crinivirus revealed by the complete genome sequence of Lettuce chlorosis virus (LCV) and the similar temporal accumulation of LCV genomic RNAs 1 and 2. Virology. 2009;390:45–55. doi: 10.1016/j.virol.2009.04.025. - DOI - PubMed

Publication types

Associated data